• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    RECOMBINANT MICROBIAL HOST CELLS AND METHODS FOR THE PRODUCTION OF ISOBUTANOL, 2-BUTANOL AND 1-BUTANOLThe present invention relates to candidate ADH enzymes suitable for the production of lower alkyl alcohols including isobutanol. The invention also relates to recombinant host cells that comprise such ADH enzymes and methods for producing lower alcohols in them.
    公开号:BR112012016042A2
    申请号:R112012016042-7
    申请日:2010-12-29
    公开日:2020-09-15
    发明作者:Sririam Satogopan；Daniel P. O'Keefe；Janardhan Gude
    申请人:Butamax Advanced Biofuels Llc；
    IPC主号:
    专利说明:

    “RECOMBINANT MICROBIAL HOSTING CELLS AND METHODS FOR THE PRODUCTION OF ISOBUTANOL, 2-BUTANOL AND 1- BUTANOL”
    FIELD OF THE INVENTION The present invention relates to the field of industrial microbiology and alcohol production. Specifically, the invention relates to alcohol dehydrogenases suitable for the production of lower alkyl alcohols by means of genetic engineering in microorganisms. More specifically, the invention relates to alcohol dehydrogenases suitable for the production of butanol, particularly isobutanol, through a route developed by genetic engineering in microorganisms.
    BACKGROUND OF THE INVENTION Butanol is an important industrial chemical, useful as a fuel additive, as a raw material for chemicals in the plastics industry, and as a food grade extractor in the food and seasoning industry. Each year, 10 to 12 billion pounds of butanol are produced by petrochemicals and the need for this chemical is likely to increase in the future. Methods for the chemical synthesis of isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, vol. 5, pp. 716-719) and the Guerbet condensation of methanol with n-propanol (Carlini et al, |. Moles. Catal. A: Chem. 220: 215-220, 2004). These processes use raw materials derived from petrochemicals, and, in general, are expensive and not ecological. Isobutane! it is biologically produced as a by-product of yeast fermentation. It is a component of "fusel oil" that forms as a result of the incomplete metabolism of amino acids by this group of fungi. Isobutanol is specifically produced from the catabolism of L-valine. After the L-valine amino group is collected as a nitrogen source, the resulting a-aceto acid is decarboxylated and reduced to isobutane! by enzymes of the so-called Ehrlich pathway (Dickinson et al. J. Biol. Chem. 275: 25 / 52-25756, 1998) The yields of fusel oil and / or its components achieved during fermentation of beverages are generally low. For example, the concentration of isobutanol produced in beer fermentation is reported to be less than 16 parts per million (Garcia et al, Process Biochemistry 2P: 303-309, 1994). The addition of exogenous L-valine to the fermentation mixture increases the yield of isobutanol, as described by Dickinson et a /, supra, in which it is reported that a yield of isobutanol of 3 g / L is obtained by supplying L- valine at a concentration of 20 g / L in the fermentation mixture. In addition, the production of n-propanol, isobutanol and isoamyl alcohol has been demonstrated by cells of Zymomonas —mobilis immobilized in calcium alginate. A medium containing 10% glucose supplemented with L-Leu, L-lle, L-Val, a-ketoisocaproic acid (a-KCA), a-ketobutyric acid (a-KBA) or a-ketoisovaleric acid (a- KVA) (Oaxaca, et al, Acta Biotechnol. 77: 523-532, 1991). A-KCA increased isobutanol levels. Amino acids also resulted in corresponding alcohols, but to a lesser extent than keto acids. An increase in the yield of C3 — Cs alcohols from carbohydrates was demonstrated when the amino acids leucine, isoleucine, and / or valine were added to the growth medium as the nitrogen source (PCT Publication: WO 2005/040392). Considering that the methods described above indicate the potential for production of isobutanol through biological means, these methods are prohibitive costs for the production of isobutanol on an industrial scale.
    For an efficient biosynthetic process, an optimal enzyme is needed in the last step to quickly convert isobutyraldehyde to isobutanol. In addition, an accumulation of isobutyraldehyde in the production host usually leads to undesirable cellular toxicity. Alcohol dehydrogenases (ADHs) are a family of proteins that comprise a large group of enzymes that catalyze the interconversion of aldehydes and alcohols (by Smidt et al, Yeast Res. FEMS., 5: 967-978, 2008), with different specificities for different alcohols and aldehydes. There is a need to identify suitable ADH enzymes to catalyze the formation of alcohol products in recombinant microorganisms. There is also a need to identify a suitable ADH enzyme that catalyzes the formation of isobutane! at a high rate, with specific affinity for isobutyraldehyde as the substrate and in the presence of high levels of isobutanol.
    BRIEF DESCRIPTION OF THE INVENTION One aspect of the invention is directed to a recombinant microbial host cell comprising a heterologous polynucleotide that encodes a polypeptide in which the polypeptide has alcohol dehydrogenase activity. In exemplary embodiments, the recombinant microbial host cell further comprises a biosynthetic pathway for the production of a lower alkyl alcohol, wherein the biosynthetic pathway comprises the conversion of substrate into product catalyzed by a polypeptide with alcohol dehydrogenase activity. In exemplary embodiments, the polypeptide has alcohol dehydrogenase activity and one or more of the following characteristics: (a) the Kyw value for a lower alkylaldehyde is lower for the —polypeptide when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26 (b) the K value for a lower alkyl alcohol for the polypeptide is higher when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO:
    26, and (c) the Ka / Km value for a lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26. In exemplary embodiments, the polypeptide having alcohol activity dehydrogenase has two more of the characteristics listed above.
    In exemplary embodiments, the polypeptide preferably uses NADH as a cofactor.
    In exemplary embodiments, the polypeptide having alcohol dehydrogenase activity has three of the characteristics listed above.
    In exemplary embodiments, the biosynthetic pathway for the production of a lower alkyl alcohol is a biosynthetic pathway of butanol, propanol, isopropanol or ethanol.
    In exemplary embodiments, the biosynthetic pathway for the production of a lower alkyl alcohol is a butanol biosynthetic pathway.
    Consequently, an aspect of the invention is a recombinant microbial host cell comprising: a biosynthetic pathway for the production of a lower alkyl alcohol, the biosynthetic pathway comprising the conversion of substrate into product catalyzed by a polypeptide with alcohol dehydrogenase activity and one or more, two or more, or all of the following: (a) the Km value for isobutyraldehyde is lower for the polypeptide when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; (b) the K value for isobutanol for said polypeptide is higher when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26, and (c) the kK.a / Km value for isobutyraldehyde for said polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26. In exemplary embodiments, the biosynthetic pathway for the production of a lower alkyl alcohol is a biosynthetic butanol pathway, propanol, isopropanol or ethanol.
    In exemplary embodiments, the polypeptide with alcohol dehydrogenase activity has at least 90% identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 31, 32, 34, 35, 36, 37 or 38. In exemplary embodiments, the polypeptide with alcohol dehydrogenase activity has the amino acid sequence of SEQ ID NO: 31. In exemplary embodiments, polypeptide 5 with alcohol dehydrogenase activity is encoded by a polynucleotide having at least 90% identity with the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 11, 12, 14, 15, 16 or 17. In embodiments, the polypeptide having alcohol dehydrogenase activity catalyzes the conversion of the isobutyraldehyde to isobutanol, in the presence of isobutanol, at a concentration of at least about 10 g / L, at least about 15 g / L, or at least about 20 g / L.
