RECOMBINANT SACCHAROMYCES CEREVISIAE AND ISOBUTANOL PRODUCTION METHOD

专利摘要:
yeast microorganisms with reduced by-product accumulation for improved production of fuels, chemicals and amino acids. the present invention relates to recombinant microorganisms comprising biosynthetic pathways and methods of using said recombinant microorganisms to produce various beneficial metabolites. in various aspects of the invention, recombinant microorganisms can further comprise one or more modifications that result in the reduction or elimination of 3-keto acid (e.g., aceto-lactate and 2-aceto-2-hydroxybutyrate) and / or derived products of aldehydes. in various embodiments described here, recombinant microorganisms can be microorganisms of saccharomyces clade, microorganisms of crabtree-negative yeast, microorganisms of crabtree-positive yeast, microorganisms of post-wgd yeast (genome duplication whole), pre-wgd yeast microorganisms (whole genome duplication) and non-fermenting yeast microorganisms.
公开号:BR112012020261B1
申请号:R112012020261-8
申请日:2011-02-11
公开日:2020-03-10
发明作者:Kent Evans；Julie Kelly；Sabine Bastian；Frances Arnold；Thomas Buelter；Andrew Hawkins；Stephanie Proter-Scheinman；Peter Meinhold；Catherine Asleson Dundon；Aristos Aristidou；Jun URANO；Doug Lies；Matthew Peters；Melissa Dey；Justas Jancauskas；Ruth Berry
申请人:Gevo, Inc.；California Institute Of Technology；
IPC主号:

专利说明:

RECOMBINANT SACCHAROMYCES CEREVISIAE AND ISOBUTANOL PRODUCTION METHOD
CROSS REFERENCE TO RELATED ORDERS
This order claims priority for Provisional Order US 61 / 304,069, filed on February 12, 2010; Provisional Application US 61 / 308,568, filed on February 26, 2010; Provisional Application US 61 / 282,641, filed on March 10, 2010; Provisional Application US 61 / 352,133, filed on June 7, 2010; Provisional Application US 61 / 411,885, filed on November 9, 2010, and Provisional Application US 61 / 430,801, filed on January 7, 2011, each of which is incorporated by reference in its entirety for all purposes.
RECOGNITION OF GOVERNMENT SUPPORT
This invention was made with government support under Contract No. 200910006-05919, granted by the United States Department of Agriculture, and under Contract No. W911NF-09-2-0022, granted by the Armed Forces Research Laboratory from United States. The government has certain rights in the invention.
TECHNICAL FIELD
Recombinant microorganisms and methods of producing such organisms are provided. Also provided are methods of producing beneficial metabolites, including fuels, chemicals, and amino acids by contacting a suitable substrate with recombinant microorganisms and enzyme preparations thereof.
DESCRIPTION OF THE TEXT FILE ELECTRONICALLY SUBMITTED
The contents of the text file sent electronically as an attachment are incorporated by reference in their entirety: A computer readable copy of the Sequence Listing (file name: GEVO_045_03WO_SeqList_ST25.txt, registration date: February 9, 2011, file size file: 306 kilobytes).
BACKGROUND The ability of microorganisms to convert pyruvate into beneficial metabolites, including fuels, chemicals, and amino acids has been widely described in the literature in recent years. See, for example, Alper et al., 2009, Nature Microbiol. Rev. 7: 715-723. Recombinant engineering techniques have allowed the creation of microorganisms that express biosynthetic pathways, capable of producing a number of useful products, such as valine, isoleucine, leucine and pantothenic acid (vitamin B5). In addition, fuels such as isobutanol have been produced recombinantly in microorganisms that express a heterologous metabolic pathway (See, for example, WO / 2007/050671 by Donaldson et al., And WO / 2008/098227 by Liao, et al.). Although the designed microorganisms represent potentially useful tools for the renewable production of fuels, chemicals, and amino acids, many of these microorganisms have fallen short of commercial relevance due to their low performance characteristics, including low productivity, low titers and low income.
One of the main reasons for the suboptimal performance observed in many existing microorganisms is the undesirable conversion of intermediates from the undesirable by-products pathway. The present inventors have identified several by-products, including 2,3-dihydroxy-2-methylbutanoic acid (DH2MB) (CAS # 14868-24-7), 2-ethyl-2,3-dihydroxybutyrate, 2,3-di -hydroxy-2-methyl-butanonate, isobutyrate, 3-methyl-1-butyrate, 2-methyl-1-butyrate, and propionate, which are derived from various intermediates in biosynthetic pathways used to produce fuels, chemicals, and amino acids. The accumulation of these by-products negatively affects the synthesis and production of desirable metabolites in a variety of fermentation reactions. Until now, the enzymatic activities responsible for the production of these undesirable by-products have not been characterized. More particularly, the present patent application shows that the activities of a 3-keto acid reductase (3-KAR) and an aldehyde dehydrogenase (ALDH) allow the formation of these by-products from important intermediates in the biosynthesis pathway. The present invention results from the study of these enzymatic activities and shows that the deletion of 3-KAR and / or ALDH enzymes considerably reduces or eliminates the formation of unwanted by-products, and, concomitantly, improves the yields and titrations of beneficial metabolites. The present application also shows that the increase in 3-KAR and / or ALDH enzymatic activities can be used to increase the production of several by-products, such as 2,3-dihydroxy-2-methylbutanoic acid (DH2MB), 2 -ethyl-2,3-hydroxybutyrate, 2,3-dihydroxy-2-methyl-butanonate, isobutyrate, 3-methyl-1-butyrate, 2-methyl-1-butyrate, and propionate.
SUMMARY OF THE INVENTION
The present inventors have found that unwanted by-products can accumulate during the various fermentation processes, including fermentation of the biofuel candidate, isobutanol. The accumulation of these undesirable by-products results from the undesirable conversion of pathway intermediates, including 3-keto, acetolactate and 2-aceto-2-hydroxybutyrate and / or aldehydes, such as isobutyraldehyde, 1-butanal, 1-propanal, 2- methyl-1-butanal, and 3-methyl-1-butanal. The conversion of these intermediates to unwanted by-products can hamper the optimal productivity and yield of a 3-keto acid and / or aldehyde-derived products. Therefore, the present inventors have developed methods to reduce the conversion of 3-keto acid and / or aldehyde intermediates to various fermentation by-products during processes in which a 3-keto acid and / or an aldehyde acts as an intermediate route.
In a first aspect, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway in which a 3-keto acid and / or an aldehyde is / are intermediate (s), wherein said recombinant microorganism is (a) substantially free of an enzyme that catalyzes the conversion of a 3-keto acid to a 3-hydroxy acid, (b) substantially free of an enzyme that catalyzes the conversion of an aldehyde to an acid by-product, (c) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of a 3-keto acid to a 3-hydroxy acid, and / or (d) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of a aldehyde with the acidic by-product. In one embodiment, 3-keto acid is acetolactate. In another embodiment, 3-keto acid is 2-aceto-2-hydroxy-butyrate.
In one embodiment, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway, which uses 3-keto acid, acetolactate, as an intermediate, wherein said recombinant microorganism is developed to reduce or eliminate expression or activity of an enzyme that catalyzes the conversion of acetolactate to the corresponding 3-hydroxy acid, DH2MB. In some embodiments, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR).
In one embodiment, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway, which uses 3-keto, 2-aceto-2-hydroxybutyrate, as an intermediate, in which said recombinant microorganism is developed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of acetolactate to the corresponding 3-hydroxy acid, 2-ethyl-2,3-dihydroxybutanoate. In some embodiments, the enzyme that catalyzes the conversion of 2-ethyl-2-hydroxy-butyrate to 2-ethyl-2,3-dihydroxybutanoate is a 3-keto acid reductase (3-KAR).
In one embodiment, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway that uses an aldehyde as an intermediate, wherein said recombinant microorganism is constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of the aldehyde to an acid by-product. In some embodiments, the enzyme that catalyzes the conversion of the aldehyde to an acid by-product is an aldehyde dehydrogenase (ALDH).
In one embodiment, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway that uses both 3-keto acid and an aldehyde, as intermediates, in which said recombinant microorganism is (a) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of a 3-keto acid intermediate to a 3-hydroxy acid by-product, and (b) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of an intermediate of aldehyde to an acid by-product. In one embodiment, 3-keto acid is acetolactate and the by-product of 3-hydroxy acid is DH2MB. In another embodiment, 3-keto acid is 2-aceto-2-hydroxy-butyrate and the 3-hydroxy acid by-product is 2-ethyl-2,3-dihydroxybutanoate. In some embodiments, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR). In some other embodiments, the enzyme that catalyzes the conversion of 2-aceto-2-hydroxy-butyrate to 2-ethyl-2,3-dihydroxybutanoate is a 3-keto acid reductase (3-KAR). In some other embodiments, the enzyme that catalyzes the conversion of the aldehyde to an acid by-product is an aldehyde dehydrogenase (ALDH). In still other modalities, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR) and the enzyme that catalyzes the conversion of the aldehyde to an acid by-product is an aldehyde dehydrogenase (ALDH). In still other embodiments, the enzyme that catalyzes the conversion of 2-aceto-2-hydroxy-butyrate to 2-ethyl-2,3-dihydroxybutanoate is a 3-keto acid reductase (3-KAR) and the enzyme that catalyzes the conversion from aldehyde to an acid by-product is an aldehyde dehydrogenase (ALDH).
In various embodiments described herein, the recombinant microorganisms of the present invention may comprise a reduction or deletion of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of a 3-keto acid intermediate to a 3-hydroxy acid by-product. In one embodiment, the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of a 3-keto acid intermediate to a 3-hydroxy acid by-product is reduced by at least about 50%. In another embodiment, the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of a 3-keto acid intermediate to a 3-hydroxy acid by-product is reduced by at least about 60%, by at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , or at least about 99% as compared to a recombinant microorganism not comprising a reduction or deletion of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of a 3-keto acid intermediate to a by-product of 3-hydroxy acid. In one embodiment, the 3-keto acid intermediate is acetolactate and the 3-hydroxy acid by-product is DH2MB. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate and the 3-hydroxy acid by-product is 2-ethyl-2,3-dihydroxybutanoate.
In the various embodiments described herein, the protein involved in the catalysis of the conversion of a 3-keto acid intermediate to a 3-hydroxy acid by-product is a ketorreductase. In an exemplary embodiment, ketorreductase is a 3-keto acid reductase (3-KAR). In another embodiment, the protein is a short-chain alcohol dehydrogenase. In yet another embodiment, the protein is a medium chain alcohol dehydrogenase. In yet another embodiment, the protein is an aldose reductase. In yet another embodiment, the protein is a D-hydroxy acid dehydrogenase. In yet another embodiment, the protein is a lactate dehydrogenase. In yet another modality, the protein is selected from the group consisting of genes YAL060W, YJR159W, YGL157W, YBL114W, YOR120W, YKL055C, YBR159W, YBR149W, YDL168W, YDR368W, YLR426W, YCR107, YCR107W, YR107 , YBR046C, YHR104W, YIR036C, YDL174C, YDR541C, YBR145W, YGL039W, YCR105W, YDL124W, YIR035C, YFL056C, YNL274C, YLR255C, YGL185C, YGL256W, YJR096W, YMR226C, YJR155W, YPL275W, YOR388C, YLR070C, YMR083W, YER081W, YJR139C, YDL243C , YPL113C, YOL165C, YML086C, YMR303C, YDL246C, YLR070C, YHR063C, YNL331C, YFL057C, YIL155C, YOL086C, YAL061W, YDR127W, YPR127W, YIL0CW, YIL0CW, YIL0C, YIL018
In one embodiment, the endogenous protein is a 3-keto acid reductase (3-KAR).
In an exemplary embodiment, 3-keto acid reductase is the protein of S. cerevisiae YMR226C (SEQ ID NO: 1), used interchangeably here with “TMA29”. In some embodiments, the endogenous protein may be the protein of S. cerevisiae YMR226C (SEQ ID NO: 1) or a homologous or variant thereof. In one embodiment, the homologue can be selected from the group consisting of Vanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomyces castellii (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomyces bayanus (SEQ ID NO: 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), Kluyveromyces lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQ ID NO: 9) NO: 10), Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12), Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14), Candida dubliniensis (SEQ ID NO: 14) NO: 15), Candida albicans (SEQ ID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO: 18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO: 19) NO: 20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQ ID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).
In one embodiment, the recombinant microorganism includes a mutation in at least one gene encoding a 3-keto acid reductase, resulting in a reduction in the 3-keto acid reductase activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism includes a partial deletion of the gene that encodes a 3-keto acid reductase, resulting in a reduction in the 3-keto acid reductase activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprises a complete deletion of the gene encoding a 3-keto acid reductase resulting in a reduction in the 3-keto acid reductase activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with the gene encoding a 3-keto acid reductase, resulting in a reduction in the expression of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator resulting in a reduction in the transcription of a gene encoding a 3-keto acid reductase. In yet another embodiment, the recombinant microorganism comprises mutations in all genes that encode a 3-keto acid reductase, resulting in a reduction in the activity of a polypeptide encoded by the gene (s). In one embodiment, the activity or expression of 3-keto acid reductase is reduced by at least about 50%. In another embodiment, the activity or expression of 3-keto acid reductase is reduced by at least about 60%, by at least about 65%, by at least about 70%, by at least about 75%, by at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% compared to a recombinant microorganism that does not comprise a decrease activity or expression of 3-keto acid reductase. In one embodiment, said 3-keto acid reductase is encoded by the S. cerevisiae TMA29 gene (YMR226C) or a homologue thereof.
In various embodiments described herein, the recombinant microorganisms of the present invention may comprise a reduction or deletion of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product. In one embodiment, the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product is reduced by at least about 50%. In another embodiment, the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product is reduced by at least about 60%, by at least about 65%, by at least about 70 %, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% compared to a non-recombinant microorganism comprising a reduction or deletion of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product.
In various embodiments described here, the endogenous protein involved in the catalysis of the conversion of an aldehyde to an acid by-product is an aldehyde dehydrogenase (ALDH). In one embodiment, aldehyde dehydrogenase is encoded by a gene selected from the group consisting of ALD2, ALD3, ALD4, ALD5, ALD6, and HFD1, and homologues and variants thereof. In an exemplary embodiment, aldehyde dehydrogenase is the protein of S. cerevisiae ALD6 (SEQ ID NO: 25). In some embodiments, aldehyde dehydrogenase is the protein of S. cerevisiae ALD6 (SEQ ID NO: 25) or a homologous or variant thereof. In one embodiment, the homologue is selected from the group consisting of Saccharomyces castelli (SEQ ID NO: 26), Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO: 28), Kluyveromyces lactis (SEQ ID NO: 27) : 29), Kluyveromyces thermotolerans (SEQ ID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQ ID NO: 33), Saccharomyces cerevisiae (EC11 SEQ ID NO: 34), Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomyces cerevisiae AWRI1631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a (SEQ ID NO: 37), Pichiapastoris (SEQ ID NO: 38) , Kluyveromyces marxianus (SEQ ID NO: 39), Schizosaccharomycespombe (SEQ ID NO: 40), and Schizosaccharomyces pombe (SEQ ID NO: 41).
In one embodiment, the recombinant microorganism includes a mutation in at least one gene encoding an aldehyde dehydrogenase, resulting in a reduction in the aldehyde dehydrogenase activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism includes a partial deletion of the gene encoding an aldehyde dehydrogenase resulting in a reduction in the aldehyde dehydrogenase activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprises a complete deletion of the gene encoding an aldehyde dehydrogenase resulting in a reduction in the aldehyde dehydrogenase activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with the gene encoding an aldehyde dehydrogenase resulting in a reduction in the expression of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator resulting in a reduction in the transcription of a gene encoding an aldehyde dehydrogenase. In yet another embodiment, the recombinant microorganism comprises mutations in all genes that encode an aldehyde dehydrogenase resulting in a reduction in the activity of a polypeptide encoded by the gene (s). In one embodiment, the activity or expression of aldehyde dehydrogenase is reduced by at least about 50%. In another embodiment, the activity or expression of aldehyde dehydrogenase is reduced by at least about 60%, by at least about 65%, by at least about 70%, by at least about 75%, by at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% compared to a recombinant microorganism, not comprising a reduction in activity or expression of aldehyde dehydrogenase. In one embodiment, said aldehyde dehydrogenase is encoded by the S. cerevisiae ALD6 gene or a homologue thereof.
In various embodiments described herein, the recombinant microorganism can comprise a biosynthetic pathway that uses a 3-keto acid, as an intermediate. In one embodiment, the 3-keto acid intermediate is acetolactate. The biosynthetic pathway that uses acetolactate as an intermediate product can be selected from a pathway for the biosynthesis of isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, acid pantothenic, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate. The biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate as an intermediate can be selected from a pathway for the biosynthesis of 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1 -hexanol, and 5-methyl-1-heptanol.
In various embodiments described here, the recombinant microorganism can comprise a biosynthetic pathway that uses an aldehyde as an intermediate. The biosynthetic pathway that uses an aldehyde as an intermediate can be selected from a pathway for the biosynthesis of isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol, 1- pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol. In several embodiments described here, the aldehyde intermediate can be selected from isobutyraldehyde, 1-butanal, 2-methyl-1-butanal, 3-methyl-1-butanal, 1-propanal, 1-pentanal, 1-hexanal, 3-methyl-1-pentanal, 4-methyl-1-pentanal, 4-methyl-1-hexanal, and 5-methyl-1-heptanal.
In various embodiments described here, the recombinant microorganism can comprise a biosynthetic pathway that uses a 3-keto acid and an aldehyde as intermediates. In one embodiment, the 3-keto acid intermediate is acetolactate. The biosynthetic pathway that uses acetolactate and an aldehyde as intermediates can be selected from a pathway for the biosynthesis of isobutanol, 1-butanol, and 3-methyl-1-butanol. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate. The biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate and an aldehyde as intermediates can be selected from a pathway for the biosynthesis of 2-methyl-1-butanol, 3-methyl-1-pentanol, 4-methyl-1 -hexanol, and 5-methyl-1-heptanol.
In one embodiment, the invention is directed to a recombinant microorganism for the production of isobutanol, in which said recombinant microorganism comprises an isobutanol-producing metabolic pathway and in which said microorganism is constructed to reduce or eliminate expression or activity of an enzyme that catalyzes the conversion of acetolactate to DH2MB. In some embodiments, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR). In a specific embodiment, a 3-keto acid reductase is encoded by the S. cerevisiae TMA29 (YMR226C) gene or a homologue thereof.
In another embodiment, the invention is directed to a recombinant microorganism for the production of isobutanol, in which said recombinant microorganism comprises an isobutanol-producing metabolic pathway and in which said microorganism is constructed to reduce or eliminate expression or activity of an enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate. In some embodiments, the enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate is an aldehyde dehydrogenase. In a specific embodiment, aldehyde dehydrogenase is encoded by the gene S. cerevisiae ALD6 or a homologue thereof.
In yet another embodiment, the invention is directed to a recombinant microorganism for the production of isobutanol, in which said recombinant microorganism comprises a metabolic pathway producing isobutanol and in which said microorganism is (i) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of acetolactate to DH2MB and (ii) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate. In some embodiments, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR). In a specific embodiment, a 3-keto acid reductase is encoded by the S. cerevisiae TMA29 gene (YMR226C) or a homologue thereof. In some embodiments, the enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate is an aldehyde dehydrogenase. In a specific embodiment, aldehyde dehydrogenase is encoded by the gene S. cerevisiae ALD6 or a homologue thereof.
In one embodiment, the isobutanol-producing metabolic pathway comprises at least one exogenous gene that catalyzes a step in the conversion of pyruvate to isobutanol. In another embodiment, the isobutanol-producing metabolic pathway comprises at least two exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol-producing metabolic pathway comprises at least three exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol-producing metabolic pathway comprises at least four exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol-producing metabolic pathway comprises at least five exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol.
In one embodiment, one or more of the genes in the isobutanol pathway encodes an enzyme that is located in the cytosol. In one embodiment, recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least one enzyme from the isobutanol pathway located in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least two enzymes from the isobutanol pathway located in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least three enzymes from the isobutanol pathway located in the cytosol. In yet another embodiment, recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least four enzymes from the isobutanol pathway located in the cytosol. In an exemplary embodiment, recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least five enzymes from the isobutanol pathway located in the cytosol.
In various modalities described here, the genes of the isobutanol pathway encode enzymes selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductisomerase (KARI), dihydroxy acid dehydratase (DHAD), 2-keto-acid decarboxylase ( KIVD), and alcohol dehydrogenase (ADH).
In another aspect, the recombinant microorganism can be constructed to reduce the conversion of isobutanol to isobutyraldehyde by reducing and / or eliminating the expression of one or more alcohol dehydrogenases. In a specific embodiment, alcohol dehydrogenase is encoded by a gene selected from the group consisting of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7, and homologues and variants thereof.
In another aspect, the present invention relates to modified alcohol dehydrogenase (ADH) enzymes that exhibit enhanced ability to convert isobutyraldehyde to isobutanol. In general, cells that express these enhanced ADH enzymes will produce increased levels of isobutanol during fermentation reactions. Although the modified ADH enzymes of the present invention are useful in isobutanol-producing fermentation reactions, it will be understood by those skilled in the art with the aid of this disclosure that the modified ADH enzymes are also useful in fermentation reactions that produce other alcohols, such as 1 -propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-methyl-1-butanol, and 3-methyl-1-butanol.
In certain aspects, the invention is directed to alcohol dehydrogenases (ADHs), which have been modified to enhance the enzyme's ability to convert isobutyraldehyde to isobutanol. Examples of such ADHs include enzymes having one or more mutations at the positions corresponding to the amino acids selected from: (a) L. lactis AdhA tyrosine 50 (SEQ ID NO: 185); (b) L. lactis AdhA glutamine 77 (SEQ ID NO: 185); (c) L. lactis AdhA valine 108 (SEQ ID NO: 185); (d) tyrosine 113 from L. lactis AdhA (SEQ ID NO: 185); (e) L. lactis AdhA isoleucine 212 (SEQ ID NO: 185); and (f) L. lactis AdhA leucine 264 (SEQ ID NO: 185), where AdhA (SEQ ID NO: 185) is encoded by the L. lactis alcohol dehydrogenase (ADH) adha gene (SEQ ID NO: 184) or an optimized codon version of it (SEQ ID NO: 206).
In one embodiment, the modified ADH enzyme contains a mutation in the amino acid corresponding to position 50 of L. lactis AdhA (SEQ ID NO: 185). In another embodiment, the modified ADH enzyme contains a mutation in the amino acid corresponding to position 77 of L. lactis AdhA (SEQ ID NO: 185). In yet another embodiment, the modified ADH enzyme contains a mutation in the amino acid corresponding to position 108 of L. lactis AdhA (SEQ ID NO: 185). In yet another embodiment, the modified ADH enzyme contains a mutation in the amino acid corresponding to position 113 of L. lactis AdhA (SEQ ID NO: 185). In yet another embodiment, the modified ADH enzyme contains a mutation in the amino acid corresponding to position 212 of L. lactis AdhA (SEQ ID NO: 185). In yet another embodiment, the modified ADH enzyme contains a mutation in the amino acid corresponding to position 264 of L. lactis AdhA (SEQ ID NO: 185).
In one embodiment, the ADH enzyme contains two or more mutations in the amino acids corresponding to the positions described above. In another embodiment, the ADH enzyme contains three or more mutations in the amino acids corresponding to the positions described above. In yet another embodiment, the ADH enzyme contains four or more mutations in the amino acids corresponding to the positions described above. In yet another embodiment, the ADH enzyme contains five or more mutations in the amino acids corresponding to the positions described above. In yet another embodiment, the ADH enzyme contains six mutations in the amino acids corresponding to the positions described above.
In a specific embodiment, the invention is directed to ADH enzymes in which the tyrosine at position 50 is replaced with a phenylalanine or tryptophan residue. In another specific embodiment, the invention is directed to ADH enzymes in which the glutamine at position 77 is replaced with an arginine or serine residue. In another specific embodiment, the invention is directed to ADH enzymes in which the valine at position 108 is replaced with a serine or alanine residue. In another specific embodiment, the invention is directed to ADH enzymes in which the tyrosine at position 113 is replaced with a phenylalanine or glycine residue. In another specific embodiment, the invention is directed to ADH enzymes in which the isoleucine at position 212 is replaced with a threonine or valine residue. In yet another specific embodiment, the invention is directed to ADH enzymes in which the leucine at position 264 is replaced with a valine residue. In one embodiment, the ADH enzyme contains two or more mutations in the amino acids corresponding to the positions described in these specific modalities. In another embodiment, the ADH enzyme contains three or more mutations in the amino acids corresponding to the positions described in these specific modalities. In yet another embodiment, the ADH enzyme contains four or more mutations in the amino acids corresponding to the positions described in these specific modalities. In yet another embodiment, the ADH enzyme contains five or more mutations in the amino acids corresponding to the positions described in these specific modalities. In yet another embodiment, the ADH enzyme contains six mutations in the amino acids corresponding to the positions described in these specific modalities.
In certain exemplary embodiments, the ADH enzyme comprises a selected sequence of SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO : 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, and homologues or variants thereof comprising corresponding mutations compared to the parent or wild-type enzyme.
As mentioned in the previous paragraph, still included in the scope of the invention are ADH enzymes, different from L. lactis AdhA (SEQ ID NO: 185), which contain changes corresponding to those established above. Such ADH enzymes can include, but are not limited to, ADH enzymes listed in Table 97.
In some embodiments, the ADH enzymes to be modified are NADH-dependent ADH enzymes. Examples of such NADH-dependent ADH enzymes are described in the commonly owned copending US Patent Publication and copending US Patent Publication No. 2010/0143997, which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the genes that originally encode ADH enzymes that use NADPH are modified to change the enzyme's cofactor preference to NADH.
As described herein, modified ADHs generally exhibit an improved ability to convert isobutyraldehyde to isobutanol, as compared to wild-type or parental ADH. Preferably, the catalytic efficiency (kcat / KM) of a modified ADH enzyme is improved by at least 5% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 15% compared to parental or wild-type ADH. Most preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 25% compared to parental or wild-type ADH. Most preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 50% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 75% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 100% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 200% compared to parental or wild-type ADH. Most preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 500% compared to parental or wild-type ADH. Most preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 1000% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 2000% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 3000% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of a modified ADH enzyme is improved by at least 3500% compared to parental or wild-type ADH.
In additional aspects, the invention is directed to modified ADH enzymes that have been codon optimized for expression in certain desirable host organisms, such as yeasts and E. coli. In other aspects, the present invention is directed to recombinant host cells comprising a modified ADH enzyme of the invention. In accordance with this aspect, the present invention is also directed to methods of using modified ADH enzymes in any fermentation process, where conversion from isobutyraldehyde to isobutanol is desired. In an embodiment according to this aspect, the modified ADH enzyme may be suitable to enhance the ability of host cells to produce isobutanol. In another embodiment according to this aspect, modified ADH enzymes may be suitable for enhancing a host cell's ability to produce 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-methyl-1 -butanol, and 3-methyl-1-butanol.
In various embodiments described here, recombinant microorganisms comprising a modified ADH can further be constructed to express an isobutanol-producing metabolic pathway. In one embodiment, the recombinant microorganism can be constructed to express an isobutanol-producing metabolic pathway comprising at least one exogenous gene. In one embodiment, the recombinant microorganism can be constructed to express an isobutanol-producing metabolic pathway comprising at least two exogenous genes. In another embodiment, the recombinant microorganism can be constructed to express an isobutanol-producing metabolic pathway comprising at least three exogenous genes. In another embodiment, the recombinant microorganism can be constructed to express an isobutanol-producing metabolic pathway comprising at least four exogenous genes. In another embodiment, the recombinant microorganism can be constructed to express an isobutanol-producing metabolic pathway comprising five exogenous genes. Thus, the present invention further provides recombinant microorganisms that comprise an isobutanol-producing metabolic pathway methods of using said recombinant microorganisms to produce isobutanol.
In various embodiments described here, the enzyme (s) of the isobutanol pathway is / are selected from acetolactate synthase (ALS), ketol-acid reductisomerase (KARI), dihydroxy acid dehydratase (DHAD), 2- keto-acid decarboxylase (KIVD), and alcohol dehydrogenase (ADH).
In various embodiments described here, enzymes from the isobutanol pathway can be derived from a prokaryotic organism. In alternative embodiments described here, enzymes from the isobutanol pathway can be derived from a eukaryotic organism. An exemplary metabolic pathway that converts pyruvate to isobutanol can consist of an acetohydroxy acid synthase (ALS) enzyme encoded, for example, by B. subtilis alsS, a ketol-acid reductisomerase (KARI) encoded, for example, by E ilvC coli, a dihydroxy acid dehydratase (DHAD), encoded, for example, by L. lactis ilvD, a 2-keto-acid decarboxylase (KIVD) encoded, for example, by L. lactis kivD, and an alcohol dehydrogenase (ADH) (for example, a modified ADH described here), encoded, for example, by L. lactis adhA with one or more mutations at positions Y50, Q77, V108, Y113, I212, and L264 as described here.
In various embodiments described here, recombinant microorganisms can be Saccharomyces clade microorganisms, Saccharomyces sensu stricto microorganisms, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD yeast (whole genome duplication), pre-WGD yeast microorganisms (whole genome duplication), and non-fermenting yeast microorganisms.
In some embodiments, the recombinant microorganisms may be recombinant microorganisms from Saccharomyces clade yeasts.
In some embodiments, recombinant microorganisms may be Saccharomyces sensu stricto microorganisms. In one embodiment, Saccharomyces sensu stricto is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum. S. carocanis and hybrids thereof.
In some embodiments, the recombinant microorganisms may be Crabtree-negative yeast recombinant microorganisms. In one embodiment, the Crabtree-negative yeast recombinant microorganism is classified into a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, or Candida. In additional embodiments, the Crabtree-negative yeast microorganism is selected from Saccharomyces kluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula anomala, Candida utilis and Kluyveromyces waltii.
In some embodiments, the recombinant microorganisms may be Crabtree-positive yeast recombinant microorganisms. In one embodiment, the yeast microorganism Crabtree-positive is classified into a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and Schizosaccharomyces. In additional modalities, the yeast microorganism Crabtree-positive is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, Kluyveromyces thermotolerans, Candida glabrata, Z. bailli, Zomy , Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.
In some embodiments, the recombinant microorganisms may be post-WGD yeast recombinant microorganisms (whole genome duplication). In one embodiment, the post-WGD yeast recombinant microorganism is classified into a genus selected from the group consisting of Saccharomyces or Candida. In additional modalities, the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.
In some embodiments, the recombinant microorganisms can be recombinant microorganisms from pre-WGD yeast (whole genome duplication). In one embodiment, the pre-WGD yeast recombinant microorganism is classified into a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Pachysolen, Yarrowia and Schizosaccharomyces. In additional modalities, the pre-WGD yeast is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia anomaen, Issisiakia , Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomyces pombe.
In some embodiments, the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to, those classified into a genus selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida. In a specific modality, the non-fermenting yeast is C. xestobii.
In another aspect, the present invention provides methods of producing beneficial metabolites, including fuels, chemicals, and amino acids using a recombinant microorganism, as described herein. In one embodiment, the method includes culturing the recombinant microorganism in a culture medium that contains a raw material providing the carbon source until a recoverable amount of the metabolite is produced and, optionally, recovery of the metabolite. In one embodiment, the microorganism produces the metabolite from a carbon source with a yield of at least about 5 percent theoretical. In another embodiment, the microorganism produces the metabolite with a yield of at least about 10 percent, at least about 15 percent, about less than about 20 percent, at least about 25 percent, at least at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5 percent theoretical. In one embodiment, the metabolite can be derived from a biosynthetic pathway that uses a 3-keto acid as an intermediate. In one embodiment, the 3-keto acid intermediate is acetolactate. Thus, the metabolite can be derived from a biosynthetic pathway that uses acetolactate as an intermediate, including, but not limited to, isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine , leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate. Consequently, the metabolite can be derived from a biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate as an intermediate, including, but not limited to, 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4 -methyl-1-hexanol, and 5-methyl-1-heptanol. In another embodiment, the metabolite can be derived from a biosynthetic pathway that uses an aldehyde as an intermediate, including, but not limited to, isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol. In yet another embodiment, the metabolite can be derived from a biosynthetic pathway that uses acetolactate and an aldehyde as intermediates, including, but not limited to, isobutanol, 1-butanol, and 3-methyl-1-butanol biosynthetic pathways. In yet another embodiment, the metabolite can be derived from a biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate and an aldehyde as intermediates, including, but not limited to, 2-methyl-1-butanol, 3- biosynthetic pathways. methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
In one embodiment, the recombinant microorganism is grown under aerobic conditions. In another embodiment, the recombinant microorganism is grown under microaerobic conditions. In yet another embodiment, the recombinant microorganism is grown under anaerobic conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
The illustrative modalities of the Invention are illustrated in the drawings, in which: Figure 1 illustrates an exemplary embodiment of an isobutanol pathway. Figure 2 illustrates exemplary reactions that can be catalyzed by 3-keto acid reductases. Figure 3 illustrates a non-limiting list of exemplary 3-keto acid reductases and their corresponding enzyme classification numbers. Figure 4 illustrates exemplary reactions that can be catalyzed by aldehyde dehydrogenases. Figure 5 illustrates a strategy for reducing the production of DH2MB and isobutyrate in recombinant isobutanol-producing microorganisms. Figure 6 illustrates a strategy for reducing the production of DH2MB and 3-methyl-1-butyrate in recombinant microorganisms producing 3-methyl-1-butanol. Figure 7 illustrates a strategy to reduce the production of 2-ethyl-2,3-dihydroxybutyrate and 2-methyl-1-butyrate in recombinant microorganisms producing 2-methyl-1-butanol. Figure 8 illustrates a stacked overlay of LC4 chromatograms showing a sample containing DH2MB and ethyl acetate (top) and a sample containing acetate and DHIV (bottom). Elution order: DH2MB followed by ethyl acetate (top); acetate, lactate, DHIV, isobutyrate, pyruvate (bottom). Figure 9 illustrates a chromatogram for the fraction of the sample collected at the retention time corresponding to the DHIV collected over LC1 and analyzed by LC4 in an AS-11 column with conductivity detection. Figure 10 illustrates a 1H-COZY spectrum of the isolated LC1 peak. The spectrum indicates that DH2MB methyl protons (doublet) at 0.95 ppm are coupled to the methyl proton (quartet) at 3.7 ppm. Figure 11 illustrates a 1 H NMR spectrum of the isolated LC1 peak. The spectrum indicates the presence of DH2MB: a singlet of methyl protons (a) at 1.2 ppm with integral value 3, a doublet of methyl protons (b) at 0.95 ppm with integral value 3 and a quartet of methino (c) proton at 3.7 ppm with the full value of 1.84. The integral value of the methino proton (c) is greater than 1 due to the overlap with glucose resonance in the same region. Figure 12 illustrates an LC-MS analysis of the isolated LC1 peak. Several molecular ions were identified in the sample as indicated in the upper portion of the figure. The additional fragmentation (MS2) of molecular ion 134 indicated that the isolated fraction of LC1 contains hydroxyl carboxylic acid due to the loss of CO2 (*) and H2O + CO2 (**) characteristics. Figure 13 illustrates the diastereomeric and enantiomeric structures of 2,3-dihydroxy-2-methylbutanoic acid (2R, 3S) -1a, (2S, 3R) -1b, (2R, 3R) -2a, (2S, 3S) - 2b. Figure 14 illustrates the 1H spectrum of DH2MB crystallized in D2O. 1H NMR (TSP) 1.1 (d, 6.5 Hz, 3H), 1.3 (s, 3H), 3.9 (q, 6.5 Hz, 3H) Figure 15 illustrates the 13C spectrum of the DH2MB crystallized in D2O. The spectrum indicates five different carbon resonances, one of which is the characteristic carboxylic acid resonance at 181 ppm. Figure 16 illustrates the fermentation profile of isobutanol and by-products of a single fermentation with GEVO3160. Production aeration was reduced from an OTR of 0.8 mM / h to 0.3 mM / h at 93 h after inoculation. Open diamond = iBuOH, square = unknown quantified as DH2MB, asterisk = dry cell weight (cdw), and closed triangle = total by-products. Figure 17 illustrates a structural alignment of the L. lactis Adha amino acid sequence with the structure of G. stearothermophilus (PyMOL). Active site mutations are shown (Y50F and L264V). Mutations in the cofactor binding domain are also shown (I212T and N219Y). Figure 18 illustrates biosynthetic pathways using acetolactate as an intermediate. Biosynthetic pathways for the production of 1-butanol, isobutanol, 3-methyl-1-butanol, and 4-methyl-1-pentanol use both acetolactate and an aldehyde as an intermediate. Figure 19 illustrates biosynthetic pathways using 2-aceto-2-hydroxybutyrate as an Intermediate. Biosynthetic pathways for the production of 2-methyl-1-butanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol use both 2-aceto-2-hydroxybutyrate and an aldehyde as an Intermediary. Figure 20 illustrates additional biosynthetic pathways using an aldehyde as an intermediate.
DETAILED DESCRIPTION
As used here and in the appended claims, the singular forms "one", "one", and "o, a" include plural referents unless the context clearly indicates otherwise. Thus, for example, the reference to "a polynucleotide" includes a plurality of such polynucleotides and the reference to "the microorganism" includes reference to one or more microorganisms, and so on. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as is normally understood by a person skilled in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the methods and compositions described, the exemplary methods, devices and materials are described herein.
The publications discussed above and throughout the text are provided for publication only prior to the filing date of this application. Nothing here should be construed as an admission that inventors are not entitled to anticipate such disclosure by virtue of prior disclosure. The "microorganism" includes prokaryotic and eukaryotic microbial species from the Archaea, bacteria and Eucarya domains, the latter including yeasts and filamentous fungi, protozoa, algae, or superior Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism. The term "genus" is defined as a taxonomic group of species related according to the taxonomic scheme of Bacteria and Archaea (Garrity, GM, Lilburn, TG, Cole, JR, Harrison, SH, Euzeby, J., and Tindall, BJ (2007) The taxonomic scheme of bacteria and Archaea TOBA release 7.7, March 2007. Michigan State University Board of Trustees. [Http://www.taxonomicoutline.org/]). The term "species" is defined as a collection of organisms closely related to more than 97% 16S ribosomal RNA sequence homology and more than 70% genomic hybridization and sufficiently different from all other organisms to be recognized as one distinct unit.
The terms "recombinant microorganism", "modified microorganism", and "recombinant host cell" are used interchangeably here and refer to microorganisms that have been genetically modified to express or overexpress endogenous polynucleotides, to express heterologous polynucleotides, such as those included in a vector, in an integration construct, or that have a change in the expression of an endogenous gene. By "alteration" is meant that the expression of the gene, or the level of an RNA molecule or equivalent RNA molecules that encode one or more polypeptides or polypeptide subunits, or the activity of one or more polypeptides or polypeptide subunits is overregulated or unregulated, such that the expression, level, or activity is greater or less than that observed in the absence of the change. For example, the term "change" may mean "inhibit", but the use of the word "change" is not limited to this definition. The term "expression" with respect to a sequence of genes refers to the transcription of the gene and, where appropriate, the translation of the resulting mRNA transcript into a protein. Thus, as will be clear from the context, the expression of a protein results from the transcription and translation of the open reading frame sequence. The level of expression of a desired product in a host cell can be determined based on either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantified by qRT-PCR or by Northern hybridization (see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). The protein encoded by a selected sequence can be quantified by various methods, for example, by ELISA, by assay for the biological activity of the protein, or by using assays that are independent of such activity, such as Western blotting or radioimmunoassay using antibodies that recognize and bind to the protein. See Sambrook et al., 1989, supra. The polynucleotide generally encodes a target enzyme involved in a metabolic pathway for the production of a desired metabolite. It is understood that the terms "recombinant microorganism" and "recombinant host cell" refer not only to the particular recombinant microorganism, but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in successive generations, due to either mutation or environmental influences, such progeny may, in fact, not be identical to the parental cell, but is still included in the scope of the term as used here. The term "overexpression" refers to a high level (for example, aberrant level) of mRNAs encoding a protein (s) (for example, a TMA29 protein or one of its counterparts), and / or high levels of proteins ( for example, TMA29) in cells, compared to the corresponding unmodified cells analogous to baseline levels of mRNAs (eg, those encoding Aft proteins) or baseline levels of proteins. In particular modalities, TMA29, or its counterparts, can be overexpressed at least 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 12 times, 15 times or more in constructed microorganisms to exhibit an increase in TMA29 mRNA, proteins and / or activity.
As used herein and as would be understood by a person skilled in the art, "reduced activity and / or expression" of a protein, such as an enzyme can mean either reduced specific catalytic activity of the protein (e.g. reduced activity) and / or decreased protein concentrations in the cell (for example, reduced expression), while "deleted activity and / or expression" or "deleted activity and / or expression" of a protein, such as an enzyme can mean none or negligible specific catalytic activity of the enzyme (for example, deleted activity) and / or none or insignificant concentrations of the enzyme in the cell (for example, deleted expression). The term "wild-type microorganism" describes a cell that occurs in nature, that is, a cell that has not been genetically modified. A wild type microorganism can be genetically modified to express or overexpress a first target enzyme. This microorganism can act as a parental microorganism in generating a microorganism modified to express or overexpress a second target enzyme. In turn, the microorganism modified to express or overexpress a first and a second target enzyme can be modified to express or overexpress a third target enzyme.
Thus, a "source microorganism" functions as a reference cell for successive events of genetic modification. Each modification event can be carried out by introducing a nucleic acid molecule into the reference cell. The introduction facilitates the expression or overexpression of a target enzyme. The term "facilitates" is understood to encompass the activation of endogenous polynucleotides that encode a target enzyme by genetic modification of, for example, a promoter sequence in a parent microorganism. It is further understood that the term "facilitates" encompasses the introduction of heterologous polynucleotides that encode a target enzyme for a parental microorganism The term "build" refers to any manipulation of a microorganism that results in a detectable change in the micro -organism, in which manipulation includes, but is not limited to, insertion of a polynucleotide and / or polypeptide heterologous to the microorganism and mutation of a polynucleotide and / or polypeptide native to the microorganism. The term "mutation" as used herein indicates any modification of a nucleic acid and / or the polypeptide, which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions or insertions of one or more polynucleotide residues, which include changes that arise within a region that encodes a gene's protein, as well as changes in regions outside a sequence that encodes the protein, such as, but limited to, regulatory or promoter sequences. A genetic change can be a mutation of any kind. For example, the mutation may constitute a point mutation, a structure-shift mutation, a nonsense mutation, an insertion or deletion of part or all of a gene. In addition, in some modalities of the modified microorganism, a portion of the microorganism's genome has been replaced by a heterologous polynucleotide. In some modalities, mutations are naturally occurring. In other modalities, mutations are identified and / or enriched through artificial selection pressure. In yet other modalities, mutations in the microorganism's genome are the result of genetic engineering. The term "biosynthetic pathway", also referred to as "metabolic pathway", refers to a set of anabolic or catabolic biochemical reactions for conversion from one chemical form to another. The gene products belong to the same "metabolic pathway" if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (ie, metabolite) between the final product of metabolite and the same substrate.
As used herein, the "isobutanol-producing metabolic pathway" refers to an enzymatic pathway that produces isobutanol from pyruvate. The term "heterologous", as used here with reference to molecules and, in particular, enzymes and polynucleotides, indicates molecules that are expressed in an organism other than the organism from which they were produced or are found in nature, regardless of the level of expression that can be lower, equal or higher than the expression level of the native molecule in the microorganism.
On the other hand, the term "native" or "endogenous", as used here with reference to molecules and, in particular, enzymes and polynucleotides, indicates the molecules that are expressed in the organism in which they originated or are found in nature, regardless of the level of expression that can be lower, equal or higher than the level of expression of the native molecule in the microorganism. It is understood that the expression of polynucleotides or native enzymes can be modified in recombinant microorganisms. The term "raw material" is defined as a raw material or a mixture of raw materials supplied to a fermentation process or to microorganisms from which other products can be made. For example, a carbon source, such as biomass or carbon compounds derived from biomass, is a raw material for a microorganism that produces a biofuel in a fermentation process. However, a raw material may contain nutrients other than a carbon source. The term "substrate" or "suitable substrate" refers to any substance or compound that is converted or that must be converted to another compound with the action of an enzyme. The term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials that contain at least one substrate, or derivatives thereof. In addition, the term "substrate" encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived from sugar, but also the metabolites of intermediate and final products used in an associated pathway with a recombinant microorganism, as described here. The term "C2-compound", as used as a carbon source for yeast microorganisms constructed with mutations in all pyruvate decarboxylase (PDC) genes that result in reduced pyruvate decarboxylase activity in said genes refers to compounds organics consisting of two carbon atoms, including, but not limited to, ethanol and acetate. The term "fermentation" or "fermentation process" is defined as a process in which a microorganism is grown in a culture medium that contains raw materials, such as raw material and nutrients, into which the microorganism converts raw materials, such as a raw material, in products. The term "volumetric productivity" or "production rate" is defined as the amount of product formed per volume of the medium per unit of time. Volumetric productivity is reported in grams per liter per hour (g / L / h). The term "specific productivity" or "specific production rate" is defined as the amount of product formed by volume of the medium per unit of time per quantity of cells. Specific productivity is reported in grams or milligrams per liter per hour per OD (g / L / h / OD). "Yield" is defined as the amount of product obtained per unit weight of crude material and can be expressed as g of product per g of substrate (g / g). The yield can be expressed as a percentage of the theoretical yield. The "theoretical yield" is defined as the maximum amount of product that can be generated by a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for a typical conversion of isobutanol to glucose is 0.41 g / g. As such, an isobutanol to glucose yield of 0.39 g / g would be expressed as 95% of the theoretical value or 95% of the theoretical yield. The term "titration" is defined as the strength of a solution or the concentration of a substance in solution. For example, the titration of a biofuel in a fermentation broth is described as g of biofuel in solution per liter of fermentation broth (g / L).