    In exemplary embodiments, the biosynthetic pathway for the production of a lower alkyl alcohol is an isobutanol biosynthetic pathway comprising heterologous polynucleotides that encode polypeptides that catalyze the substrate-to-product conversions for each step of the following steps: (a) pyruvate to acetolactate , (b) acetolactate in 2,3-dihydroxyisovalerate, (c) 2,3-dihydroxyisovalerate in α-ketoisovalerate; (d) a-ketoisovalerate in isobutyraldehyde, and (e) isobutyraldehyde in isobutanol, and wherein said microbial host cell produces isobutanol. In exemplary embodiments, (a) the polypeptide that catalyzes a conversion of substrate into pyruvate to acetolactate product is acetolactate synthase which has the EC number 2.2.1.6, (b) the polypeptide that catalyzes the conversion of a substrate to product of acetolactate for 2,3-dihydroxyisovalerate is acetohydroxy acid isomeroreductase having the EC number 1.1.186, (oc) the —polypeptide that catalyzes the conversion of a substrate into 2,3-dihydroxyisovalerate product for alpha-ketoisovalerate is acetohydroxy acid dehydratase having the EC number 4.2.1.9, and (d) the polypeptide that catalyzes the conversion of a substrate into an alpha-ketoisovalerate product to isobutyraldehyde is the branched-chain alpha-keto acid decarboxylase having the EC number
    4.1.1.72. In exemplary embodiments, the biosynthetic pathway for the production of a lower alkyl alcohol is an isobutanol biosynthetic pathway comprising heterologous polynucleotides that encode polypeptides that catalyze substrate-to-product conversions for each step of the following steps: (a) pyruvate to acetolactate , (b) acetolactate in 2,3-dihydroxyisovalerate, (c) 2,3-dihydroxyisovalerate in α-ketoisovalerate; (d) a-ketoisovalerate in isobutyryl-CoA; and (e) isobutiri-CoA in isobutyraldehyde, and wherein said microbial host cell produces isobutanol. In exemplary embodiments, the biosynthetic pathway for the production of a lower alkyl alcohol is an isobutanol biosynthetic pathway comprising heterologous polynucleotides that encode polypeptides that catalyze substrate-to-product conversions for each step of the following steps: (a) pyruvate to acetolactate , (b) acetolactate in 2,3-dihydroxyisovalerate, (c) 2,3-dihydroxyisovalerate in α-ketoisovalerate; (d) a-ketoisovalerate in valine; and (e) valine in isobutylamine, and wherein said microbial host cell produces isobutanol. Also provided in the present invention are recombinant microbial host cells comprising a biosynthetic pathway for the production of a lower alkyl alcohol and a heterologous polynucleotide that encodes a polypeptide with alcohol dehydrogenase activity having at least 85% identity with the amino acid sequence of SEQ ID NO : 21, 22, 23, 24, 25, 31, 32, 34, 35, 36, 37 or 38. In examples of realization, the biosynthetic route for the production of an alcohol! lower alkyl is an isobutanol biosynthetic pathway comprising heterologous polynucleotides that encode polypeptides that catalyze substrate-to-product conversions in each of the following steps: (a) from pyruvate to alpha-acetolactate, (b) from alpha-acetolactate to acetoin, ( c) acetoin in 2,3-
    butanediol; (d) 2,3-butanediol in 2-butanone; and (e) 2-butanone in 2-butanol, and wherein said microbial host cell produces 2-butanol. In exemplary embodiments, (a) the polypeptide that catalyzes the conversion of substrate into pyruvate product to acetolactate is acetolactate synthase - having the EC number 2.2.1.6; (b) the polypeptide that catalyzes the conversion of substrate into acetolactate to acetoin product is acetolactate decarboxylase which has the EC number 4.1.1.5; (c) the polypeptide that catalyzes the conversion of substrate in acetoin product to 2,3-butanediol is butanediol dehydrogenase which has the number EC 1.1.1.76 or EC 1.1.1.4; (d) the polypeptide that catalyzes the conversion of substrate in butanediol product to 2-butanone is butanediol dehydratase which has the EC number 4.2.1.28; and (e) the polypeptide that catalyzes the conversion of substrate in 2-butanone product to 2-butanol is 2-butanol dehydrogenase which has the EC number
    1.1.1.1. In exemplary embodiments, the polypeptide with alcohol — dehydrogenase activity comprises an amino acid sequence of at least 95% with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 36, 37, or 38. In exemplary embodiments, polypeptide having alcohol dehydrogenase activity comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 31.
    In exemplary embodiments, the biosynthetic pathway for the production of a lower alkyl alcohol is an isobutanol biosynthetic pathway comprising heterologous polynucleotides that encode polypeptides that catalyze substrate-to-product conversions in each of the following steps: (a) acetyl-CoA in acetoaceti-CoA; (b) acetoaceti-CoA in 3-hydroxybutyryl-CoA; (c) 3-hydroxybutyryl-CoA to crotonyl-CoA; (d) crotonyl-CoA in butyryl-CoA; (e) butyryl-CoA in butyraldehyde, and (f) butyraldehyde in 1-butanol; and wherein said microbial host cell produces 1-butanol. In exemplary embodiments, (a) the polypeptide that catalyzes the conversion of substrate into product from acetyl-CoA to acetoacetyl-CoA is acetyl-CoA acetyltransferase which has the number EC 2.3.1.9 or EC 2.3.1.16; (b) the polypeptide that catalyzes the conversion of substrate in product from acetoacetyl-CoA to 3-hydroxybutyryl-CoA is 3-hydroxybutyri-CoA-dehydrogenase which has the EC number 1.1.1.35,
    1.1.1.30, 1.1.1.157, or 1.1.1.36; (c) the polypeptide that catalyzes the conversion of substrate into a product of 3-hydroxybutyryl-CoA to crotonyl-CoA is the crotonase that has the EC number 4.2.1.17 or 4.2.1.55; (d) the polypeptide that catalyzes the conversion of substrate into crotonyl-CoA to butyryl-CoA product is butyryl-CoA dehydrogenase which has the EC number 1.3.1.44 or 1.3.1.38; (e) the polypeptide that catalyzes the conversion of substrate in butyryl-CoA product to butyrylaldehyde is butyraldehyde dehydrogenase which has the EC number
    1.2.1.57, and (f) the polypeptide that catalyzes the conversion of substrate in butyrylaldehyde product to 1-butane! is 1-butanol dehydrogenase. In exemplary embodiments, the polypeptide with alcohol dehydrogenase activity comprises an amino acid sequence of at least 95% with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27, 31, 32, 34, 35 , 36, 37 or
    38. In exemplary embodiments, a polypeptide having alcohol dehydrogenase activity comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 31.
    In examples of embodiments, the genetically modified microorganism is selected from the group consisting of bacteria, cyanobacteria, filamentous fungi and yeasts. In exemplary embodiments, the host cell is a bacterial cell or a cyanobacterium. In realization examples, the genus of host cells is selected from the group consisting of: Salmonella, Arthrobacter, Bacillus, Brevibacterium, Clostridium, —Corynebacterium, Gluconobacter, Nocardia, Pseudomonas, Rhodococcus, Streptomyces, Zymomonas, Escherichia, Lactobacillus,
    Enterococcus, Alcaligenes, Klebsiella, Serratia, Shigella, Alcaligenes, Enwinia, Paenibacillus and Xanthomonasl.
    In embodiments, the genus of host cells provided in the present invention is selected from the group consisting of: Saccharomyces, Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zygosaccharomyces, Galactomyces, Schizosaccharomyces, Torulasporaps, Debayomy , Dekkera,
    Kloeckera, Metschnikowia, Issatchenkia and Candida.
    Another aspect of the present invention is a method for the production of isobutanol comprising: (a) the supply of a recombinant microbial host cell comprising an isobutanol biosynthetic pathway, wherein the pathway comprises a heterologous polypeptide that catalyzes the conversion of substrate into a product of isobutyraldehyde to isobutanol, and wherein the polypeptide has at least 90% identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 36, 37, or 38, and (b) contacting the host cell of (a) with a carbon substrate under conditions where isobutane! Is Produced.
    In exemplary embodiments, the heterologous polypeptide that catalyzes the conversion of substrate into isobutyraldehyde product to isobutanol has at least 90% identity with the amino acid sequence of SEQ ID NO: 31. Another aspect of the present invention is a method for producing of 2-butanol comprising: (a) providing “a recombinant microbial host cell comprising a 2-butanol biosynthetic pathway, wherein the pathway comprises a heterologous polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27, 31, 32, 34, 35,36,37, ou38, and (b) contact of the host cell of (a) with a carbon substrate under conditions where 2-butanol is produced.
    In exemplary embodiments, the heterologous polypeptide has at least 90%> identity with the amino acid sequence of SEQ ID NO: 31. Another aspect of the present invention is a method for the production of 1-butanol comprising: (a) providing of a recombinant microbial host cell comprising a biosynthetic 1-butanol pathway, wherein the pathway comprises a heterologous polypeptide having at least 90%> identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 36, 37, or 38, and (b) contacting the host cell of (a) with a carbon substrate under conditions in which 1-butanol is produced.
    In exemplary embodiments, the heterologous polypeptide has at least 90%> identity with the amino acid sequence of SEQ ID NO: 31. Also provided in the present invention are methods for producing a lower alkyl alcohol comprising: (a) the providing a recombinant host cell provided in the present invention, (b) contacting said host cell with a fermentable carbon substrate in a fermentation medium under conditions in which the lower alkyl alcohol is produced, and (c) recovering said lower alkyl alcohol.
    In exemplary embodiments, said fermentable carbon substrate is selected from the group consisting of: monosaccharides, oligosaccharides and polysaccharides.
    In exemplary embodiments, the monosaccharide is selected from the group consisting of: glucose, galactose, mannose, rhamnose, xylose and fructose.
    In exemplary embodiments, said oligosaccharide is selected from the group consisting of: sucrose, maltose and lactose.