"Aerobic conditions" are defined as conditions in which the oxygen concentration in the fermentation medium is sufficiently high for an optional aerobic or anaerobic microorganism that is used as a terminal electron receiver.
In contrast, "anaerobic conditions" are defined as conditions in which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron receptor. Anaerobic conditions can be achieved by spraying a fermentation medium with an inert gas such as nitrogen, until oxygen is no longer available to the microorganism as a terminal electron receiver. Alternatively, anaerobic conditions can be achieved by consuming the available oxygen from fermentation by the microorganism until the oxygen is no longer available to the microorganism as a terminal electron receptor. Methods for the production of isobutanol under anaerobic conditions are described in the copending common property publication, US 2010/0143997, the descriptions of which are incorporated herein by reference in their entirety for all purposes. "Aerobic metabolism" refers to a biochemical process in which oxygen is used as a terminal electron receptor to make energy, usually in the form of ATP, from carbohydrates. Aerobic metabolism occurs, for example, through glycolysis and the TCA cycle, in which a single molecule of glucose is completely metabolized to carbon dioxide in the presence of oxygen.
In contrast, "anaerobic metabolism" refers to a biochemical process in which oxygen is not the final electron receptor contained in NADH. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron receptor, and substrate-level phosphorylation, in which NADH electrons are used to generate a reduction product by means of a "fermentative pathway".
In "fermentative pathways", NAD (P) H donates its electrons to a molecule produced by the same metabolic pathway that produced the electrons transported in NAD (P) H. For example, in one of the fermentation pathways for certain yeast strains, the NAD (P) H generated through glycolysis transfers its electrons to the pyruvate, producing ethanol. Fermentation pathways are usually active under anaerobic conditions, but can also occur under aerobic conditions, under conditions where NADH is not fully oxidized through the respiratory chain. For example, above certain concentrations of glucose, yeasts positive for Crabtree produce large amounts of ethanol under aerobic conditions. The term "by-product", or "by-product" means an undesirable product related to the production of an amino acid, amino acid precursor, chemist, chemical precursor, biofuel, or biofuel precursor. The term "substantially free" when used in reference to the presence or absence of enzyme activity (3-KAR, ALDH, PDC, GPD, etc.) in carbon pathways that compete with the desired metabolic pathway (for example, a metabolic pathway producing isobutanol), means that the level of the enzyme is substantially less than that of the same enzyme in the wild-type host, where less than about 50% of the level of the wild-type is preferred and less than about 30% is more preferred. Activity can be less than about 20%, less than about 10%, less than about 5%, or less than about 1% of wild-type activity. Microorganisms that are "substantially free" of a given enzyme activity (3-KAR, ALDH, PDC, GPD, etc.), can be created using recombinant means or identified in nature. The term "non-fermenting yeast" is a kind of yeast that fails to demonstrate an anaerobic metabolism in which NADH electrons are used to generate a reduced product via a fermentation pathway, such as the production of ethanol and CO2 from glucose . Non-fermenting yeast can be identified by the "Durham tube test" (JA BRNAett, RW Payne and D. Yarrow. 2000. Yeasts Characteristics and Identification. 3rd edition. P. 28-29. Cambridge University Press, Cambridge, UK) or by monitoring the production of fermentation productions, such as ethanol and CO2. The term "polynucleotide" is used here interchangeably with the term "nucleic acid" and refers to an organic polymer composed of two or more monomers, including nucleotides, nucleosides or their analogs including, but not limited to, deoxyribonucleic acid (DNA ) single-stranded or double-stranded, sense or antisense of any length and, where appropriate, single-stranded or double-stranded, sense, antisense or any length ribonucleic acid (RNA), including siRNA. The term "nucleotide" refers to any one of several compounds consisting of a ribose or deoxyribose sugar attached to a purine or a pyrimidine base and a phosphate group, and which are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound (such as guanosine or adenosine) that consists of a purine or pyrimidine base, combined with deoxyribose or ribose and is found mainly in nucleic acids. The term "nucleotide analog" or "nucleoside analog" refers, respectively, to a nucleoside or nucleotide in which one or more individual atoms have been replaced by a different atom or with a different functional group. Thus, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called a nucleotide oligomer or oligonucleotide.
It is understood that the polynucleotides described in the present invention include "genes" and that the nucleic acid molecules described in the present invention include "vectors" or "plasmids". Therefore, the term "gene", also called a "structural gene", refers to a polynucleotide that encodes a particular sequence of amino acids, which comprises all or part of one or more proteins or enzymes, and may include DNA sequences regulatory (not transcribed), such as promoter sequences, that determine, for example, the conditions under which the gene is expressed. The transcribed region of the gene can include untranslated regions, including introns, the 5 'untranslated region (UTR), and 3'-UTR, as well as the coding sequence. The term "operon" refers to two or more genes that are transcribed as a single transcriptional unit from a common promoter. In some embodiments, the genes that comprise the operon are contiguous genes. It is understood that the transcription of a complete operon can be modified (i.e., increased, decreased or deleted), by modifying the common promoter. Alternatively, any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide. The modification can result in an increase in the activity of the encoded polypeptide. In addition, the modification may confer new activities on the encoded polypeptide. Examples of new activities include the use of alternative substrates and / or the ability to function in alternative environmental conditions.
A "vector" is any means by which a nucleic acid can be propagated and / or transferred between organisms, cells or cellular components. Vectors include viruses, bacteriophages, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs, (artificial plant chromosomes), and the like, which are "episomes", that is, that replicate autonomously, or can integrate into a host cell chromosome. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of DNA and RNA within the same chain, a polysysin-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a DNA conjugated to liposomes, or the like, which are not episomal in nature, or can be an organism comprising one or more of the above polynucleotide constructs, such as an Agrobacterium or a bacterium. "Transformation" refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection) can be accomplished by any of a number of means including chemical transformation (for example, lithium acetate transformation), electroporation, microinjection, biobalistics (or particle bombardment-mediated distribution ), or an Agrobacterium-mediated transformation. The term "enzyme", as used herein, refers to any substance that promotes or catalyzes one or more chemical or biochemical reactions, which generally includes enzymes wholly or partially composed of a polypeptide, but may include enzymes composed of a molecule different, including polynucleotides. The term "protein", "peptide", or "polypeptide" as used herein indicates an organic polymer composed of two or more amino acid monomers and / or their analogs. As used herein, the term "amino acid" or "amino acid monomer" refers to any natural and / or synthetic amino acids including glycine and both L or D optical isomers. The term "amino acid analog" refers to an amino acid wherein one or more individual atoms have been replaced, either with a different atom, or with a different functional group. Therefore, the term polypeptide includes polymer of amino acids of any length, including full length proteins, and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called an oligomeric protein or oligopeptide The term "homologous" used in relation to an enzyme or original gene of a first family or species, refers to enzymes or genes distinct from a second family or species which are determined by functional, structural or genomic analysis to be an enzyme or gene of the second family or species that corresponds to the original enzyme or gene of the first family or species. Most of the time, the counterparts will have functional, structural or genomic similarities. Techniques are known by which the homologues of an enzyme or a gene can be easily cloned using genetic probes and PCR. The identity of cloned sequences as homologous can be confirmed using functional assays and / or by genomic mapping of genes. The protein has "homology" or is "homologous" to a second protein if the amino acid sequence encoded by one gene has an amino acid sequence similar to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined as meaning that the two proteins have similar amino acid sequences). The term "analog" or "comparable" refers to sequences of nucleic acids or proteins or protein structures that are related to each other in function only and are not of common origin or do not share a common ancestral sequence. Analogues may differ in sequence, but may share a similar structure, due to convergent evolution. For example, two enzymes are analogous or comparable if the enzymes catalyze the same conversion reaction from a substrate to a product, they are not related in sequence and, regardless of whether the two enzymes are related in terms of structure.
Recombinant Microorganisms with Reduced By-Product Accumulation Yeast cells convert sugars to produce pyruvate, which is then used in a number of cell metabolism pathways. In recent years, yeast cells have been modified to produce a number of desirable products via biosynthetic pathways driven by pyruvate. In many of these biosynthetic pathways, the initial step is the pathway for converting endogenous pyruvate to a 3-keto acid.
As used herein, a "3-keto acid" refers to an organic compound that contains a fraction of carboxylic acid on carbon C1 and a fraction of ketone on carbon C3. For example, acetolactate and 2-hydroxy-2-methyl-3-oxobutanoic acid are 3-keto acids with a ketone group on C3 carbon (See, for example, Figure 2).
An example of a 3-keto acid, which is common to many biosynthetic pathways is acetolactate, which is formed from pyruvate by the action of the enzyme acetolactate synthase (also known as acetohydroxyacid synthase). Among the biosynthetic pathways that use acetolactate as an intermediary are the pathways for the production of isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3 -methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. The biosynthetic pathways constructed for the synthesis of these beneficial acetolactate-derived metabolites are found in Table 1 and Figure 18.
Table 1. Biosynthetic pathways using acetolactate as an intermediate. a - The contents of each of the references in this table are hereby incorporated by Reference in their entirety for all purposes.
Each of the biosynthetic pathways listed in Table 1 shares the common 3-keto acid intermediate, acetolactate. Therefore, the yield of the product from these biosynthetic pathways will depend, in part, on the amount of acetolactate that is available for enzymes downstream of said biosynthetic pathways.
Another example of a 3-keto acid that is common to many biosynthetic pathways is 2-aceto-2-hydroxy-butyrate, which is formed from pyruvate and 2-ketobutyrate through the action of the enzyme acetolactate synthase (also known as acetohydroxyacid synthase ). Among the biosynthetic pathways, the use of 2-aceto-2-hydroxybutyrate as an intermediate includes the pathways for the production of 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol , and 5-methyl-1-heptanol. The biosynthetic pathways constructed for the synthesis of these beneficial 2-aceto-2-hydroxy-butyrate-derived metabolites are found in Table 2 and Figure 19.
Table 2. Biosynthetic Pathways Using 2-Aceto-2-Hydroxybutyrate as an Intermediate a - The contents of each of the references in this table are incorporated by Reference in their entirety for all purposes.
Each of the biosynthetic pathways listed in Table 2 shares the common 3-keto acid intermediate, 2-aceto-2-hydroxy-butyrate. Therefore, the yield of the product from these biosynthetic pathways will depend, in part, on the amount of acetolactate that is available for enzymes downstream of said biosynthetic pathways.
Likewise, yeast cells can be built to produce a number of desirable products via biosynthetic pathways that use an aldehyde as an intermediate pathway. The biosynthetic pathways constructed comprising an intermediate aldehyde include biosynthetic pathways for the production of isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol, 1-pentanol, 1-hexanol, 3 -methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol (See Table 3 and Figures 18, 19, and 20).
Table 3. Biosynthetic pathways using an aldehyde as an intermediate. a - The contents of each of the references in this table are hereby incorporated by Reference in their entirety for all purposes.
Each of the biosynthetic pathways listed in Table 3 has an Aldehyde intermediate. For example, the aldehyde intermediate in an isobutanol-producing metabolic pathway is isobutyraldehyde (see Figure 1), while the pathways for the production of 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1- propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol use 1-butanal, 2-methyl -1-butanal, 3-methyl-1-butanal, 1-propanal, 1-pentanal, 1-hexanal, 3-methyl-1-pentanal, 4-methyl-1-pentanal, 4-methyl-1-hexanal, and 5-methyl-1-heptanol as aldehyde intermediates, respectively. Therefore, the yield of the product on biosynthetic pathways using these aldehyde intermediates will be, in part, dependent on the amount of the intermediate aldehyde that is available for enzymes downstream of said biosynthetic pathways.
As described herein, the present inventors have verified the enzymatic activities responsible for the accumulation of undesirable by-products derived from intermediates of 3-keto acid and / or aldehyde. Specifically, they determined that 3-keto acid reductase and an aldehyde dehydrogenase are responsible for converting 3-keto acids and aldehydes, respectively, to undesirable by-products. The activities of these enzymes are shown to impede optimal productivity and yield of 3-keto acid and / or products derived from aldehyde, including, but not limited to, those listed in Tables 1-3. The present inventors have found that deleting these recently characterized enzyme activities considerably reduces or eliminates the formation of undesirable by-products and, at the same time, improves yields and titrations of beneficial metabolites.
Reduced Accumulation of 3-Hydroxy Acids from 3-Keto Acids As described here, the present inventors have found that undesirable by-products, 3-hydroxy acids, can accumulate during fermentation reactions with microorganisms comprising a pathway involving the acid intermediate 3-keto.
As used herein, a "3-hydroxy acid" is an organic compound that contains a fraction of carboxylic acid on the C1 carbon atom and a fraction of alcohol on C3 carbon. 3-hydroxy acids can be obtained from 3-keto acids by chemical reduction of the 3-keto acid ketone fraction to a fraction of alcohol. For example, reducing the ketone fraction to acetolactate or 2-hydroxy-2-methyl-3-oxobutanoic results in the formation of 2,3-dihydroxy-2-methylbutanoic 3-hydroxy acid (DH2MB) (See, for example, Figure 2).
The present inventors have found that the 3-hydroxy acid by-product, 2,3-dihydroxy-2-methylbutanoic (CAS # 14868-24-7) (DH2MB), accumulates during fermentation reactions with microorganisms comprising biosynthetic pathways involving the 3-keto acid intermediate, acetolactate. It was found that the accumulation of this by-product prevents the optimal yield and productivity of metabolites targeted by the biosynthetic pathway. The present inventors have found that the production of DH2MB is caused by the reduction of acetolactate. To reduce or eliminate the activity responsible for the production of DH2MB, the corresponding enzyme activity that catalyzes this reaction had to be identified and reduced or eliminated. The inventors have found that in S. cerevisiae, an enzyme that catalyzes the conversion of acetolactate to DH2MB is YMR226C (also known as TMA29). This is the first report of a protein in yeast that converts acetolactate to DH2MB
The present inventors have also found that the 3-hydroxy acid by-product, 2-ethyl-2,3-dihydroxybutanoate, accumulates during fermentation reactions with microorganisms comprising biosynthetic pathways involving the 3-keto acid, 2-aceto-2 intermediate -hydroxybutyrate. It was found that the accumulation of this by-product impedes the yield and optimal productivity of the target metabolite of the biosynthetic pathway. The present inventors have found that the production of 2-ethyl-2,3-dihydroxybutanoate is caused by the reduction of 2-aceto-2-hydroxy-butyrate. In order to reduce or eliminate the activity responsible for the production of 2-ethyl-2,3-dihydroxybutanoate, the corresponding enzymatic activity that catalyzes this reaction had to be identified and reduced or eliminated. The inventors found in S. cerevisiae, the enzyme YMR226C (also known as TMA29), which catalyzes the conversion of acetolactate to DH2MB also catalyzes the conversion of 2-aceto-2-hydroxy-butyrate to 2-ethyl-2,3-dihydroxybutanoate . This is the first report of a protein in yeast that converts 2-aceto-2-hydroxy-butyrate to 2-ethyl-2,3-dihydroxybutanoate.
The inventors of the present invention describe here several strategies for reducing the conversion of the 3-keto acid intermediate to the corresponding 3-hydroxy acid by-product, a process that is accompanied by an increase in the yield of desirable metabolites. In one embodiment, the 3-keto acid intermediate is acetolactate and the corresponding 3-hydroxy acid is DH2MB. As described herein, reducing the conversion of acetolactate to DH2MB allows for an increase in the production of beneficial metabolites such as isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A, which are derived from biosynthetic pathways that use acetolactate as an intermediate. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxy-butyrate and the corresponding 3-hydroxy acid is 2-ethyl-2,3-dihydroxybutanoate. As described herein, reducing the conversion of 2-aceto-2-hydroxy-butyrate to 2-ethyl-2,3-dihydroxybutanoate allows for an increase in the production of beneficial metabolites, such as 2-methyl-1-butanol, isoleucine, to 3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
Accordingly, an aspect of the present invention is directed to a recombinant microorganism comprising a biosynthetic pathway that uses a 3-keto acid, as an intermediate, wherein said recombinant microorganism is substantially free of an enzyme that catalyzes the conversion of the intermediate of 3-keto acid to a 3-hydroxy acid by-product. In one embodiment, the 3-keto acid intermediate is acetolactate and the 3-hydroxy acid by-product is DH2MB. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxy-butyrate and the 3-hydroxy acid by-product is 2-ethyl-2,3-dihydroxybutanoate.
In another aspect, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway that uses a 3-keto acid, as an intermediate, in which said recombinant microorganism is constructed to reduce or eliminate the expression or activity of a enzyme that catalyzes the conversion of the 3-keto acid intermediate to a 3-hydroxy acid by-product. In one embodiment, the 3-keto acid intermediate is acetolactate and the 3-hydroxy acid by-product is DH2MB. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxy-butyrate and the 3-hydroxy acid by-product is 2-ethyl-2,3-dihydroxybutanoate.
In various embodiments described herein, the protein involved in the catalysis of the conversion of the 3-keto acid intermediate to the 3-hydroxy acid by-product is a ketorreductase. In an exemplary embodiment, ketorreductase is a 3-keto acid reductase (3-KAR). As used herein, the term "3-keto acid reductase" refers to a ketorreductase (i.e., ketone reductase) active for the 3-oxo group of a 3-keto acid. An illustration of exemplary reactions that can be catalyzed by 3-keto acid reductases is shown in Figure 2. Suitable 3-keto acid reductases are generally found in the enzyme classification subgroups 1.1.1.X, the final X digit being substrate dependent. A non-limiting list of exemplary 3-keto acid reductases and their corresponding enzyme classification numbers are shown in Figure 3.
In an exemplary embodiment, 3-keto acid reductase is the protein of S. cerevisiae YMR226C (SEQ ID NO: 1), used interchangeably here with “TMA29”. In some embodiments, 3-keto acid reductase is the protein of S. cerevisiae YMR226C (SEQ ID NO: 1) or a homolog or variant thereof. In one embodiment, the homologue can be selected from the group consisting of Vanderwaltomzymapolyspora (SEQ ID NO: 2), Saccharomyces castellii (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomyces bayanus (SEQ ID NO: : 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), K. lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQ ID NO: 10), Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12), Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14), Candida dubliniensis (SEQ ID NO: 14) NO: 15), Candida albicans (SEQ ID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO: 18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO: 19) NO: 20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQ ID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).
In one embodiment, the recombinant microorganism of the invention includes a mutation in at least one gene encoding a 3-keto acid reductase that results in a reduction in the 3-keto acid reductase activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism includes a partial deletion of a gene encoding a 3-keto acid reductase gene that results in a reduction in the 3-keto acid reductase activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprises a complete deletion of a gene encoding a 3-keto acid reductase that results in a reduction in the 3-keto acid reductase activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with the gene encoding a 3-keto acid reductase that results in a reduction in the expression of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator that results in a reduction in the transcription of the gene encoding a 3-keto acid reductase. In yet another embodiment, the recombinant microorganism comprises mutations in all genes that encode a 3-keto acid reductase that results in a reduction in the activity of a polypeptide encoded by the gene (s). In one embodiment, said 3-keto acid reductase gene is the S. cerevisiae TMA29 gene (YMR226C) or a homologue thereof. As would be understood in the art, naturally occurring TMA29 homologues in yeast other than S. cerevisiae can be similarly inactivated using the methods of the present invention. TMA29 homologues and methods of identifying such TMA29 homologs are described here.
As understood by those skilled in the art, there are several additional mechanisms available to reduce or interrupt the activity of a protein, such as 3-keto acid reductase, including, but not limited to, the use of a regulated promoter, use of a weak constitutive promoter , rupture of one of the two copies of the gene in a diploid yeast, rupture of both copies of the gene in a diploid yeast, the expression of an antisense nucleic acid, the expression of a siRNA, the overexpression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, the use of a heterologous gene with less specific activity, or similar or combinations thereof.
As described herein, the recombinant microorganisms of the present invention are constructed to produce less of a 3-hydroxy acid by-product than an unmodified parental microorganism. In one embodiment, the recombinant microorganism produces the 3-hydroxy acid by-product from a carbon source with a carbon yield of less than about 20 percent. In another embodiment, the microorganism produces the 3-hydroxy acid by-product from a carbon source with a carbon yield of less than about 10, less than about 5, less than about 2, less than about 1, less than about 0.5, less than about 0.1, or less than about 0.01 percent. In one embodiment, the 3-hydroxy acid by-product is DH2MB, derived from 3-keto acid, acetolactate. In another embodiment, the by-product of 3-hydroxy acid is 2-ethyl-2,3-dihydroxybutanoate, derived from 3-keto acid, 2-aceto-2-hydroxy-butyrate.
In one embodiment, the carbon yield of the 3-hydroxy acid by-product derived from 3-keto acid is reduced by at least about 50% in a recombinant microorganism, compared to a parent microorganism that does not comprise a reduction or deletion of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of the 3-keto acid intermediate to the 3-hydroxy acid by-product. In another embodiment, the 3-hydroxy acid by-product derived from 3-keto acid is reduced by at least about 60%, by at least about 65%, by at least about 70%, by at least about 75%, by at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99.9%, or by at least about 100% compared to a parental microorganism that does not comprise a reduction or deletion of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of 3-keto acid with the 3-hydroxy acid by-product. In one embodiment, the 3-hydroxy acid by-product is DH2MB, derived from 3-keto acid, acetolactate. In another embodiment, the by-product of 3-hydroxy acid is 2-ethyl-2,3-dihydroxybutanoate, derived from 3-keto acid, 2-aceto-2-hydroxy-butyrate.
In an additional embodiment, the yield of a desired fermentation product is increased in recombinant microorganisms comprising a reduction or elimination of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of the 3-keto acid intermediate to the by-product of 3-hydroxy acid. In one embodiment, the production of a desired fermentation product is increased by at least about 1% compared to a parental microorganism that does not include a reduction or elimination of the activity or expression of one or more endogenous proteins involved in the catalysis the conversion of the 3-keto acid intermediate to the 3-hydroxy acid by-product. In another embodiment, the production of a desired fermentation product is increased by at least about 5%, by at least about 10%, by at least about 25%, or by at least about 50% compared to a non-parental microorganism that comprises a reduction or elimination of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of the 3-keto acid intermediate to the 3-hydroxy acid by-product. In one embodiment, the 3-hydroxy acid by-product is DH2MB, derived from 3-keto acid, acetolactate. Consequently, in one embodiment, the desired fermentation product is obtained from any biosynthetic pathway in which acetolactate acts as an intermediate, including, but not limited to, isobutanol, 2-butanol, 1-butanol, 2-butanone, 2 , 3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In another embodiment, the 3-hydroxy acid by-product is 2-ethyl-2,3-dihydroxybutanoate, derived from 3-keto acid, 2-aceto-2-hydroxy-butyrate. Therefore, in another embodiment, the desired fermentation product is obtained from any biosynthetic pathway in which 2-aceto-2-hydroxybutyrate acts as an intermediate, including, but not limited to, 2-methyl-1-butanol, the isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
In other embodiments, additional enzymes that potentially catalyze the conversion of a 3-keto acid intermediate to a 3-hydroxy acid by-product are eliminated from the genome of a recombinant microorganism comprising a biosynthetic pathway that uses a 3-keto acid as a intermediate. Endogenous yeast genes, with the potential to convert a 3-keto acid intermediate to a 3-hydroxy acid by-product include ketorreductases, short chain alcohol dehydrogenases, medium chain alcohol dehydrogenases, members of the aldose reductase family, members of the D- family hydroxy acid dehydrogenase, alcohol dehydrogenases and lactate dehydrogenases. In one embodiment, the 3-hydroxy acid by-product is DH2MB, derived from 3-keto acid, acetolactate. In another embodiment, the by-product of 3-hydroxy acid is 2-ethyl-2,3-dihydroxybutanoate, derived from 3-keto acid, 2-aceto-2-hydroxy-butyrate.
The methods for identifying additional enzymes that catalyze the conversion of a 3-keto acid intermediate to a 3-hydroxy acid by-product are described as follows: endogenous yeast genes encoding ketorreductases, short-chain alcohol dehydrogenases, medium-chain alcohol dehydrogenases , members of the aldose reductase family, members of the D-hydroxy acid dehydrogenase family, alcohol dehydrogenases, and lactate dehydrogenases are eliminated from the genome of a yeast strain that comprises a biosynthetic pathway in which the 3-keto acid (for example, acetolactate, or 2-aceto-2-hydroxybutyrate) is an intermediate. These deletion strains are compared with the original strain, by fermentation and analysis of the fermentation broth for the presence and concentration of the corresponding 3-hydroxy acid by-product (eg DH2MB or 2-ethyl-2,3-dihydroxybutanoate derived from acetolactate and 2-aceto-2-hydroxy-butyrate, respectively). In S. cerevisiae, deletions that reduce the production of 3-hydroxy acid by-products are combined by the construction of strains that carry multiple deletions. Candidate genes can include, but are not limited to, YAL060W, YJR159W, YGL157W, YBL114W, YOR120W, YKL055C, YBR159W, YBR149W, YDL168W, YDR368W, YLR426W, YCR107W, YILLCW, YR24, YR24, YDL174C, YDR541C, YBR145W, YGL039W, YCR105W, YDL124W, YIR035C, YFL056C, YNL274c, YLR255C, YGL185C, YGL256W, YJR096W, YJR155W, YPL275W, YOR388C, YLR070C, YMR083W, YER081W, YJR139C, YDL243C, YPL113C, YOL165C, YML086C, YMR303C, YDL246C, YLR070C, YHR063C, YNL331C, YFL057C, YIL155C, YOL086C, YAL061W, YDR127W, YPR127W, YCL018W, YIL074C, YIL124W, and YEL071W. Many of these deletion strains are commercially available (for example, YSC1054 from Open Biosystems). These deletion strains are transformed with a plasmid pGV2435 from which the ALS gene (for example, B. subtilis alsS) is expressed under the control of the CUP1 promoter. Transformants are cultured in YPD medium containing 150 g / L of glucose in shaking flasks at 30 ° C, 75 rpm in an incubator with shaking for 48 hours. After 48 h of shaking flask samples, they are analyzed by HPLC for the concentration of the 3-hydroxy acid by-product (for example, DH2MB and 2-ethyl-2,3-dihydroxybutanoate derived from 2-acetolactate and aceto-2-hydroxybutyrate, respectively ). As would be understood in the art, naturally occurring homologues of 3-keto acid reductase genes (e.g., TMA29) in yeast other than S. cerevisiae can similarly be inactivated. The homologues of the 3-keto acid reductase gene (e.g., TMA29) and the methods of identifying such homologs of 3-keto acid reductase genes are described herein.
Another way to search the deletion library is to incubate the yeast cells with the 3-keto acid intermediate (eg, acetolactate, or 2-aceto-2-hydroxy-butyrate) and analyze the broth for the production of by-product 3 - corresponding hydroxy acid (for example, DH2MB or 2-ethyl-2,3-dihydroxybutanoate, derived from 2-acetolactate and aceto-2-hydroxy-butyrate, respectively).
Some of the genes listed are the result of tandem duplication or doubling events of the entire genome and are expected to have similar substrate specificities. Examples are YAL061W (BDH1), and YAL060W (BDH2), YDR368W (YPR1) and YOR120W (GCY1). Deletion of just one of the duplicated genes is not likely to result in a phenotype. These pairs of genes must be analyzed in strains that carry the deletions in both genes.
An alternative approach to find additional endogenous activity responsible for the production of the 3-hydroxy acid by-product (for example, DH2MB or 2-ethyl-2,3-dihydroxybutanoate derived from 2-acetolactate and aceto-2-hydroxybutyrate, respectively) of yeasts that overexpress genes suspected of encoding the enzyme responsible for the production of the 3-hydroxy acid by-product. Such strains are commercially available for many of the candidate genes listed above (for example, YSC3870 from Open Biosystems). Strains that overexpress ORF are processed in the same way as deletion strains. They are transformed with an ALS expression plasmid and screened for levels of 3-hydroxy acid by-product production (for example, DH2MB or 2-ethyl-2,3-dihydroxybutanoate). To shorten the list of possible genes that cause the production of a 3-hydroxy acid by-product (for example, DH2MB or 2-ethyl-2,3-dihydroxybutanoate), its expression can be analyzed in fermentation samples. Genes that are not expressed during a fermentation that produced the 3-hydroxy acid by-product (for example, DH2MB or 2-ethyl-2,3-dihydroxybutanoate) can be excluded from the list of possible targets. This analysis can be done by extracting RNA from fermentation samples and subjecting the samples to analysis of the entire expression genome, for example, by Roche NimbleGen.
As described herein, strains that naturally produce low levels of one or more 3-hydroxy acid by-products may also have applicability for the production of increased levels of desirable fermentation products that are derived from biosynthetic pathways that comprise a 3- ketoacid. As would be understood by one skilled in the art with the aid of the present disclosure, strains that naturally produce low levels of one or more of the 3-hydroxy acid by-products can inherently have low or undetectable levels of endogenous enzyme activity, resulting in reduced conversion of 3 -keto acids for 3-hydroxy acids, a favorable feature for the production of a desired fermentation product such as isobutanol. Various approaches are described herein for the identification of a native host microorganism, which is substantially free of 3-keto acid reductase activity. For example, an approach to finding a host microorganism that exhibits inherently low or undetectable endogenous enzyme activity responsible for the production of the 3-hydroxy acid by-product (eg, DH2MB or 2-ethyl-2,3-dihydroxybutanoate) is to analyze the yeast strains by incubating the yeast cells with a 3-keto acid (for example, acetolactate, or 2-aceto-2-hydroxy-butyrate) and analyzing the broth for the production of the corresponding 3-hydroxy acid by-product (for example, DH2MB or 2-ethyl-2,3-dihydroxybutanoate derived from 2-acetolactate and aceto-2-hydroxy-butyrate, respectively).
The recombinant microorganisms described here, which produce a beneficial metabolite derived from a biosynthetic pathway that uses a 3-keto acid as an intermediate product, can be further modified to reduce or eliminate the enzymatic activity for converting pyruvate into products that are not 3-keto acid (for example, acetolactate, and / or 2-aceto-2-hydroxybutyrate). In one embodiment, the enzymatic activity of pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvate oxidase, pyruvate dehydrogenase, and / or glycerol-3-phosphate dehydrogenase (GPD) is reduced or eliminated.
In a specific embodiment, the beneficial metabolite is produced in a PDC-negative GPD-negative recombinant yeast microorganism that overexpresses an acetolactate synthase (ALS) gene. In another specific modality, ALS is encoded by B.subtilis as alsS.
Reduced Accumulation of Acid By-Products from Aldehyde Intermediate As described in the Examples, the present inventors also found that unwanted acid by-products (eg, isobutyrate, in the case of isobutanol), can accumulate during fermentation reactions with micro- organisms comprising a pathway involving an aldehyde intermediate (for example, isobutyraldehyde in the case of isobutanol).
As used herein, an "acid by-product" refers to an organic compound that contains a fraction of carboxylic acid. An acid by-product can be obtained by oxidizing an aldehyde. For example, oxidation of isobutyraldehyde results in the formation of isobutyric acid (See, for example, Figure 4).
The present inventors have found that the accumulation of acid in these by-products hinders the yield and optimal productivity of the biosynthetic pathway using aldehyde intermediates. The present inventors have found that the acid production of these by-products is caused by dehydrogenation of the corresponding aldehyde. In order to reduce or eliminate the activity responsible for the production of the acid by-product, the corresponding enzymatic activity that catalyzes this reaction had to be identified and reduced or eliminated. The inventors have found that in S. cerevisiae an enzyme that catalyzes the conversion of aldehydes to acid by-products is aldehyde dehydrogenase.
The present inventors describe here several strategies for reducing the formation of acid by-products, a process that is accompanied by an increase in the yield of desirable metabolites, such as isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl- 1-butanol, 1-propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
Accordingly, an aspect of the present invention is directed to a recombinant microorganism comprising a biosynthetic pathway that uses an aldehyde as an intermediate, wherein said recombinant microorganism is substantially free of an enzyme that catalyzes the conversion of an aldehyde to a by-product. of acid.
In another aspect, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway that uses an aldehyde as an intermediate, wherein said recombinant microorganism is constructed to reduce or eliminate the expression or activity of one or more enzymes that catalyze the conversion of the aldehyde to an acid by-product.
In one embodiment, the intermediate aldehyde is isobutyraldehyde and the acid by-product is isobutyrate. In another embodiment, the intermediate aldehyde is 1-butanal and the acid by-product is butyrate. In yet another embodiment, the intermediate aldehyde is 2-methyl-1-butanal and the acid by-product is 2-methyl-1-butyrate. In yet another embodiment, the intermediate aldehyde is 3-methyl-1-butanal and the acid by-product is 3-methyl-1-butyrate. In yet another embodiment, the intermediate aldehyde is 1-propanal and the acid by-product is propionate. In yet another embodiment, the intermediate aldehyde is 1-pentanal and the acid by-product is pentanoate. In yet another embodiment, the intermediate aldehyde is 1-hexanal and the acid by-product is hexanoate. In yet another embodiment, the intermediate aldehyde is 3-methyl-1-pentanal and the acid by-product is ethyl 3-methyl-1-pentanoate. In yet another embodiment, the intermediate aldehyde is 4-methyl-1-pentanal and the acid by-product is ethyl 4-methyl-1-pentanoate. In yet another embodiment, the intermediate aldehyde is 4-methyl-1-hexanal and the acid by-product is ethyl 4-methyl-1-hexanoate. In yet another embodiment, the intermediate aldehyde is 5-methyl-1-heptanal and the acid by-product is 5-methyl-1-heptanoate.
In various embodiments described here, the protein involved in the catalysis of the conversion of an aldehyde to an acid by-product is an aldehyde dehydrogenase (ALDH).
As used herein, "aldehyde dehydrogenase" refers to an enzyme that catalyzes the reaction: an aldehyde + oxidized cofactor + H2O = an acid + reduced cofactor + H + An illustration of the exemplary reactions that can be catalyzed by aldehyde dehydrogenases is shown in Figure 4. Suitable aldehyde dehydrogenases are generally found in the EC 1.2.1.X enzyme classification subgroup, where the final X digit is dependent on the substrate or cofactor. For example, EC 1.2.1.3 catalyzes the following reaction: an aldehyde + NAD + + H2O = an acid + NADH + H +); EC 1.2.1.4 catalyzes the following reaction: an aldehyde + NADP + + H2O = an acid + NADPH + H +); and EC1.2.1.5 catalyzes the following reaction: an aldehyde + NAD (P) + + H2O = an acid + NAD (P) H + H +.
As described herein, the protein involved in the catalysis of the conversion of an aldehyde to an acid by-product is an aldehyde dehydrogenase (ALDH). In one embodiment, an aldehyde dehydrogenase encodes a gene selected from the group consisting of ALD2, ALD3, ALD4, ALD5, ALD6, and HFD1, and homologues and variants thereof. In an exemplary embodiment, the aldehyde dehydrogenase is the aldehyde dehydrogenase of ALD6 S. cerevisiae (SEQ ID NO: 25) or a homologous or variant thereof. In one embodiment, the homologue can be selected from the group consisting of Saccharomyces castelli (SEQ ID NO: 26), Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO: 28), Kluyveromyces lactis (SEQ ID NO: 29), Kluyveromyces thermotolerans (SEQ ID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQ ID NO: 33), Saccharomyces cerevisiae (SEQ ID NO: 34), Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomyces cerevisiae AWRI1631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a (SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38), Kluyveromyces marxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40), and Schizosaccharomycespombe (SEQ ID NO: 41).
In one embodiment, the recombinant microorganism includes a mutation in at least one gene encoding an aldehyde dehydrogenase that results in a reduction in the aldehyde dehydrogenase activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism includes a partial deletion of the gene encoding an aldehyde dehydrogenase that results in a reduction in the aldehyde dehydrogenase activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprises a complete deletion of a gene encoding an aldehyde dehydrogenase that results in a reduction in the aldehyde dehydrogenase activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with the gene encoding an aldehyde dehydrogenase that results in a reduction in the expression of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator that results in a reduction in the transcription of a gene encoding an aldehyde dehydrogenase. In yet another embodiment, the recombinant microorganism comprises mutations in all genes that encode an aldehyde dehydrogenase that results in a reduction in the activity of a polypeptide encoded by the gene (s). In one embodiment, said aldehyde dehydrogenase is encoded by a gene selected from the group consisting of ALD2, ALD3, ALD4, ALD5, ALD6, and HFD1, and homologues and variants thereof. As would be understood in the art, naturally occurring aldehyde dehydrogenase homologues in yeast other than S. cerevisiae can be similarly inactivated using the methods of the present invention. Aldehyde dehydrogenase homologues and methods of identifying such aldehyde dehydrogenase homologs are described here.
As understood by one skilled in the art, there are several additional mechanisms available to reduce or stop the activity of a protein such as aldehyde dehydrogenase, including, but not limited to, the use of a regulated promoter, use of a weak constitutive promoter, disruption of one of two copies of the gene in a diploid yeast, disruption of both copies of the gene of a diploid yeast, the expression of an antisense nucleic acid, the expression of a siRNA, the overexpression of a negative regulator of the endogenous promoter, alteration of activity of a heterologous or endogenous gene, the use of a heterologous gene with less specific activity, similar or combinations thereof.
As would be understood by one skilled in the art, the activity or expression of more than one aldehyde dehydrogenase can be reduced or eliminated. In a specific modality, the activity or expression of ALD4 and ALD6 or homologues or variants thereof is reduced or eliminated. In another specific modality, the activity or expression of ALD5 and ALD6 or homologues or variants thereof is reduced or eliminated. In yet another specific modality, the activity or expression of ALD4, ALD5, and ALD6 or homologues or variants thereof is reduced or eliminated. In yet another specific embodiment, the activity or expression of the aldehyde dehydrogenases located cytosolically ALD2, ALD3, and ALD6 or homologues or variants thereof is reduced or eliminated. In yet another specific modality, the activity or expression of mitochondrial located aldehyde dehydrogenases, ALD4 and ALD5 or homologues or variants thereof, is reduced or eliminated.
As described here, the recombinant microorganisms of the present invention are constructed to produce less of the acid by-product than an unmodified parent microorganism. In one embodiment, the recombinant microorganism produces the acid by-product from a carbon source with a carbon yield of less than about 50 percent compared to a parent microorganism. In another embodiment, the microorganism produces the acid by-product from a carbon source with a carbon yield of less than about 25, less than about 10, less than about 5, less than about 1, less than about 0.5, less than about 0.1, or less than about 0.01 percent compared to a parental microorganism. In one embodiment, the acid by-product is isobutyrate, derived from isobutyraldehyde, an intermediate in the isobutanol biosynthetic pathway. In another embodiment, the acid by-product is butyrate, derived from 1-butanal, an intermediate of the 1-butanol biosynthetic pathway. In yet another embodiment, the acid by-product is 2-methyl-1-butyrate, derived from 2-methyl-1-butanal, an intermediate of the 2-methyl-1-butanol biosynthetic pathway. In yet another embodiment, the acid by-product is 3-methyl-1-butyrate, derived from 3-methyl-1-butanal, an intermediate of the 3-methyl-1-butanol biosynthetic pathway. In yet another embodiment, the acid by-product is propionate, derived from 1-propanal, an intermediate of the 1-propanol biosynthetic pathway. In yet another embodiment, the acid by-product is pentanoate, derived from 1-pentanal, an intermediate in the biosynthetic pathway of 1-pentanol. In yet another embodiment, the acid by-product is hexanoate, derived from 1-hexanal, an intermediate of the 1-hexanol biosynthetic pathway. In yet another embodiment, the acid by-product is 3-methyl-1-pentanoate, derived from 3-methyl-1-pentanal, an intermediate of the 3-methyl-1-pentanol biosynthetic pathway. In yet another embodiment, the acid by-product is 4-methyl-1-pentanoate, derived from 4-methyl-1-pentanal, an intermediate of the 4-methyl-1-pentanol biosynthetic pathway. In yet another embodiment, the acid by-product is 4-methyl-1-hexanoate, derived from 4-methyl-1-hexanal, an intermediate of the 4-methyl-1-hexanol biosynthetic pathway. In yet another embodiment, the acid by-product is 5-methyl-1-heptanoate, derived from 5-methyl-1-heptanal, an intermediate of the 5-methyl-1-heptanol biosynthetic pathway.
In one embodiment, the carbon yield of the acid by-product from the corresponding aldehyde is reduced by at least about 50% in a recombinant microorganism compared to a parent microorganism does not comprise a reduction or deletion of activity or expression of one or more proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product. In another embodiment, the carbon yield of the acid by-product from acetolactate is reduced by at least about 60%, by at least about 65%, by at least about 70%, by at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99.9% , or at least about 100% compared to a parent microorganism does not comprise a reduction or deletion of the activity or expression of one or more proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product. In one embodiment, the acid by-product is isobutyrate, derived from isobutyraldehyde, an intermediate in the isobutanol biosynthetic pathway. In another embodiment, the acid by-product is butyrate, derived from 1-butanal, an intermediate of the 1-butanol biosynthetic pathway. In yet another embodiment, the acid by-product is 2-methyl-1-butyrate, derived from 2-methyl-1-butanal, an intermediate of the 2-methyl-1-butanol biosynthetic pathway. In yet another embodiment, the acid by-product is 3-methyl-1-butyrate, derived from 3-methyl-1-butanal, an intermediate of the 3-methyl-1-butanol biosynthetic pathway. In yet another embodiment, the acid by-product is propionate, derived from 1-propanal, an intermediate of the 1-propanol biosynthetic pathway. In yet another embodiment, the acid by-product is pentanoate, derived from 1-pentanal, an intermediate in the biosynthetic pathway of 1-pentanol. In yet another embodiment, the acid by-product is hexanoate, derived from 1-hexanal, an intermediate of the 1-hexanol biosynthetic pathway. In yet another embodiment, the acid by-product is 3-methyl-1-pentanoate, derived from 3-methyl-1-pentanal, an intermediate of the 3-methyl-1-pentanol biosynthetic pathway. In yet another embodiment, the acid by-product is 4-methyl-1-pentanoate, derived from 4-methyl-1-pentanal, an intermediate of the 4-methyl-1-pentanol biosynthetic pathway. In yet another embodiment, the acid by-product is 4-methyl-1-hexanoate, derived from 4-methyl-1-hexanal, an intermediate of the 4-methyl-1-hexanol biosynthetic pathway. In yet another embodiment, the acid by-product is 5-methyl-1-heptanoate, derived from 5-methyl-1-heptanal, an intermediate of the 5-methyl-1-heptanol biosynthetic pathway.
In an additional embodiment, the yield of a desirable fermentation product is increased in the recombinant microorganisms comprising a reduction or elimination of the activity or expression of one or more proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product. In one embodiment, the yield of a desirable fermentation product is increased by at least about 1% compared to a parental microorganism that does not comprise a reduction or elimination of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product. In another embodiment, the yield of a desirable fermentation product is increased by at least about 5%, by at least about 10%, by at least about 25%, or by at least about 50% compared to a parental microorganism that does not comprise a reduction or elimination of the activity or expression of one or more endogenous proteins involved in the catalysis of the conversion of an aldehyde to an acid by-product. As described here, the desirable fermentation product can be derived from any biosynthetic pathway in which an aldehyde acts as an intermediate, including, but not limited to, isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl- 1-butanol, 1-propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol biosynthetic routes . Methods to identify additional enzymes that catalyze the conversion of an aldehyde to an acid by-product are described as follows: endogenous yeast genes that encode the putative aldehyde and alcohol dehydrogenases are deleted from the genome of a yeast strain. These deletion strains are compared with the original strain by enzymatic assay. Many of these deletion strains are commercially available (for example, YSC1054 from Open Biosystems).
Another way to search the deletion library is to incubate the yeast cells with an aldehyde (for example, isobutyraldehyde, or 1-butanal) and analyze the broth for the production of the corresponding by-product acid (for example, isobutyrate or butyrate, derived isobutyraldehyde or 1-butanal, respectively).
An alternative approach to find the additional endogenous activity responsible for the production of the acid by-product (eg, isobutyrate or butyrate, derived from isobutyraldehyde or 1-butanal, respectively) is to analyze the yeast strains that overexpress the genes suspected of encoding the responsible enzyme production of the acid by-product. Such strains are commercially available for many of the candidate genes mentioned above (for example, YSC3870 from Open Biosystems). Strains that overexpress ORF are screened for increased levels of acid by-product production. Alternatively, cell lysates from strains that overexpress ORF are tested for increased aldehyde oxidation activity. To narrow the list of possible genes that cause the production of acid by-products, their expression can be analyzed in fermentation samples. Genes that are not expressed during a fermentation that produces an acid by-product can be excluded from the list of possible targets. This analysis can be done by extracting RNA from fermentation samples and subjecting the samples to analysis of the entire expression genome, for example, by Roche NimbleGen.