    In exemplary embodiments, polysaccharide is selected from the group consisting of: starch, cellulose and maltodextrin.
    In exemplary embodiments, the conditions are anaerobic, aerobic or microaerobic.
    In exemplary embodiments, said lower alkyl alcohol is produced at a level of at least about 10 g / L, at least about 15 g / L, or at least about 20 g / L.
    In exemplary embodiments, said lower alkyl alcohol is selected from the group consisting of: butanol, isobutanol, propanol, isopropanol, and ethanol.
    n
    In exemplary embodiments, isobutanol is produced.
    In exemplary embodiments, the method for producing isobutanol comprises: (a) providing a recombinant host cell comprising a heterologous polypeptide that catalyzes the conversion of substrate into isobutyraldehyde product to isobutanol and which has one or more of the following characteristics : (i) the Ky value of a lower alkylaldehyde is lower for the polypeptide when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26, (ii) the K value of a lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26, (iii) the Kca / Km value of the lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the sequence of amino acids of SEQ ID NO: 26; and (b) the contact of the host cell of (a)
    with a carbon substrate under conditions where isobutane! Is Produced.
    In exemplary embodiments, 1-butanol is produced.
    In exemplary embodiments, the method for producing 1-butanol comprises: (a) providing a recombinant microbial host cell comprising a heterologous polypeptide that catalyzes the conversion of substrate in butyraldehyde product to 1-butanol and which has one or more of the following characteristics: (i) the Ky value of a lower alkylaldehyde is lower for the polypeptide when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26, (ii) the K value of an alcohol lower alkyl for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID —NO: 26, (iii) the kKca / Km value of the lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; and (b) the contact of the host cell of (a)
    with a carbon substrate under conditions where 1-butanol is produced.
    Methods are also provided for screening candidate polypeptides having alcohol dehydrogenase activity, comprising said method: a) supplying a candidate polypeptide and a cofactor selected from the group consisting of NADH and NADPH, b) o - monitoring a change in Az4o nm over time in the presence or absence of a lower alkyl alcohol for the candidate polypeptide; and c) the selection of candidate polypeptides in which the change in Azsonm was a decrease, and the decrease is more rapid in the absence of lower alkyl alcohol compared to the decrease in the presence of lower alkyl alcohol.
    In exemplary embodiments, the methods further comprise; (d) providing a control polypeptide having the amino acid sequence of any of SEQ ID NO: 21 or 26 and NADH; (e) monitoring a change in Az4 nm over time in the presence or absence of a lower alkyl alcohol for the control polypeptide; (f), the comparison of the changes observed in (e) with the changes observed in (b); and (g) the selection of candidate polypeptides where the decrease at Az40 nm in the absence of lower alkyl alcohol is faster than the decrease observed for the control polypeptide.
    In exemplary embodiments, the methods further comprise; (d) providing a control polypeptide having the amino acid sequence of any of SEQ ID NO: 21 or 26 and NADH; (e) monitoring a change in Az4 nm over time in the presence or absence of a lower alkyl alcohol for the control polypeptide; (f), the comparison of the changes observed in (e) with the changes observed in (b); and (g) the selection of candidate polypeptides where the decrease at Az40o nm in the presence of alcohol - lower alkyl is faster than the decrease observed for the control polypeptide.
    Also provided in the present invention is the use of an alcohol dehydrogenase enzyme having at least about 80% identity with an amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40; in a microbial host cell to catalyze the conversion of isobutyraldehyde to isobutanol; wherein said host cell comprises an isobutanol biosynthetic pathway.
    BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES Figure 1 shows the results of semi-physiological time curve tests showing the reduction of isobutyraldehyde by NAD (P) H, catalyzed by candidate ADH enzymes in the presence and absence of isobutanol. Enzyme activity is measured by the following changes in absorbance at 340 nm. In each panel, AB40 nm of NADH or NADPH alone, in the presence of all other reagents, except the enzyme, was used as a control. Panel A shows the change in absorbance at 340 nm over time for the SadB of Achromobacter xylosoxidans. Panel B shows the change in absorbance at 340 nm over time for horse liver ADH. Panel C shows the change in absorbance at 340 nm over time for Saccharomyces cerevisiae ADH6. Panel D shows the change in absorbance at 340 nm over time for Saccharomyces cerevisiae ADH7. Panel E shows the variation in absorbance at 340 nm over time for Beijierickia indica ADH. Panel F shows the change in absorbance at 340 nm over time for Clostridium beijerinckii ADH. Panel G shows the variation in absorbance at 340 nm over time for Rattus norvegicus ADH. Panel H shows the change in absorbance at 340 nm over time for Therm's ADH, Sp. ATN1.
    Figure 2 shows the results of semi-physiological time curve assays comparing the level of isobutanol inhibition observed with horse liver ADH and SadB of Achromobacter xylosoxidans in the same figure. The tests are as described for Figure 1.
    Figure 3 is an alignment of the polypeptide sequences of Pseudomonas putida formaldehyde dehydrogenase (IKolA) (SEQ ID NO: 79), horse liver ADH (2ohxA) (SEQ ID NO: 21), Clostridium beijerinckii (IpedA) ADH ( SEQ ID NO: 29), L-threonine 3-dehydrogenase from - Pyrococcus horikoshii (2d8aA) (SEQ ID NO: 80) and SadB from Achromobacter xylosoxidans (SEQ ID NO: 26).
    Figure 4 is a phylogenetic tree of oxidoreductase enzymes obtained as “found alignments” (hits) from (i) a BLAST protein search for similar sequences in Saccharomyces cerevisiae, Escherichia coli, Homo sapiens, C. elegans, Drosophila melanogaster and Arabidopsis thaliana, and (ii) a BLAST protein search from the protein database (Protein Data Bank (PDB)) for similar sequences using horse liver ADH and SadB from Achromobacter xylosoxidans as query strings .
    Figure 5 is a phylogenetic tree of oxidoreductase enzymes most closely related to the AchBobacter xylosoxidans SadB between the found alignments (hits) of a search made on the BLAST protein from the non-redundant protein sequence database (nr) at NCBI using the Achromobacter SabB sequence — xylosoxidans as a query string.
    Figure 6 is an illustration of exemplary biosynthetic pathways for converting pyruvate to isobutanol.
    Figure 7 shows the Michaelis-Menten plots describing the properties of the enzymes belonging to the reduction of isobutyraldehyde. Figure 7A shows the results of tests to determine K, from isobutanol to ADHB6 and Figure 7B shows the results of tests to determine K, from isobutane! for BIADH.
    Figure 8A shows the results of semi-physiological time curve tests that have been described for Figure 1. Panel A shows the change in absorbance at 340 nm over time for the Phenylobacterium zucineum ADH. Panel B shows the variation in absorbance at 340 nm over time for Methylocella silvestris BL2. Panel C —shows variation in absorbance at 340 nm over time for Acinetobacter baumannii AYE.
    Figure 9 represents the locus PdCl :: ilvD :: FBA-ALSS :: trxl A. The integration of the alsS gene into the pdcl-trxl intergenic region is considered an insertion without scar “scarless”, since the vector, the marker gene and the / oxP strings are lost.
    The following strings provided in the sequence listing, electronically deposited as an attachment and incorporated herein by reference are in accordance with Title 37 of CFR 8 $ 1,821-1,825 ("Requirements for Patent Applications Containing Nucleotide Sequences andor Amino Acid Sequence Disclosures - the Sequence Rules ") and are consistent with the World Intellectual Property Organization (OMPIMIPO), Standard ST.25 (2009) and the EPO and PCT sequence listing requirements (Rules 5.2 and 49.5 (a-bis), and section 208 and Annex C of the administrative instructions). The symbols and formats used for nucleotide and amino acid sequence data obey the rules established in Title 37 of CFR $ 1.822.
    SEQ ID NOs: 1 and 7-20 are codon-optimized polynucleotide sequences. SEQ ID NOs: 2 and 3 are polynucleotide sequences of Saccharomyces cerevisiae.
    SEQ ID NOs: 4 and 5 are polynucleotide sequences of Clostridium acetobutylicum.
    SEQ ID NO: 6 is a polynucleotide sequence of Achromobacter xylosoxidans. SEQ ID NOs: 21-40 and 79-80 are polypeptide sequences. SEQ ID NOs: 41-50 and 52-57 and 59-74 and 77-78 are primers. SEQ ID NO: 51 is the sequence of plasmid pRS423 :: TEF (M4) -xpkl + ENO1- eutD.
    SEQ ID NO: 58 is the sequence of plasmid pUC19- URAS: PdCI :: TEF (M4) -xpkl :: kan.
    SEQ ID NO: 75 is the sequence of plasmid pl H468.