As described herein, strains that naturally produce low levels of one or more acid by-products can also have application for the production of high levels of desirable fermentation products that are derived from biosynthetic pathways that comprise an intermediate aldehyde. As would be understood by one skilled in the art with the aid of the present disclosure, strains that naturally produce low levels of one or more acid by-products may inherently have low or undetectable levels of endogenous enzyme activity, resulting in reduced conversion of aldehydes to by-products of acid, a favorable characteristic for the production of a desired fermentation product such as isobutanol. Various approaches are described herein for the identification of a native host microorganism, which is substantially free of aldehyde dehydrogenase activity. For example, an approach to finding a host microorganism that exhibits inherently low or undetectable endogenous enzyme activity responsible for the production of the acid by-product (eg, isobutyrate or butyrate) is to analyze yeast strains by incubating yeast cells. with an aldehyde (for example, isobutyraldehyde, or 1-butanal) and analyze the broth for the production of the corresponding acid by-product (for example, isobutyrate, butyrate or, derived from isobutyraldehyde or 1-butanal, respectively).
As described above, a strategy for reducing the production of the acid by-product, isobutyrate, is to reduce or eliminate the activity or expression of one or more endogenous aldehyde dehydrogenase proteins present in yeast that may be converting isobutyraldehyde to isobutyrate.
Another strategy for reducing isobutyrate production is to reduce or eliminate the activity or expression of one or more endogenous yeast alcohol dehydrogenases. The reduction of the expression, or the deletion, of one or more alcohol dehydrogenases, including, but not limited to, S. cerevisiae ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1 and homologues or variants thereof, lead in overall reduced production of isobutyrate and a concomitant increase in isobutanol yield. The reduction and / or deletion of additional dehydrogenases is provided for in the present invention and is considered within the scope of the present invention. These alcohol dehydrogenases include additional dehydrogenases, such as S. cerevisiae BDH1, BDH2, SOR1, SOR2, and XYL1 and homologues or variants thereof, as well as aryl alcohol dehydrogenases such as AAD3, AAD4, AAD6, AAD10, AAD14, AAD15, AAD16, and YPL088W and counterparts or variants thereof.
In another embodiment, the present invention provides recombinant microorganisms modified to reduce and / or delete one or more additional genes encoding carbonyl / aldehyde reductases. These carbonyl / aldehyde reductases include S. cerevisiae ARI1, YPR1, TMA29, YGL039W and UGA2 and homologues or variants thereof.
An additional strategy described here to reduce the production of isobutyrate by-product is to reduce or eliminate the activity or expression of endogenous proteins present in yeast that may be producing isobutyrate from the 2-ketoisovalerate intermediate of the isobutanol pathway. Such enzymes are generally referred to as keto acid dehydrogenases (KDH). Eliminating or reducing the activity or expression of these endogenous proteins can reduce or eliminate the production of the undesirable by-product, isobutyrate. The activity of the KDH enzyme was identified in S. cerevisiae (Dickinson, JR, and IW Dawes, 1992, The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in S. cerevisiae. J. Gen. Microbiol. 138: 2029-2033). The reduction in expression, or deletion of one or more keto acid dehydrogenases and homologues or variants thereof, generally leads to less isobutyrate production and a concomitant increase in isobutanol yield. The reduction in the expression, or deletion, of genes in S. cerevisiae and other yeasts can be achieved by methods known to those skilled in the art, such as allele replacement or exchange, as well as disruption of the gene through the insertion of another gene or marker cassette. .
Another strategy described here to reduce the production of the isobutyrate by-product is to increase the activity and / or expression of an alcohol dehydrogenase (ADH) responsible for the conversion of isobutyraldehyde to isobutanol. This strategy avoids competition for endogenous enzymes for the intermediate isobutanol pathway, isobutyraldehyde. An increase in the activity and / or expression of the alcohol dehydrogenase enzyme can be achieved by several means. For example, alcohol dehydrogenase activity can be increased by using a promoter with increased promoter resistance, by increasing the number of copies of the alcohol dehydrogenase gene, or by using an alternative or modified alcohol dehydrogenase enzyme. increased specific activity.
An alternative strategy described here to reduce the production of the isobutyrate by-product is the use of an alcohol dehydrogenase (ADH) enzyme in the isobutanol pathway responsible for the conversion of isobutyraldehyde to isobutanol which exhibits a decrease in the Michaelis-Menten (KM) constant. This strategy also avoids competition for endogenous enzymes for the intermediate isobutanol pathway, isobutyraldehyde.
Another strategy described here to reduce the production of the isobutyrate by-product is the use of an alcohol dehydrogenase (ADH) in the isobutanol pathway responsible for the conversion of isobutyraldehyde to isobutanol which exhibits increased activity and a decrease in the Michaelis-Menten (KM) constant . This strategy also avoids competition for endogenous enzymes for the intermediate isobutanol pathway, isobutyraldehyde.
Furthermore, through the use of a modified ADH enzyme, the present inventors can establish a situation in which the direct reaction (ie, the conversion of isobutyraldehyde to isobutanol) is the reaction favored over the reverse reaction (that is, the conversion of isobutyraldehyde to isobutanol).
The strategies described above generally lead to a decrease in isobutyrate yield, which is accompanied by an increase in isobutanol yield. Thus, the above strategies are useful to decrease the yield and / or titration of isobutyrate and to increase the ratio of isobutanol yield to the isobutyrate yield.
In one embodiment, the production of isobutyrate (mole of isobutyrate per mole of glucose) is less than about 5%. In another embodiment, the yield of isobutyrate (mol deisobutyrate per mol of glucose) is less than about 1%. In yet another embodiment, the yield of isobutyrate (mole of isobutyrate per mole of glucose) is less than about 0.5%, less than about 0.1%, less than about 0.05%, or less than about 0.01%.
In one embodiment, the yield ratio of isobutanol to isobutyrate is at least about 2. In another embodiment, the yield of isobutanol to isobutyrate is at least about 5. In yet another embodiment, the yield ratio of isobutanol to isobutyrate is at least about 20, at least about 100, at least about 500, or at least about 1000.
The recombinant microorganisms described here, which produce a beneficial metabolite derived from a biosynthetic pathway that uses an aldehyde as an intermediate can be further modified to reduce or eliminate the enzymatic activity for converting pyruvate to products other than a 3-keto acid (for example, acetolactate, and / or 2-aceto-2-hydroxybutyrate). In one embodiment, the enzymatic activity of pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvate oxidase, pyruvate dehydrogenase, and / or glycerol-3-phosphate dehydrogenase (GPD) is reduced or eliminated.
In a specific embodiment, the beneficial metabolite is produced in a recombinant PDC-negative yeast GPD-negative microorganism that overexpresses an acetolactate synthase (ALS) gene. In another specific modality, ALS is encoded by B. subtilis alsS.
Reduced Accumulation of 3-Hydroxy Acid By-Products and Acid By-Products The present inventors describe here several strategies for reducing the conversion of a 3-keto acid intermediate to a corresponding 3-hydroxy acid by-product, a process that is accompanied by an increase in yield desirable metabolites. The present inventors also describe here several strategies for reducing the conversion of an aldehyde Intermediate to a corresponding acid by-product, a process that is accompanied by an additional increase in the yield of desirable metabolites.
Consequently, in one aspect, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway that uses a 3-keto acid as an intermediate and an aldehyde as an intermediate, wherein said recombinant microorganism is (i) substantially free an enzyme that catalyzes the conversion of the 3-keto acid intermediate to a 3-hydroxy acid by-product and (ii) substantially free of an enzyme that catalyzes the conversion of an aldehyde to an acid by-product. In one embodiment, the 3-keto acid intermediate is acetolactate. The biosynthetic pathway that uses acetolactate and an aldehyde as intermediates can be selected from a pathway for the biosynthesis of isobutanol, 1-butanol, and 3-methyl-1-butanol. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate. The biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate and an aldehyde as intermediates can be selected from a pathway for the biosynthesis of 2-methyl-1-butanol, 3-methyl-1-pentanol, 4-methyl-1 -hexanol, and 5-methyl-1-heptanol.
In another aspect, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway that uses a 3-keto acid as an intermediate and an aldehyde as an intermediate, wherein said recombinant microorganism is (i) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of the 3-keto acid intermediate to a 3-hydroxy acid by-product and (ii) constructed to reduce or eliminate the expression or activity of one or more enzymes that catalyze the conversion of aldehyde for an acid by-product. In one embodiment, the 3-keto acid intermediate is acetolactate. The biosynthetic pathway that uses acetolactate and an aldehyde as intermediates can be selected from a pathway for the biosynthesis of isobutanol, 1-butanol, and 3-methyl-1-butanol. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate. The biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate and an aldehyde as intermediates can be selected from a pathway for the biosynthesis of 2-methyl-1-butanol, 3-methyl-1-pentanol, 4-methyl-1 -hexanol, and 5-methyl-1-heptanol.
In various embodiments described here, the protein involved in the catalysis of the conversion of the 3-keto acid intermediate to the 3-hydroxy acid by-product is a ketorreductase. In an exemplary embodiment, ketorreductase is a 3-keto acid reductase (3-KAR). In an additional exemplary embodiment, 3-keto acid reductase is the protein of S. cerevisiae YMR226C (SEQ ID NO: 1) or a homolog or variant thereof. In one embodiment, the homologue can be selected from the group consisting of Vanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomyces castellii (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomyces bayanus (SEQ ID NO: 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), K. lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQ ID NO: 10), Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12), Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14), Candida dubliniensis (SEQ ID NO: 15), Candida albicans (SEQ ID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO: 18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO: 20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQ ID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).
In several embodiments described here, the protein involved in the catalysis of the conversion of an aldehyde to an acid by-product is an aldehyde dehydrogenase (ALDH). In one embodiment, aldehyde dehydrogenase is encoded by a gene selected from the group consisting of ALD2, ALD3, ALD4, ALD5, ALD6, and HFD1, and homologues and variants thereof. In an exemplary embodiment, the aldehyde dehydrogenase is S. cerevisiae aldehyde dehydrogenase ALD6 (SEQ ID NO: 25) or homologous or variant thereof. In one embodiment, the homologue can be selected from the group consisting of Saccharomyces castelli (SEQ ID NO: 26), Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO: 28), Kluyveromyces lactis (SEQ ID NO: 29), Kluyveromyces thermotolerans (SEQ ID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQ ID NO: 33), Saccharomyces cerevisiae (SEQ ID NO: 34), Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomyces cerevisiae AWRI1631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a (SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38), Kluyveromyces marxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40), and Schizosaccharomyces pombe (SEQ ID NO: 41).
The recombinant microorganisms described here, which produce a beneficial metabolite derivative of a biosynthetic pathway that uses a 3-keto acid and an aldehyde as an intermediate, can further be constructed to reduce or eliminate the enzymatic activity for converting pyruvate to products other than a 3-keto acid (for example, acetolactate and / or 2-aceto-2-hydroxybutyrate). In one embodiment, the enzymatic activity of pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvate oxidase, pyruvate dehydrogenase, and / or glycerol-3-phosphate dehydrogenase (GPD) is reduced or eliminated.
In a specific embodiment, the beneficial metabolite is produced in a recombinant GDP-negative yeast PDC-negative microorganism that overexpresses an acetolactate synthase (ALS) gene. In another specific embodiment, ALS is encoded by B. subtilis alsS.
Illustrative modalities of Strategies for Reducing the Accumulation of 3-Hydroxy Acid By-Products and / or Acid By-Products In a specific illustrative modality, the recombinant microorganism comprises an isobutanol-producing metabolic pathway of which acetolactate and isobutyraldehyde are intermediates, in which said micro - recombinant organism is substantially free of enzymes that catalyze the conversion of acetolactate intermediate to DH2MB and isobutyraldehyde intermediate to isobutyrate. In another specific embodiment, the recombinant microorganism comprises an isobutanol-producing metabolic pathway of which acetolactate and isobutyraldehyde are intermediates, in which said recombinant microorganism is (i) constructed to reduce or eliminate the expression or activity of one or more enzymes that catalyze the conversion of acetolactate to DH2MB and (ii) constructed to reduce or eliminate the expression or activity of one or more enzymes that catalyze the conversion of isobutyraldehyde to isobutyrate. In one embodiment, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR). In another embodiment, the enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate is an aldehyde dehydrogenase (ALDH). A non-limiting example of such a route in which a 3-keto acid reductase (3-KAR) and an aldehyde dehydrogenase (ALDH) are eliminated is shown in Figure 5.
In an additional specific illustrative embodiment, the recombinant microorganism comprises a 3-methyl-1-butanol-producing metabolic pathway of which acetolactate and 3-methyl-1-butanal are intermediates, wherein said recombinant microorganism is substantially free of enzymes that catalyze the conversion of acetolactate intermediate to DH2MB and the 3-methyl-1-butanal intermediate to 3-methyl-1-butyrate. In another specific embodiment, the recombinant microorganism comprises a metabolic pathway producing 3-methyl-1-butanol of which acetolactate and 3-methyl-1-butanal are intermediates, in which said recombinant microorganism is (i) constructed to reduce or eliminate the expression or activity of one or more enzymes that catalyze the conversion of acetolactate to DH2MB and (ii) built to reduce or eliminate the expression or activity of one or more enzymes that catalyze the conversion of 3-methyl-1-butanal for 3-methyl-1-butyrate. In one embodiment, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR). In another embodiment, the enzyme that catalyzes the conversion of 3-methyl-1-butanal to 3-methyl-1-butyrate is an aldehyde dehydrogenase (ALDH). A non-limiting example of such a route in which a 3-keto acid reductase (3-KAR) and an aldehyde dehydrogenase (ALDH) are eliminated is shown in Figure 6.
In an additional specific illustrative embodiment, the recombinant microorganism comprises a metabolic pathway producing 2-methyl-1-butanol of which acetolactate and 2-methyl-1-butanal are intermediates, wherein said recombinant microorganism is substantially free of enzymes that catalyze the conversion of 2-aceto-2-hydroxybutyrate intermediate to 2-ethyl-2,3-dihydroxybutyrate and 2-methyl-1-butanal intermediate to 2-methyl-1-butyrate. In another specific embodiment, the recombinant microorganism comprises a metabolic pathway producing 2-methyl-1-butanol of which 2-aceto-2-hydroxybutyrate and 2-methyl-1-butanal are intermediates, in which said microorganisms recombinant is (i) constructed to reduce or eliminate the expression or activity of one or more enzymes that catalyze the conversion of 2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrate and (ii) constructed to reduce or eliminate the expression or activity of one or more enzymes that catalyze the conversion of 2-methyl-1-butanal to 2-methyl-1-butyrate. In one embodiment, the enzyme that catalyzes the conversion of 2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrate is a 3-keto acid reductase (3-KAR). In another embodiment, the enzyme that catalyzes the conversion of 2-methyl-1-butanal to 2-methyl-1-butyrate is an aldehyde dehydrogenase (ALDH). A non-limiting example of such a pathway in which a 3-keto acid reductase (3-KAR) and an aldehyde dehydrogenase (ALDH) are eliminated is shown in Figure 7.
Overexpression of Enzymes That Convert DH2MB to Isobutanol Via Intermediates A different approach to reduce or eliminate the production of 2,3-dihydroxy-2-methylbutanoic acid (CAS # 14868-24-7) in isobutanol-producing yeast is overexpressing an enzyme that converts DH2MB into an intermediate of the isobutanol pathway. One way to accomplish this is through the use of an enzyme that catalyzes the interconversion of DH2MB and acetolactate, but favors the oxidation of DH2MB. Therefore, in one embodiment, the present invention provides a recombinant microorganism for the production of isobutanol, wherein said recombinant microorganism overexpresses an endogenous or heterologous protein capable of converting DH2MB to acetolactate.
In one embodiment, the endogenous or heterologous protein kinetically favors the oxidation reaction. In another embodiment, the endogenous or heterologous protein has a low KM for DH2MB and a high KM for acetolactate. In yet another embodiment, the endogenous or heterologous protein has a low KM for the oxidized form of its cofactor and a high KM for the corresponding reduced form of its cofactor. In yet another embodiment, the endogenous or heterologous protein has a higher kcat for the oxidation reaction than for the reduction direction. This endogenous or heterologous protein should preferably be able to use a redox cofactor with a high concentration of its oxidized form versus its reduced form.
In one embodiment, the endogenous or heterologous protein is encoded by a gene selected from the group consisting of YAL060W, YJR159W, YGL157W, YBL114W, YOR120W, YKL055C, YBR159W, YBR149W, YDL168W, YDR368W, YCRRW26, YCR024, , YMR318C, YBR046C, YHR104W, YIR036C, YDL174C, YDR541C, YBR145W, YGL039W, YCR105W, YDL124W, YIR035C, YFL056C, YNL274c, YLR255C, YGL185C, YGL256W, YJR096W, YJR155W, YPL275W, YOR388C, YLR070C, YMR083W, YER081W, YJR139C, YDL243C , YPL113C, YOL165C, YML086C, YMR303C, YDL246C, YLR070C, YHR063C, YNL331C, YFL057C, YIL155C, YOL086C, YAL061W, YDR127W, YPR127W, YCL018, YIL0C, In addition, heterologous genes can be overexpressed in isobutanol-producing yeast. For example, beta-hydroxy acid dehydrogenases (EC1.1.1.45 and EC1.1.1.60) would be candidates for overexpression.
In another modality, the endogenous or heterologous protein that kinetically favors the reducing reaction is built to favor the oxidation reaction. In another embodiment, the protein is built to have a low KM for DH2MB and a high KM for acetolactate. In yet another modality, the protein is built to have a low KM for the oxidized form of your cofactor and a high KM for the corresponding reduced form of your cofactor. In yet another embodiment, the protein is constructed to have a higher kcat for the oxidation reaction than for the reduction direction. This constructed protein preferably has the ability to use a redox cofactor with a high concentration of its oxidized form versus its reduced form.
Alternatively, an enzyme that can be overexpressed so that it isomerizes DH2MB to DHIV. This approach represents a new avenue for the production of isobutanol from pyruvate. Thus, in one embodiment, the present invention provides a recombinant microorganism for the production of isobutanol, wherein said recombinant microorganism overexpresses an endogenous or heterologous protein capable of converting DH2MB to 2,3-dihydroxy-isovalerate.
Overexpression of Enzymes That Convert 2-Ethyl-2,3-Dihydroxybutanoate to Intermediates in the Biosynthetic Route A different approach to reducing or eliminating the production of 2-ethyl-2,3-dihydroxybutanoate is to overexpress an enzyme that converts 2-ethyl-2, 3-dihydroxybutanoate in an intermediate of the biosynthetic pathway. This approach is useful for any biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate as an intermediate, including, but not limited to, 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl- 1-hexanol, and 5-methyl-1-heptanol. One way to accomplish this is through the use of an enzyme that catalyzes the interconversion of 2-ethyl-2,3-dihydroxybutanoate and 2-aceto-2-hydroxybutyrate, but favors the oxidation of 2-ethyl-2,3-dihydroxybutanoate. Therefore, in one embodiment, the present invention provides a recombinant microorganism to produce a product selected from 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5- methyl-1-heptanol wherein said recombinant microorganisms overexpress an endogenous or heterologous protein capable of converting 2-ethyl-2,3-dihydroxybutanoate to 2-aceto-2-hydroxybutyrate.
In one embodiment, the endogenous or heterologous protein kinetically favors the oxidation reaction. In another embodiment, the endogenous or heterologous protein has a low KM for 2-ethyl-2,3-dihydroxybutanoate and a high KM for 2-aceto-2-hydroxybutyrate. In yet another modality, the endogenous or heterologous protein has a low KM for the oxidized form of its cofactor and a high KM for the corresponding reduced form of its cofactor. In yet another embodiment, the endogenous or heterologous protein has a higher kcat for the oxidation reaction than for the reduction direction. This endogenous or heterologous protein should preferably have the ability to use a redox cofactor with a high concentration of its oxidized form versus its reduced form.
In one embodiment, the endogenous or heterologous protein is encoded by a gene selected from the group consisting of YAL060W, YJR159W, YGL157W, YBL114W, YOR120W, YKL055C, YBR159W, YBR149W, YDL168W, YDR368W, YCRRW26, YCR024, , YMR318C, YBR046C, YHR104W, YIR036C, YDL174C, YDR541C, YBR145W, YGL039W, YCR105W, YDL124W, YIR035C, YFL056C, YNL274c, YLR255C, YGL185C, YGL256W, YJR096W, YJR155W, YPL275W, YOR388C, YLR070C, YMR083W, YER081W, YJR139C, YDL243C , YPL113C, YOL165C, YML086C, YMR303C, YDL246C, YLR070C, YHR063C, YNL331C, YFL057C, YIL155C, YOL086C, YAL061W, YDR127W, YPR127W, YCL018, YIL0C, In addition, heterologous genes can be overexpressed in isoleucine-producing yeast. For example, beta-hydroxy acid dehydrogenases (EC1.1.1.45 and EC1.1.1.60) will be candidates for overexpression.
Alternatively, an enzyme can be overexpressed so that it isomerizes 2-ethyl-2,3-dihydroxybutanoate to 2,3-dihydroxy-3-methylvalerate. This approach represents a new route for the production of 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol from pyruvate. Thus, in one embodiment, the present invention provides a recombinant microorganism to produce a product selected from 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5- methyl-1-heptanol, wherein said recombinant microorganism overexpresses an endogenous or heterologous protein capable of converting 2-ethyl-2,3-dihydroxybutanoate to α, β-dihydroxy-P-methylvalerate.
Use of Super Expressed Ketol-Acid Redutoisomerase (KARI) and / or Modified Ketol-Acid Redutoisomerase (KARI) to Reduce DH2MB Production
As described here, the conversion of acetolactate to DH2MB competes with the isobutanol pathway to the intermediate acetolactate. In current yeast isobutanol producing strains, ketol-acid reductisomerase (KARI) catalyzes the conversion of acetolactate to DHIV.
In one embodiment, the present invention provides recombinant microorganisms having an overexpressed ketol-acid redutoisomerase (KARI), which catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate (DHIV). Overexpression of KARI has an effect of reducing DH2MB production. In one embodiment, KARI has at least 0.01 U / mg of activity in the lysate. In another modality, KARI has at least 0.03 U / mg of activity in the lysate. In yet another embodiment, KARI has at least 0.05, 0.1, 0.5, 1, 2, 5, or 10 U / mg of activity in the lysate.
In a preferred embodiment, the overexpressed KARI is built to exhibit a reduced KM for acetolactate compared to a wild-type or parental KARI. The use of modified KARI with a lower KM for acetolactate is expected to reduce the production of the DH2MB by-product. A KARI with KM of smaller substrate is identified by screening its counterparts. Alternatively, KARI can be built to exhibit reduced KM by directed evolution using techniques known in the art.
In each of these modalities, KARI can be a variant enzyme that uses NADH (instead of NADPH) as a cofactor. Such enzymes are described in the commonly owned Copending Publication US 2010/0143997, which is incorporated herein by reference in its entirety for all purposes.
Use of Overexpressed Dihydroxy Acid Dehydratase (DHAD) to Reduce DH2MB Production
As described here, the present inventors have found that overexpression through the enzyme of the isobutanol pathway, dihydroxy acid dehydratase (DHAD), reduces the production of the by-product, DH2MB.
Consequently, in one embodiment, the present invention provides recombinant microorganisms having a dihydroxy acid dehydratase (DHAD), which catalyzes the conversion of 2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV). Overexpression of DHAD has an effect of reducing DH2MB production. In one embodiment, DHAD has at least 0.01 U / mg of activity in the lysate. In another embodiment, DHAD has at least 0.03 U / mg of activity in the lysate. In yet another embodiment, DHAD has at least 0.05, 0.1, 0.5, 1, 2, 5, or 10 U / mg of activity in the lysate.
Recombinant microorganisms for the production of 3-hydroxy acids The present invention provides, in additional aspects, recombinant microorganisms for the production of 3-hydroxy acids as a product or a metabolic intermediate. In one embodiment, these recombinant 3-hydroxy acid producing microorganisms express acetolactate synthase (ALS) and a 3-keto acid reductase that catalyze the reduction of 2-acetolactate to DH2MB. In another embodiment, these 3-hydroxy acid producing recombinant microorganisms express acetolactate synthase (ALS) and a 3-keto acid reductase that catalyze the reduction of 2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrate.
These recombinant 3-hydroxy acid producing microorganisms can be further constructed to reduce or eliminate the enzymatic activity for converting pyruvate to products other than acetolactate. In one embodiment, the enzymatic activity of pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvate oxidase, pyruvate dehydrogenase, and / or glycerol-3-phosphate dehydrogenase (GPD) is reduced or eliminated.
In a specific embodiment, DH2MB is produced in a recombinant GDP-negative PDC-negative yeast microorganism that overexpresses an ALS gene and expresses 3-keto acid reductase. In one embodiment, 3-keto acid reductase is natively expressed. In another embodiment, 3-keto acid reductase is expressed heterologously. In yet another embodiment, 3-keto acid reductase is overexpressed. In a specific embodiment, a 3-keto acid reductase is encoded by the S. cerevisiae TMA29 gene or a homologue thereof. In another specific modality, ALS is encoded by B. subtilis AlsS.
In another specific embodiment, 2-ethyl-2,3-dihydroxybutyrate is produced in a recombinant GDP-negative yeast PDC-negative microorganism that overexpresses an ALS gene and expresses 3-keto acid reductase. In one embodiment, 3-keto acid reductase is natively expressed. In another embodiment, 3-keto acid reductase is expressed heterologously. In yet another embodiment, 3-keto acid reductase is overexpressed. In a specific embodiment, a 3-keto acid reductase is encoded by the S. cerevisiae TMA29 gene or a homologue thereof. In another specific modality, ALS is encoded by B. subtilis AlsS.
In accordance with these additional aspects, the present invention also provides a method for producing 2,3-dihydroxy-2-methylbutanoic acid (DH2MB), comprising: (a) providing a recombinant DH2MB producing microorganism that expresses acetolactate synthase (ALS ) and a 3-keto acid reductase that catalyze the reduction of 2-acetolactate to DH2MB, and (b) cultivate the said recombinant microorganism in a culture medium containing a raw material that provides the carbon source, up to an amount of DH2MB be produced.
In accordance with these additional aspects, the present invention also provides a method for producing 2-ethyl-2,3-dihydroxybutyrate, comprising: (a) providing a recombinant micro-organism producing 2-ethyl-2,3-dihydroxybutyrate that expresses acetolactate synthase (ALS) and a 3-keto acid reductase that catalyze the reduction of 2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrate, and (b) culturing said recombinant microorganism in a culture medium containing a raw material that supplies the carbon source, until a recoverable amount of 2-ethyl-2,3-dihydroxybutyrate is produced.
Recombinant microorganisms for the production of Acid Products The present invention provides, in additional aspects, recombinant microorganisms for the production of acid products derived from aldehydes. In one embodiment, these acid product producing recombinant microorganisms express an aldehyde dehydrogenase that catalyzes the conversion of an aldehyde to a corresponding acid product. These recombinant acid product producing microorganisms can be further constructed to reduce or eliminate competition from enzyme activity for the undesirable conversion of metabolites upstream of the desired acid product.
In a specific embodiment, the acid product is produced in a recombinant yeast microorganism that overexpresses an aldehyde dehydrogenase. In one embodiment, aldehyde dehydrogenase is natively expressed. In another embodiment, aldehyde dehydrogenase is expressed heterologously. In yet another embodiment, aldehyde dehydrogenase is overexpressed. In a specific embodiment, aldehyde dehydrogenase is encoded by the S. cerevisiae ALD6 gene or a homologue thereof.
In accordance with this additional aspect, the present invention also provides a method for producing an acid product, comprising: (a) providing an acid product producing recombinant microorganism that expresses an aldehyde dehydrogenase that catalyzes the conversion of an aldehyde to a product of acid, and (b) cultivating said recombinant microorganism in a culture medium containing a raw material that supplies the carbon source, until a recoverable amount of the desired acid product is produced. The Microorganism in General The recombinant microorganisms provided here can express a plurality of heterologous and / or native enzymes involved in pathways for the production of beneficial metabolites such as isobutanol, 2-butanol, 1-butanol, 2-butanone, 2, 3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, coenzyme A, 2-methyl-1-butanol, isoleucine, 1-pentanol, 1-hexanol, 3-methyl- 1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, and 1-propanol from a suitable carbon source. A non-limiting list of beneficial metabolites produced in constructed biosynthetic pathways is found here in Tables 1- As described here, "built" or "modified" microorganisms are produced by introducing genetic material into a host or parental microorganism the choice and / or by modifying the expression of native genes, thus modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material and / or the modification of the expression of native genes, the parental microorganism acquires new properties, for example, the ability to produce a new, or larger amounts, of an intracellular and / or extracellular metabolite. As described herein, the introduction of genetic material into and / or the modification of the expression of genes native to a parent microorganism results in a new or modified ability to produce beneficial metabolites such as isobutanol, 2-butanol, 1-butanol , 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, coenzyme A, 2-methyl-1-butanol, isoleucine, 1-pentanol, 1 -hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, and 1-propanol from a suitable carbon source. The genetic material introduced and / or the modified genes for expression in the parental microorganism contains the gene (s), or parts of genes, encoding one or more of the enzymes involved in the biosynthesis pathway for the production of one or more more metabolites selected from isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, coenzyme A, 2-methyl-1-butanol, isoleucine, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, and 1-propanol and may also include additional elements for the expression and / or regulation of the expression of these genes, for example, promoter sequences.
In addition to introducing genetic material into a parental or host microorganism, a modified or constructed microorganism may also include altering, disrupting, deleting or knocking out a gene or polynucleotide to alter the cellular and biochemical physiology of the micro- body. Through the alteration, rupture, deletion or knockout of a gene or polynucleotide, the microorganism acquires new or improved properties (for example, the ability to produce a greater amount of new metabolites or an intracellular metabolite, to improve the flow of a metabolite over desired route, and / or to reduce the production of by-products).
The recombinant microorganisms provided here can also produce metabolites in amounts that are not available in the parent microorganism. A "metabolite" refers to any substance produced by metabolism or a substance needed to, or be part of, a particular metabolic process. A metabolite can be an organic compound that is a starting material (for example, glucose or pyruvate), an intermediate (for example, 2-ketoisovalerate), or a final product (for example, isobutanol), of metabolism. Metabolites can be used to build more complex molecules, or they can be divided into simpler molecules. Intermediate metabolites can be synthesized from other metabolites, possibly used to make more complex substances, or divided into simpler compounds, often with the release of chemical energy. The disclosure identifies specific genes useful in the methods, compositions and organisms of the disclosure, however, it will be recognized that the absolute identity of such genes is not necessary. For example, changes in a particular gene or polynucleotide comprising a coding sequence for a polypeptide or enzyme can be made and tracked for activity. Usually, these changes include conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for the expression of a functional enzyme, using methods known in the art.
Due to the inherent degeneracy of the genetic code, other polynucleotides that encode substantially the same functionally equivalent polypeptides or polypeptides can also be used to clone and express the polynucleotides that encode these enzymes.
As will be understood by those skilled in the art, it may be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms generally use a subset of these codons. Codons that are used most often of a species are called ideal codons, and those that are not used are often classified as rare or little-used codons. Codons can be replaced to reflect the host's preferred use of codons, in a process sometimes called "codon optimization" or "codon bias control between species".
Optimized coding sequences that contain codons preferred by a particular prokaryotic or eukaryotic host (Murray et al, 1989, Nucl Acids Res. 17: 477-508) can be prepared, for example, to increase the translation rate or to produce transcripts of recombinant RNA having the desirable properties, such as a longer half-life, compared to the transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, the typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocot plants is UGA, whereas those for insects and E. coli generally use UAA as the stop codon (Dalphin et al, 1996, Nucl Acids Res. 24: 216-8.). The methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in US Patent 6,015,891, and the references cited therein.
Those skilled in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds that differ in their nucleotide sequences can be used to encode a given disclosure enzyme. The native DNA sequences encoding the biosynthetic enzymes described above are mentioned here only to illustrate one embodiment of the disclosure and the disclosure includes DNA compounds that encode the sequence of any of the amino acid sequences of the polypeptides and proteins of the enzymes used in the methods of the disclosure. Similarly, a polypeptide can typically tolerate one or more amino acid substitutions, deletions and insertions in its amino acid sequence without significant loss or loss of a desired activity. The disclosure includes such polypeptides with amino acid sequences other than the specific proteins described herein, provided that the modified or variant polypeptides have the anabolic or catabolic enzymatic activity of the reference polypeptide. In addition, the amino acid sequences encoded by the DNA sequences presented here merely illustrate disclosure modalities.
In addition, enzyme homologues useful for the generation of metabolites are encompassed by the microorganisms and methods provided herein.
As used herein, two proteins (or a region of proteins) are substantially homologous when the amino acid sequences are at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, gaps can be introduced into one or both of a first and a second amino acid sequence or nucleic acids for optimal alignment and non-homologous sequences can be disregarded for comparison). In one embodiment, the length of an aligned reference sequence for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the reference sequence length. Amino acid or nucleotide residues at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical in that position (as used here the "identity" of amino acids or nucleic acids is equivalent to "homology" "amino acid or nucleic acid). The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for the optimal alignment of the two sequences.
When the "homologue" is used in reference to proteins or peptides, it is recognized that the positions of the residues that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue with a side chain (group R) with similar chemical properties (for example, charge, or hydrophobicity). In general, conservative amino acid substitution does not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percent identity of the sequence or degree of homology can be adjusted upward to correct the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art (See, for example, Pearson W.R., 1994, Methods in Mol. Biol 25: 365-89).
The following six groups each contain amino acids that are conservative substitutions for another: 1) Serine (S), Threonine (T) 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), valine (V) and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See Copending and Commonly Owned Order US 2009/0226991. A typical algorithm used that compares a sequence of molecules to a large number of sequences containing a database of different organisms is the BLAST computer program. When searching a database containing sequences from a large number of different organisms, it is typical to compare the amino acid sequences. The database search using the amino acid sequences can be measured using algorithms described in the copending and jointly owned application US 2009/0226991.
It is understood that a variety of microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of beneficial metabolites from acetolactate and / or aldehyde-dependent biosynthetic pathways. In various modalities, microorganisms can be selected from yeast microorganisms. Yeast microorganisms for the production of a metabolite, such as isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl -1-butanol, coenzyme A, 2-methyl-1-butanol, isoleucine, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl- 1-heptanol, and 1-propanol can be selected based on certain characteristics: A characteristic may include the property that the microorganism is selected to convert various sources of carbon into beneficial metabolites such as isobutanol, 2- butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, coenzyme A, 2-methyl-1-butanol, isoleucine, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, and 1-propanol. The term "carbon source" generally refers to a substance suitable for use as a carbon source for the growth of prokaryotic or eukaryotic cells. Examples of suitable carbon sources are described in Copending and Common Ownership Order US 2009/0226991. Accordingly, in one embodiment, the recombinant microorganism described herein can convert a variety of carbon sources to products, including, but not limited to, glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof. . The recombinant microorganism can thus also include a pathway for the production of isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3- methyl-1-butanol, coenzyme A, 2-methyl-1-butanol, isoleucine, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol , 5-methyl-1-heptanol, 1-propanol and from five-carbon sugars (pentoses), including xylose. Most yeast species metabolize xylose via a complex route, in which xylose is first reduced to xylitol via an xylose reductase (XR) enzyme. Xylitol is then oxidized to xylulose using an xylitol dehydrogenase (XDH) enzyme. The xylulose is then phosphorylated using a xylitol xylulokinase (XK) enzyme. This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the cell. The xylose step for xylitol uses as a NADH cofactor, while the xylitol step for xylulose uses NADPH as a cofactor. Other processes must operate to restore the redox imbalance within the cell. This generally means that the body cannot grow anaerobically on xylose sugars or other pentoses. Thus, a kind of yeast that can effectively ferment xylose and other pentose sugars into a desired fermentation product is, therefore, very desirable.
Thus, in one aspect, the recombinant microorganism is constructed to express a functional exogenous xylose isomerase. Functional exogenous xylose isomerases in yeasts are known in the art. See, for example, Rajgarhia et al., US2006 / 0234364, which is incorporated herein by reference in its entirety. In one embodiment, according to this aspect, the exogenous xylose isomerase gene is operationally linked to the promoter and terminator sequences that are functional in the yeast cell. In a preferred embodiment, the recombinant microorganism still contains a deletion or disruption of a native gene that encodes an enzyme (for example, XR and / or XDH), which catalyzes the conversion of xylose to xylitol. In an even more preferred embodiment, the recombinant microorganism also contains a functional, exogenous xylulokinase (XK) gene operably linked to promoter and terminator sequences that are functional in the yeast cell. In one embodiment, the xylulokinase (XK) gene is overexpressed.
In one embodiment, the microorganism has reduced pyruvate decarboxylase (PDC) activity or no activity at all. PDC catalyzes the decarboxylation of pyruvate in acetaldehyde which is then reduced to ethanol by means of an ADH through the oxidation of NADH to NAD +. Ethanol production is the main route to oxidize NADH from glycolysis. Deleting this pathway increases pyruvate and reduction equivalents (NADH) available for the biosynthetic pathway. Therefore, deletion of the PDC genes can further increase the yield of the desired metabolites.
In another embodiment, the microorganism has reduced glycerol-3-phosphate dehydrogenase (GPD) activity or no activity at all. GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) through the oxidation of NADH to NAD +. Glycerol is then produced from G3P by glycerol-3-phosphatase (GPP). Glycerol production is a secondary way to oxidize excess NADH from glycolysis. The reduction or elimination of this pathway would increase the pyruvate and reduction equivalents (NADH) available for the biosynthetic pathway. Thus, deletion of GPD genes can further increase the yield of desired metabolites.
In yet another embodiment, the microorganism has reduced or no PDC activity and reduced GPD activity or no activity. PDC-negative / GPD-negative yeast-producing strains are described in the common ownership copending publications US 2009/0226991 and US 2011/0020889, both of which are incorporated herein by reference in their entirety for all purposes.
In one embodiment, yeast microorganisms can be selected from the "Yeast Saccharomyces Clade", as described in the common property copending application US 2009/0226991. The term taxonomy group of "Saccharomyces stricto sensu" is a group of yeast species that are highly related to S. cerevisiae (Rainieri et al., 2003, J. Biosci Bioengin 96: 1-9). Saccharomyces stricto sensu yeast species include, but are not limited to, S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrid derivatives of these species (Masneuf et al., 1998 , Yeast 7: 61-72).
An old whole genome duplication (WGD) event occurred during the evolution of the hemiascomycete yeast and was identified using comparative genomic tools (Kellis et al., 2004, Nature 428: 617-24; Dujon et al., 2004, Nature 430 : 35-44; Langkjaer et al., 2003, Nature 428: 848-52; Wolfe et al., 1997, Nature 387: 708-13). Using this main evolutionary event, yeast can be divided into species that diverged from a common ancestor after the WGD event (called "post-WGD yeast" here) and species that diverged from the yeast strain before the WGD event ( called "pre-WGD yeast here").
Consequently, in one embodiment, the yeast microorganism can be selected from a post-WGD yeast genus, including, but not limited to, Saccharomyces and Candida. The favored post-WGD yeast species include: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, and C. glabrata.
In another embodiment, the yeast microorganism can be selected from a whole genome pre-duplication yeast gene (pre-WGD) including, but not limited to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces. Representative pre-WGD yeast species include: S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K. lactis, C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, D. hansenii, H. anomala, Y. lipolytica, and S. pombe.
A yeast microorganism can be either Crabtree-negative or Crabtree-positive as described in copending and proprietary application US 2009/0226991. In one embodiment, the yeast microorganism can be selected from yeast with a Crabtree-negative phenotype including, but not limited to, the following genera: Saccharomyces. Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida. Crabtree-negative species include but are not limited to: S. kluyveri, K. lactis, K. marxianus, P. anomala. P. stipitis. I. orientalis. I. occidentalis. I. scutulata. H. anomala. and C. utilis. In another embodiment, the yeast microorganism can be selected from yeast with a Crabtree-positive phenotype, including, but not limited to Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia and Schizosaccharomyces. Crabtree-positive yeast species include, but are not limited to: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, K. thermotolerans, C. glabrata, Z. bailli, Z. rouxii , D. hansenii, P. pastorius, and S. pombe.
Another feature that may include the property of the microorganism is that it is not a fermenter. In other words, it cannot metabolize a carbon source anaerobically while yeast is able to metabolize a carbon source in the presence of oxygen. Non-fermenting yeast refers to both naturally occurring yeasts and genetically modified yeasts. During anaerobic fermentation with fermentation yeast, the main way to oxidize NADH from glycolysis is through the production of ethanol. Ethanol is produced by the enzyme alcohol dehydrogenase (ADH) through the reduction of acetaldehyde, which is generated from pyruvate by pyruvate decarboxylase (PDC). In one embodiment, a fermentation yeast can be constructed to be non-fermentative by reducing or eliminating native PDC activity. Thus, most of the pyruvate produced by glycolysis is not consumed by the PDC and is available for the isobutanol route. Deleting this pathway increases pyruvate and the reduction equivalents available for the biosynthetic pathway. The fermentative pathways contribute to the low yield and low productivity of desired metabolites, such as isobutanol. Therefore, deletion of the PDC genes can increase the yield and productivity of the desired metabolites, such as isobutanol.
In some embodiments, the recombinant microorganisms may be microorganisms that are microorganisms from non-fermenting yeasts, including, but not limited to, classified into a genus selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida. In a specific modality, the non-fermenting yeast is C. xestobii.
Isobutanol-Producing Yeast Microorganisms As described here, in one embodiment, a yeast microorganism is constructed to convert a carbon source, such as glucose, to glycolysis pyruvate and pyruvate is converted to isobutanol via a metabolic pathway isobutanol producer (See, for example, WO / 2007/050671, WO / 2008/098227, and Atsumi et al., 2008, Nature 45: 86-9). Alternative routes for the production of isobutanol have been described in WO / 2007/050671 and in Dickinson et al, 1998, JBiol Chem 273: 25751-6.
Consequently, in one embodiment, the isobutanol-producing metabolic pathway to convert pyruvate to isobutanol can comprise the following reactions: 1. 2 pyruvate acetolactate + CO2 2. acetolactate + NAD (P) H 2,3-dihydroxyisovalerate + NAD (P) + 3. 2,3-dihydroxyisovalerate alpha-ketoisovalerate 4. alpha-ketoisovalerate isobutyraldehyde + CO2 5. isobutyraldehyde + NAD (P) H isobutanol + NAD (P) + These reactions are carried out by the enzymes 1) Acetolactate Synthase (ALS), 2) Ketol-acid Reduct-Isomerase (KARI), 3) Dihydroxy-acid dehydratase (DHAD), 4) Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) (Figure 1). In another embodiment, the yeast microorganism is designed to express these enzymes. For example, these enzymes can be encoded by native genes. Alternatively, these enzymes can be encoded by heterologous genes. For example, ALS can be encoded by the B. subtilis alsS gene, L. lactis alsS, or K. pneumonia ilvK gene. For example, KARI can be encoded by E. coli ilvC genes, C. glutamicum, M. maripaludis, or Piromyces sp E2. For example, DHAD can be encoded by ilvD, C. glutamicum, or E. coli L. lactis genes. For example, KIVD can be encoded by the L. lactis kivD gene. ADH can be encoded by ADH2, ADH6, or ADH7 from S. cerevisiae or adhA from L. lactis.
In one embodiment, steps 2 and 5 can be performed by KARI and ADH enzymes that use NADH (instead of NADPH) as a cofactor. Such enzymes are described in the copending and commonly owned publication, US 2010/0143997, which is incorporated herein by reference in its entirety for all purposes. The present inventors have found that the use of NADH-dependent KARI and ADH enzymes to catalyze steps 2 and 5, respectively, surprisingly allows for the production of isobutanol under anaerobic conditions. Thus, in one embodiment, the recombinant microorganisms of the present invention can use a NADH-dependent KARI to catalyze the conversion of acetolactate (+ NADH) to produce 2,3-dihydroxyisovalerate. In another embodiment, the recombinant microorganisms of the present invention can use a NADH-dependent ADH to catalyze the conversion of isobutyraldehyde (+ NADH) to produce isobutanol. In yet another embodiment, the recombinant microorganisms of the present invention can use both a NADH-dependent KARI to catalyze the conversion of acetolactate (+ NADH) to produce 2,3-dihydroxyisovalerate, and a NADH-dependent ADH to catalyze the conversion of isobutyraldehyde (+ NADH) to produce isobutanol.
In another embodiment, the yeast microorganism can be constructed to have an increased capacity to convert pyruvate to isobutanol. In one embodiment, the yeast microorganism can be constructed to have an increased ability to convert pyruvate to isobutyraldehyde. In another embodiment, the yeast microorganism can be built to have an increased capacity to convert pyruvate to keto-isovalerate. In another embodiment, the yeast microorganism can be constructed to have an increased capacity to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeast microorganism can be constructed to have an increased capacity to convert pyruvate to acetolactate.
In addition, any of the genes encoding the previous enzymes (or any others referred to here (or any of the regulatory elements that control or modulate their expression)) can be optimized using genetic / protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to the ordinary versed in the technique. Such action allows the common ones versed in the technique to optimize the enzymes for the expression and activity in yeast.