    SEQ ID NO: 76 is the coding region for BIADH (codon-optimized for yeast) plus 5 'homology for the GPM promoter and 3' homology for the ADH1 terminator.
    SEQ ID NO: 81 is the sequence of plasmid pRS426 :: GPD- xXpkI + ADH-eutD.
    DETAILED DESCRIPTION OF THE INVENTION The indicated problems are solved as described in the present invention by designing and using a suitable screening strategy to evaluate several candidate ADH enzymes. The screening strategy can be used to identify ADH enzymes that have desirable characteristics. These identified ADH enzymes can be used to increase the biological production of lower alkyl alcohols, such as isobutanol. Recombinant host cells expressing the identified desirable ADH enzymes are also provided and methods are provided for producing lower alkyl alcohols using them.
    The present invention describes a method for screening large numbers of alcohol dehydrogenase (ADH) enzymes for its ability to quickly convert isobutyraldehyde to isobutanol, in the presence of high concentrations of isobutanol. Also described in the present invention is a new ADH that is present in the bacterium Beijerínckia indica subspecies ATCC 9039. The ADH enzyme from Beijerinckia indica can be used in the production of isobutanol from isobutyraldehyde in a recombinant microorganism having an isobutyraldehyde source.
    The present disclosure fulfills a number of commercial and industrial needs. Butanol is an important industrial chemical that has a variety of applications, where its potential as a fuel or fuel additive is particularly significant. Although being an alcohol with only four carbons, butanol has an energy content similar to that of gasoline and can be mixed with any fossil fuel. Butanol is favored as a fuel or fuel additive, as it produces only CO, and little or no SO, or NO, when burned in a standard internal combustion engine. In addition, butanol is less corrosive than ethanol, the fuel used as a fuel additive to date.
    In addition to its usefulness as a biofuel or fuel additive, butanol has the potential to impact hydrogen distribution problems in the emerging fuel cell industry. Fuel cells are currently affected by safety issues associated with the transport and distribution of hydrogen. Butanol can be easily regenerated due to its hydrogen content and can be distributed through existing fuel stations in the necessary purity for both cells and vehicles.
    The present invention produces butanol from carbon sources derived from vegetables, avoiding the negative environmental impact associated with standard petrochemical processes for the production of butanol. In one embodiment, the present invention provides a method for the selection and identification of ADH enzymes that increase the flow in the last reaction of the isobutanol biosynthesis pathway; converting isobutyraldehyde to isobutanol. In one embodiment, the present invention provides a method for the selection and identification of ADH enzymes that increase flow in the last reaction of the 1-butanol biosynthesis pathway; the conversion of butyrylaldehyde to 1-butanol.
    In one embodiment, the present invention provides a method for the selection and identification of ADH enzymes that increase flow in the last reaction of the 2-butanol biosynthesis pathway; the conversion of 2-butanone to 2-butanol.
    Particularly useful ADH enzymes are those that are better able to increase the flow in the reaction of conversion of isobutyraldehyde to isobutanol when compared to the already known ADH control enzymes.
    The present invention also provides recombinant host cells that express such identified ADH enzymes and methods of using them.
    The following definitions and abbreviations should be used for the interpretation of the specification and claims.
    The term "invention" or "present invention" as used herein means that it is generally applicable to all examples of carrying out the invention, such as those described in the claims in the form that are presented, amended and completed later, or in the specification.
    The term "isobutanol biosynthetic pathway" refers to an enzymatic pathway to produce isobutanol from pyruvate.
    The term "1-butanol biosynthetic pathway" refers to an enzymatic pathway to produce 1-butanol from pyruvate.
    The term "2-butanol biosynthetic pathway" refers to an enzymatic pathway to produce 2-butanol from acetyl-CoA.
    The term "NADH consumption test" refers to an enzymatic test to determine the specific activity of the alcohol dehydrogenase enzyme, which is measured as a stoichiometric disappearance of NADH, a cofactor for the enzymatic reaction, as described in Racker, J Biol .
    Chem., 184: 313-319 (1950).
    "ADH" is the abbreviation for the enzyme alcohol dehydrogenase.
    The terms "isobutyraldehyde dehydrogenase", "secondary alcohol dehydrogenase", "butanol dehydrogenase", "branched chain alcohol dehydrogenase" and "alcohol dehydrogenase" will be used interchangeably in the present invention and refer to the enzyme that has the EC number : EC 1.1.1.1 (Enzyme Nomenciature 1992, Academic Press, San Diego). The pdited branched chain alcohol dehydrogenases are known by the EC number
    1.1.1.265, but can also be classified under other alcohol dehydrogenases (more specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes use NADH (nicotinamide-reduced adenine dinucleotide) and / or NADPH as an electron donor.
    As used herein, "heterologous" refers to a polynucleotide, gene or polypeptide that is not normally found in the host organism, but that is introduced or otherwise modified.
    "Heterologous polynucleotide" includes a native coding region from the host organism, or a portion thereof, which is reintroduced or otherwise modified into the host organism, so that it is different from the corresponding native polynucleotide, as well as a coding region from a different organism, or a portion of this region. “heterologous gene” includes a native coding region, or portion thereof, so that it is reintroduced or otherwise modified from the source organism in a form that is different of the corresponding native gene, as well as a coding region from a different organism. For example, a heterologous gene can include a native coding region that is a portion of a chimeric gene, including non-native regulatory regions that are reintroduced into the native host. "Heterologous polypeptide" includes a native polypeptide that is reintroduced or otherwise modified into the host organism in a form that is different from the corresponding native polypeptide, as well as a polypeptide from another organism.
    The term "carbon substrate" or "fermentable carbon substrate" refers to a carbon source capable of being metabolized by host organisms of the present invention. Non-limiting examples of carbon sources that can be used in the invention include monosaccharides, oligosaccharides, polysaccharides, and substrates of a carbon or mixtures thereof.
    The terms "kKcat" and "Km and" * are terms known to those skilled in the art and are described in "Enzyme Structure and Mechanism", 2nd ed (Ferst, WH Freeman: NY, 1985; pages 98-120). The term “kKca ', often referred to as the“ tumover number, ”is defined as the maximum number of substrate molecules converted to product molecules per active site per unit of time, or the number of times that the enzyme regenerates per unit time. Kcat = Vmaw [E], where [E] is the - enzyme concentration (Ferst, supra).
    The term "catalytic efficiency" is defined as the kKca / Km of an enzyme. The "catalytic efficiency" is used to quantify the specificity of an enzyme for a substrate.
    The term "specific activity" means units of enzyme / mg of protein, where an enzyme unit is defined as moles of product formed / minute under specific conditions of pH, temperature, [S], etc.
    The terms "slow", "slower", "faster", or "fast" when used in reference to an enzyme activity relate to the number of enzyme renewal compared to a standard.
    The term "control polypeptide" refers to a known polypeptide having known alcohol dehydrogenase activity. Non-limiting examples of control polypeptides suitable for use in the present invention include SadB from Achromobacter xylosoxidans and horse liver ADH.
    The term "lower alkyl alcohol" refers to any straight or branched, saturated or unsaturated alcohol molecule with 1-10 carbon atoms.
    The term "lower alkylaldehyde" refers to any straight or branched chain, saturated or unsaturated aldehyde molecule with 1-10 carbon atoms.
    The term "butanol" as used herein, refers to 1-butanol, 2-butanol isobutanol, or mixtures thereof.
    The term “biosynthetic route for the production of an alcohol! lower alkyl ”as used in the present invention, refers to an enzymatic pathway to produce lower alkyl alcohols. For example, isobutane biosynthetic pathways! are disclosed in Patent Application Publication US2007 / 0092957, which is hereby incorporated by reference.
    As used herein, the term "yield" refers to the amount of product by the amount of carbon source in 9 / g. The yield can be exemplified for glucose as a carbon source. Unless stated otherwise, it is understood that the yield is expressed as a percentage of the theoretical yield. In reference to a microorganism or metabolic pathway, "theoretical yield" is defined as the maximum amount of product that can be generated by the total amount of substrate as dictated by the stoichiometry of the metabolic pathway used to produce the product. For example, the theoretical yield for a typical conversion of glucose to isopropanol is 0.33 µg. In this way, an isopropanol yield from glucose of 29.7 µg will be expressed as 90% of the theoretical or 90%> theoretical yield. It is understood that although in the present description the yield is exemplified for glucose as a carbon source, the invention can be applied to other carbon sources and the yield may vary depending on the carbon source used.
    A subject technician can calculate the yield on different carbon sources.
    The term "NADH" means nicotinamide adenine reduced dinucleotide.
    The term "NADPH" means nicotinamide-adenine dinucleotide reduced phosphate.