In addition, the genes encoding these enzymes can be identified from other species of fungi and bacteria and can be expressed to modulate this pathway. A wide variety of organisms can serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., Including S. cerevisiae and S. uvarum, Kluyveromyces spp., Including K. thermotolerans, K. lactis, and K. marxianus , Pichia spp., Hansenula spp., Including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., Including Y. spp. stipitis, Torulaspora pretoriensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of anaerobic fungi genes include, but are not limited to, Piromyces spp., Orpinomyces spp., Or Neocallimastix spp. Sources of prokaryotic enzymes that are useful include, but are not limited to, Escherichia. coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., and Salmonella spp.
In one embodiment, the invention is directed to a recombinant microorganism for the production of isobutanol, in which said recombinant microorganism comprises an isobutanol-producing metabolic pathway and in which said microorganism is constructed to reduce or eliminate expression or activity of an enzyme that catalyzes the conversion of acetolactate to DH2MB. In some embodiments, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR). In a specific embodiment, a 3-keto acid reductase is encoded by the S. cerevisiae TMA29 gene (YMR226C) or a homologue thereof. In one embodiment, the homologue can be selected from the group consisting of Vanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomyces castellii (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomyces bayanus (SEQ ID NO: 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), Kluyveromyces lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQ ID NO: 9) NO: 10), Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12), Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14), Candida dubliniensis (SEQ ID NO: 14) NO: 15), Candida albicans (SEQ ID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO: 18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO: 19) NO: 20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQ ID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).
In another embodiment, the invention is directed to a recombinant microorganism for the production of isobutanol, in which said recombinant microorganism comprises an isobutanol-producing metabolic pathway and in which said microorganism is constructed to reduce or eliminate expression or activity of an enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate. In some embodiments, the enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate is an aldehyde dehydrogenase. In an exemplary embodiment, the aldehyde dehydrogenase is S. cerevisiae aldehyde dehydrogenase ALD6 (SEQ ID NO: 25) or a homologous or variant thereof. In one embodiment, the homologue is selected from the group consisting of Saccharomyces castelli (SEQ ID NO: 26), Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO: 28), Kluyveromyces lactis (SEQ ID NO: 27) : 29), Kluyveromyces thermotolerans (SEQ ID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQ ID NO: 33), Saccharomyces cerevisiae (EC11 SEQ ID NO: 34), Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomyces cerevisiae AWRI1631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a (SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38) ), Kluyveromyces marxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40), and Schizosaccharomyces pombe (SEQ ID NO: 41).
In yet another embodiment, the invention is directed to a recombinant microorganism for the production of isobutanol, in which said recombinant microorganism comprises a metabolic pathway producing isobutanol and in which said microorganism is (i) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of acetolactate to DH2MB and (ii) constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate. In some embodiments, the enzyme that catalyzes the conversion of acetolactate to DH2MB is a 3-keto acid reductase (3-KAR). In a specific embodiment, a 3-keto acid reductase is encoded by the S. cerevisiae TMA29 gene (YMR226C) or a homolog or variant thereof. In one embodiment, the homologue is selected from the group consisting of Vanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomyces castellii (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomyces bayanus (SEQ ID NO: : 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), Kluyveromyces lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQ ID NO: : 10), Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12), Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14), Candida dubliniensis (SEQ ID NO: : 15), Candida albicans (SEQ ID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO: 18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO: NO) : 20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQ ID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23). In some embodiments, the enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate is an aldehyde dehydrogenase. In a specific embodiment, the aldehyde dehydrogenase is S. cerevisiae aldehyde dehydrogenase ALD6 (SEQ ID NO: 25) or a homologous or variant thereof. In one embodiment, the homologue is selected from the group consisting of Saccharomyces castelli (SEQ ID NO: 26), Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO: 28), Kluyveromyces lactis (SEQ ID NO: 27) : 29), Kluyveromyces thermotolerans (SEQ ID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQ ID NO: 33), Saccharomyces cerevisiae (EC11 SEQ ID NO: 34), Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomyces cerevisiae AWRI1631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a (SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38) ), Kluyveromyces marxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40), and Schizosaccharomyces pombe (SEQ ID NO: 41).
In one embodiment, the isobutanol-producing metabolic pathway comprises at least one exogenous gene that catalyzes a step in the conversion of pyruvate to isobutanol. In another embodiment, the isobutanol-producing metabolic pathway comprises at least two exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol-producing metabolic pathway comprises at least three exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol-producing metabolic pathway comprises at least four exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol-producing metabolic pathway comprises at least five exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol.
In one embodiment, one or more of the genes in the isobutanol pathway encodes an enzyme that is located in the cytosol. In one embodiment, recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least one enzyme from the isobutanol pathway located in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least two enzymes from the isobutanol pathway located in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least three enzymes from the isobutanol pathway located in the cytosol. In yet another embodiment, recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least four enzymes from the isobutanol pathway located in the cytosol. In an exemplary embodiment, recombinant microorganisms comprise an isobutanol-producing metabolic pathway with at least five enzymes from the isobutanol pathway located in the cytosol. The isobutanol-producing metabolic pathways in which one or more genes are located in the cytosol are described in the Copent and Commonly Owned Application US 12 / 855.276, which is incorporated herein by Reference in its entirety for all purposes.
Expression of Modified Alcohol Dehydrogenases in Isobutanol Production Another strategy described here to reduce the production of the isobutyrate by-product is to increase the activity and / or the expression of an alcohol dehydrogenase (ADH) enzyme, responsible for the conversion of isobutyraldehyde to isobutanol. This strategy avoids competition for endogenous enzymes for the intermediate isobutanol pathway, isobutyraldehyde. An increase in ADH activity and / or expression can be accomplished in several ways. For example, ADH activity can be increased through the use of a promoter with increased promoter strength or through the number of copies of the alcohol dehydrogenase gene.
In alternative modalities, the production of the isobutyrate by-product can be reduced through the use of an ADH with increased specific activity for isobutyraldehyde. Such ADH enzymes with increased specific activity for isobutyraldehyde can be identified in nature, or may result from modifications to the ADH enzyme, such as the modifications described herein. In some embodiments, these modifications will produce a decrease in the Michaelis-Menten (KM) constant for isobutyraldehyde. Through the use of such modified ADH enzymes, competition for endogenous enzymes for isobutyraldehyde is even more limited. In one embodiment, the yield of isobutyrate (mole of isobutyrate per mole of glucose) in a recombinant microorganism comprising a modified ADH as described herein is less than about 5%. In another embodiment, the yield of isobutyrate (mole of isobutyrate per mole of glucose) in a recombinant microorganism comprising a modified ADH as described herein is less than about 1%. In yet another embodiment, the yield of isobutyrate (mole of isobutyrate per mole of glucose) in a recombinant microorganism comprising a modified ADH as described herein is less than about 0.5%, less than about 0.1% , less than about 0.05%, or less than about 0.01%.
In addition, through the use of a modified ADH enzyme, the inventors of the present invention can establish a situation in which the direct reaction (ie, the conversion of isobutyraldehyde to isobutanol) is the favored reaction over the reverse reaction (ie, the conversion of isobutyraldehyde to isobutanol).
The strategies described above generally lead to a decrease in isobutyrate yield which is accompanied by an increase in isobutanol yield. Thus, the above strategies are useful for decreasing isobutyrate yield and / or titration and for increasing the ratio of isobutanol yield to isobutyrate yield.
Thus, in one aspect, the present application describes the generation of modified ADHs with enhanced activity, which can facilitate the production of improved isobutanol when coexpressed with the remaining four enzymes from the isobutanol pathway. In an embodiment according to this aspect, the present application is directed to recombinant microorganisms that comprise one or more modified ADHs. In one embodiment, the recombinant microorganism is further constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of acetolactate to DH2MB as described herein. In another embodiment, the recombinant microorganism is still constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of isobutyraldehyde to isobutyrate, as described herein.
In addition to the biosynthetic pathway of isobutanol, other biosynthetic pathways use ADH enzymes to convert an aldehyde to an alcohol. For example, ADH enzymes convert various aldehydes to alcohols, as part of the biosynthetic pathways for the production of 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-methyl-1-butanol, 3 -and methyl-1-butanol.
As used herein, the terms "ADH" or "ADH enzyme" or "alcohol dehydrogenase" are used interchangeably herein to refer to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. ADH sequences are available from a wide variety of microorganisms, including, but not limited to, L. lactis (SEQ ID NO: 175), Streptococcus pneumoniae, Staphylococcus aureus, and Bacillus cereus. ADH enzymes modifiable by the methods of the present invention include, but are not limited to, disclosed in copending and commonly owned Patent Publication US 2010/0143997. A representative list of ADH enzymes modifiable by the methods described here can be found in Table 97.
Modified ADH enzymes According to the invention, any number of mutations can be made for ADH enzymes, and in one embodiment, multiple mutations can be made to result in an increased ability to convert isobutyraldehyde to isobutanol. Such mutations include point mutations, structure displacement mutations, deletions, and insertions, with one or more (e.g., one, two, three, four, five, or six, etc.) preferred point mutations. In an exemplary embodiment, the modified ADH enzyme comprises one or more mutations at the positions corresponding to the selected amino acids from: (a) L. lactis AdhA tyrosine 50 (SEQ ID NO: 185); (b) L. lactis AdhA glutamine 77 (SEQ ID NO: 185); (c) L. lactis AdhA valine 108 (SEQ ID NO: 185); (d) L. lactis AdhA tyrosine 113 (SEQ ID NO: 185); (e) L. lactis AdhA isoleucine 212 (SEQ ID NO: 185); and (f) L. lactis AdhA leucine 264 (SEQ ID NO: 185), where AdhA (SEQ ID NO: 185) is encoded by the L. lactis adhA alcohol dehydrogenase (ADH) gene (SEQ ID NO: 184) or an optimized codon version thereof (SEQ ID NO: 206).
Mutations can be introduced into the ADH enzymes of the present invention using any methodology known to those skilled in the art. Mutations can be introduced randomly, for example, by conducting a PCR reaction, in the presence of manganese as a divalent metal ion cofactor. Alternatively, oligonucleotide-directed mutagenesis can be used to create the modified ADH enzymes that allow all possible classes of base pairs to change at any given location along the encoding DNA molecule. In general, this technique involves hybridizing a complementary oligonucleotide (with the exception of one or more incompatibilities) to a single stranded nucleotide sequence encoding the ADH enzyme of interest. The incompatible oligonucleotide is then extended by the DNA polymerase, generating a double-stranded DNA molecule that contains the desired change in a chain sequence. Changes in the sequence can, for example, result in the deletion, replacement or insertion of an amino acid. The double-stranded polynucleotide can then be inserted into an appropriate expression vector, and a mutated or modified polypeptide can thus be produced. The oligonucleotide-directed mutagenesis described above can, for example, be performed by PCR.
Enzymes for use in the compositions and methods of the invention include any enzyme that has the ability to convert isobutyraldehyde to isobutanol. Such enzymes include, but are not limited to, Adha from L. lactis, Adha from S. pneumoniae, Adha from S. aureus, and Adha from Bacillus cereus, among others. Additional ADH enzymes modifiable by the methods of the present invention include, but are not limited to, those disclosed in the copending and commonly owned Patent Publication US 2010/0143997. A representative list of ADH enzymes modifiable by the methods described herein can be found in Table 16. As will be understood by one skilled in the art, modified ADH enzymes can be obtained by routine and well-known genetic engineering or recombinant techniques in the technique. Modified ADH enzymes can, for example, be obtained by mutating the gene or genes encoding the ADH enzyme of interest by site-directed or random mutagenesis. Such mutations can include point mutations, deletion mutations, insertion mutations. For example, one or more point mutations (for example, substitution of one or more amino acids, with one or more different amino acids), can be used to construct the modified ADH enzymes of the invention. The invention also includes homologous ADH enzymes, which are 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% , 96%, 97%, 98% or 99% identical at the amino acid level to a wild-type ADH enzyme (for example, Adha from L. lactis or Adha from E. coli) and exhibit an increased ability to convert isobutyraldehyde to isobutanol . Also included in the scope of the invention are ADH enzymes, which are 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical at the amino acid level of an ADH enzyme comprising the amino acid sequence set out in SEQ ID NO: 185, and exhibiting an increased ability to convert isobutyraldehyde to isobutanol, compared to the unmodified wild-type enzyme. The invention also includes nucleic acid molecules that encode the ADH enzymes described above. The invention also includes fragments of ADH enzymes that comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 amino acid residues and maintains one or more activities associated with ADH enzymes. Such fragments can be obtained by deletion mutation, by recombinant techniques that are routine and well known in the art, or by enzymatic digestion of the ADH enzyme (s) of interest, using any of a number of known proteolytic enzymes. The invention further includes nucleic acid molecules, which encode the modified ADH enzymes and fragments of the ADH enzyme described above.
By a protein or protein fragment having an amino acid sequence of at least 50%, for example, "identical" to a reference amino acid sequence, it is meant that the protein's amino acid sequence is identical to the reference sequence except that the protein sequence can include up to 50 amino acid changes for every 100 amino acids of the amino acid sequence of the reference protein. In other words, to obtain a protein with an amino acid sequence at least 50% identical to a reference amino acid sequence, up to 50% of the amino acid residues in the reference sequence can be deleted or replaced with another amino acid, or a number of amino acids up to 50% of the total amino acid residues in the reference sequence can be inserted into the reference sequence. These changes in the reference sequence can occur at the amino (N-) and / or carboxy (C-) terminal positions of the reference amino acid sequence and / or anywhere between those terminal positions, interspersed either individually between the residues in the sequence reference and / or in one or more contiguous groups within the reference sequence. As a practical matter, the fact that whether a given amino acid sequence is, for example, at least 50% identical to the amino acid sequence of a reference protein can be determined conventionally using known computer programs, such as those described above for the nucleic acid sequence identity determinations, or using the CLUSTAL W program (Thompson, JD, et al., Nucleic Acids Res. 22: 4673 4680 (1994)).
In one aspect, amino acid substitutions are made in one or more of the positions identified above (that is, the positions of amino acids equivalent to or corresponding to Y50, Q77, V108, Y113, I212, L264 or Adha from L. lactis (SEQ ID NO: 185)). Thus, the amino acids in these positions can be replaced by any other amino acid, including Ala, Asn, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp , Tyr, and Val. A specific example of an ADH enzyme that has a greater ability to convert isobutyraldehyde to isobutanol is an ADH in which (1) the tyrosine at position 50 has been replaced by a phenylalanine or tryptophan residue, (2) a glutamine at position 77 has been replaced by an arginine or serine residue, (3) valine at position 108 has been replaced by an alanine or serine residue (4), tyrosine at position 113 has been replaced by a phenylalanine or glycine residue, (5), the isoleucine at position 212 has been replaced by a threonine or valine residue, and / or (6), the leucine at position 264 is replaced by a valine residue.
Polypeptides that have the ability to convert isobutyraldehyde to isobutanol for use in the present invention can be isolated from their natural prokaryotic or eukaryotic sources, according to standard procedures for isolating and purifying natural proteins that are well known to one skilled in the art. (see, for example, Houts, GE, et al., J. Virol. 29: 517 (1979)). In addition, polypeptides that have the ability to convert isobutyraldehyde to isobutanol can be prepared by recombinant DNA techniques that are familiar to anyone skilled in the art (see, for example, Kotewicz, ML, et al., Nucl. Acids Res. 16 : 265 (1988); Soltis, DA, and Skalka, AM, Proc. Natl. Acad. Sci. USA 85: 3372 3376 (1988)).
In one aspect of the invention, modified ADH enzymes are made by recombinant techniques. To clone a gene or other nucleic acid molecule that encodes an ADH enzyme that will be modified according to the invention, the isolated DNA that contains the ADH enzyme gene or the open reading frame can be used to build a DNA library recombinant. Any vector, well known in the art, can be used to clone the ADH enzyme of interest. However, the vector used must be compatible with the host into which the recombinant vector will be transformed.
Prokaryotic vectors for constructing the plasmid library include plasmids such as those capable of replication in E. coli, such as, for example, vectors pBR322, ColE1, pSC101, pUC (pUC18, pUC19, etc .: In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982); and Sambrook et al., In: Molecular Cloning A Laboratory Manual (2d ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) ). Bacillus plasmids include pC194, pUB110, pE194, pC221, pC217, etc. Such plasmids are disclosed by Glyczan, T. In: The Molecular Biology Bacilli, Academic Press, York (1982), 307 329. Suitable Streptomyces plasmids include pIJ101 (Kendall et al., J. Bacteriol. 169: 4177 4183 (1987)) . Pseudomonas plasmids are reviewed by John et al., (Rad. Insec. Dis. 8: 693 704 (1986), and Igaki, (Jpn. J. Bacteriol. 33: 729 742 (1978)). A wide variety of plasmids and Host cosmids, such as pCP13 (Darzins and Chakrabarty, J. Bacteriol. 159: 9 18 (1984)) can also be used for the present invention.
Suitable hosts for cloning ADH nucleic acid molecules of interest are prokaryotic hosts. An example of a prokaryotic host is E. coli. However, the desired ADH nucleic acid molecules of the present invention can be cloned into other prokaryotic hosts, including, but not limited to, hosts in the genus Escherichia, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and Proteus.
Eukaryotic hosts for the cloning and expression of the ADH enzyme of interest include fungal and yeast cells. A particularly preferred host is eukaryotic yeast. Expression of the desired ADH enzyme in such eukaryotic cells may require the use of eukaryotic regulatory regions, which include eukaryotic promoters. The cloning and expression of the ADH nucleic acid molecule in eukaryotic cells can be performed by well-known techniques, using well-known eukaryotic vector systems.
According to the invention, one or more mutations can be made in any ADH enzyme of interest, in order to increase the enzyme's ability to convert isobutyraldehyde to isobutanol, or to confer other properties described here on the enzyme, according to the invention. Such mutations include point mutations, structure displacement mutations, deletions and insertions. Preferably, one or more point mutations, which result in one or more amino acid substitutions, are used to produce the ADH enzymes having an enhanced ability to convert isobutyraldehyde to isobutanol. In a preferred aspect of the invention, one or more mutations at positions equivalent or corresponding to the position (for example, Y50, Y50W or Y50F), Q77 (for example, Q77S or Q77R), V108 (for example, V108S or V108A), Y113 (for example, Y113F or Y113G), I212 (for example, I212T or I212V), and / or L264 (for example, L264V) of the L. lactis Adha enzyme (SEQ ID NO: 185) can be made to produce the result desired in other ADH enzymes of interest.
The corresponding positions of the ADH enzymes identified herein (for example, Adha from L. lactis of SEQ ID NO: 185) can be readily identified for other ADH enzymes by one skilled in the art. Thus, taking into account the defined region and the assays described in the present application, one skilled in the art can make one or more modifications, which would result in an increased ability to convert isobutyraldehyde to isobutanol in any ADH enzyme of interest.
In a preferred embodiment, the modified ADH enzymes have 1 to 6 amino acid substitutions selected from positions that correspond to Y50, Q77, Y113, V108, I212, L264 or compared to wild-type ADH enzymes. In other embodiments, the modified ADH enzymes have additional amino acid substitutions in other positions, compared to the respective wild-type ADH enzymes. Thus, modified ADH enzymes can have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 different residues in other positions, compared to the respective wild-type ADH enzymes. As will be appreciated by those skilled in the art, the number of additional positions that may have amino acid substitutions will depend on the wild-type ADH enzyme used to generate the variants. Thus, in some cases, up to 50 different positions may have amino acid substitutions.
It is understood that several microorganisms can act as "sources" for the genetic material encoding ADH enzymes suitable for use in a recombinant microorganism provided herein. For example, in addition, the genes encoding these enzymes can be identified from other species of fungi and bacteria and can be expressed to modulate this pathway. A variety of organisms can serve as sources for these enzymes, including, but not limited to, Lactococcus sp., Including L. lactis, Lactobacillus sp., Including L. brevis, L. buchneri, L. hilgardii, L. fermentum, L reuteri, L. vaginalis, L. antri, L. oris, and L. coleohominis, Pediococcus sp., including P. acidilactici, Bacillus sp., including B. cereus, B. thuringiensis, B. coagulans, B. anthracis, B. weihenstephanensis, B. mycoides, and B. amyloliquefaciens, Leptotrichia sp., Including L. goodfellowii, L. buccalis, and L. hofstadii, Actinobacillus sp., Including A. pleuropneumoniae, Streptococcus sp., Including S. sanguinis, S parasanguinis, S. gordonii, S. pneumoniae, and S. mitis, Streptobacillus sp., including S. moniliformis, Staphylococcus sp., including S. aureus, Eikenella sp., including E. corrodens, Weissella sp., including W. paramesenteroides, Kingella sp., including K. oralis, and Rothia sp., including R. dentocariosa, and Exiguobacterium sp.
The nucleotide sequences for various ADH enzymes are known. For example, ADH enzyme sequences are available from a wide variety of microorganisms, including, but not limited to, L. lactis (SEQ ID NO: 185), S. pneumoniae, S. aureus and Bacillus cereus. ADH enzymes modifiable by the methods of the present invention include, but are not limited to those disclosed in the copending and commonly owned Patent Publication US 2010/0143997. A representative list of ADH enzymes modifiable by the methods described here can be found in Table 97.
In addition, any method can be used to identify genes that encode ADH enzymes with a specific activity. Generally, homologous or analogous genes with similar activity can be identified by functional, structural and / or genetic analysis. In most cases, homologous or analogous genes with similar activity will have functional, structural or genetic similarities. Techniques known to those skilled in the art may be suitable for identifying homologous genes and homologous enzymes. Generally, analogous genes and / or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable for identifying analogous genes and analogous enzymes. For example, to identify homologous or analogous genes, proteins or enzymes, techniques may include, but are not limited to, cloning a gene by PCR using primers based on a published gene / enzyme sequence or by degenerate PCR using primers degenerate cells designed to amplify a conserved region between a gene. In addition, one skilled in the art can use techniques to identify homologous or analogous genes, proteins or enzymes, with homology or functional similarity. Techniques include examining a cell or cell culture for catalytic efficiency or specific activity of an enzyme through in vitro enzymatic assays for said activity and then isolating the enzyme with said activity by means of purification, determination of the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers for the probable nucleic acid sequence, amplification of said DNA sequence through PCR and cloning of said nucleic acid sequence. To identify homologous or analogous genes with similar activity, the techniques also include comparing data relating to a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme can be identified within the databases mentioned above, according to the present teaching. In addition, enzyme activity can be determined phenotypically. Methods for Preparing ADH Enzymes with Enhanced Catalytic Efficiency The present invention further provides methods for building ADH enzymes to improve their catalytic efficiency.
One approach to increase the catalytic efficiency of ADH enzymes is by saturation mutagenesis with NNK libraries. These libraries can be screened for increases in catalytic efficiency in order to identify which individual mutations contribute to an increased ability to convert isobutyraldehyde to isobutanol. Combinations of mutations in the aforementioned residues can be investigated by any method. For example, a combinatorial library of mutants can be designed based on the results of saturation mutagenesis studies.
Another approach is the use of random oligonucleotide mutagenesis to generate diversity by incorporating random mutations, encoded in a synthetic oligonucleotide, into the enzyme. The number of mutations in individual enzymes within the population can be controlled by varying the length of the target sequence and the degree of randomization during oligonucleotide synthesis. The more definite advantages of this approach are that all possible amino acid mutations and also the coupled mutations can be found.
If the best variants of the experiments described above do not show enough activity, the evolution directed by error-prone PCR can be used to obtain further improvements. Error-prone PCR mutagenesis of the ADH enzyme can be performed, followed by screening for ADH activity.
Improved Catalytic Efficiency of ADH In one aspect, the catalytic efficiency of the modified ADH enzyme is improved. As used herein, the phrase "catalytic efficiency" refers to the property of the ADH enzyme that allows to convert isobutyraldehyde to isobutanol.
In one embodiment, the catalytic efficiency of modified ADH is improved compared to parental or wild-type ADH. Preferably, the catalytic efficiency of the modified ADH enzyme is improved by less than about 5% compared to parental or wild-type ADH. Most preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 15% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 25% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 50% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 75% compared to parental or wild-type ADH. Most preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 100% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 200% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 500% compared to parental or wild-type ADH. More preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 1000% compared to wild-type or parental ADH. More preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 2000% compared to wild-type or parental ADH. More preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 3000% compared to wild-type or parental ADH. Most preferably, the catalytic efficiency of the modified ADH enzyme is improved by at least about 3500% compared to wild-type or parental ADH.
Gene Expression of Modified ADH Enzymes Here are provided methods for expressing one or more of the genes of modified ADH enzymes involved in the production of beneficial metabolites and recombinant DNA expression vectors useful in the method. Thus, included within the scope of the disclosure are recombinant expression vectors that include such nucleic acids. The expression vector refers to a nucleic acid that can be introduced into a host microorganism or free cell transcription and translation system. An expression vector can be maintained permanently or transiently in a microorganism, either as part of the chromosome or other DNA in the microorganism or in any cell compartment, such as a replication vector in the cytoplasm. An expression vector further comprises a promoter that directs the expression of an RNA, which is usually translated into a polypeptide in the microorganism or cell extract. For efficient translation of RNA into protein, the expression vector also typically contains a ribosome binding site sequence positioned upstream of the codon initiating the coding sequence of the gene to be expressed. Other elements, such as enhancers, secretion signal sequences, transcription termination sequences, and one or more marker genes, through which host microorganisms containing the vector can be identified and / or selected, may also be present in an expression vector. Selectable markers, for example, genes that confer resistance or sensitivity to antibiotics are used and confer a selectable phenotype on transformed cells, when the cells are grown in an appropriate selective medium.
The various components of an expression vector can vary widely, depending on the intended use of the vector and the host cell (s) in which the vector is intended to replicate or conduct expression. The components of the expression vector suitable for the expression of genes and maintenance of the vectors in E. coli, yeast, Streptomyces, and other commonly used cells are widely known and are commercially available. For example, promoters suitable for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can include regulatory sequences that allow regulation of expression in relation to the growth of the host microorganism, or that cause the expression of a gene to be activated or deactivated in response to a chemical or physical stimulus. For E. coli and other bacterial host cells, promoters derived from genes for biosynthetic enzymes, enzymes that confer resistance to antibiotics, and phage proteins can be used and include, for example, promoters of galactose, lactose (lac), maltose , tryptophan (trp), beta-lactamase (bla), bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (US Patent 4,551,433) can also be used. For E. coli expression vectors, it is useful to include an E. coli origin of replication, such as from pUC, p1P, p1, and pBR.
Thus, recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of a biosynthetic gene coding sequence operably linked to a promoter and, optionally, termination sequences that operate to effect the expression of the coding sequence in compatible host cells. Host cells are modified by transformation with the recombinant DNA expression vectors of the disclosure to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome.
In addition, methods for expressing a polypeptide from a nucleic acid molecule that are specific to a particular microorganism (i.e., a yeast microorganism) are well known. For example, the nucleic acid constructs that are used for the expression of heterologous polypeptides within Kluyveromyces and Saccharomyces are well known (see, eg, US Patent 4,859,596 and US 4,943,529, each of which is incorporated herein by reference. entirely for Kluyveromyces and, for example, Gellissen et al., Gene 190 (1): 87-97 (1997) for Saccharomyces. Yeast plasmids have a selectable marker and origin of replication also known as autonomous replication sequences (ARS). In addition, certain plasmids may also contain a centromere sequence. These centromere plasmids are generally a single or low copy plasmid. Plasmids without a centromere sequence and using either a 2 micron origin of replication (S. cerevisiae) or 1.6 microns (K. lactis} are high copy plasmids. The selectable marker can be either prototrophic, such as HIS3, TRP1, LEU2, URA3 or ADE2, or d and resistance to antibiotics, such as, bar, ble, hph, or kan.
A nucleic acid of the present disclosure can be amplified using cDNA, synthetic mRNA DNA, or alternatively, genomic DNA, as an appropriate oligonucleotide template and primers according to conventional PCR amplification techniques and the procedures described in the Examples section below. The nucleic acid thus amplified can be cloned into a suitable vector and characterized by analysis of the DNA sequence. In addition, oligonucleotides that correspond to nucleotide sequences can be prepared by standard synthetic techniques, for example, using an automatic DNA synthesizer. It is also understood that an isolated nucleic acid molecule encoding a homologous polypeptide for the enzymes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence that encodes the particular polypeptide in such a way that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the polynucleotide using standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In contrast to the positions where it may be desirable to make non-conservative amino acid substitutions (see above), in some positions it is preferable to make conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid) uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine , tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Although the effect of an amino acid change varies depending on factors, such as phosphorylation, glycosylation, intrachain bonds, the tertiary structure and function of the amino acid at the active site or a possible allosteric site, it is generally preferred that the substituted amino acid is the from the same group as the amino acid to be replaced. To a certain extent, the following groups contain amino acids, which are interchangeable: the basic amino acids like lysine, arginine, histidine and, the amino acids like aspartic acid and glutamic acid; neutral polar amino acids such as serine, threonine, cysteine, glutamine, asparagine and, to a lesser extent, methionine; non-polar aliphatic amino acids glycine, alanine, valine, isoleucine and leucine (however, due to size, glycine and alanine are more closely related and valine, isoleucine and leucine are more closely related), and the aromatic amino acids like phenylalanine, tryptophan and tyrosine . In addition, although classified into different categories, alanine, glycine, and serine appear to be interchangeable to some extent, and cysteine additionally fits into this group or can be classified with the polar neutral amino acids. General Methods Identification of 3-keto acid Reductase Homologs Any method can be used to identify genes encoding enzymes with 3-keto acid reductase activity, including, but not limited to, S. cerevisiae TMA29. Generally, genes that are homologous or similar to 3-keto acid reductases, such as TMA29 can be identified by functional, structural and / or genetic analyzes. In most cases, homologous or similar genes and / or homologous or similar enzymes will have functional, structural or genetic similarities. The S. cerevisiae TMA29 gene is also known as YMR226C. The YMR226C open reading frame (ORF) is found on the S. cerevisiae XIII chromosome at positions 722395 ... 721592. The YMR226C chromosomal location is a region that is highly synthetic for chromosomes in many related yeasts [Byrne, K.P. and K. H. Wolfe (2005) “The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species.” Genome Res. 15 (10): 1456-61. Scannell, D. R., K. P. Byrne, J. L. Gordon, S. Wong, and K. H. Wolfe (2006) “Multiple rounds of speciation associated with reciprocal gene loss in polyploidy yeasts.” Nature 440: 341-5. Scannell, D. R., A. C. Frank, G. C. Conant, K. P. Byrne, M. Woolfit, and K. H. Wolfe (2007) "Independent sorting-out of thousands of duplicated gene pairs in two yeast species descended from a whole-genome duplication." Proc Natl Acad Sci U S A 104: 8397-402.].
For example, the locations of YMR226C synthetic versions of other yeast species can be found on chromosome 13 in Candida glabrata, Chromosome 1 in Zygosaccharomyces rouxii, Chromosome 2 in K. lactis, Chromosome 6 in Ashbya gossypii, Chromosome 8 in S. kluyveri , Chromosome 4 in K. thermotolerance and Chromosome 8 of the inferred ancestral yeast species [Gordon, JL, KP Byrne, and KH Wolfe (2009) “Additions, losses, and rearrangements on the evolutionary route from a reconstructed ancestor to the modern Saccharomyces cerevisiae genome ”PLoS Genet. 5: e1000485.].
Using this synthetic relationship, species-specific versions of this gene are readily identified and examples can be found in Table 4.
Table 4. YMR226C and their counterparts.
In addition to synteny, fungal homologues for the S. cerevisiae TMA29 gene can be identified by one skilled in the art using tools such as BLAST and sequence alignment. These other homologues can be similarly excluded from the respective yeast species to eliminate accumulation of 3-hydroxyacid by-product. Examples of homologous proteins can be found in Vanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomyces castellii (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomyces bayanus (SEQ ID NO: 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), K. lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQ ID NO: 10), Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12), Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14), Candida dubliniensis (SEQ ID NO: 15), Candida albicans (SEQ ID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO: 18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO: 20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQ ID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).
The techniques known to those skilled in the art may be suitable for identifying other homologous genes and homologous enzymes. Generally, analogous genes and / or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable for identifying analogous genes and analogous enzymes. For example, to identify homologous or analogous genes, proteins or enzymes, techniques may include, but are not limited to, cloning a dehydratase gene by PCR using primers based on a published sequence of a gene / enzyme or by degenerate PCR using degenerate primers designed to amplify a conserved region between dehydratase genes. In addition, one skilled in the art can use techniques to identify homologous or analogous genes, proteins or enzymes, with homology or functional similarity. Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzymatic assays for that activity (for example, as described here or in Kiritani, K. Branched-Chain Amino Acids Methods Enzymology , 1970) and then isolation of the enzyme with said activity by means of purification, determination of the protein sequence of the enzyme using techniques such as Edman degradation, design of PCR primers for the probable nucleic acid sequence, amplification of said DNA sequence by PCR and cloning of said nucleic acid sequence. To identify homologous or similar genes and / or homologous or similar enzymes, analogous genes and / or analogous enzymes or proteins, the techniques also include comparing data relating to a candidate gene or enzyme with databases, such as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme can be identified in the databases mentioned above, in accordance with the present teaching.
Identification of Aldehyde Dehydrogenase Homologs Any method can be used to identify genes encoding enzymes with aldehyde dehydrogenase activity, including, but not limited to, S. cerevisiae ALD6.
Generally, genes that are homologous or similar to aldehyde dehydrogenases such as ALD6, can be identified by functional, structural and / or genetic analyzes. In most cases, homologous or similar genes and / or homologous or similar enzymes will have functional, structural or genetic similarities. The S. cerevisiae ALD6 gene is also known by its systematic name YPL061W. The YPL061W open reading frame (ORF) is found on chromosome 5. cerevisiae XVI at positions 432585 ... 434087. The chromosomal location of YPL061W is a region that is highly synthetic of chromosomes in many related yeasts [Byrne, K.P. and K. H. Wolfe (2005) “The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species.” Genome Res. 15: 1456-61. Scannell, D. R., K. P. Byrne, J. L. Gordon, S. Wong, and K. H. Wolfe (2006) “Multiple rounds of speciation associated with reciprocal gene loss in polyploidy yeasts.” Nature 440: 341-5. Scannell, D. R., A. C. Frank, G. C. Conant, K. P. Byrne, M. Woolfit, and K. H. Wolfe (2007) "Independent sorting-out of thousands of duplicated gene pairs in two yeast species descended from a whole-genome duplication." Proc Natl Acad Sci U S A 104: 8397-402.].
For example, the locations of YPL061W synthetic versions of other yeast species can be found on Chromosome 8 in Candida glabrata, Chromosome 5 in K. lactis, Chromosome 5 in K. thermotolerans and Chromosome 8 from the inferred ancestral yeast species [ Gordon, JL, KP Byrne, and KH Wolfe (2009) “Additions, losses, and rearrangements on the evolutionary route from a reconstructed ancestor to the modern Saccharomyces cerevisiae genome.” PLoS Genet. 5: e1000485.].
Using this synthetic relationship, species-specific versions of this gene are easily identified and examples can be found in Table 5 ..
Table 5. ALD6 and its counterparts.
In addition to synteny, fungal homologues for the ALD6 gene of 5. cerevisiae can be identified by one skilled in the art using tools such as BLAST and sequence alignment. These other counterparts can be similarly deleted from the respective yeast species to eliminate the accumulation of aldehyde by-product. Examples of homologous proteins can be found in Saccharomyces castelli (SEQ ID NO: 26), Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO: 28), Kluyveromyces lactis (SEQ ID NO: 29), Kluyveromyces thermotolerans (SEQ ID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQ ID NO: 33), Saccharomyces cerevisiae EC1118 (SEQ ID NO: 34) , Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomyces cerevisiae AWRI1631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a (SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38), Kluyveromyces marxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40), and Schizosaccharomyces pombe (SEQ ID NO: 41).
Identification of an ADH or KDH in a Microorganism Any method can be used to identify genes that encode enzymes with alcohol dehydrogenase (ADH), or keto acid dehydrogenase (KDH) activity. Alcohol dehydrogenase (ADH) can catalyze the reversible conversion of isobutanol to isobutyraldehyde. Keto acid dehydrogenases (KDH) can catalyze the conversion of 2-ketoisovalerate to isobutyryl-CoA, which can be further converted to isobutyrate by the action of carboxylic acid kinase and transacetylase enzymes. Generally, genes that are homologous or similar to known alcohol dehydrogenases and keto acid dehydrogenases can be identified by functional, structural and / or genetic analyzes. In most cases, homologous or similar alcohol dehydrogenase genes and / or homologous or similar alcohol dehydrogenase enzymes will have functional, structural or genetic similarities. Likewise, homologous or similar keto acid dehydrogenase genes and / or homologous or analogous keto acid dehydrogenase enzymes will have functional, structural or genetic similarities.
Identification of PDC and GPD in a Yeast Microorganism Any method can be used to identify the genes encoding enzymes with pyruvate decarboxylase (PDC) activity or glycerol-3-phosphate dehydrogenase (GPD) activity. Suitable methods for the identification of PDC and GPD are described in copendent and commonly owned publications, US 2009/0226991 and US 2011/0020889, both of which are incorporated herein by reference in their entirety for all purposes.
Genetic Insertions and Deletions Any method can be used to introduce a nucleic acid molecule into yeast and many such methods are well known. For example, transformation and electroporation are the common methods for introducing nucleic acid into yeast cells.
See, for example, Gietz et al., 1992, Nuc Acids Res.Π: 69-74; Ito et al., 1983, J. Bacteriol. 153: 163-8; and Becker et al., 1991, Methods in Enzymology 194: 182-7.
In one embodiment, the integration of a gene of interest into a DNA fragment or target gene of a yeast microorganism occurs according to the principle of homologous recombination. According to this modality, an integration cassette containing a module comprising at least one yeast marker gene and / or the gene to be integrated (internal module) is flanked on both sides by DNA fragments homologous to those at the ends of the site target integration (recombinogenic sequences). After yeast transformation with the cassette by appropriate methods, a homologous recombination between the recombinogenic sequences can result in the replacement of the internal module in the chromosomal region between the sites in the genome that correspond to the recombinogenic sequences of the integration cassette. (Orr-Weaver et al, 1981, PNAS USA 78: 6354-58).
In one embodiment, the integration of the cassette for the integration of a gene of interest in a yeast microorganism includes the heterologous gene under the control of a suitable promoter and a terminator, together with the selectable marker flanked by recombinogenic sequences for integration of a heterologous gene on the yeast chromosome. In one embodiment, the heterologous gene includes an appropriate native gene desired to increase the copy number of a native gene (s). The selectable marker gene can be any marker gene used in yeast, including, but not limited to, HIS3, TRP1, LEU2, URA3, bar, ble, hph and kan. Recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.
In another embodiment, the integration of a gene into the chromosome of the yeast microorganism can occur through random integration (Kooistra et al, 2004, Yeast 21: 781-792).
In addition, in one embodiment, certain marker genes introduced are removed from the genome using techniques well known to those skilled in the art. For example, the loss of the URA3 marker can be achieved by plating cells containing URA3 in FOA (5-fluoro-orotic acid) containing the medium and selection for FOA-resistant colonies (Boeke et al, 1984, Mol. Gen. Genet 197: 345-47). The exogenous nucleic acid molecule contained within a yeast cell of the disclosure can be maintained within the cell in any form. For example, exogenous nucleic acid molecules can be integrated into the cell's genome or maintained in an episomal state, which can be transmitted stably ("inherited") to daughter cells. Such extrachromosomal genetic elements (such as plasmids, mitochondrial genomes, etc.) may additionally contain selection markers that ensure the presence of such genetic elements in the daughter cells. In addition, yeast cells can be stably or transiently transformed. In addition, the yeast cells described herein can contain a single copy, or several copies of an exogenous nucleic acid molecule, in particular, as described above.
Reduction of Enzyme Activity Yeast microorganisms, within the scope of the invention, may have reduced enzyme activity, such as reduced activity of 3-keto acid reductase, PDC, ALDH, or glycerol-3-phosphate dehydrogenase (GPD). The term "reduced" as used herein in relation to a given enzyme activity refers to a lower level of enzyme activity than that measured in a comparable yeast cell of the same species. The term reduced also refers to the elimination of enzymatic activity compared to a comparable yeast cell of the same species. Thus, yeast cells that lack 3-keto acid reductase, PDC, ALDH or glycerol-3-phosphate dehydrogenase (GPD) are considered to have reduced activity of 3-keto acid reductase, PDC, ALDH or glycerol-3-phosphate dehydrogenase ( GPD) since most, if not all, comparable yeast strains have at least some activity of 3-keto acid reductase, PDC, ALDH, or glycerol-3-phosphate dehydrogenase (GPD). Such reduced enzyme activities may be the result of a lower concentration of enzyme, lower specific activity of an enzyme, or a combination of these. Many different methods can be used to make yeasts having reduced enzyme activity. For example, a yeast cell can be built to have a disrupted enzyme coding site using common mutagenesis or knockout technology. See, for example, Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser and Stems, Cold Spring Harbor Press (1998). In addition, certain point mutations can be introduced, which result in an enzyme with reduced activity. Also included within the scope of the present invention are yeast strains which, when found in nature, are substantially free of one or more activities selected from the activity of 3-keto acid reductase, PDC, ALDH, or glycerol-3-phosphate dehydrogenase ( GPD).
Alternatively, antisense technology can be used to reduce enzyme activity. For example, yeast can be constructed to contain a cDNA that encodes an antisense molecule that prevents an enzyme from being made. The "antisense molecule" as used herein encompasses any nucleic acid molecule that contains sequences that correspond to the coding strand of an endogenous polypeptide. An antisense molecule can also have flanking sequences (for example, regulatory sequences). Thus, antisense molecules can be antisense oligonucleotides or ribozymes. The ribozyme can have any general structure, including, without limitation, hairpin (or hairpin), hammerhead, or axhead (ax) structures, as long as the molecule cleaves the RNA. Yeast with reduced enzyme activity can be identified using several methods. For example, yeast that has reduced activity of 3-keto acid reductase, PDC, ALDH, or glycerol-3-phosphate dehydrogenase (GPD) can be easily identified through common methods, which may include, for example, measuring the formation of glycerol, using liquid chromatography.
Overexpression of Heterologous Genes Methods for overexpression of a polypeptide from a heterologous or native nucleic acid molecule are well known. Such methods include, without limitation, the construction of a nucleic acid sequence such that a regulatory element promotes the expression of a nucleic acid sequence that encodes the desired polypeptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. Thus, regulatory elements include, without limitation, promoters, enhancers and the like. For example, exogenous genes may be under the control of an inducible promoter or a constitutive promoter. In addition, methods for expressing a polypeptide from an exogenous nucleic acid molecule in yeast are well known. For example, the nucleic acid constructs that are used for the expression of exogenous polypeptides within Kluyveromyces and Saccharomyces are well known (see, for example, US Patent 4,859,596 and 4,943,529, to Kluyveromyces and, for example, Gellissen et al ., Gene 190 (1): 87-97 (1997) for Saccharomyces). Yeast plasmids have a selectable marker and an origin of replication. In addition, certain plasmids may also contain a centromere sequence. These centromeric plasmids are generally a single or low copy plasmid. Plasmids without a centromere sequence and using either a 2 micron (S. cerevisiae) or 1.6 micron (K. lactis) origin of replication are high copy plasmids. The selectable marker can be either prototrophic, such as HIS3, TRP1, LEU2, URA3 or ADE2, or antibiotic resistance, such as, bar, ble, hph, or kan.
In another embodiment, heterologous control elements can be used to activate or repress the expression of endogenous genes. In addition, when the expression is to be repressed or eliminated, the gene for the corresponding enzyme, protein or RNA can be eliminated using known deletion techniques.
As described herein, any yeast within the scope of the disclosure can be identified by selecting specific techniques for the particular enzyme being expressed, overexpressed or suppressed. Methods of identifying strains with the desired phenotype are well known to those skilled in the art. Such methods include, without limitation, PCR, RT-PCR, and nucleic acid hybridization techniques, such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound , a selection agent and the like. In some cases, immunohistochemistry and biochemistry techniques can be used to determine whether a cell contains a particular nucleic acid by detecting the expression of the encoded polypeptide.
For example, an antibody having specificity for an encoded enzyme can be used to determine whether or not there is a special yeast cell that contains the encoded enzyme. In addition, biochemical techniques can be used to determine whether a cell contains a nucleic acid molecule that encodes a particular enzyme polypeptide by detecting a product produced as a result of the expression of the enzyme polypeptide. For example, the transformation of a cell with a vector that encodes acetolactate synthase and detects increased concentrations of acetolactate compared to a cell without the vector indicates that the vector is present and the gene product is active. Methods for detecting specific enzyme activities or the presence of certain products are well known to those skilled in the art. For example, the presence of acetolactate can be determined as described by Hugenholtz and Starrenburg, 1992, Appl. Micro. Biot. 38: 17-22.
Increased Enzyme Activity The yeast microorganisms of the present invention can be further constructed to have increased enzyme activity (for example, increased activity of the enzymes involved in an isobutanol-producing metabolic pathway). The term "augmented" as used herein in relation to a given enzyme activity refers to a higher level of enzyme activity than that measured in a comparable yeast cell of the same species. For example, overexpression of a specific enzyme can lead to an increase in the level of activity in cells for the enzyme. Increased activities for enzymes involved in glycolysis or the isobutanol pathway would result in increased productivity and isobutanol yield.