    The term "NAD (P) H" is used to refer to NADH or NADPH.
    POLYPEEPTIDS AND POLYNUCLEOTIDS FOR USE IN THE INVENTION The ADH enzymes used in the invention comprise polypeptides and fragments thereof.
    As used in the present invention, the term "polypeptide" is intended to encompass a single "polypeptide", as well as several "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds) ). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
    Thus, peptides, dipeptides, tripeptides and oligopeptides, "proteins", "amino acid chains", or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" can be used in place of, or alternatively with, any of these terms.
    The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, "protein-cleavage, or modification by non-naturally occurring amino acids.
    A polypeptide of the invention can be of a size of about 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids.
    Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such a structure.
    Polypeptides with a defined three-dimensional structure are said to be folded, and polypeptides that do not have a defined three-dimensional structure, but which instead can adopt a large number of different conformations are said to be not folded.
    Also included as polypeptides of the present invention are the derivatives, analogs, or variants of the polypeptides mentioned above, and any combination thereof.
    The terms "active variant", "active fragment", "active derivative", and "analog" refer to the polypeptides of the present invention and include all polypeptides that are capable of catalyzing the reduction of a lower alkylaldehyde.
    Variant forms of polypeptides of the present invention include polypeptides with amino acid sequences altered due to amino acid substitutions, deletions and / or insertions.
    Variants may occur naturally or may be unnatural.
    Unnaturally occurring variants can be produced using mutagenesis techniques known in the prior art.
    Variant polypeptides may comprise conservative or non-conservative substitutions, deletions and / or additions of amino acids.
    The polypeptide derivatives of the present invention are polypeptides that have been altered to exhibit additional characteristics that are not found in the native polypeptide.
    Examples are fusion proteins.
    Variant polypeptides can also be said in the present invention as "polypeptide analogs". As used in the present invention, a "derivative" refers to a "polypeptide object of the invention that has one or more residues chemically derivatized by the reaction of a functional side group.
    Also included as “derivatives” are those peptides that contain one or more derivatives of naturally occurring amino acids among the twenty amino acids.
    For example, 4-hydroxyproline can be replaced with proline; 5-hydroxylysine can be replaced by lysine; 3-methylhistidine can be replaced by histidine; homoserine can be replaced by serine; and ornithine can be replaced by lysine.
    A "fragment" is a single portion of an ADH enzyme that is identical in sequence, but shorter in length than the parental sequence. A fragment can comprise up to the entire length of the defined sequence, minus an amino acid residue. For example, a fragment can comprise from 5 to 1000 contiguous amino acid residues.
    A fragment can be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues in length. The fragments can preferably be selected from certain regions of a molecule. For example, a polypeptide fragment can comprise a given length of contiguous amino acids selected from the first 100 or 200 amino acids of a polypeptide as shown in a given defined sequence. Clearly these lengths are exemplary, and any length supported by the specification, including the List of Sequences, Tables and Figures, can be covered by the present examples of realization.
    Alternatively, recombinant variants that encode these identical or similar polypeptides can be synthesized or selected, using “redundancy” in the genetic code. Several codon substitutions, such as silent changes that produce multiple restriction sites, can be introduced to optimize cloning in a plasmid or viral vector or expression in a host cell system. Mutations in the polynucleotide sequence can be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, change characteristics such as Km for lower alkylaldehyde, Ku for lower alkyl alcohol, for a lower alkyl alcohol, and etc.
    Preferably, amino acid "substitutions" are - results of replacing one amino acid with another amino acid having similar structural and / or chemical properties, that is, conservative amino acid substitutions.
    The "conservative" substitutions of amino acids can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues involved.
    For example, nonpolar (hydrophobic) include the amino acids alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; neutral polarity amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged amino acids (acids) include aspartic acid and glutamic acid.
    The "insertions" or "deletions" are preferably in the range of about | to about 20 amino acids, more preferably from 1 to 10 amino acids.
    The allowed variation can be determined experimentally by systematically making - insertions, deletions or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and evaluating the resulting recombinant variants by activity.
    A polypeptide having an amino acid sequence, or polypeptide that is at least, for example, 95% "identical" to a query amino acid sequence of the present invention, means that the amino acid sequence of the polypeptide object of the invention is identical to the sequence of query (reference) except that the polypeptide sequence object of the invention can include up to five amino acid changes for each
    100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the object sequence of the invention can be inserted, deleted or replaced with a other amino acids. These changes in the reference sequence can occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually between residues in the reference sequence or in one or more contiguous groups within the sequence of reference.
    As a practical matter, if any specific polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference polypeptide this can be conventionally determined using known computer. A proposed method for determining the best overall match between a query sequence (a sequence of the present invention) and an object sequence of the invention, also referred to as global sequence alignment, can be determined using the FASTDB computer program based on the Brutlag algorithm et al., Comp. Appl. Biosci. (5: 237-245 (1990)). In a sequence alignment the query and object sequences of the invention are nucleotide sequences or amino acid sequences. The result of said global sequence alignment is in percent identity. The recommended parameters used in an amino acid alignment with FASTDB are: Matrix = PAM 0, k-tuple = 2, Penalty for mismatch (substitution) (Mismatch - Penalty) = 1, Penalty for joining (Joining Penalty) = 20, Randomization Group Length = 0, Cutoff Score = 1, Window Size Window Size = sequence length, Gap Penalty = 5, Gap Size Penalty
    Penalty) = 0.05, Window Size = 500 or the length of the amino acid sequence object of the invention, whichever is shorter.
    If the sequence object of the invention is shorter than the query sequence due to N- or C-terminal deletions, and not due to - internal deletions, a manual correction must be made to the results.
    This is because the FASTDB program does not take into account N- and C-terminal truncations of the object sequence when calculating the percentage of global identity.
    For sequences object of the invention truncated at the N- and C-terminal ends, in relation to the query sequence, the percentage of identity is corrected by calculating the number of residues in the query sequence that are N- and C- terminals of the sequence object of invention, which are not paired / aligned with a residue of the corresponding object sequence, as a percentage of the total bases of the query sequence.
    Whether a residue is correctly paired / aligned, this is determined by the results of FASTDB string alignment.
    This percentage is then subtracted from the percentage of identity, calculated by the FASTDB program above using the parameters specified to arrive at a final score of the percentage of identity.
    This percentage of final identity score is that used for the purposes of the present invention.
    Only residues of the N- and C-terminal ends of the sequence object of the invention that are not correctly paired / aligned with the query sequence are considered in order to manually adjust the percentage identity score.
    That is, only the positions in the query residues that are outside the N- and C-terminal residues furthest from the sequence object of the invention.
    For example, a sequence object of the invention of 90 amino acid residues is aligned with a query sequence of 100 residues to determine the percent identity.
    The deletion occurs at the N-terminal of the sequence object of the invention and, therefore, the FASTDB alignment does not show a correct match / alignment of the first 10 residues at the N-terminal. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-terminus that are not correctly matched / total number of residues in the query sequence), so> 10% is subtracted from the percentage identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly aligned, the percentage of final identity would be 90%. In another example, a sequence that is the subject of the invention of 90 residues is compared to a query sequence of 100 residues. This time the deletions are internal deletions, so that there are no residues at the N- or C-terminal end of the sequence object of the invention that are not correctly paired / aligned in relation to the query sequence. In this case, the percentage of identity calculated by FASTDB is not corrected manually. Again, only the positions of residues outside the N- and C-terminal ends of the sequence object of the invention, as shown in the FASTDB alignment, which are not correctly paired / aligned with the query sequence are corrected manually. No other manual correction should be made for the purposes of the present invention.
    Polypeptides useful in the present invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences shown in Table 5, including active variants, fragments, or derivatives thereof. The invention also encompasses polypeptides comprising amino acid sequences from Table 5 with conservative amino acid substitutions.
    In an example of an embodiment of the invention, polypeptides having alcohol dehydrogenase activity that will be expressed in the recombinant host cells of the present invention have amino acid sequences that are at least about 80%, 81%, 82%, 83%, 84%, 85 %,
    86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, —SEQIDNO 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40. In another embodiment of the invention, a polypeptide having alcohol dehydrogenase activity that will be expressed in the recombinant host cells of the present invention has the amino acid sequence selected from the group consisting of : SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, or an active variant, fragment or derivative thereof.
    In one embodiment, polypeptides having alcohol dehydrogenase activity are encoded by polynucleotides that have been codon-optimized for expression in a specific host cell.