Methods for increasing enzyme activity are known to those skilled in the art. Such techniques may include increasing the expression of the enzyme by increasing the number of copies and / or using a strong promoter, introducing mutations to alleviate negative regulation of the enzyme, introducing specific mutations to increase specific activity and / or decrease the KM for the substrate, or by directed evolution. See, for example, Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003). Methods for Using Recombinant Microorganisms for High Yield Ferments For a biocatalyst to produce a more economically advantageous metabolite, it is desirable to produce said metabolite in high yield. Preferably, the only product produced is the desired metabolite, as additional products (ie, by-products) lead to a reduction in the yield of the desired metabolite and an increase in capital and operating costs, particularly if the additional products have little or no no value. These extra products also require additional capital and operating costs to separate these products from the desired metabolite.
In one aspect, the present invention provides a method of producing a beneficial metabolite derived from a recombinant microorganism comprising a biosynthetic pathway.
In one embodiment, the method includes culturing a recombinant microorganism comprising a biosynthetic pathway that uses a 3-keto acid as an intermediate in a culture medium containing a raw material that provides the carbon source until a recoverable amount of the beneficial metabolite is produced and, optionally, the recovery of the metabolite. In one embodiment, the 3-keto acid intermediate is acetolactate. In an exemplary embodiment, said recombinant microorganism is constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of acetolactate to DH2MB. The beneficial metabolite can be derived from any biosynthetic pathway that uses acetolactate as an intermediate, including, but not limited to, biosynthetic pathways for the production of isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin , diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In a specific embodiment, the beneficial metabolite is isobutanol. In another embodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxy-butyrate. In an exemplary embodiment, said recombinant microorganism is constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of 2-aceto-2-hydroxy-butyrate to 2-ethyl-2,3-hydroxybutyrate. The beneficial metabolite can be derived from any biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate as an intermediate, including, but not limited to, biosynthetic pathways for the production of 2-methyl-1-butanol, isoleucine, 3-methyl-1 -pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
In another embodiment, the method includes culturing a recombinant microorganism comprising a biosynthetic pathway that uses an aldehyde as an intermediate in a culture medium containing a raw material that provides the carbon source until a recoverable amount of the beneficial metabolite produced and, optionally, recovery of the metabolite. In an exemplary embodiment, said recombinant microorganism is constructed to reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of an aldehyde to the acid by-product. The beneficial metabolite can be derived from any biosynthetic pathway that uses an aldehyde as an intermediate, including, but not limited to, biosynthetic pathways for the production of isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1- butanol, 1-propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol. In a specific embodiment, the beneficial metabolite is isobutanol.
In another embodiment, the method includes culturing a recombinant microorganism comprising a biosynthetic pathway that uses acetolactate and an aldehyde as intermediates in a culture medium containing a raw material that supplies the carbon source until a recoverable amount of the metabolite beneficial effect is produced and, optionally, recovery of the metabolite. In an exemplary embodiment, said recombinant microorganism is constructed to (i) reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of acetolactate to DH2MB and (ii) reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of an aldehyde to an acid by-product. The beneficial metabolite can be derived from any biosynthetic pathway that uses acetolactate and an aldehyde as an intermediate, including, but not limited to, biosynthetic pathways for the production of isobutanol, 1-butanol and 3-methyl-1-butanol. In a specific embodiment, the beneficial metabolite is isobutanol.
In another embodiment, the method includes culturing a recombinant microorganism comprising a biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate and an aldehyde as intermediates in a culture medium containing a raw material that provides the carbon source up to a recoverable amount of the beneficial metabolite is produced and, optionally, recovery of the metabolite. In an exemplary embodiment, said recombinant microorganism is constructed to (i) reduce or eliminate the expression or activity of an enzyme that catalyzes the conversion of 2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-hydroxybutyrate and (ii) reducing or eliminating the expression or activity of an enzyme that catalyzes the conversion of an aldehyde to the acid by-product. The beneficial metabolite can be derived from any biosynthetic pathway that uses 2-aceto-2-hydroxybutyrate and an aldehyde as intermediates, including, but not limited to, biosynthetic pathways for the production of 2-methyl-1-butanol, 3-methyl- 1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
In another embodiment, the present invention provides a method of producing a beneficial metabolite derived from a biosynthetic pathway that requires alcohol dehydrogenase (ADH). In one embodiment, the method includes culturing a recombinant microorganism comprising a modified ADH described herein, in a culture medium containing a raw material that provides the carbon source until a recoverable amount of the beneficial metabolite is produced and , optionally, recovery of the metabolite. The beneficial metabolite can be derived from any biosynthetic pathway that requires ADH, including, but not limited to, biosynthetic pathways for the production of 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-methyl -1-butanol and 3-methyl-1-butanol. In a specific embodiment, the beneficial metabolite is isobutanol.
In a method for producing a beneficial metabolite from a carbon source, the yeast microorganism is grown in an appropriate culture medium that contains a carbon source. In certain embodiments, the method also includes isolating the beneficial metabolite from the culture medium. For example, isobutanol can be isolated from the culture medium by any method known to those skilled in the art, such as distillation, pervaporation, or liquid-liquid extraction.
In one embodiment, the recombinant microorganism can produce the beneficial metabolite from a carbon source with a theoretical yield of at least 5 percent. In another embodiment, the microorganism can produce the beneficial metabolite from a carbon source with a yield of at least about 10 percent, at least about 15 percent, at least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5% of the theoretical value. In a specific embodiment, the beneficial metabolite is isobutanol. The present invention is further illustrated by the following examples which are not to be considered as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and sequence listing, are hereby incorporated by reference for all purposes.
EXAMPLES General Methods for Examples 1-26 Sequences: The amino acid and nucleotide sequences described here are shown in Table 6.
Table 6. Amino acid and nucleotide sequences of genes and enzymes disclosed in various examples.
Medium: The medium used was the standard yeast medium (see, for example, Sambrook, J., Russel, DW Molecular Cloning, A Laboratory Manual. 3rd ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press and Guthrie, C. and Fink, GR eds. Methods in Enzymology Part B: Guide to Yeast Genetics and Molecular and Cell Biology 350: 3-623 (2002)). The YP medium contains yeast extract at 1% (w / v), peptone at 2% (w / v). YPD is YP containing 2% glucose unless otherwise specified. YPE is YP containing 25 mL / L of ethanol. SC medium is 6.7 g / L Difco ™ yeast nitrogen base, 14 g / L Synthetic Dropout Sigma ™ medium supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 0.076 g / L of histidine, 0.076 g / L of tryptophan, 0.380 g / L of leucine, and 0.076 g / L of uracil. SCD is SC containing 2% glucose (w / v), unless otherwise noted. The Dropout versions of SC and SCD media are made by omitting one or more of histidine (-H), tryptophan (-W), leucine (-L), or uracil (U). Solid versions of the media described above contain 2% (w / v) agar. Cloning techniques: Standard molecular biology methods for cloning and constructing the plasmid were generally used, unless otherwise indicated (Sambrook, J., Russel, DW Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). Cloning techniques included restriction enzyme digestion, PCR to generate DNA fragments (KOD Hot Start Polymerase, Cat # 71086, Merck, Darmstadt, Germany), ligation of two DNA fragments using the DNA ligation kit (Mixture powerful Cat # TAK 6023, Clontech Laboratories, Madison, WI), and bacterial transformations into competent E. coli cells (Xtreme Efficiency DH5a Competent Cells, Cat # ABP-CE-CC02096P, Allele Biotechnology, San Diego, CA). Plasmid DNA was purified from E. coli cells using the Qiagen QIAprep Spin Miniprep Kit (Cat # 27106, Qiagen, Valencia, CA). DNA was purified from agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA; Catalog # D4002) according to the manufacturer's protocols. Cologne PCR: Yeast colony PCR used the FailSafe ™ PCR System (EPICENTRE® Biotechnologies, Madison, WI; Catalog # FS99250) according to the manufacturer's protocols. A PCR cocktail containing 15 pL of master mix buffer E, 10.5 pL of water, 2 pL of each primer at a concentration of 10 μΜ, 0.5 pL of polymerase enzyme mixture in the kit was added to a 0.2 mL PCR tube for each sample (30 pL each). For each candidate a small amount of cells were added to the reaction tube using a sterile pipette tip. The presence of the positive PCR product was assessed using agarose gel electrophoresis. PCR SOE: PCR reactions were incubated in a thermocycler using the following PCR conditions: 1 cycle of 94 ° C x 2 min, 35 cycles of 94 ° C x 30 s, 53 ° C x 30 s, 72 ° C x 2 min and 1 cycle of 72 ° C x 10 min. A master mix was made such that each reaction contained the following: 3 pL of MgSO4 (25 mM), 5 pL of 10X KOD buffer, 5 pL of 50% DMSO, 5 pL of dNTP mixture (2 mM each), 1 pL KOD, 28 pL dH2O, 1.5 pL forward primer (10 pM), 1.5 pL reverse primer (10 pM), 0.5 pL template (plasmid or genomic DNA).
Genomic DNA Isolation: The Zymo Research ZR fungal / bacterial DNA kit (Zymo Research Orange, CA; Catalog # D6005) was used for the isolation of genomic DNA according to the manufacturer's protocols, with the following modifications. After resuspension of the pellets, 200 μL were transferred to 2 separate ZR BashingBead ™ Lysis Tubes (to maximize yield). Following lysis by beating the granule, 400 μL of supernatant from each of the ZR BashingBead ™ lysis tubes were transferred to 2 separate Zymo-Spin ™ IV Spin Filters and centrifuged at 7000 rpm for 1 min. Following centrifugation, 1.2 ml of fungal / bacterial DNA binding buffer was added to each of the filtrates. In 800 μL aliquots, the filtrate from both filters was transferred to a single Zymo-Spin ™ IIC column in a collection tube and centrifuged at 10,000 x g for 1 min. For the elution step, instead of eluting in 100 μL of EB (elution buffer, Qiagen), 50 μL of EB were added, incubated 1 min and then the columns were centrifuged for 1 min. This elution step was repeated for a final elution volume of 100 μL
Transformations of S. cerevisiae. Strains of 5. cerevisiae were grown in YPD medium containing 1% ethanol. Competent transformation cells were prepared by resuspending 5. cerevisiae cells in 100 mM lithium acetate. Once the cells were prepared, from a mixture of DNA (final volume of 15 μL with sterile water), 72 μL of 50% PEG, 10 mL of 1M lithium acetate, and 3 μL of denatured salmon sperm DNA (10 mg / ml) were prepared for each transformation. In a 1.5 mL tube, 15 μL of the cell suspension was added to the DNA mixture (100 μL), and the transformation suspension was vortexed for 5 short pulses. The transformation was incubated for 30 min at 30 ° C, followed by incubation for 22 min at 42 ° C. The cells were harvested by centrifugation (18,000 x g, 10 seconds, 25 ° C). The cells were resuspended in 350 μL and YPD after an overnight recovery with shaking at 30 ° C and 250 rpm, the cells were seeded on YPD plates containing 0.2 g / L of selective G418 plates. The transformants were then purified from single colonies on selective G418 plates.
Transformations of K. marxianus: strains of K. marxianus were grown in 3 ml of an appropriate culture medium at 250 rpm and 30 ° C overnight. The next day, the cultures were diluted in 50 ml of the same medium and grown at an OD600 between 1 and 4. The cells were collected in a 50 ml sterile conical tube for centrifugation (1600 x g, 5 min at room temperature). The cells were resuspended in 10 ml of electroporation buffer (10 mM Tris-C1, 270 mM sucrose, 1 mM MgCE, pH 7.5), and collected at 1600 x g for 5 min at room temperature. The cells were resuspended in 10 mL of IB (YPE, 25 mM DTT, 20 mM HEPES, pH 8.0; freshly prepared, diluting 100 μL of 2.5 M DTT and 200 mL of 1 M HEPES, pH 8.0 in 10 mL of YPD). The cells were incubated for 30 min, 250 rpm, 30 ° C (in a vertical support tube). The cells were harvested at 1600 x g for 5 min at room temperature and resuspended in 10 ml of cold electroporation buffer. The cells were pelleted at 1600 x g for 5 min at 4 ° C. The cells were resuspended in 1 ml of cold electroporation buffer and transferred to a microcentrifuge tube. The cells were harvested by centrifugation at> 10,000 x g for 20 seconds at 4 ° C. The cells were resuspended in an appropriate amount of cold electroporation buffer to a final biomass concentration of 30-38 OD600 / mL. 400 pL of cells were added to a cold electroporation cuvette (0.4 cm gap), 50 pL of SOE PCR product (or control water) was added and mixed by pipetting up and down, and the cuvette was incubated on ice for 30 min. The samples were electroporated at 1.8 kV, 1000 Ohm, 25 pF. The samples were then transferred to a 50 ml tube with 1 ml of an appropriate culture medium, and the samples were incubated overnight at 250 rpm, at 30 ° C. After incubation, the cells were plated on suitable agar plates.
K. lactis transformations: K. lactis strains were grown in 3 mL of YPD at 250 rpm and 30 ° C overnight. The next day, the cultures were diluted in 50 ml of YPD and allowed to grow until they reached an OD600 of ~ 0.8. Cells from 50 ml of YPD cultures were collected for centrifugation (2700 rcf, 2 min, 25 ° C). The cells were washed with 50 ml of sterile water and collected by centrifugation at 2700 rcf for 2 min at room temperature. The cells were washed again with 25 ml of sterile water and collected by centrifugation at 2700 rcf for 2 min at room temperature. The cells were resuspended in 1 ml of 100 mM lithium acetate and transferred to a 1.5 ml Eppendorf tube. The cells were harvested by centrifugation for 10 seconds at 18,000 rcf at RT. The cells were resuspended in a volume of 100 mM lithium acetate, which was approximately 4x the volume of the cell pellet. A volume of 10-15 pL of DNA, 72 pL of 50% PEG (3350), 10 pL of 1 M lithium acetate, 3 pL of denatured salmon sperm DNA, and sterile water were combined to a final volume of 100 pL for each transformation. In a 1.5 mL tube, 15 µl of the cell suspension was added to the DNA mixture and the transformation suspension was vortexed with 5 short pulses. The transformation was incubated for 30 min at 30 ° C, followed by incubation for 22 min at 42 ° C. The cells were harvested by centrifugation for 10 seconds at 18,000 rcf at RT (Room Temperature). The cells were resuspended in 400 µl of a suitable medium, and seeded on agar plates containing an appropriate medium to select the transformed cells.
Analytical Chemistry: Gas Chromatography (GC1 method). The analysis of volatile organic compounds, including ethanol and isobutanol, was performed on an Agilent 5890/6890/7890 gas chromatograph equipped with an Agilent 7673 autosampler, a ZB-FFAP column (J&W; 30 m long, 0.32 mm ID 0.25 μΜ film thickness) or equivalent, connected to a flame ionization detector (FID). The temperature program was as follows: 200 ° C for the injector, 300 ° C for the detector, oven at 100 ° C for 1 minute, gradient from 70 ° C / minute to 230 ° C, and then keep for 2.5 min. The analysis was performed using authentic standards (> 99%, obtained from Sigma-Aldrich, and a 5-point calibration curve with 1-pentanol as an internal standard).
High Performance Liquid Chromatography (LC1 method): Analysis of organic acid metabolites including 2,3-dihydroxyisovalerate (DHIV), 2,3-dihydroxy-2-methylbutanoic acid (DH2MB), isobutyrate and glucose was performed on a chromatography system Equivalent High Efficiency Liquid or Agilent 1200 equipped with a Bio-Rad Microguard Cation Cartridge and two Phenomenex Rezex RFQ-Fast Fruit H + (8%), 100 x 7.8 mm columns in series, or equivalent. Organic acid metabolites were detected using an equivalent UV detector or Agilent 1100 (210 nm) and a refractive index detector. The column temperature was 60 ° C. This method was isocratic with 0.0180 N H2SO4 in Milli-Q water as a mobile phase. The flow was adjusted to 1.1 ml / min. The injection volume was 20 pL and the execution time was 16 min. Quantification of organic acid metabolites was performed using a 5-point calibration curve with authentic standards (> 99% or higher available purity), with the exception of DHIV (2,3-dihydroxy-3-methyl-butanoate , CAS 175618-9), which was synthesized according to Cioffi et al. (Cioffi, E. et al. Anal Biochem 1980, 104, pp.485) and DH2MB which was quantified based on the assumption that DHIV and DH2MB exhibit the same response factor. In this method, DHIV and DH2MB therefore coelute, their concentrations are presented as the sum of the two concentrations.
High Performance Liquid Chromatography (LC4 method): Analysis of oxo acids, including 2,3-dihydroxyisovalerate (DHIV, CAS 1756-18-9), 2,3-dihydroxy-2-methylbutyrate (DH2MB), lactate, acetate, acetolactate, isobutyrate and pyruvate were performed in a High Performance Liquid Chromatography or Agilent 1100 system equipped with an IonPac AS11-HC analytical column (Dionex: 9 pm, 4.6 x 250 mm) coupled with an IonPac AG11-HC guard column (Dionex: 13 pm, 4.6 x 50 mm) and an IonPac ATC-3 Anion Trap column (Dionex: 9 x 24 mm). Acetolactate was detected using a 225nm UV detector, while all other analytes were detected using a conductivity detector (ED50 with conductivity deletion with 4 mm ASRS in AutoSuppression recycle mode, 200 mA suppressor current). The column temperature was 35 ° C. Injection size was 10 pL. This method used the following elution profile: 0.25 mM NaOH over 3 min, followed by a linear gradient of 0.25 to 5 mM NaOH in 22 min and the second linear gradient from 5 mM to 38.25 mM in 0, 1 min, followed by 38.25 mM NaOH for 4.9 min and a final linear gradient of 38.25 mM and 0.25 mM over 0.1 minutes before re-equilibrating with 0.25 mM NaOH over 7 minutes. The flow was adjusted to 2 ml / min. The analysis was performed using a 4-point calibration curve, with authentic standards (> 99%, or highest available purity), with the following exceptions: DHIV was synthesized according to Cioffi et al. (Cioffi, E. et al. Anal Biochem 1980, 104, pp.485). DH2MB was synthesized as described in Example 8 and quantified based on the assumption that DHIV and DH2MB exhibit the same response factor. Racemic acetolactate was made by hydrolysis of Ethyl-2-acetoxy-2-methylacetoacetate (EAMMA) with NaOH (Krampitz, L.O. Methods in Enzymology 1957, 3, 277-283.). In this method, DHIV and DH2MB are separated (Figure 8) Enzyme Assays Determination of protein concentration: The protein concentration (yeast lysate or purified protein) was determined using the BioRad Bradford Protein Assay Reagent kit (Cat # 500 -0006, BioRad Laboratories, Hercules, CA) and using BSA for the standard curve. A standard curve for the assay was made with a series of dilutions from a standard Protein stock of 500 pg / mL BSA. An appropriate dilution of cell lysate was made in water to obtain OD595 measurements of each lysate that fell within the linear range of the standard BioRad Protein curve. Ten pL of a lysate dilution was added to 500 pL of diluted BioRad Protein assay dye, the samples were mixed by vortexing and incubated at room temperature for 6 min. The samples were transferred to cuvettes and read at 595 nm in a spectrophotometer. Linear regression analysis of the patterns was used to calculate the protein concentration of each sample.
Alcohol dehydrogenase (ADH) assay. The cells were thawed on ice and resuspended in lysis buffer (100 mM Tris-HCl pH 7.5). 1000 pL of glass microspheres (0.5 mm in diameter) were added to a 1.5 ml Eppendorf tube and 875 pL of cell suspension were added. The yeast cells were lysed using a Retsch MM301 mixing mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each at full speed, with a 1 min incubation on ice between each microsphere tapping step. The tubes were centrifuged for 10 minutes at 23500 x g at 4 ° C and the supernatant was removed for use. These lysates were kept on ice until testing. Protein concentrations of yeast lysates were determined as described.
The sample dilutions were made in such a way that an activity reading can be obtained. Generally, samples from strains expected to have low ADH activity were diluted 1: 5 in lysis buffer (100 mM Tris-HCl pH 7.5) and samples from strains with high ADH activity expected, as strains in which the ADH gene is expressed from a high copy number of the plasmid, were diluted from 1:40 to 1: 100. The reactions were performed in triplicate, using 10 μl of cell extract appropriately diluted with 90 μl of reaction buffer (100 mM Tris-HCl, pH 7.5, 150 mM NADH, 11 mM isobutyraldehyde) in a 96-well plate in a SpectraMax® 340PC multi-card reader (Molecular Devices, Sunnyvale, CA). The reaction was followed at 340 nm for 5 minutes, with absorbance readings every 10 seconds. The reactions were carried out at 30 ° C. The reactions were carried out in complete buffer and also in buffer without substrate.
Isobutyraldehyde Oxidation Assay (ALD6 Assay): Cell pellets were thawed on ice and resuspended in lysis buffer (10 mM sodium phosphate, pH 7.0, 1 mM dithiothreitol, 5% w / v glycerol). One ml of glass microspheres (0.5 mm in diameter) was added to a 1.5 ml Eppendorf tube for each sample and 850 µl of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each at full speed with 1 min incubation on ice, between intervals. The tubes were centrifuged for 10 minutes at 21,500 xg at 4 ° C and the supernatant was transferred to a new tube. The extracts were kept on ice until the test. Yeast lysate Protein concentrations were determined as described. The method used to measure the enzymatic activity of enzymes that catalyze the oxidation of isobutyraldehyde to isobutyrate in cell lysates has been modified from Meaden et al. 1997, Yeast 13: 1319-1327 and Postma et al. 1988, Appl. Environ. Microbiol. 55: 468-477. Briefly, for each sample, 10 µL of diluted cell lysate was added to 6 wells of a UV microtiter plate. Three wells received 90 pL of assay buffer containing 50 mM HEPES-NaOH at pH 7.5, NADP + 0.4 mM, 3.75 mM MgCT, and 0.1 mM, 1 mM, or 10 mM isobutyraldehyde. The other 3 wells received 90 pL of buffer without substrate (same as the assay buffer, but without isobutyraldehyde). The buffers were mixed with the lysate in the wells, pipetting up and down. The reactions were then monitored at 340 nm for 5 minutes, with absorbance readings obtained every 10 seconds on a SpectraMax® 340PC plate reader (Molecular Devices, Sunnyvale, CA). The reactions were carried out at 30 ° C. The Vmax of each sample was determined by subtracting the background reading from the control without substrate. An unbound control was also performed in triplicate for each concentration of substrate ALS Assay: For the ALS assays described in Examples 1-18, the cells were thawed on ice and resuspended in lysis buffer (50 mM potassium phosphate at pH 6.0 and 1 mM MgSO4). 1000 pL of glass microspheres (0.5 mm in diameter) were added to a 1.5 ml Eppendorf tube and 875 pL of cell suspension were added. The yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each at full speed, with 1 min incubations on ice between each step of the microsphere beating. The tubes were centrifuged for 10 minutes at 23,500 x g at 4 ° C and the supernatant was removed for use. These lysates were kept on ice until testing. The protein content of the lysates was measured as described. All ALS assays were performed in triplicate for each lysate, both with and without substrate. For each lysate assay, 15 pL of lysate was mixed with 135 pL of buffer (50 mM potassium phosphate buffer pH 6.0, 1 mM MgSO4, 1 mM thiamine-pyrophosphate, 110 mM pyruvate), and incubated for 15 minutes at 30 ° C. The buffers were prepared at room temperature. A control without substrate (buffer without pyruvate) and control without lysate (lysis buffer instead of lysate) were also included. After incubation, 21.5 μL of 35% H2SO4 was added to each reaction and incubated at 37 ° C for 1 h.
For the ALS assays described in Examples 19-25, the cells were thawed on ice and resuspended in lysis buffer (100 mM NaPO4, pH 7.0, 5 mM MgC.'l ·, and 1 mM DTT). One ml of glass microspheres (0.5 mm in diameter) was added to a 1.5 ml Eppendorf tube and 800 μL of the cell suspension was added to the tube containing glass microspheres. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA) and a cooling block by mixing six times for 1 minute each at 30 cycles / second with 1 min cooling between the mixture. The tubes were centrifuged for 10 minutes at 21,500 x g at 4 ° C and the supernatant was removed. The extracts were kept on ice until the test. The protein concentration of yeast lysate was determined using the BioRad Bradford Protein Assay reagent kit (Cat # 500-0006, BioRad Laboratories, Hercules, CA) and using BSA for the standard curve, as described. All ALS assays were performed in triplicate for each lysate. All buffers, lysates and reaction tubes were pre-cooled on ice. For the assay of each lysate, 15 μL of lysate (diluted with lysis buffer, as needed) was mixed with 135 μL of assay buffer (50 mM KPi, pH 7.0, 1 mM MgSO4, 1 mM thiamine-pyrophosphate, 110 mM pyruvate) and incubated for 15 min at 30 ° C. A control without substrate (buffer without pyruvate) and control without lysate (lysis instead of lysate) were also included. After incubation, each reaction was mixed with 21.5 μL of 35% H2SO4, incubated at 37 ° C for 1 hour and centrifuged for 5 minutes at 5,000 x g to remove any insoluble precipitants.
All test samples were analyzed for the test substrate (pyruvate) and product (acetoin) using high performance liquid chromatography with an HP-1200 High Performance Liquid chromatography system equipped with two 150 x Restek RFQ series columns 4.6 mm. The organic acid metabolites were detected using an HP 1100 UV (210 nm) detector and refractive index. The column temperature was 60 ° C. This method was isocratic with H2SO4 0.0180 N (in Milli-Q water), as the mobile phase. The flow was adjusted to 1.1 ml / min. The injection volume was 20 μL and the execution time was 8 min. The analysis was performed using authentic standards (> 99%, obtained from Sigma-Aldrich) and a 5-point calibration curve.
TMA29 Enzyme Assay: Cell pellets were thawed on ice and resuspended in lysis buffer (10 mM sodium phosphate, pH 7.0, 1 mM dithiothreitol, 5% w / v glycerol). One ml of glass microspheres (0.5 mm in diameter) was added to a 1.5 ml Eppendorf tube for each sample and 850 μL of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixing mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each full speed, with 1 min incubation on ice, between intervals. The tubes were centrifuged for 10 minutes at 21,500 x g at 4 ° C and the supernatant was transferred to a new tube. The extracts were kept on ice until the test. The protein concentration of yeast lysate was determined using the BioRad Bradford protein assay reagent kit (Cat # 500-0006, BioRad Laboratories, Hercules, CA) and using BSA for the standard curve, as described. The enzymatic synthesis of (S) -2-acetolactate ((S) -AL) was performed in an anaerobic flask. The reaction was carried out in a total volume of 55 ml containing 20 mM potassium phosphate, pH 7.0, 1 mM MgCl2, 0.05 mM thiamine pyrophosphate (TPP), and 200 mM sodium pyruvate. The synthesis was initiated by the addition of 65 units of purified B. subtilis ALsS, and the reaction was incubated at 30 ° C in a static incubator for 7.5 h. The chemical synthesis of racemic 2-acetolactate ((R / S) -2-AL) was performed by mixing 50 ml of ethyl-2-acetoxy-2-methylacetoacetate (EAMMA) with 990 pL of water. 260 mL of 2 N NaOH was then added in 10 µl increments with 15 seconds of vortexing after each addition. The solution was then mixed on an orbital shaker for 20 minutes. The chemical synthesis of racemic AHB ((R / S) -AHB) was carried out by mixing 50 pL of ethyl-2-acetoxy-2-ethyl-3-oxobutanoate with 990 pL of water. 2N NaOH was then added in 10 µl increments with 15 seconds of vortexing after each addition. NaOH was added until the pH of the solution was 12 (~ 180 pL of 2 N NaOH). The solution was then mixed on an orbital shaker for 20 minutes.
For the determination of the reduction activity of (S) -AL, (R / S) -AL or (R .S ') - AHB, 10 pL of undiluted cell lysate was added to 6 wells of a microtiter plate of UV. Three wells received 90 pL of assay buffer containing 100 mM KPO4, pH 7.0, 150 mM NADPH, and (S) -AL 5 mM or (R / S) -AL 10 mM or (R / S) -AHB 10 mM as substrate. The other 3 wells received 90 pL of assay buffer, but without substrate. The buffers were mixed with the lysate in the wells, pipetting up and down. The reactions were then monitored at 340 nm, with absorbance readings obtained every 10 seconds on a SpectraMax® 340PC plate reader (Molecular Devices, Sunnyvale, CA). The reactions were carried out at 30 ° C. The reduction activity of (S) -AL, (R / S) -AL or (R / S) -AHB for each sample was determined by subtracting the background reading from the control without substrate. A control without lysate was also performed in triplicate.
DHAD Enzyme Assay: Cell pellets were thawed on ice and resuspended in lysis buffer (50 mM Tris pH 8.0, 5 mM MgSO4, and Yeast G Biosciences / ProteaseArrest ™ fungus (St. Louis, MO, USA, Catalog # 788-333)). One ml of glass microspheres (0.5 mm in diameter) was added to a 1.5 ml Eppendorf tube for each sample and 850 µl of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each at full speed, with 1 min incubation on ice, between intervals. The tubes were centrifuged for 10 minutes at 21,500 x g at 4 ° C and the supernatant was transferred to a new tube. The extracts were kept on ice until the test. The protein concentration of yeast lysate was determined as described. The protein in each sample was diluted in DHAD assay buffer (50 mM Tris pH 8, 5 mM MgSO4) to a final concentration of 0.5 pg / pL. Three samples of each lysate were analyzed, along with controls without lysates. 10 µl of each sample (or DHAD assay buffer) was added to 0.2 ml PCR tubes. Using a multichannel pipette, 90 pL of substrate was added to each tube (mix of substrate was prepared by adding 4 ml of DHAD assay buffer to 0.5 ml of 100 mM DHIV). The samples were placed in a thermocycler (Eppendorf Mestrecycler) at 35 ° C for 30 min, followed by a 5 min incubation at 95 ° C. The samples were cooled to 4 ° C in the thermocycler, then centrifuged at 3,000 x g for 5 minutes. Finally, 75 µl of supernatant was transferred to new PCR tubes and analyzed by HPLC as follows. 100 pL of DNPH reagent (12 mM 2,4-dinitrophenyl hydrazine, 10 mM citric acid, pH 3.0, 80% acetonitrile, 20% H2O MilliQ) was added to 100 pL of each sample. The samples were incubated for 30 min at 70 ° C in a thermocycler (Eppendorf, Mestrecycler). The analysis of isobutyraldehyde and keto-isovalerate was performed in an HP-1200 high-performance liquid chromatography system equipped with an Eclipse XDB C-18 reverse phase column (Agilent) and a C-18 reverse phase guard column (Phenomenex ). Ketoisovalerate and isobutyraldehyde were detected using an HP-1100 UV (210 nm) detector. The column temperature was 50 ° C. This method was isocratic with acetonitrile at 70% and water as a mobile phase with phosphoric acid diluted to 2.5% (4%). The flow was adjusted to 3 ml / min. The injection size was 10 pL and the execution time was 2 min.
Example 1: Isobutanol / Isobutyrate ratio increased by increasing ADH activity in, S '. cerevisiae The purpose of this example is to demonstrate that the increase in alcohol dehydrogenase activity results in an increased isobutanol yield, with a decreased isobutyrate yield, and an increase in the ratio of isobutanol yield to isobutyrate yield.
The strains and plasmids described in this example are shown in Tables 7 and 8, respectively.
Table 7. Genotype of Strains Disclosed in Example 1.
Table 8. Plasmids Disclosed in Example 1. The strain of S. cerevisiae GEVO2843, which expresses a unique alcohol dehydrogenase (D. melanogaster ADH, Dm_ADH) from its chromosomal DNA was transformed with 2μ of plasmids pGV2011 that carry only KARI and DHAD (Ec_ilvC_Q110V and LlilvDcoSc, respectively) or pGV2485 carrying KARI, DHAD and ADH (Ec_ilvC_Q110V, Ll ilvD coSc, and LladhA, respectively), as described.
To start fermentation cultures, small cultures of strains transformed overnight were started in YPD medium containing 1% ethanol and 0.2 g / L of G418 and incubated overnight at 30 ° C and 250 rpm. Three biological replicates of each strain were tested. The following morning, the OD600 of these cultures was determined and an appropriate amount used to inoculate 50 ml of the same medium in a 50 ml bottle with baffles for an OD600 of about 0.1. These pre-cultures were incubated at 30 ° C and 250 rpm overnight. When the cultures reached an OD600 of approximately 5-6 they were centrifuged at 2700 rpm for 5 min at 25 ° C in 50 ml Falcon tubes. The cells from a 50 mL culture (a clone) were resuspended in YPD medium containing 8% glucose, 0.2 g / L of G418, 1% (v / v) ethanol (containing 3 g / L of ergosterol and 132 g / L of Tween-80) and buffered to pH 6.5 with 200 mM MES. The cultures were then transferred to 250 ml flasks without baffles and incubated at 30 rpm and 75 ° C.
At the 72 h time point, samples from each of the fermentation flasks were taken to determine OD600, ADH activity, and for analysis by GC1 and LC1. To prepare the samples for GC1 and LC1 analysis, an appropriate volume of cell culture was centrifuged in a microcentrifuge for 10 minutes at maximum speed and the supernatant was removed for analysis of GC1 and LC1. Cell pellets were prepared for ADH assays by centrifuging 14 ml of culture medium at 3,000 x g for 5 minutes at 4 ° C. The supernatant was removed and the cells were washed in 3 ml of cold, sterile water. The tubes were then centrifuged as above for 2 minutes, the supernatant was removed, and the tubes were weighed again to determine the total cell weight. Falcon tubes were stored at -80 ° C. ADH assays were performed as described. Table 9 shows the OD600 for each strain, during the course of fermentation. During the 72 h of this fermentation, the OD600 of the strains was similar: they started with an OD600 of about 7 and ended with an OD600 of about 9. The in vitro ADH enzymatic activity of GEVO2843 lysates transformed with the two plasmids was measured for the 72 h time point. Table 9 shows the activity of ADH in lysates as measured in vitro. The strain carrying the plasmid without ADH (pGV2011) showed an activity of about 0.04 U / mg. The strain that carries the plasmid with the LladhA gene, (pGV2485), had about 7 times more ADH activity.
Table 9. OD600 and Alcohol Dehydrogenase Activity of GEVO2843 Strain Transformed with Plasmids pGV2011 or pGV2485 After 72h of Fermentation.
The titers of Isobutanol and isobutyrate after 72 h of fermentation are shown in Table 10. The titration of isobutanol in the strain with low ADH activity of 0.04 U / mg was significantly lower compared to the strain with high ADH activity of 0 , 29 U / mg. The isobutyrate titration in the strain with low ADH activity of 0.04 U / mg was significantly higher compared to the strain with high ADH activity of 0.29 U / mg. Table 6 also shows the yield for isobutyrate and isobutanol after 72 h of fermentation. The isobutanol yield in the strain with low ADH activity of 0.04 U / mg was significantly lower compared to the strain with high ADH activity of 0.29 U / mg. The isobutyrate yield in the strain with low ADH activity of 0.04 U / mg was significantly higher compared to the strain with high ADH activity of 0.29 U / mg.
Table 10. Titrations and Yields for Isobutanol and Isobutyrate in GEVO2843 Strain Transformed with Plasmids pGV2011 or pGV2485 After 72h of Fermentation.
Example 2: Additional Increased Isobutanol / Isobutyrate Ratio Using ADH Ll AdhARE1 Variant in S. cerevisiae The purpose of this example is to demonstrate that the expression of an alcohol dehydrogenase with increased kcat and decreased KM results in an additional increase in isobutanol yield, decrease in isobutyrate yield, and increase in the ratio of isobutanol yield to isobutyrate yield.
Table 11. Genotype of Strains Disclosed in Example 2.
Table 12. Plasmids Disclosed in Example 2. The strain of S. cerevisiae GEVO2843, which expresses a unique alcohol dehydrogenase (D. melanogaster ADH, DmADH) from its chromosomal DNA was transformed with 2μ of plasmids pGV2543 that carry KARI, DHAD, KIVD and codon-optimized wild-type ADH, with tag-his (Ec_ilvCQ110V, LlilvDcoSc, and Ll_adhA_coSchls6, respectively) or pGV2545 carrying KARI, DHAD, KIVD and codon-optimized mutant ADH, with tag-his (Ec_ilvCQD10 and Ll_adhARE1_coSchts6, respectively). These strains were cultured and evaluated for ADH enzyme activity and extracellular metabolite production by GC1 and LC1 as described.
The kinetic parameters of the Ll_adhA_coSchls6 gene products Ll_adhARE1_coSchls6 (Ll_adhAhis6 and Ll_adhARE1-his6, respectively) are shown in Table 13.
Table 13. Comparison of wild-type Ll_adhAhis6 kinetic parameters with Modified Ll_adhARE1 Measured for Isobutyraldehyde with NADH as Cofactor. Table 14 shows the OD600 for each strain during the course of fermentation.
During 72 h of this fermentation, the OD600 of the strains was similar: they started with an OD600 of about 6 and ended with an OD600 of about 9. The in vivo ADH enzymatic activity of GEVO2843 lysates transformed with two plasmids was measured for the 72 h time point. Table 14 shows ADH activity in lysates as measured in vitro, as described above. The strain carrying the plasmid with Ll_adhA_coSchls6 (pGV2543) showed an activity of about 0.38 U / mg. The strain that carries the plasmid with the Ll_adhAR ^ 1_coSchls6 gene, (pGV2545), had approximately 7 times more ADH activity.
Table 14. OD600, and Alcohol dehydrogenase activity of GEVO2843 strain transformed with plasmids pGV2543 or pGV2545 after 72 h of fermentation.
The titers and yield of Isobutanol and isobutyrates after 72 h of Fermentation are shown in Table 15. The titration and yield of isobutanol in the strain carrying pGV2543 was lower compared to the strain carrying pGV2545. The titre and yield of isobutyrate in the strain carrying pGV2543 was significantly higher compared to the strain carrying pGV2545.
Table 15. Titrations and yields for isobutanol and isobutyrate in GEVO2843 strain transformed with plasmids pGV2453 or pGV2485 after 72 h of fermentation.
Example 3: Additional Increased Isobutanol / Isobutyrate Ratio in S. cerevisiae by Expression of RE1 The purpose of this example is to demonstrate that the expression of an alcohol dehydrogenase with increased kcat and decreased KM results in an increase in isobutanol yield and a decrease in yield of isobutyrate in fermentations carried out in fermentation containers. Fermentation was carried out to compare the performance of S. cerevisiae strains of GEVO3519 GEVO3523. The titers and yields of isobutanol and isobutyrate were measured during fermentation. GEVO3519 carries 2μ of plasmid pGV2524 that contains the genes encoding the following enzymes: KARI, DHAD, KIVD and ADH of codon-optimized wild-type Lactococcus lactis, with tag-his. GEVO3523 carries 2μ of plasmid pGV2524 that contains the genes encoding the following enzymes: KARI, DHAD, KIVD and an improved variant of Lactococcus lactis codon-optimized ADH, with tag-his having decreased KM and increased kcat. These strains were evaluated for the consumption of isobutanol, glucose, isobutyraldehyde, by LC1 and GC1, as well as for OD600 during fermentation in DasGip fermentation containers.
Table 16. Genotype of Strains Disclosed in Example 3.
Table 17. Plasmids Disclosed in Example 3. The S. cerevisiae GEVO3128 strain was transformed with plasmid or 2μ plasmid pGV2524 or pGV2546, to generate GEVO3519 and GEVO3523 strains, respectively, as described. Inoculum cultures of GEVO3519 and GEVO3523 were initiated by inoculating 500 mL flasks with baffles containing 80 mL of YPD medium with 0.2 g / L of G418 antibiotic, 1% v / v ethanol, and 0.019 g / L of tryptophan . The cultures were incubated for approximately 34 h. The orbital shaker was set at 250 rpm and 30 ° C in both experiments. Similar cell mass was obtained for strains GEVO3519 and GEVO3523. The cell density obtained after incubation was 8.0 OD600. Batch fermentations were carried out in YPD medium containing 80 g / L of glucose, 0.2 g / L of G418, 1% v / v ethanol, and 0.019 g / L of tryptophan using DasGip engines with an overhead 2 motor L with a working volume of 0.9 L per container. The containers were sterilized, together with suitable pH and dissolved oxygen probes, for 60 minutes at 121 ° C. The dissolved oxygen probes were calibrated after sterilization, in order to allow polarization, however, the pH probes were calibrated before sterilization. The pH was controlled to pH 6.0 using 6N KOH and 2N H2SO4. During the growth phase of the culture the oxygen transfer rate (OTR) was 10 mM / h and during the culture production phase the OTR was 0.2 to mM / h. Table 18 shows the titration and yield of isobutanol (in theoretical%), as calculated for the production phase of the culture. Both titration and isobutanol yield are increased in GEVO3523 strains that transport alcohol dehydrogenase with decreased KM and increased kcat. Table 18 also shows the isobutyrate titration, reported as the maximum achieved titration, and yield as carbon yield as%. Both the titration and the isobutyrate yield are decreased in the GEVO3523 strain that transport alcohol dehydrogenase with decreased KM and increased kcat.
Table 18. Titration and Yield of Isobutanol and Isobutyrate.
Example 4: Decreased Isobutyrate and Acetate Production in Ferments with ALD6 Gene Deletion in S. cerevisiae The following example shows that deletion of the ALD6 gene leads to a decrease in the production of isobutyrate and acetate in fermentations.
Construction of ALD6 Deletion Strains: PCR was used to generate a DNA fragment that contained an ALD6 deletion allele for S. cerevisiae ALD6 deletion. A PCR reaction amplified a DNA fragment (A) comprising the flanking region upstream of the ALD6 region and an overlapping region at the 3 'end of the DNA fragment with the 5' end of the Psc_ccwi2 promoter region of pGV1954, using the oGV2834 and oGV2835 primers. Another PCR reaction amplified a DNA fragment (D) comprising the flanking region downstream of ALD6 and an overlapping region at the 5 'end of the DNA fragment with the 3' end of pGV2074 hph hygromycin resistance ORF using primers oGV2836 and oGV2837. Another PCR reaction amplified a DNA fragment (B) comprising the Psc_ccwi2 promoter region from pGV1954 with an overlapping region, at the 5 'end of the DNA fragment with the 3' end of the flanking region upstream of ALD6 (fragment) A) and the 3 'end region of the DNA fragment with 5' ends of hyphromycin resistance ORF from pGV2074, using primers oGV2631 and oGV2632. Another PCR reaction amplified a DNA fragment (C) comprising the hygromycin resistance ORF hph from pGV2074 with an overlapping region at the 5 'end of the DNA fragment with the 3' end of the Psc_ccwi2 promoter region of pGV1954 (fragment B ) and the overlapping region at the 3 'end of the DNA fragment with the 5' end of the downstream flanking region of ALD6 (fragment D), using primers oGV2633 and oGV2634. The DNA fragments of A and B were combined by PCR using primers oGV2834 and oGV2632 to generate fragments of DNA AB and fragments of DNA C and D were combined by PCR using primers oGV2633 and oGV2837 to generate fragment of CD DNA. The DNA fragments AB and CD were combined by PCR using primers oGV2834 and oGV2837 to generate the final DNA fragment ABCD that contained the ALD6 deletion allele.
Table 19. Starters disclosed in Example 4.
To demonstrate decreased isobutyrate and acetate production by the ALD6 deletion, the strains were constructed by transforming GEVO3198 with the ABCD DNA fragment that contained the ALD6 deletion allele. Transformants were selected for resistance to 0.1 g / L of hygromycin and transformant colonies were screened by colony PCR for the correct integration of the DNA fragment using ABCD primer pairs oGV2840 / oGV2680, oGV968 / oGV2841, and oGV2838 / oGV2839 . Strains GEVO3711, GEVO3712 and GEVO3713 were identified by this colony PCR as having ALD6 deleted by correct integration of the ABCD DNA fragment.
Strains that contain an isobutanol production pathway to demonstrate decreased production of isobutyrate and acetate by the ALD6 deletion were constructed by transforming GEVO3711, GEVO3712 and GEVO3713 with a 2μ plasmid replication origin, pGV2247, which carry genes that express KARI, DHAD, KIVD and ADH (Ec ilvC coScP '', Ll_ilvD_coSc, LlkivD2coEc, and Ll adhA, respectively). The transformants were selected for resistance to 0.2 g / L of G418 and 0.1 g / L of hygromycin and purified by crosslinking in media containing 0.1 g / L of hygromycin and 0.2 g / L of G418, generating strains GEVO3714, GEVO3715 and GEVO3716. An ALD6 control strain containing an isobutanol production pathway, GEVO3466, was generated by transforming GEVO3198 with plasmid pGV2247. The transformants were selected for resistance to 0.2 g / L of G418 and purified by crosslinking in media containing 0.2 g / L of G418.
Construction of Deletion Strains ald2 , ald3 , ald4 , ald5 and hfdlA: PCR was used to generate separate DNA fragments that contained individual deletion alleles ALD2, ALD3, ALD4, ALD5 and HFD1 for deletion of ALD2, ALD3 , ALD4, ALD5 and HFD1 individually from S. cerevisiae in separate strains. In addition, PCR was used to generate a DNA fragment that contained the deletion allele spanning both ALD2 and ALD3 together, which are adjacent genes in the S. cerevisiae genome, for deletion of ALD2 and ALD3 together (ald2A ald3N) from S. cerevisiae in an individual strain. The four-component fragments containing the upstream flanking region, the Psc_ccw12 promoter region of pGV1954, the hyphromycin resistance ORF of pGV2074 and the downstream flanking region for each individual gene were generated by PCR, as for the generation of the ABCD fragment for ALD6 deletion except using the primer pairs shown in Table 20. The four-component fragment for ALD2 and ALD3 deletion together contained the flanking region upstream of ALD2 and the flanking region downstream of ALD3 and was equally constructed through PCR using the primer pairs indicated in Table 20. The Psc_ccw12 promoter region of pGV1954 has always been amplified with the primer pairs oGV2631 / oGV2632 and the hygromycin resistance ORF of pGV2074 has always been amplified with the primer pairs oGV2633 / oGV2634.