    In an example of an embodiment of the invention, the polypeptides having alcohol dehydrogenase activity to be expressed in the recombinant host cells of the present invention comprise a sequence of amino acids having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 22. In another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 22 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the polypeptides having alcohol dehydrogenase activity to be expressed in the recombinant host cells of the present invention comprise a sequence of amino acids having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 23. In another example, the polypeptide comprises the amino acid sequence of SEQ ID NO: 23 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the polypeptides having alcohol dehydrogenase activity to be expressed in the recombinant host cells of the present invention comprise a sequence of amino acids having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 31. In another example, the polypeptide comprises the amino acid sequence of SEQ ID NO: 31 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the polypeptides having alcohol dehydrogenase activity to be expressed in the recombinant host cells of the present invention comprise a sequence of amino acids having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 29. In another example, the polypeptide comprises the amino acid sequence of SEQ ID NO: 29 or a variant, fragment or active derivative thereof.
    ADH enzymes suitable for use in the present invention and fragments thereof can be encoded by polynucleotides. The term "polynucleotide" is intended to encompass a single nucleic acid as well as several nucleic acids, and refers to an isolated or constructed nucleic acid molecule, for example, messenger RNA (mMRNA), viral-derived RNA or plasmid DNA (PDNA). A polynucleotide can comprise a conventional phosphodiester bond or an unconventional bond (for example, an amide bond, as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one of one or more nucleic acid segments, for example, fragments of DNA or RNA present in a polynucleotide. The polynucleotides according to the present invention further include such synthetically produced molecules. The polynucleotides of the invention can be native to the host cell or they can be heterologous. In addition, a polynucleotide or nucleic acid can be or can include a regulatory element, such as a promoter, ribosome binding site, or a transcription terminator.
    As used in the present invention, a "coding region" or "ORF" is a portion of nucleic acid that consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered as part of a coding region, if present, but any flanking sequences, for example, ribosome-binding site promoters, transcription terminators, introns, 5 'and 3' untranslated regions, and the like are not part of a coding region.
    The term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3 'from a promoter sequence. The promoters can be obtained in their entirety, from a native gene, or be composed of several elements from different promoters found in nature, or even comprise segments of synthetic DNA. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types are, in most cases, commonly referred to as "constitutive promoters". It is also recognized that in most cases the exact limits of the regulatory sequences have not been completely defined, DNA fragments of - different lengths can have identical promoter activity.
    In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid that encodes a polypeptide can usually include a promoter and / or other transcription or translation control elements - operably linked to one or more coding regions. An operable association is when a coding region of a gene product, for example, a polypeptide, is associated with one or more regulatory sequences, so this association puts the expression of the gene product under the influence or control of the sequence (s) ( s) regulator (s) Two DNA fragments (such as a polypeptide coding region and a promoter associated with it) are "operationally associated" if induction of the promoter function results in the transcription of mRNA that encodes the desired gene product and the nature of the link between the two DNA fragments does not interfere with the ability of regulatory sequences to direct expression of the gene product, or does not interfere with the ability of the template DNA to be transcribed. Thus, a promoter region would be operationally associated with a nucleic acid that encodes a polypeptide if the promoter were able to affect the transcription of the nucleic acid. Other elements of transcription control, in addition to a promoter, for example, enhancers, operators, repressors, and transcription termination signals, can be operationally associated with the polynucleotide. Suitable promoters and other transcription control regions are disclosed in the present invention.
    A variety of translation control elements are known to those skilled in the art. These elements include, but are not limited to, ribosome binding sites and translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as ISCED sequence).
    In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (MRNA). The RNA of the present invention can be single or double stranded.
    The polynucleotide and nucleic acid coding regions of the present invention can be associated with additional coding regions that encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
    As used herein, the term "transformation" refers to the transfer of a fragment of nucleic acid into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are said to be "recombinant" or "modified" organisms.
    The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. The expression can also refer to the translation of mRNA into a polypeptide.
    The terms "plasmid", "vector" and "cassette" refer to an extrachromosomal element that usually carries genes that they do not - part of the cell's central metabolism and are usually in the form of circular double-stranded DNA fragments. Such elements can be sequences that replicate autonomously, sequences of genome integration, phage or nucleotide sequences, linear or circular, single-stranded or double-stranded, of DNA or RNA, from any source, in which a variety of Nucleotide sequences have been joined or recombined into a single construct that is capable of introducing a promoter fragment and DNA sequence from a selected gene product, —unjoint with the appropriate 3 ”untranslated sequence into a cell. A "transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates the transformation of a specific host cell. An "expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allows for improved expression of that gene in a different cell.
    The term "artificial" refers to a composition derived from a cell that is not the synthetic host cell, for example, a chemically synthesized Oligonucleotide.
    By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the sequence reference, except that the polynucleotide sequence can include up to five point mutations for every 100 nucleotides in the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or a number nucleotides of up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence.
    As a practical matter, to find out if any specific nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptides of the present invention, this can be determined conventionally using known computer programs.
    A proposed method for determining the best overall correspondence between a query sequence (a sequence of the present invention) and an object sequence, also referred to as global sequence alignment, can be determined using the FASTDB computer program based on the Brutlag et algorithm al, Comp.
    Appl.
    Biosci. 6: 237-245 (1990). In a sequence alignment, the query and object sequences are two DNA sequences.
    An RNA sequence can be compared by exchanging U's for T's.
    The result of said global sequence alignment is in percent identity.
    The default parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuplez4, Mismatch Penalty = ll Joining Penalty-30, Randomization Group Length = 0, Cutoff Score = |, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500 Matrix = or the length of the nucleotide sequences object of the invention, whichever is shorter.
    If the object sequence is shorter than the query sequence due to 5 'or 3' deletions, and not due to internal deletions, a manual correction must be made to the results.
    This is because the FASTDB program does not take into account truncations in 5 'and 3' of the sequence object of the invention when calculating the percentage of global identity.
    For sequences in question truncated at the 5 'or 3' ends, in relation to the query string, the percent identity is corrected by calculating the number of bases of the query string that are 3 'and 5' from the object string that is not correctly paired / aligned, as a percentage of the total base of the query sequence. If a nucleotide is correctly matched / aligned, this is determined by the results of the FASTDB sequence alignment.
    This percentage is then subtracted from the percentage of identity, calculated by the FASTDB program above using the parameters specified to arrive at a final score of the percentage of identity.
    This corrected score (score) is the one used for the purposes of the present invention.
    Only the bases outside the bases in the region 5 'and 3' of the sequence object of the invention, as shown by the FASTDB alignment, that are not correctly paired / aligned with the query sequence are considered for the purposes of manually adjusting the percentage score of identity.
    For example, a 90-base sequence of the invention is aligned with a 100-base query sequence to determine the percent identity.
    The deletion occurs at the 5 'end of the sequence object of the invention and, therefore, the FASTDB alignment does not exhibit a “correct match / alignment of the first 10 bases at end 5. The 10 unpaired bases represent 10% of the sequence (number of bases in 5 'and 3' ends that are not correctly matched / total number of bases in the query sequence), thus 10%> is subtracted from the percentage identity score calculated by the FASTDB program.
    If the remaining 90 bases were perfectly aligned, the percentage of final identity would be 90%. In another example, a 90-base sequence of the invention is compared to a 100-base query sequence.
    This time the deletions are internal deletions, so that there are no bases at the 5 'and 3' ends of the object sequence that are not correctly paired / aligned in relation to the query sequence.
    In this case, the percentage of identity calculated by FASTDB is not corrected manually.
    Again, only the bases 5 'and 3' of the sequence object of the invention that are not correctly paired / aligned with the query sequence are corrected manually.
    No other manual correction should be made for the purposes of the present invention.
    Polynucleotides useful in the present invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set out in Table 4, including variants, fragments or derivatives thereof that encode polypeptides with active alcohol dehydrogenase activity.
    The terms "active variant", "active fragment", "active derivative", and "analog" refer to the polynucleotides of the present invention and include all polynucleotides that encode polypeptides capable of catalyzing the reduction of a lower alkylaldehyde.
    Variant forms of polynucleotides of the present invention include polynucleotides with nucleotide sequences altered due to base pair substitutions, deletions and / or insertions.
    Variants may occur naturally or may be unnatural.
    Unnaturally occurring variants can be produced using mutagenesis techniques known in the prior art.
    The polynucleotides derived from the present invention are polynucleotides that have been altered so that the polypeptides they encode exhibit additional characteristics that are not found in a native polypeptide.
    Examples include polynucleotides that encode fusion proteins.
    Variant polynucleotides can also be said in the present invention as "analogous polynucleotides". As used in the present invention, a "derivative" of a polynucleotide refers to a polynucleotide object of the invention that has one or more nucleotides chemically derivatized by the reaction of a functional side group.
    Also included as "derivatives" are those polynucleotides that contain one or more naturally occurring derived nucleotides.