Table 20. Initiators used to amplify upstream and downstream regions for gene deletions.
Strains with ALD2, ALD3, ALD4, ALD5 and HFD1 deletion individually and with ALD2 and ALD3 deletion together were constructed by transforming GEVO3198 or GEVO3466 with the individual four-component DNA fragment that contained the ALD2, ALD3 individual deletion allele , ALD4, ALD5 or HFD1 or with the four-component DNA fragment that contained the deletion allele of ALD2 and ALD3 together. The transformants were selected for resistance to 0.1 g / L of hygromycin and the transformant colonies were screened by colony PCR for the correct insertion of the four-component DNA fragment using the pairs of primers indicated in Table 21. The GEVO3567 strain was identified by this colony PCR as having correctly deleted ALD2, strain GEVO3568 was identified by this colony PCR as having correctly deleted ALD3, strain GEVO3569 was identified by this colony PCR as having correctly deleted and ALD2 together, the strain GEVO3579 was identified by this colony PCR as having correctly deleted ALD4, strains GEVO3705, GEVO3706 and GEVO3707 were identified by this colony PCR as having ALD5 correctly deleted, and strains GEVO3720, GEVO3721 and GEVO3722 were identified by this colony PCR as having HFD1 correctly deleted.
Strains containing an isobutanol production pathway with ALD2, ALD3 and ALD5 deletion individually or with ALD2 and ALD3 deletion together were constructed by transforming strains GEVO3567, GEVO3568, GEVO3569, GEVO3705, GEVO3706 and GEVO3707 with plasmid pGV2247. The transformants were selected for resistance to 0.2 g / L of G418 and 0.1 g / L of hygromycin and purified by crosslinking in media containing 0.1 g / L of hygromycin and 0.2 g / L of G418, generating strains GEVO3586, GEVO3587, GEVO3588, GEVO3590, GEVO3591, GEVO3592, GEVO3593, GEVO3594, GEVO3595, GEVO3708, GEVO3709 and GEVO3710. The strains GEVO3579, GEVO3720, GEVO3721 and GEVO3722 were generated from GEVO3466 and, therefore, contained the plasmid pGV2247.
Table 21. Primers Used to Track Colonies for Checking for Gene Deletions.
Table 22. Genotype of Strains Disclosed in Example 4.
Table 23. Plasmids Disclosed in Example 4.
Fermentation in Flasks with Shaking: Fermentations were performed to compare the performance of GEVO3466 from strains containing the deletion mutations ald2A, ald3A, ald2A ald3 , ald4A, ald5A, hfdl and aldóA. The yeast strains were inoculated from cell stains or from simple colonies purified from YPD agar plates containing 0.2 g / L of G418 in 3 mL of YPD containing 0.2 g / L of G418 and 1% v / v ethanol medium in tubes with a 14 mL round bottom pressure cap. The cultures were incubated overnight up to 24 h with shaking at an angle of 250 rpm at 30 ° C. Separately for each strain, these overnight cultures were used to inoculate 50 ml of YPD containing 0.2 g / L of G418 and 1% v / v ethanol medium in a 250 ml bottle with baffles with a mango closure. an OD600 of 0.1. These flask cultures were incubated overnight for up to 24 hours with shaking at 250 rpm at 30 ° C. The cells of these cultures in vials were harvested separately for each strain by centrifugation at 3,000 xg for 5 minutes and each cell pellet was resuspended separately in 5 mL of YPD containing 80 g / L of glucose, 1% v / v stock solution of 3 g / L of ergosterol and 132 g / L of Tween 80 dissolved in ethanol, 200 mM MES buffer, pH 6.5, and 0.2 g / L of G418 medium. Each cell suspension was used to inoculate 50 mL of YPD containing 80 g / L of glucose, 1% v / v stock solution of 3 g / L of ergosterol and 132 g / L of Tween 80 dissolved in ethanol, 200 mM MES buffer, pH 6.5, and 0.2 g / L of G418 medium in a 250 ml bottle without baffles with a vented screw cap for an OD600 of about 5. These fermentations were incubated with shaking at 250 rpm at 30 ° Ç. Periodically, samples from each stirred fermentation vial were removed to measure OD600 and prepare for gas chromatography (GC1) analysis, for isobutanol and other metabolites, and for high performance liquid chromatography (LC1) analysis. for organic acids and glucose. The 2 ml samples were removed into a microcentrifuge tube and centrifuged in a microcentrifuge for 10 min at maximum rpm. One ml of the supernatant was for the analysis of extracellular metabolites by GC1 and LC1 as described.
Deletion of ALD6 decreased the production of isobutyrate and acetate in fermentation of flasks with agitation: The results of fermentation of flasks with agitation for 52 h for GEVO3466 and the GEVO3714, GEVO3715 and GEVO3716 strains are summarized in Table 24. The ald6 strains GEVO3714, GEVO3715 and GEVO3716 produced 71% less isobutyrate than the ALD6 GEVO3466 strain. The aldóA strains GEVO3714, GEVO3715 and GEVO3716 also produced 86% less acetate than the ALD6 GEVO3466 strain. The isobutanol yield in the ald6 GEVO3714, GEVO3715 and GEVO3716 strains was not significantly different from that of the ALD6 GEVO3466 strain. The isobutanol titration in the GEVO3714, GEVO3715 and GEVO3716 strains was 23% higher than that of the ALD6 GEVO3466 strain.
Table 24. Fermentation Results in Flasks with Shaking Demonstrating Decreased Production of Isobutyrate and Acetate by Deletion of ALD6 The results of fermentation in flasks with shaking for 72 h for GEVO3466 and the strains ald2A, ald3A, ald2A, ald3A, ald4A, ald5A and hdd are summarized in Table 25 and Table 26. Strains with deletions in ALD3, ALD2 and ALD3 together or ALD4 had no decrease in isobutyrate production compared to the wild type ALDH strain GEVO3466. Strains with deletions in ALD2, ALD5 or HFD1 had no appreciable decrease in isobutyrate production compared to the wild type strain ALDH GEVO3466. Strains with deletions in ALD2 and ALD3 together produced 19% less acetate than the wild type ALDH strain GEVO3466, but strains with individual deletions of ALD2, ALD3, ALD4, ALD5 or HFD1 had no appreciable decrease in acetate production compared to wild type ALD strain GEVO3466.
Table 25. Fermentation Results in Flasks with Shaking Showing No Decrease in Isobutyrate and Acetate Production by Deleting ALD2, ALD3, ALD4 or ALD2 and ALD3 Together.
Table 26. Fermentation Results in Flasks with Shaking Showing No Decreased Production of Isobutyrate and Acetate by Deletion of ALD5 or HFD1.
Bench fermentation fermentations: Bench fermentation fermentations were carried out to compare the performance of GEVO3466 (ALD6) with GEVO3714 and GEVO3715 (aldóN). Glucose consumption, isobutanol production, isobutyrate production, and OD600 were measured during fermentation. For these fermentations, the strains purified from streak plates were transferred to flasks with 500 ml baffles containing 80 ml of YPD medium containing 1% v / v ethanol, CuSO4.5H2O 100 μΜ and 0.2 g / L of G418 and incubated for 32 h at 30 ° C on an orbital shaker at 250 rpm. The flask cultures were transferred to motor-driven fermentation vessels on top of 2 L with a working volume of 0.9 L of YPD medium containing 80 g / L of glucose, 1% v / v ethanol, 100 μΜ and 0 CUSO4ÓH2O 0 , 2 g / L of G418 per container for an initial OD600 of 0.5. The fermenters were operated at 30 ° C and pH 6.0, controlled with 6N KOH and 2N H2SO4 in a 2-phase aerobic condition based on the oxygen transfer rate (OTR). Initially, the fermenters were operated at a growth phase OTR of 10 mM / h by fixed agitation of 700 rpm and an air overlap of 5 sL / h. Cultures were grown for 24 hours at about 9-10 OD600 then immediately transferred to an aeration of OTR production = 2.0 mM / h by agitation reduction from 700 rpm to 450 rpm, over the period of 24 h to 86 , 5 h. Periodically, samples from each fermentor were removed to measure OD600 and to prepare for gas chromatography (GC1) analysis, for isobutanol and other metabolites, and for high performance liquid chromatography (LC1) analysis for organic acids and glucose. The 2 mL samples were removed into a microcentrifuge tube and centrifuged in a microcentrifuge for 10 min at maximum rpm. One ml of the supernatant was subjected to GC1 and LC1 analysis, as described.
Deletion of ALD6 decreased the production of isobutyrate and acetate and decreased the yield of isobutanol in fermentations in bench fermenters: The results of fermentation in bench fermenters for 86.5 h are summarized in Table 27. The AldóA strains GEVO3714 and GEVO3715 produced 38% less isobutyrate than the ALD6 GEVO3466 strain. The aldóA strains GEVO3714 and GEVO3715 also produced 61% less acetate than the ALD6 GEVO3466 strain. The isobutanol yield in the GEVO3714 and GEVO3715 aldóA strains was 25% higher than the ALD6 GEVO3466 strain. Isobutanol titration in the GEVO3714 and GEVO3715 aldóA strains was also 35% higher than the ALD6 GEVO3466 strain.
Table 27. Bench Fermenter Fermentation Results Demonstrating Decreased Isobutyrate and Acetate Production and Increased Isobutanol Yield by ALD6 Deletion.
Example 5: Determination of ALD6 Activity in S. cerevisiae The following example illustrates that isobutyraldehyde oxidation activity is significantly decreased in an AldóA strain.
Table 28. Genotype of Strains Disclosed in Example 5.
The GEVO3940 yeast strains from which the ALD6 gene (YPL061W) was deleted and its original GEVO3527 strain were cultured, in triplicate, by inoculating 3 ml of YPD medium in a 14 ml culture tube, in triplicate for each strain. Cultures were initiated from spots on the YPD agar plate for GEVO3527 and on YPD agar plates containing 0.2 g / L of G418 plates for GEVO3940. The cultures were incubated overnight at 30 ° C and 250 rpm. On the following day, the OD600 of the cultures overnight was measured and the volume of each culture to inoculate a 50 ml culture at an OD600 of 0.1 was calculated. The calculated volume of each culture was used to inoculate 50 ml of YPD in a 250 ml bottle with baffles and the cultures were incubated at 30 ° C and 250 rpm. The cells were harvested during the mid-log phase at ODs of 1.6-2.1 after 7 h of growth. The cultures were transferred to 50 ml pre-weighed Falcon tubes and the cells were harvested by centrifugation for 5 minutes at 3000 x g. After removing the medium, the cells were washed with 10 ml of H20 MilliQ. After removing the water, the cells were again centrifuged at 3000 x g for 5 minutes and the remaining water was carefully removed using a 1 ml pipette tip. The cell pellets were weighed and then stored at -80 ° C, until they were lysed and tested for isobutyraldehyde oxidation activity, as described.
As shown in Table 29, the specific activity of ALD6 of S. cerevisiae in GEVO3527 lysates for oxidation of 10 mM isobutyraldehyde was 13.9 mU / mg. The same strain with an ALD6 deletion had a specific activity of 0.6 mU / mg, which is 22 times less. The specific activity of ALD6 of S. cerevisiae in GEVO3527 lysates for the oxidation of 1.0 mM isobutyraldehyde was 17.6 mU / mg. The same strain with an ALD6 deletion had a specific activity of 2.1 mU / mg, which is 8 times less. The specific activity of ALD6 of S. cerevisiae in GEVO3527 lysates for the oxidation of 0.1 mM isobutyraldehyde was 6.7 mU / mg. The same strain with an ALD6 deletion had a specific activity of 1.3 mU / mg, which is 5 times less. These data demonstrate that the endogenous ALD6 enzyme is responsible for the isobutyrate by-product of the isobutanol pathway, in S. cerevisiae.
Table 29. Isobutyraldehyde Oxidation Activities Specific Strains GEVO3527 and GEVO3940 using Various Isobutyraldehyde Concentrations. Specific Activities were Measured in Lysates of 3 Parallel Cultures of GEVO3527 and GEVO3940. The Means and Standard Deviations of the Measured Activities in the Biological Replicate Cultures are shown.
Example 6: Isobutyrate production decreased further with ALD6 gene deletion and overexpression of an improved alcohol dehydrogenase in S. cerevisiae The following example illustrates that the combination of an ALD6 deletion and overexpression of an ADH with better kinetic properties further decrease in isobutyrate production and an additional increase in isobutanol production. Isobutyrate is a by-product of yeast isobutyraldehyde metabolism and can comprise a significant fraction of the carbon yield. The following yeast strains were constructed: GEVO3466 was constructed by transforming the GEVO3198 strain with a 2μ plasmid, pGV2247, which carry genes encoding the following enzymes: KARI, DHAD, KIVD and wild type ADH (Ec_ilvC_coScP2D1-A1, Ll ilvD coSc, Ll_kivD2_coEc, and Ll adhA, respectively). GEVO3198 expresses a single copy of alcohol dehydrogenase (L. lactis ADH, Ll adhA) from its chromosomal DNA. The second strain, of which the biological replicas are called GEVO3714 and GEVO3715, was built by transforming two independent strains, GEVO3711 and GEVO3712, with a 2μ plasmid, pGV2247 that carry genes that encode the following enzymes: KARI, DHAD, KIVD and ADH wild type (Ec ilvC coScP2 '', LlilvDcoSc, Ll_kivD2_coEc, and Ll_adhA, respectively). GEVO3711 and 3712 express a single alcohol dehydrogenase (L. lactis ADH, LIadhA) and have the ALD6 gene deleted from chromosomal DNA. A third strain, of which the biological replicas are called GEVO3855 and GEVO3856, was built by transforming a strain, GEVO3711, with the 2μ plasmid pGV2602 that carry genes that encode the following enzymes: KARI, DHAD, KIVD and a mutant ADH ( Ec_ilvC_coScP21D1 ~ -A1'hls6 ,, LlilvDcoSc, Ll kivD2 coEc, and Ll adhARE1, respectively).
Table 30. Genotype of Strains Disclosed in Example 6.
Table 31. Plasmids Disclosed in Example 6.
Two different sets of fermentations were carried out. Fermentation A set was performed to compare the performance of GEVO3466 (LI_adhA) with GEVO3714-GEVO3715 (LI-adhA, aldóN). The Fermentation B set was performed to compare the performance of GEVO3714 (LIadhA, aldóN) with GEVO3855-GEVO3856 (LI_adhARE1, aldóN), respectively. Glucose consumption, isobutanol production, isobutyrate production, and OD600 were measured during fermentation. For these fermentations, colonies of single isolate cells grown on YPD agar plates were transferred to flasks with 500 mL baffles containing 80 mL of YPD medium containing 1% v / v ethanol, CuSO4.5H2O 100 μΜ, and 0.2 g / L of G418 and incubated for 32 h at 30 ° C on an orbital shaker at 250 rpm. The flask cultures were transferred to motor-driven fermentation vessels on top of 2 L with a working volume of 0.9 L of YPD medium containing 80 g / L of glucose, 1% v / v ethanol, CuSO4.5H2O 100 μΜ , and 0.2 g / L of G418 per container for an initial OD600 of 0.5. The fermenters were operated at 30 ° C and pH 6.0 controlled by 6N KOH in a 2-phase aerobic condition based on the oxygen transfer rate (OTR). Initially, the fermenters were operated at a growth phase OTR of 10 mM / h by fixed agitation of 700 rpm and an air overlap of 5 sL / h in both experiments. The cultures were grown for 24 hours at about 9-10 ° C and then immediately transferred to aeration production conditions for 48.5 h. In the first experiment, an OTR of 2.5-3.0 mM / h was sustained by reducing agitation from 700 rpm to 425 rpm while in the second experiment, an OTR of 2.0 - 2.5 mM / h was sustained by reducing agitation from 700 rpm to 400 rpm. Periodically, samples from each fermenter were removed to measure ODOoo and prepared for gas chromatography (GC1) and liquid chromatography (LC1) analysis. For GC1 and LC1, 2 mL of sample was removed into an Eppendorf tube and centrifuged in the microcentrifuge for a maximum of 10 minutes. One ml of the supernatant was analyzed by GC1 (isobutanol, other metabolites) and one ml was analyzed by high performance liquid chromatography (LC1) for organic acids and glucose.
The 72.5 h data from two separate fermentation sets A and B are summarized in Tables 32 and 33. The fermentation set compared AGEVO3466 (WT ADH) with GEVO3714 and 3715 (WT ADH, aldóN), while the set fermentation B compared GEVO3714 (WT ADH, aldóN) with GEVO3855 and 3856 (Ll_adhARE1, aldóN).
The data referring to fermentation set A (Table 32) show that the isobutanol titration and the theoretical yield in the strain that carries LladhA with the deletion of the ALD6 gene was 1.4 and 1.3 times higher, respectively, in comparison with the strain that carries LIadhA without deletion of the ALD6 gene. The strain that carries LIadhA without deletion of the ALD6 gene (GEVO3466) had an isobutyrate yield (gram isobutyrate produced / gram glucose consumed) while strains that carry LI adhA with the deletion of the ALD6 gene (GEVO3714 , GEVO3715) had a lower yield of 0.017 g / g isobutyrate. The strain that carries the L. lactis Adha without deletion of the ALD6 gene produced 2.3 g / L of acetate, while the strain that carries the L. lactis Adha with the deletion of the ALD6 gene produced 0.6 g / L acetate.
Table 32. Fermentation Set A data
The data for fermentation set B (Table 33) show that the isobutanol titration and theoretical yield in the strain that carries L. lactis adhARE1 with the ALD6 gene deletion was 1.2 and 1.1 times higher, respectively, compared to the strain that carries Adha de L. lactis with the deletion of the ALD6 gene. The strains carry L. lactis adhARE1 with the deletion of the ALD6 gene (GEVO3855, GEVO3856) had the lowest deisobutyrate yield (gram isobutyrate produced / gram of glucose consumed), 0.005 g / g, and produced 0.0 g / L of acetate compared to the strainer carries Adha de L. lactis with deletion of the ALD6 gene (GEVO3714) which had a higher isobutyrate yield of 0.014 g / g and a similar acetate titration of 0.0 g / L (Table 33).
Table 33. Fermentation Set B data.
Example 7: Identification of DH2MB as a by-product of Isobutanol Fermentation During fermentation of isobutanol-producing yeast strains, an unknown peak was found, coeluting with 2,3-dihydroxy isovalerate (DHIV) in the LC1 method, and quantified based on that, it was acting as a sink for a substantial portion of the carbon being used.
Initially, it was believed that this peak was only 2,3-dihydroxy-isovalerate (DHIV), but subsequent studies indicated that inhibition of the KARI product would have occurred at these levels of DHIV, making such concentrations impossible. Additional experiments showed that this recovered peak was not reactive with DHAD in enzymatic assays, thus eliminating the possibility that significant amounts of DHIV were present.
High performance liquid chromatography LC1: The analysis of organic acid metabolites was performed in an Agilent-1200 high performance liquid chromatography system equipped with two Rezex RFQ-Fast Fruit H + columns (8%) 150 x 4.6 mm (Phenomenex ) in series. The organic acid metabolites were detected using an Agilent UV-1100 detector (210 nm) and the detector's refractive index (IR). The column temperature was 60 ° C. This method was isocratic with 0.0128 N H2SO4 (25% 0.0512 N H2SO4 in Milli-Q water), as a mobile phase. The flow was adjusted to 1.1 ml / min. The injection volume was 20 pL and the reaction time was 16 min.
High performance liquid chromatography LC3: For samples containing a maximum of 10 mM aldehydes, ketones and keto acids (combined) intermediates, DNPH reagent was added to each sample in a 1: 1 ratio. 100 pL of DNPH reagent (2.4 - 12 mM Dinitrophenyl hydrazine, 20 mM citric acid, pH 3.0 to 80% acetonitrile, 20% MilliQ H2O) were added to 100 pL of each sample. The samples were incubated for 30 min at 70 ° C in a thermocycler (Eppendorf, Mestrecycler). The analysis of acetoin, diacetyl, ketoisovalerate and isobutyraldehyde was performed in an Agilent 1200 high performance liquid chromatography system equipped with a 5 pm reverse phase column of Eclipse XDB C-18 150 x 4 mm (Agilent) particle size and a C-18 reverse phase guard column (Phenomenex). All analytes were detected using an Agilent-1100 UV (360 nm) detector. The column temperature was 50 ° C. This method was isocratic with 60% phosphoric acid to 2.5% acetonitrile (0.4%), 37.5% water as the mobile phase. The flow was adjusted to 2 ml / min. The injection size was 10 pL and the execution time was 10 min.
LC4 High Performance Liquid Chromatography: Oxo acid analysis was performed on an Agilent-1100 high performance liquid chromatography system equipped with an IonPac AG11-HC, AS11-HC IonPac Analytical guard column (3-4 mm for column IonPac ATC, Dionex), or equivalent, and an IonPac ATC-1 Anion Trap column or equivalent. Oxo acids were detected using a conductivity detector (ED50 with conductivity deletion, Suppressor type: 4 mm ASRS in AutoSuppression recycle mode, Suppressor current: 300 mA). The column temperature was 35 ° C. This method used the following elution profile: Maintained at 0.25 mM for 3 min, linear gradient from 5 mM to 25 min, linear gradient from 38.5 mM to 25.1 min, maintained at 38.5 mM for 4, 9 min, 0.25 mM linear gradient at 30.1 min; held for 7 min to balance. The flow was adjusted to 2 ml / min. The injection size is 5 mL and the execution time is 37.1 min. GC-MS: Varian 3800CP GC system equipped with a single column DB-5 ms, 320MS single quad; injection port from 1.079 to 250 ° C; 1.0mL / min of constant flow in split ration 100; oven profile: initial temperature 40 ° C, maintained for 5 min, ramp from 20 ° C / min to 235 ° C and maintained for 2 minutes; combiPAL self-sampler dispensing 0.5 pL of sample, the masses being collected between 35 and 100. BSTFA bypass: (1) Evaporate the sample to dryness under nitrogen in a GC flask, (2) add 0.5 mL of acetonitrile and 0.5 mL of BSTFA reagent, (3) Incubate at 50 ° C for 30 minutes, (4) Inject GC-MS. LC-MS: For the LC-MS analysis of the sample's LC1 peak fraction it was injected into an Agilent 1100 Series high performance liquid chromatographic (HPLC) system, which was equipped with a multiple wavelength detector and a trapping mass spectrometer LC / MSD (trapping ion). The separations were monitored by mass spectrometry to provide identification of the component in the sample. The mass spectrometer was operated in chemical ionization at atmospheric pressure (APCI) mode for sample injection. The analyzes were performed using the positive and negative APCI modes. Detection of the "unknown" was observed only in the negative ionization mode. The analysis was performed using MSN to obtain fragmentation data about the sample analyte. Separations were achieved using Agilent Zorbax SB 4.6 x 150 mm C-18 column with 5 pm particles. The sample was run using an isocratic method that used an eluent of 90% HPLC water and 10% methanol. A 10 pL injection was used for the analysis of the sample solution. The sample was also analyzed with a chromatographic column bypass. DHIV and its isomer, DH2MB, elute at the same retention time in LC1. The peak related to these compounds is separated from other compounds in the fermentation samples. The peak was collected from the HPLC and used for further analysis. The signal ratio of the RI detector signal to the UV detector signal seen in LC1 for DHIV (and DH2MB) is characteristic of common organic acids (eg acetate, lactate, etc.); Conjugated acids (eg, pyruvate) have very different UV / IR signal ratios. The "DHIV peak" recovered had the characteristics of an unconjugated acid: Ratio (RI / UV): DHIV / DH2MB peak recovered (130); DHIV Std (150); Pyruvate (14). The absence of a carbonyl fraction at the "mysterious peak" was confirmed by the total absence of reaction between the peak fraction recovered from LC1 and DNPH: None of the adduct peaks were evident in the LC3 chromatographic system. The peak fraction recovered from LC1 was then analyzed by the LC4 method, which is performed under alkaline conditions, and is capable of separating DHIV and acetolactate. This result is shown in Figure 9, together with a coating of conventional mixtures. This clearly shows the separation between DH2MB (as it was later identified), and DHIV. Some pyruvate was also considered in the DH2MB peak collection.
NMR analysis: The sample peak recovered from the LC1 method was neutralized and lyophilized and sent for NMR analysis. The 2-D connectivity analysis by 1H-COZY NMR (Figure 10) and the proton NMR spectrum (Figure 11) produced good results.
2-D analysis of the "mysterious peak" eluting with DHIV (Figure 10): A methyl group, displaced in the middle, is not divided by any adjacent protons, where the 0.95 ppm methyl group is divided into a doublet with a proton adjacent to a hydroxyl group. The proton, in turn, is divided into a quartet by the adjacent methyl group. Complex patterns between 3.1 and 3.7 ppm indicate the different glucose anomers performed during the peak collection of "DHIV".
The NMR peak designations are shown in the spectrum below (Figure 11), clearly indicating that the "mysterious peak" identity is 2,3-dihydroxy-2-butyrate (DH2MB).
The 1H NMR and COZY spectra support the presence of 2,3-dihydroxy-2-methylbutanoic acid, a structural isomer of dihydroxy-isovaleric acid. Other signals from these spectra support the presence of anomeric proteins and, therefore, a sugar component. In addition, complex signal grouping between 3.1-3.8 ppm is often seen with oligosaccharides. The 13C NMR spectrum is very weak, and appears to be a fixed proton test (APT) experiment based on the signal at 45 ppm, which drops below the baseline. LC-MS was also performed on the peak fraction LC1. LC-MS was sufficient to demonstrate that the compound had a mass of 134 (both DHIV and DH2MB) (Figure 12).
This analysis conclusively identified the unknown by-product as 2,3-dihydroxy-2-methylbutanoic (CAS # 14868-24-7). This compound exists in four different stereoisomeric forms. 2,3-Dihydroxy-2-methylbutanoic acid exists as a set of cis and trans diastereomers, each of which exists as a set of enantiomers. The four compounds are shown in Figure 13.
As described herein, DH2MB is derived from (2S) -2-hydroxy-2-methyl-3-oxobutyrate (acetolactate). The product of this reaction would be (2S, 3R) -2,3-dihydroxy-2-methylbutanoic acid, or (2S, 3S) -2,3-dihydroxy-2-methylbutanoic acid, or a mixture of the two diastereomers depending on the stereoselectivity of the endogenous enzyme (s) that catalyzes this conversion.
Example 8: DH2MB Production and Purification The purpose of this example is to illustrate how DH2MB was produced and purified.
A CEN.PK2 strain of S. cerevisiae constructed comprising ALS activity (GEVO3160, CEN.PK2 of S. cerevisiae: MATa URA3 leu2 his3 trpl gpd1A :: Pccwi2: Hph gpd2A :: TKi_URA3_short: Pfba1 Kl_URA3: Tki_ura3: Tki_ura3 BsalsScoSc: Tcyc1: Ppgk1: LlkivD: Peno2_Sp_HIS5 pdc5A :: LEU2: bla: Ptef1: ILV3AN: Ptdh3: ilvC_coSc_Q110V pdc6A :: PTEF1: LI_íIvD_C_P_H glucose and faster growth} expressing plasmid pGV2247 (2-micron, plasmid resistant to G418 for the expression of EcilvC_P2D1-A1, LlilvD, Ll_kivD2, and Ll_adhA) was used to produce approximately 10 g / L of DH2MB a batch fermentation using a 2L DasGip engine powered top equipped with 1L of culture medium (10 g / L yeast extract, 20 g / L peptone, 80 g / L glucose, 1% v / v ethanol, CuSO4.5H20 100 μΜ, 0.2 g / L G418) at 30 ° C, pH 6.0, and an OTR of approximately 10 mmol / h. cell-free fermentation broth was acidified to pH 2 using concentrated H2SO4. The acidified broth was concentrated to 350 mL under reduced pressure (0-100 mbar) using Rotovapor Büchi R-215. The flask containing the broth was heated in the water bath to 20-30 ° C during evaporation. A volume of 70 ml MeOH was added to the concentrated broth and the mixture was transferred to a 500 ml liquid-liquid extractor (Sigma-Aldrich cat. # Z562432), which was configured according to the manufacturer's specifications for continuous extraction with ethyl acetate (EtOAc). Continuous extraction was performed for 3 days, replacing the EtOAc extract with fresh EtOAc daily.
After extraction, the first two batches of DH2MB extract in EtOAc were combined and dried with anhydrous MgSO4, followed by filtration. The dry extract was concentrated in vacuo to 500 ml and treated with 3 g of activated carbon (Fluka cat # 05105), during 30 min of stirring at room temperature. The bleached solution was filtered and concentrated to approximately 50 mL under vacuum (0-100 mbar using Rotovapor Büchi R-215). The solution was incubated at 4 ° C for two days. The obtained crystals were filtered and washed with ice-cold diethyl ether and acetone. The crystals were dried using a lyophilizer under reduced pressure (0.05 mbar) for one day. Isolated DH2MB was analyzed by 1H (Figure 14) and (Figure 15) 13C NMR. 1H NMR (TSP) 1.1 (d, 6.5 Hz, 3H), 1.3 (s, 3H), 3.9 (q, 6.5 Hz, 3H). A 13C spectrum indicated five different carbon atoms present in the sample. Resonance at 181 ppm indicated carbon of carboxylic acid present in the sample. In conclusion, based on the NMR spectra, 99% purity of isolated DH2MB can be estimated.
Example 9: Impact of DH2MB Production on Isobutanol Yield in Fermentation The purpose of this example is to demonstrate that DH2MB accumulates at substantial levels in the yeast strains that comprise ALS and TMA29 activity.
The strains and plasmids described in this example are shown in Tables 34 and 35, respectively.
Table 34. S. cerevisiae strain GEVO3160 strain.
Tabe a 35. Plasmid Genotype pGV2247. The GEVO3160 strain of S. cerevisiae was transformed with pGV2247 as described. Fermentation was carried out to characterize the transformed strain. A single colony of isolated cells grown on a YPD agar plate containing 0.2 g / L of G418 was transferred to 5 mL of YPD medium containing 80 g / L of glucose, 1% v / v ethanol, CuSO4.5H20 100 μΜ , and 0.2 g / L of G418 and incubated for 24 hours at 30 ° C, 250 rpm. Then, this culture was transferred to flasks with 500 ml baffles containing 80 ml of the same medium and incubated for 24 hours at 30 ° C on an orbital shaker at 250 rpm. The culture flask was transferred to a motor fermentor container driven on top of 2L with a working volume of 0.9 L of the same medium from an initial OD of 0.5. The fermenter was operated at 30 ° C and pH 6.0 controlled by 6N KOH in a 2-phase aerobic condition based on the oxygen transfer rate (OTR). Initially, the fermenter was operated at a growth phase OTR of 10 mM / h of fixed agitation at 700 rpm and an air overlay of 5sL / h in both experiments. The cultures were grown for about 20 h at an OD of about 8, and then immediately moved to aeration production. An OTR of 1 mM / h was sustained by reducing agitation from 700 rpm to 350 rpm. After 93 h after inoculation, a replicate container for each strain was further reduced to an OTR = 0.3 mM / h, reducing agitation from 350 rpm to 180 rpm. Periodically, samples from each fermentor were removed to measure ODOoo and to prepare for gas chromatography (GC1) and liquid chromatography (LC1) analysis. For GC1 and LC1, 2 mL of sample was removed into an Eppendorf tube and centrifuged in the microcentrifuge for a maximum of 10 minutes. One ml of the supernatant was analyzed by GC1 (isobutanol, other metabolites) and one ml was analyzed by high performance liquid chromatography (LC1) for organic acids and glucose. Figure 16 shows the product and by-product profiles of GEVO3160 from S. cerevisiae transformed with pGV2247. These profiles are representative for Pdc-negative, Gpd-negative isobutanol-producing yeast strains. Pdc-negative / Gpd-negative yeast-producing strains are described in copendent and common-owned publications, US 2009/0226991 and US 2011/0020889, both of which are incorporated herein by reference in their entirety for all purposes. Figure 16 shows that isobutanol (13.9 g / L) and the unknown compound quantified as "DHIV" and now identified as DH2MB (8.4 g / L) are the main products produced during the microaerobic production OTR. Assuming that the quantification using the DHIV response factor leads to an accurate quantification of DH2MB, about 12-13% of the carbon consumed is diverted to the production of DH2MB. If the acetolactate that is converted to DH2MB instead of being converted to isobutanol, then the yield of isobutanol throughout the fermentation time shown in Figure 16 would be significantly higher.
Example 10: Expression of ALS is Necessary for the Production of DH2MB The purpose of this example is to demonstrate that the exogenously expressed ALS activity is necessary for accumulation of DH2MB in S. cerevisiae.
This experiment was carried out to determine if ALS is necessary for the production of DH2MB. The strains used in this experiment were GEVO1187 (CEN.PK2 of S. cerevisiae; MATa URA3-52 leu2-3112 his3A1 trp1-289 ADE2) and GEVO2280 (CEN.PK2 of S. cerevisiae; MATa URA3 leu2 his3 trp1 ADE2 pdc1A :: PCUP1 -1: Bs_alsS2: TRP1). Before fermentation, both strains were transformed with the 2 micron plasmid pGV2082 (Ptdh3: EcíIvCcoScQ110V, Ptefí.LI_íIvD_coSc, PpGK1: Ll kivD coEc, and PENO2: Dm ADH, 2μ ori, bla, g) as described.
To measure ALS activity, extracts of yeast cells from GEVO1187 and GEVO2280 were prepared. The cells were cultured at an OD600 of about 1, induced with 1 mM CuSO4, for 2 hours and then harvested. To prepare the cells for the assays, 50 ml of cells were collected by centrifugation at 2700 x g. After removing the medium, the cells were resuspended in sterile dH2O, centrifuged at 2700 X g and the remaining medium was carefully removed with a 1 ml pipette tip. Cell pellets were weighed (empty tubes were previously weighed) and then frozen at -80 ° C until use. Cell lysates were made using the following SOP, as described below. The cells were thawed on ice and resuspended in lysis buffer (250 mM KPO4, pH 7.5, 10 mM MgCL and 1 mM DTT), such that the result was a 20% mass cell suspension. A 1000 µl volume of glass microspheres (0.5 mm in diameter) was added to a 1.5 ml Eppendorf tube and 875 µl of cell suspension were added. Yeast cells were lysed using a Retsch MM301 mixing mill (Retsch Inc. Newtown, PA) by mixing 6 X 1 min at full speed, with 1 min cooling steps between them. The tubes were centrifuged for 10 minutes at 23,500 x g at 4 ° C and the supernatant was removed. The extracts were kept on ice until the test. The concentration of lysate protein was determined using the BioRad Protein Bradford assay reagent kit (Cat # 500-0006, BioRad Laboratories, Hercules, CA) and using BSA for the standard curve, as described. Briefly, all ALS assays were performed in triplicate for each lysate, both with and without substrate. For each lysate assay, 100 pl of lysate diluted 1: 2 with lysis buffer was mixed with 900 pl of buffer (50 mM potassium phosphate buffer, pH 6.0, MgSO41 mM, 1 mM thiamine pyrophosphate, pyruvate 110 mM) and incubated for 15 minutes at 30 ° C. The buffers were prepared at room temperature. A control without substrate (buffer without pyruvate) and a control without lysate (lysis buffer instead of lysate) were also included. After incubating 175 μl of each reaction mixture, they were mixed with 25 ml of 35% H2SO4 and incubated at 37 ° C for 30 min. The samples were subjected to analysis for analysis by LC1. Using this method, it was determined that the GEVO1187 wild type strain had no detectable activity, whereas the GEVO2280 strain expressing ALS had 0.65 units / mg of ALS activity lysate. The performance of the two strains (with or without the heterologous expression construct integrated with ALS) was compared using the following flask fermentation conditions. The strains were stained on YPD plates containing 0.2 mg / mL of G418. After growing overnight, the cells were removed from the plate with a sterile toothpick and resuspended in 4 mL of YPD with 0.2 g / L of G418. OD600 was determined for each culture. The cells were added to 50 ml YP with 50 g / L dextrose and 0.2 mg / ml G418 such that a final OD600 of 0.1 was obtained. To induce ALS expression driven by the CUP1 promoter, 1 mM copper sulfate was added at the 24 hour time point. Unused material was stored at 4 ° C to act as a blank medium for GC and LC, and to act as the sample of t = 0 for fermentation. At t = 24, 48 and 72 hours the samples were prepared for analysis by GC1 and the samples for 72 hours were additionally analyzed by LC1. In 24 and 48 hours a 1:10 dilution of the supernatant from each culture was analyzed by YSI. If necessary, 50% glucose containing 0.2 g / L of G418 was added to a final concentration of 100 g / L of glucose. Fermentations were carried out at 30 ° C with agitation at 250 RPM. The DH2MB titration achieved in 72 hours of fermentation in a stirred flask was determined using the LC1 method, both for the WT strain (BUD1187) without ALS and for a strain that expresses Pcupi: Bs_alsS2 in PDC1 (BUD2280). Each strain was transformed with the 4-component plasmid pGV2082. Fermentation was carried out as described. Without the exogenous expression of ALS, the strain produced no DH2MB, whereas the strain with ALS expression produced up to 1.4 g / L of DH2MB of DHIV.
Example 11: Only ALS Expression is Necessary for Production of DH2MB The purpose of this example is to demonstrate that the activity of ALS individually is responsible for the accumulation of DH2MB in S. cerevisiae.
This experiment was performed to determine whether ALS individually or in combination with a KARI expression plasmid, DHAD, kivD, ADH is responsible for the production of DH2MB. The strain used in the experiment was GEVO2618 (MATa URA3 read2 his3 trpl pdc! A :: [Pcupi: BsalsS1coSc: TRP1). The plasmids tested in this experiment were pGV2227 which contains the remaining four pathway genes (-Ptefí-LIíIvD._coSc: Ptdh3: Ec_íIvC_coScQ110V: Psc_tp71: G418: Ppgkí.
Ll_kivD2_coEc: PDC1 -3'region: Peno2: Ll adhA 2μ bla, pUC-ori), and pGV2020, the empty vector control (Psc_tef1, Psc_tpi1, G418R, APr, 2μ).
Shake flask cultures of GEVO2618 transformed with pGV2020 and GEVO2618 transformed with pGV2227 were started in YPD medium (15% glucose) containing 200 mM MES, pH 6.5, and 0.4 g / L of G418 at an OD600 ~ 0.1, and were performed at 30 ° C and 75 rpm in a shaking incubator. The samples were collected at 24 h and 48 h and the samples were analyzed for metabolite levels by HPLC (LC1) and GC (GC1). After 48 hours, all of the glucose was consumed from the middle of both strains. The strain containing the empty vector (GEVO2618 + pGV2020) produced 4.6 g / L of DHIV + DH2MB representing 3.8% yield. The strain containing the vector expressing four additional gene pathways (GEVO2618 + pGV2227) produced a similar titer of 5.6 g / L of DHIV + DH2MB representing 3.1% yield.
Example 12: Effect of increased KARI activity on DH2MB production The purpose of this example is to demonstrate that increased KARI activity results in decreased DH2MB production in yeast comprising ALS activity.
The strains and plasmids disclosed in this example are shown in Tables 36 and 37, respectively.
Table 36. Genotype of Strains Disclosed in Example 12.
Table 37. Plasmids Disclosed in Example 12. The GEVO2843 strain of S. cerevisiae was transformed with the 2μ plasmids pGV2377, pGV2466, pGV2398, and pGV2400 as described, to determine whether the expression of wild-type or constructed KARIs led to greater DH2MB accumulation.
Pre-cultures of GEVO2843 transformed with 2μ plasmids (pGV2377, 2466, 2398, 2400) were created in YPD medium containing 1% ethanol and 0.2 g / L of G418 and incubated overnight at 30 ° C and 250 rpm. These pre-cultures were used to inoculate 50 ml of the same medium in a bottle with baffles and incubated at 30 ° C and 250 rpm, until reaching an OD600 of ~ 5. They were pelleted in 50 ml Falcon tubes at 2700 rcf for 5 minutes at 25 ° C. Then, the cells of each 50 mL culture were resuspended in 50 mL of YPD containing 8% glucose, 1% (v / v) ethanol, ergosterol, Tween-80, 0.2 g / L of G418, and MES 200 mM, pH 6.5. The cultures were added to 250 ml flasks without baffles and placed in an incubator at 30 ° C and 75 rpm. Samples were collected after 72 h to determine OD600 and analyze the fermentation broth for the extracellular metabolite pathway by analyzing GC1 and LC1. Table 38 shows that the strain transformed with pGV2377 (without overexpression of any plasmid KARI gene) produced the highest 15% carbon yield for combined DH2MB + DHIV, whereas strains with pGV2466 (containing Ec_ilvC_coSchis6), pGV2398 (containing Ec_ilvC_coScQ110- his6), and pGV2400 (containing Ec_ilvC_coScP2D1-A1-his6) had a similar combined 8-10% DH2MB + DHIV carbon yield. Likewise, the strain transformed with pGV2377 produced isobutanol with a lower carbon yield of 6%. The remaining strains that comprise KARI genes in a plasmid produced isobutanol with higher carbon yields. The observation that decreased DH2MB production correlates with increased isobutanol production is consistent with the conclusion that DH2MB is produced from acetolactate through a reaction that does not involve KARI.
Table 38. Combined DH2MB + DHIV and Isobutanol Carbon Yields A second experiment was carried out on strains that expressed either no KARI from a plasmid, a low level of KARI, or a high level of KARI. In this experiment the KARI activity of cell lysates was measured. The GEVO2843 strain of S. cerevisiae was transformed as described with combinations of plasmids, as described in Table 37, the strain without KARI contained pGV2377 + pGV2196 and had no KARI of plasmid origin, the strain with low KARI contained pGV2377 + pGV2406 and expressed KARI from a low copy plasmid, and a high KARI strain contained pGV2398 + pGV2196 and expressed KARI from a high copy plasmid. Fermentations and sampling were carried out as described. GC1 and LC1 methods were performed as described. The cells for KARI assays were lysed, as described, except that the lysis buffer was 250 mM KPO4, pH 7.5, 10 mM MgCl2 and 1 mM DTT. The protein concentration of lysates was determined as described.
To measure KARI activity in vitro, the acetolactate substrate was made by mixing 50 pl of ethyl-2-acetoxy-2-methyl-acetoacetate with 990 pl of water. Then, 10 pl of 2N NaOH were added sequentially, with a vortex mixture between the 15 sec additions, up to 260 pl of NaOH. The acetolactate was stirred at room temperature for 20 min and kept on ice. NADPH was prepared in 0.01 N NaOH to a concentration of 50 mM. The concentration was determined by reading the OD of a sample diluted at 340 nm on a spectrophotometer and using the molar extinction coefficient of 6.22 M-1cm-1 to determine the exact concentration. Three buffers were prepared and kept on ice. The reaction buffer contained 250 mM KPO4, pH 7.5, 10 mM MgCl2, 1 mM DTT, 10 mM acetolactate, and 0.2 mM NADPH. No substrate buffer was missing acetolactate. No NADPH buffer was without NADPH.
The reactions were performed in triplicate, using 10 μΐ of cell extract with 90 μΐ of reaction buffer in a 96-well plate in a SpectraMax 340PC multiplate reader (Molecular Devices, Sunnyvale, CA). The reaction was followed at 340 nm by measuring a kinetic curve for 5 minutes, with the DO reading every 10 seconds at 30 ° C. The Vmax of each extract was determined after subtracting the background reading from the control without substrate from the complete reading in buffer. Table 39 shows the data for KARI activity, as well as carbon yield in% for isobutanol and DH2MB + DHIV combined. As KARI activity increased the carbon yield of isobutanol increased and the carbon yield in combined DH2MB + DHIV decreased.
Table 39. Combined carbon yields from KARI, Isobutanol and DH2MB + DHIV Activity. * These data comprise only one sample Example 13: Effect of Increased DHAD Activity The purpose of this example is to demonstrate that increased DHAD activity results in decreased production of DH2MB in yeast comprising ALS activity.
The strains and plasmids disclosed in this example are shown in Tables 40 and 41, respectively. GEVO2843 was transformed with different pairs of plasmids. Strain A contains pGV2227 plus pGV2196. Strain B contains pGV2284 plus pGV2196. Strain C contains pGV2284 plus pGV2336. The unique BUD2843 transformants with one of three combinations of 2 plasmids were single colonies purified on YPD plates containing hygromycin and the corrected cells were used to inoculate 3 mL of YPD containing 1% (v / v) ethanol, 0.2 g / L of G418, and 0.1 g / L of hygromycin. Cultures were incubated at 30 ° C, 250 rpm overnight, before using to inoculate 3 ml of YPD containing 1% (v / v) ethanol, 0.2 g / L G418, hygromycin and 0.1 g / L. The cultures were incubated at 30 ° C, 250 rpm overnight. The next day, cultures were used to inoculate 50 ml of YPD containing 8% glucose, 200 mM MES, pH 6.5, ergosterol, and Tween80 to an OD600 of about 0.1. These cultures were incubated at 30 ° C, 250 rpm overnight. The next day, the cultures were diluted in 50 ml of the same medium at an OD600 of ~ 0.1. Cultures were incubated at 30 ° C, 250 rpm, and 1.5 mL of samples were taken after 0, 24, 47, 70, and 92 hours of incubation. The samples were prepared for analysis by GC and LC, as described. After 92 hours, the rest of all samples were centrifuged and the pellets were weighed and stored at -80 ° C. DHAD assays were performed with lysates prepared from frozen pellets as described. LC1 and GC1 analysis was performed as described.