    For example, 3-methylcytidine can be replaced with cytosine; ribotimidine can be replaced by thymidine; and N4-acetylcytidine can be replaced by cytosine.
    A "fragment" is a single portion of the polypeptide encoding the ADH enzyme that is identical in sequence, but shorter in length than the parental sequence.
    A fragment can comprise up to the entire length of the defined sequence, minus one nucleotide.
    For example, a fragment can comprise from 5 to 1000 contiguous nucleotides.
    A fragment used as a probe, primer, or for other purposes, can be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides.
    The fragments can preferably be selected from certain regions of a molecule.
    For example, a polynucleotide fragment can comprise a given length of contiguous nucleotides selected from the first 100 or 200 nucleotides of a polynucleotide as shown in a given defined sequence.
    Clearly these lengths are exemplary, and any length supported by the specification, including the List of Sequences, Tables and Figures, can be covered by the present examples of realization.
    In an example of an embodiment of the invention, polynucleotide sequences suitable for expression in host cells — recombinants of the present invention comprise nucleotide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99% or 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. In another example of the invention, the polynucleotide sequence suitable for expression in recombinant host cells of the The present invention can be selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14, SEQ ID N O: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQIDNO: 19eSEQID NO: 20, or a variant, fragment or active derivative thereof. In one embodiment, the polynucleotides were codon-optimized for expression in a specific host cell.
    In an example of an embodiment of the invention, the polynucleotide sequence suitable for expression in recombinant host cells of the present invention has a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the polynucleotide sequence suitable for expression in recombinant host cells of the present invention has a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 3. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the sequence of -polyucleotides suitable for expression in recombinant host cells of the present invention has a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO : 11. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 11 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the sequence of -polyucleotides suitable for expression in recombinant host cells of the present invention has a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO : 9. In another exemplary embodiment, the -pollucleotide comprises the nucleotide sequence of SEQ ID NO: 9 or a variant, fragment or active derivative thereof.
    As used herein, "codon degeneration" refers to the nature of the genetic code that allows variation in the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Those skilled in the art are aware of the "codon-bias" exhibited by a specific host cell in the use of nucleotide codons to specify a particular amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene so that the frequency of codons of preferred use (codon usage) approximates the frequency of codons of preferential use of the host cell.
    As used herein, the term "codon-optimized coding region" means a region of coding nucleic acid that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, or a number significant number of codons, by one or more codons that are most often used in the genes of that organism.
    Deviations in the nucleotide sequence that comprise the codons coding for the amino acids of any polypeptide chain allow variations in the coding sequence of the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals that end the translation). The “genetic code”, which displays which codons encode amino acids is reproduced in the present as the Table
    1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are encoded by four triplets, the amino acids serine and arginine by six, while the amino acids tryptophan and methionine are encoded by only one triplet. This degeneration allows the base DNA composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA. TABLE 1 THE STANDARD GENETIC COPY A and TTT Fen (F) ITOT Ser (S) ITATTir (Y) | TGTCis (C) T TIC "TCC" TAC "TGC TTA Leu (L) ITCA" TAA Ter TGA Ter TTG "TCG" TAG Have TGG Trp (W) CTT Leu (L) [CCT Pro (P) [CAT His (H) [CGT Arg (R) c CTC "CTA" jJCCC "CAC" cGec "cTG" CCA "CAA GINn (Q) | CGA "CccG" CAG "cec" ATT Ile (Il) [ACT Ter (T) J | AAT Asn (N) | AGT Ser (S) ATC "ACC" AAC "AGC" A IATA "ACA" AAA Lis (K ) JAGA Arg (R) ATG Met —jACG "AAG" AGG "(M) GTT Val (V) [GCT Wing (A) [GAT Asp (D) [GGT GI (G) G GTC" GCC "GAC" GGc " GTA "GCA" GAA GIlu (L) | GGA "GTG" GCG "GAG" GGG "
    Many organisms exhibit a tendency to use specific codons when encoding the insertion of a specific amino acid in the peptide chain in formation.
    The preference for codon or codon-bias, differences in the use of codons between organisms, is provided by the —degeneration of the genetic code, and is well documented among many organisms.
    The bias codon often correlates with the translation efficiency of messenger RNA (mMRNA), which in turn is believed to be dependent, inter alia, on the properties of the codons being translated and on the availability of certain transfer RNA molecules (t (RNA) The predominance of selected tRNAs in a cell is usually a reflection of the codons most frequently used in peptide synthesis.
    Consequently, genes can be adapted for optimal gene expression in a given organism based on codon optimization.
    Due to the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. The codon usage tables are readily available, for example, in the codon usage database available on the web page —http: /Www.kKazusa.or.jp/codon/, and these tables can be adapted in several ways.
    See, Nakamura, Y., et al.
    Nucl.
    Acids Res. 28: 292 (2000). The tables of preferential use of codons for yeasts, calculated from the publication GenBank 128.0 [February 15, 2002], are reproduced below as Table 2. This table uses the nomenclature of mMRNA, and therefore, instead of thymine ( T) that is found in DNA, the table uses uracil (U) that is found in RNA.
    The Table has been adapted so that the frequencies are calculated for each amino acid, instead of being calculated for all 64 codons.
    TABLE 2 TABLE OF PREFERENTIAL USE OF CODON (CoDON USAGE) FOR
    GENES OF SACCHAROMYCES CEREVISIAE Amino Acid Codon Number Frequency by a to a A Fatal E | fotar - [| q “- P u = | mea o6BAa | ot mec ADE ZA ra | me - AUA | 11654 | 178 | rare At Fatal | And the | rat
    The being “= | you guys | 6550661 | ag | be = | AGC | 63726 | og straight Lo
    Codon Amino Acid Number Frequency per thousands 68203 [Pro ee | 4306 68 -) Ti9647 34597
    RR DT A 132522 83207 116084 52045 | 8 | rar "A 136358 82357 105910 40358
    BC TOO ATI T22728 965986
    BR TOTO 89007 50785 Ta Do A T76251 79121 Ba TO TO 233724 162199 Fa DA 273678 201361 Pa TOO IO AI 245641
    Codon Amino Acid Number Frequency per thousands 132048 rat 2079544 125717 rat 52908 31065 [| 48 Rr A CC EE 67789 aa amo 6a 16993 19562 11357 139081 60289 rata 156108 ess | 8 | 71216 39369 | 60 aa 6973 3312 aaa By using this table or similar tables, a person skilled in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment from a codon-optimized coding region that encodes the polypeptide, but that uses optimal codons for a given species.
    i 46 The random assignment of codons with a frequency optimized to encode a given polypeptide sequence, can be done manually by calculating the codon frequencies for each amino acid, and then assigning the codons to the - polypeptide at random. In addition, various computer software algorithms and software are readily available to those skilled in the art. For example, the “EditSeq” function in the Lasergene program package, available from DNAstar, Inc., Madison, WI, the “backtranslation” function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, MD, and “backtranslation” function in the GCG - Wisconsin package, available from Accelrys, Inc., San Diego, CA. In addition, several resources are publicly available to perform codon-sequence optimization of the coding region, for example, the “backtranslation” function on the page “http: /MWww.entelechonxorn/bioinformatics/backtranslation.php lang = eng" (visited on April 15, 2008) and the “backtranseq” function available on the page “http: //bioinfo.pbi.nrc. ca: 8090 / EMBOSS / index.html ”. The construction of a rudimentary algorithm to assign codons based on a given frequency can also be easily accomplished with basic mathematical functions by a technician skilled in the subject.
    Codon-optimized coding regions can be designed by various methods known to those skilled in the art, including software packages such as the “synthetic gene designer” (http://phenotype.biosci.urnbc.edu/codon/sgd/index.php ).
    Standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and are described by Sambrook, et al. (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Edition; Cold Spring Harbor Laboratory Laboratory Press: Cold Spring Harbor, NY (1989) (above as “Maniatis”)); and by Silhavy et al. (Silhavy et al ., Experiments with Gene Fusions; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and by Ausubel, FM et al., (Ausubel, et al. Current Protocols in Molecular Biology, published by Greene Publishing Assoc. And Wiley-Interscience, 1987) ALCOHOL DEHYDROGENASE (ADH) ENZYMES Alcohol dehydrogenases (ADH) are a broad class of enzymes that catalyze the interconversion of aldehydes into alcohols as part of several pathways in the cell medium. in several families based on the length of the amino acid sequence or type of metal cofactors they use.
    More than 150 structures are available in the Protein Data Bank (PDB) protein database for a variety of ADH enzymes. The enzymes are highly divergent and different ADHs exist as oligomers with different subunit compositions. Figures 4 shows the relationship phylogenetics of oxidoreductase enzymes in Saccharomyces cerevisiae, Escherichia coli Homo sapiens, C. elegans, Drosophila melanogaster, and Arabidopsis thaliana which are related to horse liver ADH and Achromobacter xylosoxidans SadB.