Table 40. Genotype of Strains Disclosed in Example 13.
Table 41. Plasmids Disclosed in Example 13. Table 42 shows the DHAD activity, isobutanol yield and DHIV + DH2MB yield combined. The strain transformed with pGV2284 + pGV2196 (without DHAD expressed from a plasmid) produced the highest carbon yield, from 19% for combined DH2MB + DHIV and the lowest carbon yield of 9% isobutanol. The strain transformed with pGV2227 + pGV2196 (highest DHAD expression from a plasmid) had the lowest carbon yield of 9% for DH2MB + DHIV and the highest carbon yield of 18% isobutanol. The strain transformed with pGV2284 + pGV2336 (low copy DHAD expression from a plasmid) had an intermediate carbon yield of 16% for combined DH2MB + DHIV and 12% for isobutanol.
Table 42. Carbon yield of DHAD, Isobutanol and DH2MB + DHIV Activity Combined in Fermentation for 92 h.
In a second experiment, GEVO2843 was transformed with different pairs of plasmid (Table 43) and evaluated in a shaking bottle fermentation as above. Strain D contains pGV2196 plus pGV2589. Strain E contains pGV2529 plus pGV2589. Strain F contains pGV2196 plus pGV2485. The strain transformed with pGV2196 + pGV2589 (without plasmid origin DHAD) produced 1.25 g / L of isobutanol and 5.67 g / L DH2MB + DHIV. The DHAD strain expressed from a high copy plasmid (pGV2196 + pGV2485) produced 2.74 g / L of isobutanol and 3.71 g / L DH2MB + DHIV, indicating that an increase in DHAD expression led to a decreased accumulation of DH2MB + DHIV. The DHAD strain expressed from a low copy plasmid (pGV2529 + pGV2485) produced an intermediate level of both metabolites, consistent with an intermediate level of DHAD activity.
Table 43. Additional Plasmids Disclosed in Example 13.
Table 44. DHAD activities, isobutanol titration and yield, and combined DH2MB + DHIV titrations in fermentation for 72 h.
Example 14: Deletion of TMA29 in S. cerevisiae By Target Deletion The following example illustrates that deletion of the TMA29 gene from the S. cerevisiae genome eliminates DH2MB production when acetolactate synthase is overexpressed.
Several enzyme reductase candidates that can catalyze the production of DH2MB have been identified in the S. cerevisiae genome, including the TMA29 gene product. The genes encoding these reductases were deleted in the GEVO2618 strain of S. cerevisiae, a strain known to produce g / L amounts of DH2MB, using the integration of a URA3 marker. Fermentations were carried out with these strains to determine whether the deletion of any of the candidate genes, including TMA29, reduced or eliminated the production of DH2MB.
The strains, plasmids, and primer sequences are listed in Tables 45, 46, and 47, respectively.
Table 45. Genotype of Strains Disclosed in Example 14.
Table 46. Plasmids Disclosed in Example 14.
Table 47. Oligonucleotide sequences disclosed in Example 14.
Strain Construction: Strains of GEVO3638 GEVO3639 and GEVO3640 of S. cerevisiae, were constructed by transforming GEVO2618 with bipartid-integrating SOE PCR products to replace TMA29 with a URA3 marker. Primers to amplify the 5 'and 3' target sequences for reductase genes were designed with a 20 bp homologous sequence for a URA3 fragment. This was done so that the SOE PCR can be used to create the fragments containing the URA3 marker and homologous regions that flank the reductase gene of interest. PCR was performed on an Eppendorf Mestrecycler® (Cat # 71086, Novagen, Madison, WI). The following PCR program was followed for sets of primers used to generate PCR fragments SOE: 94 ° C for 2 min then 30 cycles (94 ° C 30 sec, 53 ° C 30 sec, 72 ° C 1.5 min ), then 72 ° C for 10 min. The following pairs of primers and models were used for the first stage of SOE reactions.
To generate the 5 'URA3 fragment, oGV2232 and oGV2862 were used to amplify the 5' URA3 fragment using pGV2129 as a model. The 1364 bp fragment was purified by gel electrophoresis. To generate the 3 'URA3 fragment, oGV2231 and oGV893 were used to amplify the 3' URA3 fragment using pGV1299 as a model. The 1115 bp fragment was purified by gel electrophoresis.
To generate the 5 'TMA29 fragment, oGV2867 and oGV2891 were used to amplify the 5' TMA29 fragment using S. cerevisiae genomic DNA S288c as a model. The S. cerevisiae S288c strain was purchased from ATCC (ATCC # 204508). The 412 bp fragment was purified by gel electrophoresis. To generate the 3 'TMA29 fragment, oGV2869 and oGV2870 were used to amplify the 3' TMA29 fragment using S. cerevisiae S288c genomic DNA as a model. The 305 bp fragment was purified by gel electrophoresis.
The following pairs of primers and models were used to generate the SOE PCR products. To generate the SOE PCR 5 'TMA29 product, oGV2232 and oGV2867 were used. The 5 'URA3 fragment and the 5' TMA29 fragment were used as a model. To generate the SOE PCR 3 'TMA29 product, oGV2231 and oGV2870 were used. The 3 'URA3 fragment and the 3' TMA29 fragment were used as a model. The transformation of the S. cerevisiae strain GEVO2618 with the bipartid integration SOE PCR products was carried out as described. After transformation, the cells were harvested by centrifugation (18,000 x g, 10 seconds, 25 ° C) and resuspended in 400 pL of SCD-HLWU medium. Integrative transformants were selected by plating the transformed cells on SCD-Ura agar medium. Since the transformants were purified single colonies they were maintained on SCD-Ura plates. Colony PCR was used to verify correct integration. For URA3 screening, from the correct 5 'end,: the 5' junction primers TMA29 oGV2915 and oGV2902 were used to give an expected 991 bp band. For URA3 screening, from the correct 3 'end,: the 3' TMA29 junction primers oGV2904 and oGV2916 were used to give an expected band of 933 bp. To track the deletion of TMA29 genes, the primers oGV2913 and oGV2914 were used, in the expectation of a 288bp shortage, if the CDS was deleted.
Fermentations: Fermentations were carried out with the TMA29A GEVO3638, GEVO3639 and GEVO3640 strains and the TMA29 strain of GEVO2618 origin. Cultures were started in YPD with agitation at 30 ° C and 250 rpm. After four duplications, OD600 was determined for each culture. The cells were added to 50 ml YPD with 15% glucose in such a way that a final OD600 of 0.05 was obtained. At t = 24 h, 2 ml of medium was removed and 25 pL used at a 1:40 dilution to determine OD600. The remaining culture was centrifuged in a microcentrifuge at maximum speed for 10 minutes and 1 ml of supernatant was removed and subjected to LC1 and LC4 analysis. At t = 48 h, 2 ml of medium was removed and 25 pL used at a 1:40 dilution to determine OD600. 1 mL of the supernatant was subjected to LC1 analysis. In addition, 14 mL was collected by centrifugation at 2700 x g. After removing the medium, the cells were resuspended in sterile DH2O, centrifuged at 2700 X g and the remaining medium was carefully removed with a 1 mL pipette tip. Cell pellets were weighed (empty tubes were previously weighed) and then frozen at -80 ° C until thawing for ALS assays, as described. The production of DH2MB is dependent on the expression of heterologous ALS, for example, the BsalsSlcoSc gene. The ALS activity of cell lysates was measured as described to demonstrate that the TMA29 deletion had no impact on ALS expression and / or activity. The ALS activity of extracts from strains that carry the TMA29 deletion is no less than, and is slightly more than the activity of extracts from the source strain. The 24-hour results (48h for ALS activity) are summarized in Table 48 and clearly demonstrate the lack of DH2MB production in the strain with the TMA29 deletion. LC4 analysis confirmed that GEVO3527 did not produce DHIV.
Table 48. Production of DH2MB in the strain with TMA29 deletion Example 15: Deletion of TMA29 in S. cerevisiae by Deletion Library The following example illustrates that deletion of the TMA29 gene from the S. cerevisiae genome eliminates DH2MB production when acetolactate synthase is overexpressed.
Strains, ORF deletions, and plasmids are described in Tables 49, 50, and 51.
Table 49. Genotype of Strains Disclosed in Example 15.
Table 50. ORF Deletion Disclosed in Example 15.
Table 51. Plasmid Disclosed in Example 15.
A commercial library of Strains of S. cerevisiae that has a gene / ORF deleted by strain was used to screen for a deletion that can catalyze the production of DH2MB. The candidate strain containing the TMA29 ORF deletion (ie YMR226C) was selected. Since the expression of exogenous ALS is necessary for the production of DH2MB, a CEN plasmid (pGV2435) containing the BsalsSlcoSc gene carried by the CUP1 promoter was transformed into Strains as described. The transformations were recovered overnight at 30 ° C, 250 rpm before plating on YPD plates containing 0.2 g / L of hygromycin. The transformants were then corrected on YPD plates containing 0.2 g / L of hygromycin and incubated at 30 ° C.
Fermentations were performed with these strains to determine whether the deletion of TMA29 (YMR226C) reduced or eliminated the production of DH2MB. Three independent transformants from each strain were used to inoculate fermentation pre-cultures that were grown overnight for YPD saturation containing 0.2 g / L of hygromycin at 30 rpm and 250 ° C. The following day, the OD600 of the pre-cultures was measured and the volume of the overnight culture required to inoculate a 50 ml culture at an OD600 of 0.1 was calculated for each culture. 50 mL of YPD containing 150 g / L of glucose, 200 mM MES, pH 6.5, and 0.2 g / L of hygromycin in a 250 mL bottle with baffles were inoculated with the calculated amount of overnight culture. The cells were incubated at 30 ° C and 75 rpm on an orbital shaker. Within 24 hours, all cultures were fed an additional 75 g / L of glucose by adding 8.8 mL of a 50% glucose solution to each flask and then returning to incubation at 30 rpm and 75 ° C. In 72 h, 1.5 mL was sampled from each vial (750 pL divided between two Eppendorf tubes). OD600 was measured for each culture (1:40 dilution in H2O). The cells were removed from samples by centrifugation at> 14000 x g for 10 minutes in a microcentrifuge. Sample supernatants were collected and stored at 4 ° C until analysis by LC1, and cell pellets were stored at -80 ° C until thawed for ALS assays, as described.
There was some variation in growth between the two strains, with OD600 values of 13.7 for GEVO3527 and 15.7 for the TMA29 deletion strain at 72 h (Table 52). Strains consumed the same amount of glucose of about 223 g / L in 72 h (Table 52). GEVO3527 produced 2.8 g / L of DH2MB for 72 h. The YMR226C deletion strain (TMA29A) did not produce detectable levels of DH2MB. The GE23527 specific DH2MB titration was 0.2 g / L / OD; the YMR226C deletion strain (TMA29A) did not produce detectable levels of DH2MB. LC4 analysis confirmed that GEVO3527 did not produce DHIV.
Table 52. Cell Growth, Glucose Consumed, and DH2MB Production at 72 h.
Example 16: Improved Isobutanol Rate, Yield, and TMA29 Gene Deletion Titer in S. cerevisiae The following example illustrates that deletion of the TMA29 gene from the S. cerevisiae genome leads to an increase in yield, productivity, and titer of the desired product, isobutanol. In addition, it leads to a decrease in productivity, yield and DH2MB titration. DH2MB is a by-product of the metabolism of yeast acetolactate. In isobutanol fermentations, DH2MB can comprise 10% or more of the carbon yield. Strains with wild type TMA29 produce DH2MB in the presence of expressed acetolactate synthase (ALS), encoded by Bs alsSl coSc (SEQ ID NO: 23). Strains deleted by TMA29 do not produce DH2MB in the presence of Bs alsSl coSc expressed. A yeast strain deleted for all PDC and GPD genes expressing ALS (Bs_alsS1_coSc) from the chromosome was suppressed by TMA29 and transformed with a high copy four component isobutanol pathway, pGV2550 with genes for DHAD (Ll ilvD coSc), KARI (EcilvC_coScP21D1 ~ -A1'his6), KIVD (Ll_kivD2_coEc) and ADH (Ll_adhA_coScRE1'íhs6). The isobutanol titration, yield and productivity of this strain were compared with the original strain that was not deleted by the TMA29 gene, both in fermentation in shaken flasks and in fermenters. Strains and plasmids are listed in Tables 53 and 54, respectively.
Table 53. Genotype of Strains Disclosed in Example 16.
Table 54. Plasmids Disclosed in Example 16.
Yeast Strain Construction: GEVO3663 was constructed by transforming GEVO3351 with bipartid-integrated SOE PCR products described in Example 14 to replace TMA29 with a URA3 marker, as described, except after transformation, the cells were resuspended in 350 pL of SCD-Ura medium before being spread onto SCD-Ura plates.
The GEVO3690 GEVO3691 and GEVO3692 strains of S. cerevisiae were constructed by transforming GEVO3351 with plasmid pGV2550. The strains GEVO3694 GEVO3695 and GEVO3697 from S. cerevisiae were constructed by transforming GEVO3663 with plasmid pGV2250. Briefly, competent cells were prepared by removing the cells from a freshly prepared plate in 100 µl of 100 mM lithium acetate. The cell suspension was incubated at room temperature for 30 min. Plasmid DNA was transformed, as described. After transformation, the cells were resuspended in 400 µL of YPD containing 1% ethanol and incubated at 30 ° C for 6 hours with shaking at 250 rpm. The cells were then seeded on YPD plates containing 0.2 g / L of G418. The transformants were single colonies purified on YPD plates containing plates with 0.2 g / L of G418. Since the transformants were purified single colonies they were kept on YPD plates containing 0.2 g / L of G418.
Fermentations: A flask fermentation with agitation was performed comparing the performance of GEVO3690-GEVO3692 (TMA29) with GEVO3694-GEVO3695 and GEVO3697 (TMA29N). Cultures (3 mL) were started in YPD containing 1% ethanol and 0.2 g / L of G418 and incubated overnight at 30 ° C and 250 rpm. The OD600 of these cultures was measured after about 20 h. An adequate amount of each culture was used to inoculate 50 mL of YPD containing 1% ethanol and 0.2 g / L of G418, in a 250 mL bottle with baffles for an OD600 of approximately 0.1. These pre-cultures were incubated at 30 ° C and 250 rpm overnight. When the cultures reached an OD600 of approximately 5 they were centrifuged at 2700 rcf for 5 min at 25 ° C in 50 ml Falcon tubes. The cells of each 50 ml culture were resuspended in 50 ml of fermentation medium, as described. The cultures were then transferred to 250 ml screw cap flasks without baffles with small holes and incubated at 30 rpm and 75 ° C. At 24 and 48 h, samples from each flask were removed to measure OD600 and prepared for GC1 analysis. For GC1, 2 mL of sample was removed into an Eppendorf tube and centrifuged in the microcentrifuge for a maximum of 10 minutes. One ml of the supernatant was analyzed by GC1. In 72 hours the same procedures were used to collect cells for OD600 and GC analysis and, in addition, the samples were analyzed by high performance liquid chromatography (LC1) for organic acids, including DH2MB and DHIV, and glucose.
The 72 h results are summarized in Table 55. The titration, yield and increased rate of isobutanol with deletion of the TMA29 gene, while DH2MB production decreases.
Table 55. Increase in Titration, Yield, and Isobutanol Rate in 72 h.
In addition, the performance of GEVO3690-GEVO3691 (TMA29} to GEVO3694-GEVO3696 (TMA29N) was also compared in fermentations carried out in fermentation vessels.The plated cultures were transferred to 500 mL flasks with baffles containing 80 mL of YP medium with 20 g / L glucose, 1% v / v ethanol, CuSO4.5H20 100 μΜ, and 0.2 g / L G418 and incubated for 34.5 hours at 30 ° C on an orbital shaker at 250 rpm. in vials, they were transferred to fermentation containers with a motor driven on top of 2 with a working volume of 1.2 L of 80 mL of YP medium with 20 g / L of glucose, 1% v / v ethanol, CuSO4.5H2Ü 100 μΜ and 0.2 g / L of G418 for an initial OD600 of 0.2 The fermenters were operated at 30 ° C and pH 6, controlled with 6N KOH in a two-stage aerobic fermentation. Initially, the fermenters were operated at a growth phase oxygen transfer rate (OTR) of 10 mM / h by fixed agitation of 850 rpm and an air overlay of 5 sL / h The cultures were grown for 31 h at approximately 6-7 OD600 then immediately transferred to a production aeration OTR of 0.5 mM / h by agitation reduction from 850 rpm to 300 rpm for the rest of the fermentation. 111 h. Periodically, samples from each fermentor were removed to measure OD600 and prepared for gas chromatography (GC1) analysis. By GC, 2 mL of sample was removed into an Eppendorf tube and centrifuged in the microcentrifuge for a maximum of 10 minutes. One ml of the supernatant was analyzed by GC1 (isobutanol, other metabolites). In 72 hours the same procedures were used to collect cells for ODOoo and GC analysis and, in addition, the samples were analyzed by high performance liquid chromatography (LC1) for organic acids and glucose.
The 111-hour results are summarized in Table 56. The increase in titre, yield and rate of Isobutanol with the deletion of the TMA29 gene. DH2MB production decreased to undetectable levels.
Table 56. Increase in Titration, Yield, and Isobutanol Rate in 111 h. Glucose, isobutanol, and DH2MB titrations are the final titrations, that is, in 111 h of fermentation. b Yield and isobutanol rate are calculated based on the production phase only, that is, from 31 to 111 h of fermentation.
Example 17: Determination of TMA29 Activity in S. cerevisiae The following example illustrates that the reduction in (S) -2-acetolactate activity is significantly decreased in a TMA29A strain.
Table 57. Genotype of Strains Disclosed in Example 17.
The GEVO3939 yeast strains from which the TMA29 gene (YMR226C) was deleted and its origin GEVO3527 strain were grown, in triplicate, by inoculating 3 ml of YPD in a 14 ml culture tube, in triplicate for each strain. Cultures were initiated from spots on the YPD agar plate for GEVO3527 and on YPD plates containing 0.2 g / L of G418 for GEVO3939 and GEVO3940. The cultures were incubated overnight at 30 ° C and 250 rpm. On the following day, the OD of cultures overnight was measured and the volume of each culture to inoculate a 50 ml culture at an OD of 0.1 was calculated. The calculated volume of each culture was used to inoculate 50 ml of YPD in a 250 ml bottle with baffles and the cultures were incubated at 30 ° C and 250 rpm.
The cells were harvested during the mid-log phase in ODs of 1.6-2.1 after 7 h of growth. The cultures were transferred to pre-weighed 50 ml Falcon tubes and the cells were harvested by centrifugation for 5 minutes at 3000 x g. After removing the medium, the cells were washed with 10 ml of H2O MilliQ. After removing the water, the cells were again centrifuged at 3000 x g for 5 minutes and the remaining water was carefully removed using a 1 ml pipette tip. Cell pellets were weighed and then stored at -80 ° C until use.
The cell pellets were thawed on ice and resuspended in lysis buffer (10 mM sodium phosphate, pH 7.0, 1 mM dithiothreitol, 5% w / v glycerol) so that the result was a 20% cell suspension in pasta. One ml of glass microspheres (0.5 mm in diameter) was added to a 1.5 ml Eppendorf tube for each sample and 850 µl of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each at full speed, with 1 min incubation on ice, between intervals. The tubes were centrifuged for 10 minutes at 21500 x g at 4 ° C and the supernatant was transferred to a freshly prepared tube. The extracts were kept on ice until tested using the TMA29 test as described. The specific activity of TMA29 of S. cerevisiae in lysates of GEVO3527, a strain of S. cerevisiae MATa wild type, for the reduction of (S) -2-acetolactate was 6.9 ± 0.2 mU / mg. The ima29A GEVO3939 strain had a specific activity of 0.7 ± 0.3 mU / mg. The GEVO3527 wild type strain had specific TMA29 activity about 10 times greater than the deletion strain.
Example 18. Determination of TMA29 Activity in Kluyveromyces lactis The following example illustrates that (S) -2-acetolactate reduction activity is significantly decreased in a TMA29A strain.
Table 58. Genotype of Strains Disclosed in Example 18.
Table 59. Oligonucleotide sequences disclosed in Example 18.
K. lactis strain GEVO4458 was constructed from GEVO1742 as follows. DNA constructs were made to eliminate the K. lactis TMA29 locus using PCR SOE. The 5 'target sequence was amplified by PCR using GEVO1287 genomic DNA as a model and with the primers oGV3103 and oGV3065. The 376 bp fragment was purified by gel electrophoresis. The 3 'target sequence was amplified by PCR using GEVO1287 genomic DNA as a model and with the primers oGV3106 and oGV3067. The 405 bp fragment was gel purified. The Hph marker was amplified by PCR using pGV2701 (PTEFi-Hph, CEN / ARS, pUC-ori, bla) as a model and with the primers oGV3066 and oGV3068. The 1,165 bp fragment was gel purified. Then, the 5 'target sequence and the hph marker were joined using the PCR products described as a template. The reaction mixture was amplified using the oGV3068 and oGV3103 primers. The 1,984 bp fragment was gel purified. Then, the 5 'target sequence plus the Hph marker PCR fragment was joined with the 3' target sequence using PCR with primers oGV3103 and oGV3106. The 2,331 bp was gel purified and used for transformation. Yeast DNA was isolated using the Zymo Research ZR Fungal / Bacterial DNA Kit (Zymo Research Orange, CA; Catalog # D6005). GEVO1287 was grown to saturation in 12.5 mL of YPD in flasks with 125 mL baffles. THE
whole culture was collected in 15 ml Falcon tubes and cells collected at 2700 rcf for 5 min. Genomic DNA was isolated according to the manufacturer's instructions. The DNA concentration was measured and all genomic DNA preps were diluted to a final concentration of 25 ng / pL GEVO1742 was transformed as follows. 50 mL of YPD medium, in flasks with 250 mL baffles, were inoculated with GEVO1742 cells from a freshly prepared plate. The cultures were incubated overnight at 30 ° C and 250 rpm. The following morning, the culture was diluted 1:50 in YPD medium and allowed to grow for 6 h. The cells were harvested by centrifugation at 2700 rcf for 2 min at 30 ° C. The cells were washed by completely resuspending the cells with 50 ml of sterile MilliQ water. The cells were harvested by centrifugation at 2700 rcf for 2 min at 30 ° C. The cells were washed by resuspension with 25 ml of sterile MilliQ water. The cells were harvested by centrifugation at 2700 rcf for 2 min at 30 ° C. The cells were resuspended in 1 ml of 100 mM lithium acetate, transferred to an Eppendorf tube and collected by centrifugation at 14,000 rcf for 10 seconds. The supernatant was removed and the cells were resuspended with a volume of 4X the volume of the pellet in 100mM LiOAc. A mixture of DNA (15 μl of PCR product), 72 μl of 50% PEG, 10 μl of 1 M lithium acetate, and 3 μl of denatured salmon sperm DNA (10 mg / μl) was prepared for each transformation. In a 1.5 mL tube, 15 µl of the cell suspension was added to the DNA mixture (170 µl) and the transformation suspension was vortexed for 5 short pulses. The transformation was incubated for 30 min at 30 ° C, followed by incubation for 22 min at 42 ° C. The cells were harvested by centrifugation (18,000 x g, 10 sec, 25 ° C). The cells were resuspended in 400 µL of YPD medium and allowed to recover overnight at 30 ° C and 250 rpm. The following morning, the cells were seeded in plates of YPE 1% (w / v) yeast extract, 2% (w / v) peptone, 25 mL / L of ethanol), supplemented with 0.1 g / L of hygromycin. The transformants were single colonies purified on YPE plates supplemented with 0.1 g / L of hygromycin.
Isolates from single colonies were corrected in YPE supplemented with 0.1 g / L hygromycin plates and the stains were tested for correct integration by colony PCR. The presence of the correct PCR product was confirmed using agarose gel electrophoresis. To trace the internal TMA29 coding region, primers oGV3103 and oGV3106 were used. To track the 5 'integration junction, primers oGV3069 and oGV821 were used. To trace the 3 'integration junction, primers oGV2320 and oGV3070 were used.
Yeast cells were cultured by inoculating 3 ml of YPD medium (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose) in a tube of culture of 14 mL in triplicate for each strain. Cultures were started from spots on a YPD plate with 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, 2% agar) . The cultures were incubated overnight at 30 ° C and 250 rpm. The next day, the OD600 of the cultures overnight, were measured and the volume of each culture to inoculate a 50 ml culture at an OD600 of 0.1 was calculated. The calculated volume of each culture was used to inoculate 50 ml of YPD in a 250 ml bottle with baffles and the cultures were incubated at 30 ° C and 250 rpm overnight. The cells were harvested during mid-log phase in ODs of 1.8 - 2.2. The cultures were transferred to 50 ml pre-weighed Falcon tubes and the cells were harvested by centrifugation for 5 minutes at 3000 x g. After removing the medium, the cells were washed with 10 ml of H2O MilliQ. After removing the water, the cells were again centrifuged at 3000 x g for 5 min, and the remaining water was carefully removed with a 1 ml pipette tip. Cell pellets were weighed and then stored at -80 ° C.
The cell pellets were thawed on ice and resuspended in lysis buffer (10 mM sodium phosphate, pH 7.0, 1 mM dithiothreitol, 5% w / v glycerol) so that the result was a 20% cell suspension in pasta. One ml of glass microspheres (0.5 mm in diameter) was added to a 1.5 ml Eppendorf tube of each sample and 850 µL of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each at full speed, with 1 min incubation on ice, between intervals. The tubes were centrifuged for 10 minutes at 21,500 x g at 4 ° C and the supernatant was transferred to a fresh tube. The extracts were kept on ice until tested using the TMA29 assay as described. The specific activity of Gevo1742 with the TMA29 gene for the reduction of (S) -2-acetolactate was 0.0043 ± 0.0005 gmol / min / mg of lysate. The specific activity of Gevo4459 deleted for the TMA29 gene was 0.0019 ± 0.0003 gmol / min / mg of lysate.
Example 19: Increased Yield of Isobutanol in Strains Comprising a Deletion of ALD6, a Deletion of TMA29 and an Alcohol Dehydrogenase with Increased kcat and Decreased Km in S. cerevisiae The following example illustrates that the combination of an ALD6 deletion, deletion of TMA29 and overexpression of a gene encoding an ADH with better kinetic properties leads to increased production of isobutanol and theoretical yield. The CEN.PK2 strain of S. cerevisiae, GEVO3991, was built by transforming a CEN.PK2 strain of S. cerevisiae, GEVO3956, which expresses an improved alcohol dehydrogenase (L. lactis ADH *, LIADH *) and a decarboxylase (L lactis KIVD, LI_kivD2 ') from its chromosomal DNA with a 2μ Plasmid, pGV2603 (Ptdh3: Ec_ ilvC_ coSc' 'A1 ~ his6 · Ptefi: LI_íIvD_coSc, PENO2: Ll_adhARE1, 2μ-or, p G418R), which expresses genes encoding enzymes: KARI, DHAD, and improved ADH (Ec_ilvC_coScP2'D1 ~ -A1 ~ his6, Ll ilvD coSc, and LladhA1 ^ 1, respectively).
Table 60. Genotype of Strains Disclosed in Example 19. Fermentation was performed to determine the performance of GEVO3991 (LI adhA1 ^ 1, ALD6A, TMA29N) in four replicate fermenters. Glucose consumption, isobutanol production, isobutyrate production, acetate production and OD600 were measured during fermentation. For these fermentations, colonies of single isolated cells cultured on YPD agar plates were transferred to flasks with 500 mL baffles containing 80 mL of YPD containing 80 g / L of glucose, 5 g / L of ethanol, 0.5 g / L of MgSO4, and 0.2 g / L of G418 and incubated for 30 h at 30 ° C on an orbital shaker at 250 rpm. The flask cultures were transferred to four motor driven fermentation containers on top of 2 individual L with a working volume of 0.9 L YPD containing 80 g / L glucose, 5 g / L ethanol, 0.5 g / L of MgSO4, and 0.2 g / L of G418 per container for an initial OD600 of 0.3. The fermenters were operated at 30 ° C and pH 6.0 controlled by 6N KOH in a 2-phase aerobic condition based on the oxygen transfer rate (OTR). Initially, the fermenters were operated at a growth phase OTR of 10 mM / h by fixed agitation of 700 rpm and an air overlap of 5 sL / h. Cultures were grown for 22.5 h at approximately 10-11 OD600 then immediately transferred to production aeration conditions for 40.7 h. Cell density during the production phase approached 13-14 OD600. The production phase was operated at an OTR of 0.5 mM / h by fixed agitation of 300 rpm. Periodically, samples from each fermenter were removed to measure OD600 and prepared for gas chromatography (GC) and liquid chromatography (LC) analysis. For GC and LC, 2 mL of sample was removed into an Eppendorf tube and centrifuged in the microcentrifuge for a maximum of 10 minutes. One mL of the supernatant was analyzed by GC1 (isobutanol, other metabolites) and one mL was analyzed by high performance liquid chromatography (LC1) for organic acids and glucose, as described. GEVO3991 reached a cell density of 13.8, during the growth phase of 22.5 h. The isobutanol produced during the entire duration of the experiment (63.2 h) was 18.6 ± 0.9 g / L with 0.84 ± 0.10 g / L isobutyrate and 0.15 ± 0.02 g / L of acetate produced. The theoretical yield of isobutanol achieved during the production phase of the experiment (22.5-63.5 h) was 80.3 ± 1.1%, while the yield of isobutyrate was only 0.013 ± 0.001 g / g of glucose. DH2MB production was not detected.
In addition, three GEVO3991 independent transformants were also characterized in shake flasks. The strain was grown overnight in 3 mL of YPD containing 1% ethanol and 0.2 g / L of G418 at 30 ° C at 250 rpm. These cultures were diluted to an OD600 of 0.1 in 50 ml of the same medium in a flask with 250 ml baffles and grown overnight. OD600 was measured and a volume of cells approximately equal to 250. ODooo was collected for each culture by centrifugation at 2700 rcf for 2 minutes and the cells were resuspended in 50 ml of fermentation medium (YPD containing 80 g / L of glucose, 0.03 g / L of ergosterol, 1.32 g / L of Tween80, 1% v / v ethanol, 200 mM MES, pH 6.5), and transferred to a 250mL vented screw cap bottle . ODooo was checked and cultures were placed at 30 ° C at 75 rpm to start microaerobic fermentation. The samples for analysis of liquid chromatography (LC), gas chromatography (GC) and ODOoo were taken at intervals of about 24 h. The samples (2 ml) were centrifuged at 18,000 x g for 10 min and 1.5 ml of the clarified supernatant was used for analysis by GC1 and LC1.
Fermentations started with an OD of about 4. The cells grew to an OD of about 8 for 72 h of microaerobic fermentation. After 72 h, the isobutanol titration was 12.3 g / L and the isobutanol yield was 67.2% of the theoretical value. Isobutyrate titration and yield were low: 0.1 g / L isobutyrate was produced with a 0.013 g / g glucose yield. DH2MB production was not detected.
Example 20: Deletion Effect of TMA29 in K. marxianus The purpose of this example is to demonstrate that deletion of a TMA29 in a Kluyveromyces marxianus strain comprising ALS activity results in reduced DH2MB production.
The strains, plasmids, and oligonucleotide sequences described in this example are indicated in Tables 61, O2, and 63, respectively.
Table 61. Genotype of Strains Disclosed in Example 20.
Table 62. Plasmid Disclosed in Example 20.
Table 63. Oligonucleotide sequences disclosed in Example 20.
Construction of the strain: The homologue of the K. marxianus TMA29 gene encoding the K. marxianus TMA29 protein (SEQ ID NO: 23) was deleted from the GEVO2348 strain of K. marxianus origin as follows, resulting in the GEVO6403 and GEVO6404 strains. Genomic DNA was isolated from GEVO1947 as described. The constructs were made to integrate the E. coli hph (hygromycin resistance) cassette into the GEVO2348 TMA29 locus by SOE PCR as described. PCR step # 1 consisted of three reactions that result in the 5 'TMA29 target sequence, 3' TMA29 target sequence and the hph marker. The 5 'target sequence was amplified from genomic DNA prepared with GEVO1947 Primers oGV3498 and oGV3137. The 385 bp fragment was purified by gel electrophoresis. The 3 'target sequence was amplified from genomic DNA prepared with GEVO1947 Primers oGV3140 and oGV3499. The 473 bp fragment was gel purified. The PTEFi: hph: TcYCi cassette (partial) was amplified from pGV2701 with oGV3138 and oGV3139 primers. The 1651 bp fragment was gel purified. The final SOE PCR step joined the three products from step # 1 (5 'target sequence / hph marker / 3' target sequence). The reaction mixture was amplified using oGV3498 and oGV3499 primers. The 2414 bp fragment was gel purified as described and used for the transformation of GEVO2348 as described. The medium used to grow the cells during the transformation was YPE. After transformation, 150 pL of the transformation culture was seeded on YEP plates containing 0.1 g / L of hygromycin. The plates were incubated at 30 ° C and the transformed colonies were isolated from single colonies and then stained for colony PCR on YPE plates containing 0.1 g / L of hygromycin. Yeast colony PCR was used to screen the appropriate 3 'integration junction, the 5' integration junction, as well as the lack of the TMA29 coding region as described. The proper 3 'integration junction has been confirmed using the oGV3501 and 2320 primers. The proper 5' integration junction has been confirmed using the oGV3500 and oGV0821 primers. Finally, screening for deletion of the internal coding region of TMA29, and primers oGV3500 and oGV3141 were used.
Fermentation: Agitated flask fermentations were performed in triplicate for each of the GEVO2348 (TMA29), GEVO6403 (tma29A), and GEVO6404 (tma29A) strains, as described, to determine whether deleting TMA29 in strains expressing BsalsS would result in decreased DH2MB production. Transformants isolated from a single colony of tma29A strains were stained for YPE plates containing 0.1 g / L of hygromycin, while strains of origin were stained for YPE plates. The spot cells were used to inoculate 3 ml YPE cultures. The cultures were incubated overnight at 30 ° C and 250 rpm. After overnight incubation, the OD600 of these cultures was determined by 1:40 dilution in water. The appropriate amount of culture was added to 50 ml of YPE to obtain an OD600 of 0.1, in flasks with 250 ml baffles and incubated at 30 ° C and 250 rpm. After a 24 h incubation, the OD600 of these cultures was determined by 1:40 dilution in water. The appropriate amount of culture was added to 50 mL of YPD containing 8% glucose and 200 mM MES, pH 6.5 to obtain an OD600 of 5. Fermentation cultures were incubated at 30 ° C and 75 rpm in flasks of 250 mL without baffles. A 15 mL aliquot of medium was also collected for use as a blank for LC4 analysis and was kept at 4 ° C until the sample was presented. After 72 h, 1.5 ml of the culture was removed and samples were prepared as described above for OD600 and LC4 analysis. In addition, samples for enzymatic assays were collected in 72 h, transferring ODs 80 of the appropriate sample to two 15 ml Falcon tubes centrifuged at 3000 x g for 5 min at 4 ° C. The pellets were resuspended in 3 ml of cold, sterile water and were centrifuged at 5000 x g for 2 min at 4 ° C in a rotating rotor in the bench centrifuge. The water was removed by vacuum aspiration. The conical tubes were stored at -80 ° C.
The in vitro ALS enzymatic activities of the lysates were measured as described. Table 64 shows the average in vitro ALS enzymatic activity of strain lysates after 72 h. ALS activity is measurable in GEVO2348 (average of 3.14 Units / mg of lysate), as well as in both tma29A strains GEVO6403 and GEVO6404 (averages of 1.63 and 1.58 units / mg of lysate respectively). Table 64 also shows the titers of DH2MB and DHIV by LC4 for these strains. The GEVO2348 (TMA29) strains produced mean DH2MB titers of 0.89 g / L, while DHIV was not detected. DH2MB titrations were significantly decreased in the tma29 GEVO6403 and GEVO6404 strains that measured 0.16 and 0.15 g / L respectively. Although ALS activity is decreased in tma29 strains, this does not account for the> 80% decrease in DH2MB titers in deletion strains. For example, a GEVO2348 replication technique exhibited an ALS activity of 2.5 Units / mg and the lysates produced 0.83 g / L of DH2MB, while one of the replication techniques of the GEMA6404 tma29A strain had a similar activity of 1 , 9 Units / mg of lysates and produced only 0.16 g / L of DH2MB.
Table 64. DH2MB and DHIV titrations, ALS activity, and decreased DH2MB percentage in TMA29A strains after 72h of fermentation. n.d. = not detected Example 21: Effect of TMA29 Deletion on Kluyveromyces lactis The purpose of this example is to demonstrate that deletion of TMA29 in a Kluyveromyces lactis strain comprising ALS activity results in reduced DH2MB production.
The strain, plasmid, and oligonucleotide primers shown in this example are listed in Tables 65, 66, and 67, respectively.
Table 65. Genotype of Strains Disclosed in Example 21.
Table 66. Plasmids Disclosed in Example 21.
Table 67. Oligonucleotide sequences disclosed in Example 21.
Strain Construction: The homologue of the K. lactis TMA29 gene encoding the K. lactis TMA29 protein (SEQ ID NO: 7) was deleted from the GEVO1742 strain of K. lactis origin as follows, resulting in the GEVO4458 strain as described in Example 18.
K. lactis strains GEVO1742 (origin, TMA29) and GEVO4458 (tma29N) were transformed with plasmid pGV1429 (empty control vector), pGV1645 (expressing BsalsS) or with plasmid pGV1726 linearized by AhdI (resulting from random integration of BsalsS) as described, resuspended in 400 pL of 1.25 x SC-HWLU and plated on SCD-W plates to select the transformed cells. The random integration of linearized pGV1726 by Ahdl in both GEVO1742 and ΤΜΑ29Δ GEVO4458 strains was confirmed by colony PCR with oGV1321 and oGV1324 primers that are specific to the BsalsS internal coding region, as described. The strains GEVO6316, GEVO6317, GEVO6324 and GEVO6325 were positive for gene integration.
Fermentation: Shaken flask fermentation was performed on several GEVO strains (Table 65), as described to determine whether deletion of TMA29 in strains expressing Bs alsS would result in decreased DH2MB production. Isolated single colony transformants were spotted for SCD-W plates, untransformed originals were spotted in YPD. The cells from the spots were used to inoculate 3 ml cultures in either YPD (original strains and integrated strains) or 3 ml of SCD-W. The cultures were incubated overnight at 30 ° C and 250 rpm. After overnight incubation, the OD600 of these cultures was determined by 1:40 dilution in water. The appropriate amount of culture was added to 50 mL of YPD containing 5% glucose or SCD-W containing 5% glucose to obtain an OD600 of 0.1, in flasks with 250 mL baffles and incubated at 30 ° C and 250 rpm. After 24 h of incubation, the OD600 of these cultures was determined by 1:40 dilution in water. The appropriate amount of culture was added to 50 mL of YPD containing 8% glucose, 200 mM MES at pH 6.5 or SCD-W containing 8% glucose to obtain an OD600 of 5. When ODs of 250 were not available for start fermentation, the entire 50 mL culture was used. Fermentation cultures were incubated at 30 ° C and 75 rpm in 250 ml flasks without baffles. A 15 mL conical tube was also collected for blanks from the media for the analysis of LC1 and LC4 as described and maintained at 4 ° C until sample presentation. At the 72 h time point, 1.5 ml of the culture was collected. The OD600 values were determined and the samples were prepared for analysis of LC4 and LC1 by centrifugation for 10 min at 14,000 rpm and removal of 1 mL of the supernatant to be analyzed. In addition, samples for enzymatic assays were collected at the 72 h time point. The Ods of 60 from the appropriate sample were transferred to a 15 ml Falcon tube and centrifuged at 3000 X g for 5 min at 4 ° C. The pellets were resuspended in 3 ml of cold, sterile water and transferred to 3 Eppendorf tubes of 1.5 ml (1 ml each) to make replicates of 3 x 20 OD. The tubes were centrifuged at 5000 x g for 2 min at 4 ° C in a tilting rotor in the bench centrifuge. The water was removed by vacuum aspiration. Eppendorf tubes were stored at -80 ° C.
The in vitro ALS enzymatic activities of the lysates were measured as described. Table 68 shows the average in vitro ALS enzymatic activity of strains lysates after 72 h. ALS activity is measurable only in strains with BsalsS randomly integrated (GEVO6316, GEVO6317, GEVO6324, 6325) or expressed from plasmid (GEVO6313-6315, GEVO6321-6323). ALS activity in strains with integrated Bs alsS is less than in strains expressing plasmid Bs alsS. However, the activity of 0.25 Units / mg of lysate in the TMA29 strains with Bs alsS integrated (GEVO6316, GEVO6317) was still sufficient to produce a titration of 1.06 g / L of combined DHIV + DH2MB. Table 68 shows the combined DHIV + DH2MB titrations for the various strains after 72 h of fermentation based on the LC1 analysis. The GEVO1742 strains (source, TMA29) produced measurable DHIV + DH2MB titrations combined only when Bs alsS was randomly integrated (1.06 g / L), or expressed from the plasmid pGV1645 (0.45 g / L). These DHIV + DH2MB titrations were abolished in the ΤΜΑ29Δ GEVO4458 strain when expressing Bs alsS via random integration (GEVO6324, GEVO6325) or plasmid (GEVO6321-6323). The LC4 analysis indicated that most of the combined DHIV + DH2MB was in fact DH2MB.
Table 68. ALS Activity, Combined DHIV + DH2MB Titration, and DH2MB Percentage DHIV + DH2MB Combined. n / a = not applicable, samples had no peaks detectable by LC1 therefore were not analyzed by LC4 Example 22: Effect of TMA29 Deletion on I. orientalis The following example illustrates that the deletion of the TMA29 gene from I. orientalis results in decreased activity of TMA29 and also results in decreased DH2MB production in Strains comprising ALS activity.
Table 69. Genotype of Strains Disclosed in Example 22.
Strain Construction: Issatchenkia orientalis strains derived from PTA-6658, which were constructed were wild-type for the TMA29 gene (GEVO4450, GEVO12425), heterozygous for the deletion of a copy of the TMA29 gene (GEVO6155), or completely deleted for the gene TMA29 (GEVO6158, GEVO12473, GEVO12474) using standard yeast molecular and genetic biology methods. These strains also carry a copy of the Bacillus subtilis alsS gene.
TMA29 Enzyme Assay: For the in vitro TMA29 assay, strains I. orientalis GEVO4450 (TMA29 / TMA29), GEVO6155 (tma29A / TMA29), and GEVO6158 (tma29A / tma29A complete) were grown by inoculating 25 mL in flasks with 125 ml YPD baffles with cells from a freshly prepared YPD plate. The cultures were grown overnight at 30 ° C and 250 rpm. These cultures were used to inoculate 50 mL of YPD, in flasks with 250 mL baffles to an OD600 of 0.05. Cultures were grown at 30 ° C and 250 rpm until they reached an OD600 of approximately 5-8 (final log phase). The cells were harvested by collecting OD 80 of cells in a 50 ml Falcon tube and centrifuging at 2700 x g for 3 min. After removing the supernatant, the cells were placed on ice and washed with 5 ml of cold water. The cells were centrifuged at 2700 x g for 3 minutes and the water was removed. Cell pellets were stored at -80 ° C until use. In addition, the same strains were grown by inoculating 3 ml of YPD from freshly prepared plates and growing for 8 hours at 30 ° C and 250 rpm. These cultures were used to inoculate 50 mL of YPD, in flasks with 250 mL baffles to an OD600 of 0.01 and the cultures were grown at 30 ° C and 250 rpm until they reached an OD600 of approximately 4-8. This culture was used to inoculate 50 mL of YPD containing 8% glucose, 200 mM MES, pH 6.5, at a final OD600 of 4-5 by centrifuging an appropriate amount of culture at 2700 xg for 3 minutes, in a 50 ml Falcon tube and then resuspend cell pellets in 50 ml of the indicated medium. The cells were incubated in 250 ml flasks without baffles at 30 ° C and 75 rpm for 48 h (fermentation phase). Eighty OD cell pellets were collected as described. The cells were resuspended, lysed and tested for TMA29 activity, as described. Table 70 shows the specific TMA29 activity of lysates of strains I. orientalis GEVO4450, 6155, and 6158 in U / mg of total protein. The specific activity of TMA29 is reduced in GEVO6155 (lma29l'MA29) and GEVO6158 (complete tma29 deletion) in relation to GEVO4450 (TMA29 / TMA29).
Table 70. Activity of TMA29 in Strains I. orientalis Fermentation: For fermentation, I. orientalis Strains GEVO12425 (TMA29 / TMA29), GEVO12473 (TMA29 / TMA29), and GEVO12474 (TMA29 / TMA29) were grown by inoculation of 12 mL of YPD in flasks with 125 mL baffles with cells from a freshly prepared YPD plate. The cultures were grown overnight at 30 ° C and 250 rpm. OD600 of 12 mL overnight cultures were determined and the appropriate amount was used to inoculate 50 mL of YPD containing 5% glucose in flasks with 250 mL baffles to an OD600 of 0.1. The flasks were incubated at 30 ° C and 250 rpm overnight. The OD600 of 50 ml of cultures was determined. The appropriate amount of culture was centrifuged at 2700 rcf for 5 min at 25 ° C in 50 ml falcon tubes and the supernatant was removed. The cells of each 50 ml culture were resuspended in 50 ml of YPD containing 8% glucose, 200 mM MES, pH 6.5. The cultures were then transferred to 250 ml flasks with 250 ml baffles and incubated at 30 rpm and 75 ° C. In 72 h of samples from each flask were removed, OD600 was measured and samples prepared for LC4 analysis by transferring 1 ml of sample to an Eppendorf tube and centrifugation at 18,000 x g, 10 seconds, 25 ° C. After centrifugation, 0.75 mL of the supernatant was transferred to a microtiter plate and analyzed by LC4. Also in 72 h the enzymatic assay cells were harvested by transferring ODs 80 to 15 ml Falcon tubes as described. The cells for ALS assays were resuspended, lysed, and assayed as described. Table 71 shows the production of DH2MB and ALS activities for GEVO12425, 12473, 12474 and in 72 hours. DH2MB titration was determined by LC4. ALS activity was similar in all strains.
Table 71. DH2MB Production and ALS Activity in Strains I. orientalis in 72h of Fermentation.
Example 23: Effect of TMA29 deletion on S.. pombe The following example illustrates that (S) -2-acetolactate reduction activity is significantly decreased in a S. pombe tma29A strain compared to a S. pombe TMA29 strain.
Table 72. Genotype of Strains Disclosed in Example 23.
The yeast strains GEVO6444 that have an intact TMA29 gene (SEQ ID NO: 161) and GEVO6445 that have the deleted TMA29 gene, were grown overnight in 12 mL of YPD in bottles with 125 mL baffles at 250 rpm and 30 ° C. The following day, OD600 values were measured and cultures in triplicate techniques were started in 50 mL of YPD with 5% glucose at an OD600 of approximately 0.3. The cultures were allowed to grow at 250 rpm and 30 ° C throughout the day. At the end of the day, the cultures were diluted in YPD with 5% glucose to an OD600 of approximately 0.15 and incubated overnight at 250 rpm and 30 ° C. The cells were harvested after reaching an OD600 of between 4 and 6. To harvest pellets by enzymatic assays ODs 80 of the appropriate sample were transferred to two 15 ml Falcon tubes (for duplicate samples) and centrifuged at 3000 xg for 5 min at 4 ° C. The pellets were resuspended in 3 ml of cold, sterile water and were centrifuged at 5000 x g for 2 min at 4 ° C in a rotating rotor in the bench centrifuge. The water was removed by vacuum aspiration. The pellets were stored at -80 ° C. Lysates were prepared and TMA29 enzymatic assays were performed as described. The specific activity of GEVO6444 lysates from S. pombe for the reduction of (S) -2-acetolactate was 0.018 ± 0.002 U / mg of the total protein. The lysates of the tma29A GEVO6445 strain had a specific activity of 0.001 ± 0.002 U / mg of total protein.
Example 24: Effect of ALD6 Deletion on K. marxianus The purpose of the present example is to demonstrate that deletion of ALD6 in a Kluyveromyces marxianus strain results in reduced isobutyraldehyde oxidation activity and isobutyrate production.
The strain, plasmid, and oligonucleotide primers disclosed in this example are listed in Tables 73, 74, and 75, respectively.
Table 73. Genotype of K. marxianus Strains Disclosed in Example 24.
Table 74. Plasmids Disclosed in Example 24.
Table 75. Oligonucleotide sequences disclosed in Example 24.
Construction of the strain: The homologue of the K. marxianus ALD6 gene encoding the K. marxianus ALD6 protein (SEQ ID NO: 39) was deleted from the GEVO1947 and GEVO2087 strains of K. marxianus origin as follows, resulting in the strains GEVO6264 / GEVO6265 and GEVO6270 / GEVO6271, respectively. Genomic DNA was isolated from GEVO1947 as described. The constructs were made to integrate the E. coli hph (hygromycin resistance) cassette into the GEVO1947 and GEVO2087 ALD6 locus by SOE PCR as described. PCR step # 1 consisted of three reactions: the 5 'ALD6 target sequence, the 3' ALD6 target sequence, and the hph marker. The 5 'target sequence was amplified from genomic DNA prepared with GEVO1947 primers oGV3490 and oGV3492. The 635 bp fragment was purified by gel electrophoresis. The 3 'target sequence was amplified from GEVO1947 genomic DNA prepared with primers oGV3493 and oGV3495. The 645 bp fragment was gel purified. The PTEF1: hph: TcYCi (partial) cassette was amplified from pGV2701 with the primers of oGV3491 and oGV3494. The 1,665 bp fragment was gel purified. The final SOE PCR step joined the three products from step # 1 (5 'ALD6 target sequence / hph / marker / 3' ALD6 target sequence). The reaction was amplified using oGV3490 and oGV3495 primers. The 2826 bp fragment was gel purified and used for the transformations of GEVO1947 and GEVO2087 as described. Medium used to grow the cells for transformation was YPD. After transformation, 150 pL of each transformation culture was seeded on plates of YPD supplemented with 0.2 g / L of hygromycin. The plates were incubated at 30 ° C. The transformed colonies were stained for initial colony PCR screening, then the single colony was isolated and stained again in YPD plates supplemented with 0.2 g / L of hygromycin. Yeast Colony PCR was used to screen for the appropriate 3 'integration junction, the 5' integration junction, as well as the lack of the ALD6 coding region as described. The proper 3 'integration junction was confirmed using the primers oGV3497 and oGV2320. The proper 5 'integration junction was confirmed using the primers oGV3496 and oGV0821. Finally, the deletion of the ALD6 internal coding region was confirmed using the primers oGV3495 and oGV0706.
Fermentation: A flask shake with 2 g / L of isobutyraldehyde was carried out as described using techniques of aldóA strains GEVO6264 / GEVO6265 and GEVO6270 / GEVO6271 and their corresponding ALD6 strains GEVO1947 and GEVO2087.
Transformants isolated from single colony of aldóA strains were stained for YPD plates supplemented with 0.2 g / L hygromycin plates and those of origin were stained for YPD plates. The cells from the spots were used to inoculate 3 ml cultures of YPD triplicate techniques. The cultures were incubated overnight at 30 ° C and 250 rpm. After overnight incubation, the OD600 of these cultures was determined by 1:40 dilution in water. The appropriate amount of culture was added to 50 mL of YPD with 5% glucose to obtain an OD600 of 0.1 in flasks with 250 mL baffles and the cultures were incubated at 30 ° C and 250 rpm. After 24 h of incubation, the OD600 of these cultures was determined by 1:40 dilution in water. The appropriate amount of culture was added to 50 mL of YPD containing 8% glucose, 200 mM MES at pH 6.5, and 2 g / L of isobutyraldehyde to obtain an OD600 of 5. Fermentation cultures were incubated at 30 ° C and 75 rpm in 250 mL bottles without baffles. The unused medium was collected as a blank for LC analysis and kept at 4 ° C until sample presentation. In 48 h, samples from each flask were run as follows. 1.5 ml of the culture was removed into 1.5 ml Eppendorf tubes. OD600 values were determined and samples were prepared for LC1 analysis. Each tube was centrifuged for 10 min at 14,000 rpm and the supernatant was analyzed by LC1. In addition, samples for enzymatic assays were collected after 48 h. ODs 80 of the appropriate sample were transferred to two 15 ml Falcon tubes (for duplicate samples) and centrifuged at 3000 X g for 5 min at 4 ° C. The pellets were resuspended in 3 mL of cold, sterile water and were centrifuged at 5000 x g for 2 min at 4 ° C in a tilting rotor. The water was removed by vacuum aspiration. The conical tubes were stored at -80 ° C. Table 76 shows the isobutyrate titration after 48 h of fermentation. The strain of origin ALD6 GEVO1947 produced average total and specific isobutyrate titrations of 0.19 g / L and 0.013 g / L / OD, respectively. These total and specific isobutyrate titrations were significantly decreased in the αϊά6Δ GEVO6264 strain (0.06 g / L and 0.004 g / L / OD, respectively), and also in the αϊά6Δ GEVO6265 strain (0.05 g / L and 0.003 g / L / OD respectively). The strain of origin ALD6 GEVO2087 produced total and specific titers of isobutyrate of 0.15 g / L and 0.008 g / L / OD, respectively. Total and specific isobutyrate titrations were significantly decreased in the GEVO6270 aldóA strain (0.05 g / L and 0.003 g / L / OD), and also in the GEVO6271 aldóA strain (0.08 g / L and 0.005 g / L / OD) , respectively).
Table 76. Production of Isobutyrate from Strains of ALD6 origin and Strains aldóA Derived from said Strains of Origin ALD6 Example 25: Effect of Deletion of ALD6 on K. lactis The purpose of this example is to demonstrate that the deletion of ALD6 in a strain of Kluyveromyces lactis results in decreased isobutyraldehyde oxidation activity and isobutyrate production.
The strain, plasmid, and oligonucleotide primers disclosed in this example are listed in Tables 77, 78, and 79, respectively.
Table 77. K. lactis Strains Genotype Disclosed in Example 25.
Table 78. Plasmid Disclosed in Example 25.
Table 79. Oligonucleotide sequences disclosed in Example 25.
Construction of the strain: The homologue of the K. lactis ALD6 gene encoding the K. lactis ALD6 protein (SEQ ID NO: 29) was deleted from the K. lactis strains of GEVO1287 and GEVO1830 as follows, resulting in strains GEVO6242 and GEVO6244 / GEVO6245, respectively. Genomic DNA was isolated from GEVO1287 as described. The constructs were made to integrate the E. coli hph (hygromycin resistance) cassette into the GEVO1287 and GEVO1830 ALD6 locus by SOE PCR as described. Step # 1 PCR consisted of three reactions: the 5 'ALD6 target sequence, the 3' ALD6 target sequence, and the hph marker. The 5 'target sequence was amplified from GEVO1287 genomic DNA prepared with oGV3502 and oGV3504 primers. The 639 bp fragment was purified by gel electrophoresis. The 3 'target sequence was amplified from GEVO1287 genomic DNA prepared with oGV3505 and oGV3507 primers. The 628 bp fragment was gel purified. The PTEFi: hph: TcYCi (partial) cassette was amplified from pGV2701 with the oGV3503 and oGV3506 primers. The 1663 bp fragment was gel purified. The final SOE PCR step joined the three products from step # 1 (5 'target sequence / hph marker / 3' target sequence). The reaction was amplified using oGV3502 and oGV3507 primers. The 2,810 bp fragment was gel purified and used for the transformations of GEVO1287 and GEVO1830 as described. Colonies were selected for resistance to hygromycin on YPD plates supplemented with 0.1 g / L of hygromycin. Yeast colony PCR was used to screen for the appropriate 3 'integration junction, the 5' integration junction, as well as the lack of the ALD6 coding region as described. The proper 3 'integration junction was confirmed using the primers oGV3509 and oGV2320. The proper 5 'integration junction was confirmed using the primers oGV3508 and oGV0821. Finally, the deletion of the ALD6 internal coding region was confirmed using the primers oGV3508 and oGV3510.
Fermentation: The first fermentation in a flask with 2 g / L of isobutyraldehyde in the medium was carried out using triplicate techniques of the GEVO6242 aldóA strain and wild type strain ALD6 GEVO1287. Transformants isolated from single colony of confirmed aldóA deletion strains were stained for YPD plates supplemented with 0.1 g / L hygromycin plates, the original strains were stained in YPD. The cells from the spots were used to inoculate 3 ml cultures of YPD triplicate techniques. The cultures were incubated overnight at 30 ° C and 250 rpm. After overnight incubation, the OD600 of these cultures was determined by 1:40 dilution in water. The appropriate amount of culture was added to 50 mL of YPD with 5% glucose to obtain an OD600 of 0.1 in flasks with 250 mL baffles and the cultures were incubated at 30 ° C and 250 rpm. After 24 h of incubation, the OD600 of these cultures was determined by 1:40 dilution in water. The appropriate amount of culture was added to 50 mL of YPD containing 8% glucose, 200 mM MES at pH 6.5, and 2 g / L of isobutyraldehyde to obtain an OD600 of 5. Fermentation cultures were incubated at 30 ° C and 75 rpm in 250 mL bottles without baffles. The unused medium was collected as a blank of the medium for LC1 analysis and kept at 4 ° C until sample presentation. After 24 h, samples from each flask were run as follows. 1.5 ml of the culture was removed into 1.5 ml Eppendorf tubes. OD600 values were determined and samples were prepared for LC1 analysis as described. Each tube was centrifuged for 10 min at 14,000 rpm and the supernatant was collected for LC1 analysis as described.
A second fermentation in a flask with 2 g / L of isobutyraldehyde was carried out as described using the aldóA deletion strains GEVO6244 / GEVO6245 and its corresponding strain of origin ALD6 GEVO1830. This fermentation was sampled at 24 and 48 h, as described. Table 80 shows the isobutyrate titration of both fermentations. Isobutyrate titrations are significantly decreased in aldóA strains compared to strains of ALD6 origin.
Table 80. Production of Isobutyrate from Strains of Origin ALD6 and Strains aldóA Derived from said Strains of Origin ALD6 ._________________________________________ n.d. = not determined in this experiment * based on LOQ for 0.025 g / L isobutyrate
Example 26: TMA29 activity for 2-aceto-2-hydroxybutyrate The following example illustrates that the S. cerevisiae TMA29 protein is active for (S) -2-acetolactate ((S) -AL) and 2-aceto-2-hydroxybutyrate (AHB).
Table 81. Genotype of Strains Disclosed in Example 26.
The GEVO3939 yeast strains from which the TMA29 gene (YMR226C) was deleted and its strain of GEVO3527 origin were grown in triplicate by inoculating 3 ml of YPD in a 14 ml culture tube in triplicate for each strain. Cultures were initiated from spots on the YPD agar plate for GEVO3527 and on YPD plates containing 0.2 g / L of G418 for GEVO3939. The cultures were incubated overnight at 30 ° C and 250 rpm. The next day, the OD600 of the cultures overnight was measured and the volume of each culture to inoculate a 50 ml culture at an OD600 of 0.1 was calculated. The calculated volume of each culture was used to inoculate 50 ml of YPD in a 250 ml bottle with baffles and the cultures were incubated at 30 ° C and 250 rpm.
The cells were harvested during the mid-log phase of 2.2-2.7 ODs after 8 h of growth. The cultures were transferred to 50 ml pre-weighed Falcon tubes and the cells were harvested by centrifugation for 5 minutes at 3000 x g. After removing the medium, the cells were washed with 10 ml of H2O MilliQ. After removing the water, the cells were again centrifuged at 3000 x g for 5 minutes and the remaining water was carefully removed using a 1 ml pipette tip. Cell pellets were weighed and then stored at -80 ° C until use.
The cell pellets were thawed on ice and resuspended in lysis buffer (10 mM sodium phosphate, pH 7.0, 1 mM dithiothreitol, 5% w / v glycerol) so that the result was a 20% cell suspension in pasta. One ml of glass microspheres (0.5 mm in diameter) was added to a 1.5 ml Eppendorf tube for each sample and 850 µl of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixing mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each at full speed, with 1 min incubation on ice, between intervals. The tubes were centrifuged for 10 minutes at 21,500 xg at 4 ° C and the supernatant was transferred to a fresh tube. The extracts were kept on ice until tested using the TMA29 assay as described to determine the TMA29 activity for (RS) -AHB and (R / S) -AL The specific TMA29 activity of S. cerevisiae in GEVO3527 lysates, a wild type S. cerevisiae MATa S strain for the reduction of (R / S) -AHB was 10.5 ± 0.6 mU / mg. The tma29A GEVO3939 strain had a specific activity of 4.8 ± 0.1 mU / mg. The GEVO3527 wild type strain had a specific TMA29 activity about 2 times greater than the deletion strain. The specific activity of TMA29 S. cerevisiae in lysates of GEVO3527, a S. cerevisiae MATa wild type strain, for the reduction of (R / S) -AL was 12.3 ± 0.2 mU / mg. The tma29A GEVO3939 strain had a specific activity of 2.9 ± 0.3 mU / mg. The GEVO3527 wild type strain had a specific TMA29 activity about 4 times higher than the deletion strain. General Methods for Examples 27-30 The strains, plasmids, gene / amino acid sequences, and Primer sequences described in Examples 27-30 are listed in Tables 82, 83, 84, and 85, respectively.
Table 82. Genotype of Strains Disclosed in Examples 27-30.
Table 83. Plasmids Disclosed in Examples 27-30.
Table 84. Nucleic Acid and Protein Sequences Disclosed in Examples 27-30.
Table 85. Starters (shown from 5 'to 3') Disclosed in Examples 27-30. * A (Adenine), G (Guanine), C (Cytosine), T (Thymine), U (Uracil), R (Purine - A or G), Y (Pyrimidine - C or T), N (any nucleotide), W (Weak - A or T), S (Strong - G or C), M (Amino - A or C), K (Keto - G or T), B (Without A - G or C or T), H ( Without G - A or C or T), D (Without C - A or G or T), and V (Without T - A or G or C) Medium and Buffers: SC-URA: 6.7 g / L of Base Difco ™ Yeast Nitrogen, 14 g / L Sigma ™ Synthetic Dropout Medium Supplement (includes amino acids and nutrients excluding histidine, tryptophan, and leucine), 10 g / L casamino acids, 20 g / L glucose, 0.018 g / L adenine hemisulfate, and 0.076 g / L tryptophan. SD-URA: Commercially available from MP Biomedicals (Irvina, CA). Composition: 1.7 g / L of Yeast Nitrogen Base (YNB), 5 g / L of ammonium sulfate, 20 g / L of glucose, with casamino acids without uracil CSM-URA.
YPD medium (yeast peptone dextrose) medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose.
Tris-DTT: 0.39 g 1,4-dithiothreitol per 1 ml of 1 M TrisHCl, pH 8.0, sterile filter.
Buffer A: 20 mM Tris, 20 mM imidazole, 100 mM NaCl, 10 mM MgCE, adjusted to pH 7.4, sterile filter.
Buffer B: 20 mM Tris, 300 mM imidazole, 100 mM NaCl, 10 mM MgCE, adjusted to pH 7.4, sterile filter.
Buffer E: 1.2 g of Tris base, 92.4 g of glucose, and 0.2 g of MgCE per 1 L of deionized water, adjusted to pH 7.5, sterile filter.
Construction of pET1947: The L. lactis AdhA gene (Ll_adhA) was cloned from pGV1947 using His_Not1_1947_fwd and Sal1_rev primers and ligated into pET22b (+), yielding Plasmid pET1947.
Construction of pGV2476: Plasmid pGV2274 served as a model for PCR using adhAcoSc_SalIin_for direct primer and adhAcoSC_NotIin_his_rev reverse primer. The PCR product was purified, digested by restriction with Notl and Sal, and ligated into pGV1662, which was cut with Notl and Sal and purified.
Transformation of S. cerevisiae: The night before a planned transformation, a YPD culture was inoculated with a single colony of S. cerevisiae CEN.PK2 and incubated at 30 ° C and 250 rpm overnight. The following morning, a 20 ml YPD culture was started in a 250 ml Erlenmeyer flask without baffles with the culture overnight at an OD600 of 0.1. This culture was incubated at 30 ° C and 250 rpm until reaching an OD600 of 1.3-1.5. When the culture reached the desired OD600, 200 µL of Tris-DTT buffer was added and the culture was allowed to incubate at 30 ° C and 250 rpm for an additional 15 min. The cells were then pelleted at 4 ° C and 2500 x g for 3 min. After removing the supernatant, the pellet was resuspended in 10 ml of ice-cooled buffer E and centrifuged again as described above. Then, the cell pellet was resuspended in 1 ml of ice-cooled buffer E and centrifuged once again as before. After removing the supernatant with a pipette, 200 μl of ice-cold buffer E was added, and the pellet was resuspended gently. The 6 µl insert / structure mixture was split in half and added to 50 µl of the cell suspension. The DNA / cell mixtures were transferred to 0.2 cm electroporation cuvettes (BIORAD) and electroporated without a pulse controller at 0.54 kV and 25 μF. Immediately, 1 ml of preheated YPD was added, and the transformed cells were allowed to regenerate at 30 ° C and 250 rpm in 15 ml round-bottom culture tubes (Falcon). After 1 hour, the cells were centrifuged at 4 ° C and 2,500 x g for 3 minutes, and the pellets were resuspended in 1 ml of pre-heated SD-URA medium. Different amounts of transformed cells were seeded in SD-URA plates and incubated at 30 ° C for 1.5 days, or until the colonies were large enough to be chosen with sterile toothpicks.
Mini Yeast Cell Plasmid Preparation: The Zvmoprep ™ II - The Mini Yeast Plasmid Kit (Zymo Research, Orange, CA) was used to prepare plasmid DNA from 5. cerevisiae cells according to the protocol manufacturer for liquid cultures, which was slightly changed. An aliquot of 200 μL of yeast cells was centrifuged at 600xg for 2 minutes. After decanting the supernatant, 200 μL of Solution 1 was added to resuspend the pellets. For the samples, 3 μL of Zvmoivase ™ was added and the cell / enzyme suspensions were gently mixed by touching with a finger. After incubating the samples for 1 hour at 37 ° C, Solutions 2 and 3 were added and mixed well after each addition. The samples were then centrifuged at maximum speed and 4 ° C for 10 min. Then cleaning on Zymo columns was performed according to the manufacturer's instructions. Plasmid DNA was eluted with 10 ml of PCR grade water. Half of this volume was used to transform E. coli DH5a.
Expression of heterologous ADH in E. coli: Erlenmeyer flasks (500 mL) containing 50 mL of Luria-Bertani (LB) medium (10 g of tryptone, 10 g of NaCl, 5 g of yeast extract per liter) with ampicillin (final concentration of 0.1 mg / ml) were inoculated with an initial OD600 of 0.1 using 0.5 ml of the LBamp culture overnight from a single colony carrying pET1947 plasmid. The 50 ml LB expression culture was allowed to grow for 3-4 hours at 250 rpm and 37 ° C. Protein expression was induced at OD600 of about 1 with the addition of IPTG to a final concentration of 0.5 mM. Protein expression was allowed to continue for 24 hours at 225 rpm and 25 ° C. The cells were harvested at 5300xg and 4 ° C for 10 min and then the cell pellets were frozen at -20 ° C until use.
Heterologous expression in CEN.PK2 of 5. cerevisiae: Flasks (1000 mL Erlenmeyer) filled with 100 mL of SC-URA, were inoculated with 1 mL of culture overnight (5 mL of SC-URA inoculated with a single colony of CEN.PK2, grown at 30 ° C and 250 rpm). Expression cultures were grown at 30 ° C and 250 rpm for 24 hours. The cells were pelleted at 5300xg for 5 min. The supernatant was discarded and the pellets were spun again. The residual supernatant was then removed with a pipette. The pellets were frozen at -20 ° C until use.
Heterologous Expression in CEN.PK2 in 96-Well Plates for High Productivity Assays: Flat 96-well plates, 1 mL capacity per well, filled with 300 μL of SC-URA were inoculated with a single CEN.PK2 colony that carries the plasmids encoding Ll_adhAhis6 or their variants. 96-well deep plates, 2 mL capacity per well, filled with 600 μL of SC-URA per well were inoculated with 50 μL of these cultures overnight. The plates were cultured at 30 ° C and 250 rpm for 24 h and were then harvested at 5300xg for 5 min at 4 ° C and stored at -20 ° C.
Preparation of Extracts Containing E. coli ADH: E. coli cell pellets containing expressed ADH were thawed and resuspended (0.25 g wet weight / ml buffer) in buffer A. The resuspended cells were lysed by sonication, for 1 minute with a 50% duty cycle and pelletized at 11000xg and 4 ° C for 10 min. The extracts were stored at 4 ° C.
Preparation of Extracts Containing CEN.PK2 ADH of 5. cerevisiae: The cell pellets of CEN.PK2 of 5. cerevisiae containing expressed ADH were thawed and weighed to obtain the wet weight of the pellets. The cells were then resuspended in buffer A in such a way that the result was a 20% mass cell suspension. Glass microspheres 0.5 mm in diameter were added up to the 1000 μL (0.5 mm diameter) mark of a 1.5 ml Eppendorf tube, before 875 μL of cell suspension were added. The yeast cells were lysed by tapping the microspheres using a Retsch MM301 mixing mill (Retsch Inc. Newtown, PA), mixing 6 x 1 min each, at full speed, with cooling steps of 1 min between intervals. The tubes were centrifuged for 10 min at 23500 xg and 4 ° C, and the supernatant was removed. The extracts were stored at 4 ° C.
ADH purification: ADH was purified by IMAC (immobilized metal affinity chromatography) on a 1 ml High Histrap high efficiency column (HP histrap) from Nickel preloaded (GE Healthcare) using an Akta FPLC ( GE Healthcare). The column was equilibrated with four column volumes (CV) of buffer A. After injection of the crude extracts on the column, the column was washed with buffer A for 2 hp, followed by a linear gradient from B to 100% buffer. elution by 15 hp and collected in 96-well plates. The fractions containing the protein were collected and stored at 4 ° C.
ADH Cell Assay: ADH activity was assayed kinetically by monitoring the decrease in NADH concentration by measuring absorbance at 340 nm. A reaction buffer was prepared containing 100 mM Tris / HCl, pH 7.0, 1 mM DTT, 11 mM isobutyraldehyde, and 200 μM NADH. The reaction was initiated by adding 100 μL of crude extract or purified protein in an appropriate dilution to 900 μL of reaction buffer.
ADH microtiter plate activity assay: The measurement of microtiter plate activity is an under-scaled cuvette assay. The total volume was 100 μL. Ten μL of crude lysates or purified enzymes, properly diluted, were placed on assay plates. The reaction buffer was prepared as described above (isobutyraldehyde substrate only) and 90 μL of it was added to the enzyme solutions on the plates. The consumption of NADH was recorded at 340 nm in an infinite plate reader M200 (TECAN Trading AG, Switzerland).
High Produced ADH Activity Assay: Yeast cell pellets frozen in 96-well plates were thawed at room temperature for 20 min and then 100 μL of Y-Per (Pierce, Cat # 78990) was added. The plates were briefly centrifuged to resuspend cell pellets. After an incubation period of 60 min at room temperature and 130 rpm, 300 μL of 100 mM Tris-HCl (pH 7.0) was added to the plates to dilute the crude extract. After a centrifugation step at 5,300 xg and 4 ° C for 10 min, 40 μL of the resulting crude extract were transferred to assay plates (flat bottom, Rainin), using a liquid handling robot. The assay plates were briefly centrifuged at 4000 rpm and room temperature. Twelve mL of assay buffer per plate was prepared (100 mM Tris-HCl, pH 7.0, 1 mM isobutyraldehyde, 0.5 mM, 0.25 mM or 0.125 mM, 1 mM DTT, 200 μΜ NADH) and 100 μL of it were added to each well to start the reaction. NADH depletion was monitored at 340 nm in an infinite M200 plate reader (TECAN Trading AG, Switzerland) for 2 min.
Determination of Specific Activity Based on Data Obtained from Activity Assays: Protein concentrations of samples containing heterologically expressed L. lactis Adha, as crude extract and purified proteins, were measured using the Bradford Quick Start ™ Kit (Bio- Rad, Hercules, CA) following the manufacturer's instructions. A unit of enzyme activity (1 U) is defined as the amount of enzyme that catalyzes the conversion of one substrate micromol per minute under the conditions specified in the test method.
Thermostability Measures: The T50 values (temperature, at which 50% of the enzyme activity is maintained after an incubation time of 15 min) of the original Ll_adhA and its variants were measured to obtain the thermostability data. Aliquots of thirty μL of purified enzymes were transferred to PCR tubes. Each tube was assigned a specific incubation temperature, which corresponds to a crack in the block of a Mestrecycler®ep PCR machine (Eppendorf, Hamburg, Germany), programmed with a gradient that covers a temperature range of 20 ° C . The tubes were incubated for 15 min in respective slits. Then, the reaction was quenched with ice. Residual activity was determined with the ADH microtiter plate activity assay as described above.
Use of His-Tag for Purification: In each of the examples described below, reference is made to an ADH enzyme comprising a his-tag. As is understood in the art, such his-tags facilitate protein purification. As would be understood by one skilled in the art with the aid of the present description, ADH enzymes that do not have said his-tag are equally or more suitable for the conversion of isobutyraldehyde to isobutanol. Examples of modified ADH enzymes described here that do not have purifiable his-tags are found in SEQ ID NOs: 206-224 Example 27: Directed Evolution Through Random Mutagenesis The following example illustrates a method for improving the kinetic properties of an ADH and also describes the kinetic properties of such improved ADH enzymes. Plasmid pGV2476, a derivative of plasmid pGV1662, which carries the gene Ll_adhA_coSchls6 served as a model for error-prone PCR using direct primer pGV1994ep_for and reverse primer pGV1994_rev. These primers are specific for the pGV1662 structure and 50 bp binding upstream and downstream of the ADH insert to create an overlap for homologous recombination in yeast. The compositions of the three error prone PCR reactions are summarized in Table 86. The temperature profile was as follows: 95 ° C 3 min initial denaturation, 95 ° C 30s denaturation, 55 ° C 30s annealing, 72 ° C 2 min elongation, 25 cycles, 5 min final elongation at 72 ° C.
Table 86. PCR Conditions for Error-Prone Libraries PCR products were checked on a 1% analytical TAE agarose gel, Dpnl digested for 1 h at 37 ° C to eliminate traces of model DNA, and then cleaned with a 1% of preparative TAE agarose gel. The agarose pieces containing the PCR products were cleaned using frozen Squeeze 'n' tubes (BioRad, Hercules, CA, catalog # 732-6166), followed by a pellet painting procedure (Novagen, catalog # 69049-3) according to with manufacturers' protocols. In the meantime, plasmid pGV1662 was digested by restriction with Notl and Sall before running out of the digestion mixture in an agarose gel and pellet paint. The plasmid and insert, 500 ng each, were mixed together, precipitated with pellet paint, resuspended in 6 µl of PCR grade water and used to transform electrocompetent 5. cerevisiae cells as described in the General Methods.
A total of 88 clones from each of the libraries of 100, 200 and 300 μΜ of MnCU were doped in 96-well plates along with the four clones containing plasmids of origin pGV2476 and three clones containing pGV1102 as a control without ADH. A well was left empty and served as a sterility control. After screening these libraries, as described in General Methods (heterologous expression in CEN.PK2 in 96-well plates for high productivity assays, high productivity ADH activity assay), the 300 μΜ library was chosen and an additional 4,000 clones were selected. tracked in the same way. A total of 24 variants improved more than 1.5 times compared to the wild type and were chosen for a new screening in triplicate. The ten best variants were grown and expressed in 100 mL cultures, as described in the General Methods (heterologous expression in CEN.PK2 of 5. cerevisiae), and their specific activities in crude yeast extracts were determined as described in the General Methods ( ADH microtiter plate assay). Two variants, Ll_AdhA28E7-his6 and Ll_AdhA30C11-his6 exhibited a more than 2-fold improvement in activity (0.3 and 0.25 U / mg of total lysate protein, respectively) compared to the wild-type enzyme Ll_AdhAhis6 (0 , 1 U / mg of total lysate protein) and were characterized in greater detail. Plasmid DNA from these two variants was extracted as described in the General Methods (mini-yeast cell plasmid preparation), and subjected to DNA sequencing (Laragen, Los Angeles, CA), which revealed two mutations per variant, as listed in Table 87. Two of these mutations (Y50F and L264V) are located in close proximity to the active site, which is an interval between the substrate binding domain (cyan) and the cofactor binding domain (green). The I212T and N219Y mutations are located on the surface of the binding cofactor domain (as shown in Figure 17). In order to highlight the location of the mutation cofactor at the binding site, Figure 17 involves two points of view on structure alignment.
Table 87. List of Mutations Found in Two Improved Variants of the First Error-Prone Library (Generation 1).
The two variants of enzymes, Ll_AdhA28E7-his6 and Ll_AdhA30C11-his6, were expressed from plasmids pGV2475 and pGV30C11, respectively, on a larger scale (100 mL of each culture), purified and characterized in greater detail, as described in the General Methods ( heterologous expression in CEN.PK2S.de cerevisiae, Preparation of extracts containing ADH from CEN.PK2 of 5. cerevisiae, Purification of ADH). The Ll_AdhAhls6 wild-type enzyme was expressed from plasmid pGV2476 and purified in the same way. The enzymes were characterized by the kinetic properties as described in the General Methods (ADH cuvette assay). Table 88 shows the kinetic parameters measured with isobutyraldehyde and NADH. The decrease in the KM value was observed for both variants, while the kcat was only improved for Ll_AdhA28E7.
Table 88. Kinetic parameters of two variants (Generation 1) on Isobutyraldehyde compared to the source enzyme. The thermostability of the wild type enzyme and the two variants was determined as described in the General Methods (measurements of thermostability). The mutations found had a positive impact on the stability of the variants in addition to the beneficial effects on their catalytic efficiency. Table 89 summarizes the T50s measured for the source enzyme and variants.
Table 89. Summary of Enzyme T50s of Origin and Variants Example 28: Directed Evolution by Recombination The following example illustrates a method for improving the kinetic properties of an ADH and also illustrates the kinetic properties of such improved ADH enzymes.
A second gene library (generation 2) was built to recombine the beneficial mutations found in the first error-prone library and the wild-type residue at each of these sites (Table 90).
Table 90. Amino Acid Mutations Included in the Recombinate Library.
Four PCR fragments were generated using the primers RecombADHY50_rev and pGV1994ep_for (fragment 1), RecombADHY50_for and RecombADHI212_Y219_rev (fragment 2), RecombADHI212_Y219_for and RecombADHL264_rev (fragment 3), and RecombADHL264_rev (fragment 3), and RecombADHL264_rev (fragment 3) and e19 The fragments were analyzed in a 1% analytical preparative TAE gel, digested in Dpnl, separated over a 1% TAE preparative agarose gel, treated Freeze'n'Squeeze (BIORAD), and finally painted pellet (Novagen). The cleaned fragments served as a model for the assembly PCR. After successful assembly PCR, the PCR products were treated as described in Example 27, mixed with pGV1662 structure, as described in Example 27, and the mixture was used to transform 5. cerevisiae, as described in the General Methods for Examples 27-30. Eighty individual clones from the recombination library were harvested and compared from a high-throughput screening for the wild type and the two variants found in the error-prone library.
A total of 80 individual clones were harvested from a 96-well plate together with the original parent and the two improved variants. After screening the recombination plate, as described in the General Methods (heterologous expression in CEN.PK2 in 96-well plates for high productivity assays, high productivity ADH activity assay), twelve variants, all exhibiting activity at least twice higher compared to any of the Ll_AdhA28E7-his6 or Ll_AdhA30C11-his6 origin were grown and expressed in 100 mL cultures, as described in the General Methods (heterologous expression in CEN.PK2 of 5. cerevisiae), and their activities in Crude yeast extracts were determined as described in the General Methods (ADH microtiter plate assay). Two variants had very similar specific activity in the crude extract. Ll_AdhARE1 was chosen for further modifications, as its activity was 40% better than Ll_AdhA28E7-his6 and 64% better than Ll_AdhA30C11-his6. Plasmid DNA from this variant was extracted as described in the General Methods (mini-yeast cell plasmid preparation), and subjected to DNA sequencing (Laragen, Los Angeles, CA) which revealed that the Y50F, I212T, and L264V (found in Ll_AdhARE1) contributed to the observed improvements, while the mutation at position 219 was deleterious for the activity of the variants and was not found in any of the improved variants of the recombination library. The Ll_AdhARE1-his6 variant was expressed from plasmid pGV2477, on a larger scale (100 ml of each culture), purified, and characterized in greater detail as described in the General Methods (heterologous expression in CEN.PK2 of 5. cerevisiae, Preparation of extracts containing CEN.PK2 ADH from 5. cerevisiae, Purification of ADH). The wild-type Ll_AdhAhis6 enzyme was expressed from plasmid pGV2476 and purified in the same way. The enzymes were characterized by the kinetic properties as described in the General Methods (ADH cuvette assay). Table 91 shows the kinetic parameters measured with isobutyraldehyde and NADH.
Compared with Ll_AdhAhls6, Ll_AdhA28E7-his6, Ll_AdhA30C11-his6 a decrease in Km and an increase in kcat were observed for Ll_AdhARE1-his6.
Table 91. Biochemical Properties of Ll_AdhARE1 as Measured in Isobutyraldehyde.
Variant Ll_adhARE1-his6, exhibited a T50 value of 61.6 ± 0.1 ° C which is 5 degrees higher than T50 in weight and about 1 degree less than the most stable parent of the recombination round, Ll_AdhA28E7-his6.
Example 29: Adha's Directed Evolution of L. lactis Via Random Mutagenesis, Saturation Site Mutagenesis, and Recombination The following example illustrates a method for improving the kinetic properties of an ADH and also describes the kinetic properties of such improved ADH enzymes. The Ll_ adhÁRKI-his6 gene served as a model for a second round of error-prone and screening PCR (Generation 3). The screening assay used 0.125 mM isobutyraldehyde. Approximately 3,000 clones from a library generated using error-prone PCR, with MnCE 200 μΜ according to Example 1 above, were expressed and screened in a high throughput mode. Several groups were chosen for a new screening in triplicate and two variants, Ll_AdhA7A4-his6 and Ll_AdhA4A3-his6, were identified with improved activity. The mutations of the variants are shown in Table 92.
Table 92. List of Cumulative Mutations in Generation 3 Variants Ll_AdhA7A4-his6 and Ll_AdhA4A3-his6.
The specific activities (U / mg) in Ll_AdhA7A4-his6 and Ll_AdhA4A3-his6 lysates, as well as those of origin, were measured in biological triplicates at pH 7.0 (Table 93).
Table 93. Biochemical Properties of Ll_AdhA7A4-his6 and Ll_AdhA4A3-his6 at pH 7.0.
The T50 values of Ll_AdhA7A4-his6 (59.4 ° C) and Ll_AdhA4A3-his6 (57.6 ° C) were both greater than Ll_AdhAhis6 and less than Ll_AdhARE1-his6.
After two rounds of error-prone PCR and a round of saturation, recombination site mutagenesis, was performed at each of the six sites, generating six libraries (library of 50, 77, 108, 113, 212, and 264). The original parent, Ll_AdhAhis6, was used as a model for each NNK fragment. Two fragments from each library were amplified using primers listed in Table 4 (pGV1994ep_for and NNKADHF50_rev for fragment 1 of library 50, NNKADHF50_for and pGV1994ep_rev for fragment 2 of library 50; pGV1994ep_for and NNKADHR77_rev for fragment 1 of e77, for fragment 2 of library 77; pGV1994ep_for and NNKADHA108_rev for fragment 1 of library 108, NNKADHA108_for and pGV1994ep_rev for fragment 2 of library 108; 113; pGV1994ep_for NNKADHT212_rev and for fragment 1 of library 212, and NNKADHT212_for and pGV1994ep_rev for fragment 2 of library 212; pGV1994ep_for and NNKADHV264_rev for fragment 1 of library 264, NNKADHV4 and e4 for were then used as models for PCR assembly. The assembly PCR products were treated as described above to generate the yeast NNK libraries. Ninety clones were chosen for each NNK library, and screened separately. After re-screening, nine clones from six libraries were mini-prepared from yeast, the plasmids were used to transform E. coli and the resulting plasmids were sequenced. Their results of lysate and sequencing activities are summarized in Table 94.
Table 94. Summary of Site Saturation Mutagenesis (Generation 4).
A variety of mutations found in the site saturation mutagenesis libraries have been combinatorially recombined using SOE PCR and the library was constructed using the original non-codon optimized, pGV1947his. The primers described in Table 85 allowed for the wild type sequence for the six target sites as well. Six fragments were generated using Recomb2F50Minilib_rev and pGV1994ep_for (fragment 1), Recomb2F50Minilib_for and mixing Recomb2Q77Gen5_rev4, Recomb2R77Gen5_rev6 and Recomb2S77Gen5_rev8 (fragment 2) mixing Recomb2Q77Gen5_for3, Recomb2R77Gen5_for5 and Recomb2S77Gen5_for7 and mixing Recomb2Y113 Gen5_rev10, Recomb2F113 Gen5_rev12 and Recomb2G113 Gen5_rev14 (fragment 3) mix of Recomb2Y113 Gen5_for9, Recomb2F113 Gen5_for11 and Recomb2G113 Gen5_for13 and Recomb2T212 Mini_rev16 (fragment 4), Recomb2T212 Mini_for15 and Recomb2V264 Mini_rev18 (fragment 5), and Recomb2V264 Mini_for17 and pGV19. The fragment PCRs were analyzed in 1% analytical TAE gel and then the products were digested in Dpnl for 1 h at 37 ° C, separated over 1% preparative TAE agarose gel, Freeze'n'Squeeze (BIORAD) treated, and finally the pellets were painted (Novagen). The cleaned fragments served as a model for the assembly PCR. After successful assembly PCR, homologous recombination (as described above) was used to create the library. More than 1,000 individual clones were screened using an isobutyraldehyde concentration of 0.125 mM. A new tracing plate was compiled consisting of the 60 best variants and tested with 0.125 mM isobutyraldehyde.
Ten variants were chosen for expression in 100 ml of SC-URA medium to determine their specific activities in the lysate. Four of them were sequenced (see Table 95 for the mutations), purified and characterized in greater detail (Table 96). The new variants showed similar specific activities in the lysate as Ll_AdhARE1. Notably, variant 4A3 stood out as an enzyme with high specific activity.
Table 95. List of mutations in Variants of Generation 5.
Table 96. Biochemical Properties of Generation 5 Variants.
Example 30: Construction of ADH Enzyme Homologues The following example illustrates how additional ADH enzymes are identified and developed to improve the kinetic properties of additional ADH enzymes.
The enzymes homologous to Adha from L. lactis were identified through BlastP searches of the publicly available databases, using the amino acid sequence of Adha from L. lactis (SEQ ID NO: 185) with the following search parameters: Threshold to Wait = 10, word size = 3, matrix = Blosum62, gap opening, = 11, gap extension, = 1. The best 100 groups, representing counterparts with about more than 60% sequence identity, were selected and are listed in Table 97. The sequences were aligned using the AlignX multiple sequence alignment tool, a component of NTI Advance Vector 10.3.1 with the following parameters: gap gap penalty = 10, gap extension penalty = 0, 05, gap separation penalty interval = 8. The multiple sequence alignment showed very high levels of conservation among the residues corresponding to (a) L. lactis Adha tyrosine 50 (SEQ ID NO: 185), ( b) L. lactis Adha glutamine 77 (SEQ ID NO: 185), (L.) L. lactis Adha valine 108 (SEQ ID NO: 185), (d) L. lactis Adha tyrosine 113 (SEQ IDha: NO) 185), (e) L. lactis Adha isoleucine 212 (SEQ ID NO: 185), and (f) L. lactis Adha leucine 264 (SEQ ID NO: 185), where Adha (SEQ ID NO: 185) is encoded by the L. lactis alcohol dehydrogenase Adha (ADH) gene (SEQ ID NO: 184) or an optimized codon version (SEQ ID NO: 206).
Table 97. Homologous enzymes with> 60% Sequence Identity for L. lactis AdhA The above detailed description was given for clarity of understanding only and no unnecessary limitations should be understood from it, as modifications will be obvious to the skilled in the art.
Although the invention has been described in relation to specific modalities thereof, it will be understood that it is capable of other modifications and this application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including such deviations from the present disclosure as being within the known or usual practice in the technique to which the invention belongs and as being able to be applied to the essential characteristics defined hereinbefore and as they follow in the scope of the appended claims.
The disclosures, including the claims, figures and / or drawings of each and every patent, patent application, and publications cited herein are hereby incorporated by reference in their entirety.

权利要求:
Claims (3)
[1]
1. Recombinant Saccharomyces cerevisiae characterized by comprising a biosynthetic pathway that uses acetolactate as an intermediate, in which said yeast comprises a deletion of the YMR226C gene (SEQ ID NO: 1), in which the yeast still expresses an exogenous acetolactate synthase to convert pyruvate for acetolactate, and where the recombinant yeast produces isobutanol.
[2]
2. Recombinant Saccharomyces cerevisiae, according to claim 1, characterized by the fact that said microorganism contains mutations introduced to reduce or eliminate: (a) pyruvate decarboxylase (PDC) activity; and / or (b) glycerol-3-phosphate dehydrogenase (GPD) activity.
[3]
3. Isobutanol production method characterized by comprising: (a) providing a recombinant microorganism as defined in claim 1; (b) cultivating the recombinant microorganism in a culture medium containing a raw material that supplies the carbon source until a recoverable amount of isobutanol is produced; and (c) recovering the isobutanol.

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法律状态:
2018-03-06| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-02-19| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-02-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-03-10| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/02/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2020-04-14| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REF. RPI 2566 DE 10/03/2020 QUANTO AO INVENTOR. |

优先权:

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申请号 | 申请日 | 专利标题

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US30406910P| true| 2010-02-12|2010-02-12|

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