    Figure 5 shows the phylogenetic relationship of specific sequences of the ADH enzyme most closely related to the SadB of Achromobacter xylosoxidans by the sequence.
    In one embodiment, ADH enzymes suitable for use in the present invention have a higher Kcat for converting lower alkylaldehyde to a corresponding lower alkyl alcohol. In another embodiment, ADH enzymes suitable for use have a very low Kcat for converting a lower alkyl alcohol to a corresponding lower alkylaldehyde. In another embodiment, ADH enzymes suitable for use have a low Kyv for lower alkylaldehydes. In another embodiment, suitable ADH enzymes have a high Ky for lower alkyl alcohols. In another embodiment, suitable ADH enzymes preferably use NADH as a cofactor during the reduction reactions. In another embodiment, suitable ADH enzymes have one or more of the following characteristics: a very high Kcat for converting a lower alkylaldehyde to a corresponding lower alkyl alcohol; a very low Kcat for converting a lower alkyl alcohol to a corresponding lower alkylaldehyde; a low Km for lower alkylaldehydes; a high Km for lower alkyl alcohols; and the preferential use of NADH as a cofactor during reduction reactions. In another embodiment, suitable ADH enzymes have a high for lower alkyl alcohols. In another embodiment, suitable ADH enzymes have two or more of the above characteristics.
    In one embodiment, ADH enzymes suitable for use in the present invention oxidize cofactors in the presence and absence of lower alkyl alcohol more quickly when compared to control polypeptides. In one example, the control polypeptide is the SadB of Achromobacter xylosoxidans having the amino acid sequence of SEQ ID NO: 26.
    In another embodiment, suitable ADH enzymes have a Ku for lower alkylaldehyde which is less when compared to that of a control polypeptide. In another embodiment, suitable ADH enzymes have a Km for a lower alkylaldehyde which is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60 %, 70%, 80%, or 90% lower when compared to that of a control polypeptide. In one embodiment, the control polypeptide is the SadB of Achromobacter xylosoxidans having the amino acid sequence of SEQ ID NO: 26. In one embodiment, the lower alkylaldehyde is isobutyraldehyde.
    In another example, suitable ADH enzymes have one for an alcohol! lower alkyl which is greater when compared to that of a control polypeptide. In another example, ADH enzymes — suitable for use with lower alkyl alcohol that is at least about 10%, 50%, 100%, 200%, 300%, 400%, or 500% higher when compared to a control polypeptide. In one embodiment, the control polypeptide is the SadB of Achromobacter xylosoxidans having the amino acid sequence of SEQ ID NO: 26. In one embodiment, the lower alkyl alcohol is isobutanol.
    In another embodiment, suitable ADH enzymes have a kca / Km for a lower alkaldehyde which is greater when compared to that of a control polypeptide. In another example, suitable ADH enzymes have a ka / Km that is at least about 10%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 800% or 1000% higher when compared to that of a control polypeptide. In one embodiment, the control polypeptide is the SadB of Achromobacter xylosoxidans having the amino acid sequence of SEQ ID NO: 26. In one embodiment, the lower alkylaldehyde is isobutyraldehyde.
    In another embodiment, suitable ADH enzymes have two or more of the above characteristics. In another embodiment, suitable ADH enzymes have three or more of the above characteristics. In another embodiment, suitable ADH enzymes have all four characteristics above. In one embodiment, suitable ADH enzymes use NADH as a cofactor.
    In one embodiment, ADH enzymes suitable for use in the present invention catalyze reduction reactions optimally in the physiological conditions of the host cell. In another example of realization,
    ADH enzymes suitable for use in the present invention optimally catalyze reduction reactions at a pH of about 4 to about 9. In another embodiment, ADH enzymes suitable for use in the present invention catalyze reduction reactions optimally at a pH of about 6 to 9. about 7. In another embodiment, ADH enzymes suitable for use in the present invention catalyze reduction reactions optimally at a pH of about 6.5 to about 7. In another example, ADH enzymes suitable for use in the present invention invention catalyze reduction reactions optimally at a pH of about 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5 or 9. In another example, ADH enzymes suitable for use in the present invention catalyze reduction reactions optimally at approximately pH 7.
    In one embodiment, ADH enzymes suitable for use in the present invention catalyze reduction reactions optimally at up to about 70 ° C. In another example, suitable ADH enzymes — catalyze reduction reactions optimally at about 10 ºC, 15 ºC, 20 ºC, 25 ºC, 30 ºC, 35 ºC, 40 ºC, 45 ºC, 50 ºC, 55 ºC, 60 ºC, 65 ºC, or 70 ºC. In another embodiment, suitable ADH enzymes optimally catalyze reduction reactions at about 30 ° C.
    In one embodiment, ADH enzymes suitable for use in the present invention catalyze the conversion of an aldehyde to an alcohol in the presence of a lower alkyl alcohol, at a concentration of up to about 50 g / L. In another embodiment, suitable ADH enzymes catalyze the conversion of an aldehyde to an alcohol in the presence of an alkyl alcohol below a concentration of at least about 10 g / L, 15 g / L, 20 g / L, 250gL , 30 gL, 35 g / L, 40 g / L, 45 g / L or 50 g / L. In another embodiment, suitable ADH enzymes catalyze the conversion of an aldehyde to an alcohol in the presence of an alkyl alcohol of less than a concentration of at least about 20 g / L. In some embodiments, the lower alkyl alcohol is isobutanol.
    In some embodiments, the lower alkylaldehyde is isobutyraldehyde and alcohol! lower alkyl is isobutanol.
    RECOMBINANT HOSTING CELLS FOR EXPRESSION OF ADH ENZYME One aspect of the present invention is directed to recombinant host cells that express ADH enzymes having the activities described above.
    Non-limiting examples of host cells for use in the present invention include bacteria, cyanobacteria, filamentous fungi and yeasts.
    In one embodiment, the recombinant host cell of the invention is a bacterial cell or a cyanobacterial cell.
    In another example, the recombinant host cell is selected from the group consisting of: Salmonella, Arthrobacter, Bacillus, Brevibacterium, Clostridium, “Corynebacterium, Gluconobacter, Nocardia, Pseudomonas, —Rhodococcus, Streptomyces, Zymomonas, Escherichia, —Lacer Enterococcus, Alcaligenes, Klebsiella, Serratia, Shigella, Alcaligenes, Envinia, Paenibacillus and Xanthomonas.
    In some examples, the recombinant host cell is E. coli, S. cerevisiae or L. plantarum.
    In another embodiment, the recombinant host cell of the invention is a filamentous fungus cell or a yeast cell.
    In another example of the embodiment, the recombinant host cell is selected from the group consisting of: Saccharomyces, Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zygosaccharomyces, Galactomyces, Schizosaccharomyces, Torulaspora, Debayisecka , Metschnikowia, Issatchenkia and Candida.
    In one embodiment, the recombinant host cell of the present invention produces a lower alkyl alcohol with a yield greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, or> 90% of the theoretical value. In one embodiment, the recombinant host cell of the present invention produces a lower alkyl alcohol with a yield greater than about 25% of the theoretical value. In another embodiment, the recombinant host cell of the present invention | produces a lower alkyl alcohol with a yield greater than about 40% of the theoretical value. In another embodiment, the recombinant host cell of the present invention produces a lower alkyl alcohol with a yield greater than about 50% of the theoretical value. In another embodiment, the recombinant host cell of the present invention produces a lower alkyl alcohol with a yield greater than about 75% of the theoretical value. In another embodiment, the recombinant host cell of the present invention produces a lower alkyl alcohol with a yield greater than about 90% of the theoretical value. In some embodiments, the lower alkyl alcohol is butanol. In some embodiments, the lower alkyl alcohol is isobutanol.
    Non-limiting examples of lower alkyl alcohols produced by the recombinant host cells of the present invention include butanol, propanol, isopropanol and ethanol. In an example of an embodiment, the recombinant host cells of the present invention produce isobutanol. In another embodiment, the recombinant host cells of the present invention do not produce ethanol.
    US Publication 2007/0092957 A1 discloses the engineering of microorganisms - recombinants for the production of isobutanol (2-methylpropan-1-ol). US Publication 2008/0182308 A1 discloses the engineering of microorganisms - recombinants for the production of 1-butanol. US Publications 2007/0259410 A1 and 2007/0292927 A1 disclose the engineering of recombinant microorganisms for the production of 2-butanol. Several pathways have been described for the biosynthesis of isobutanol and 2-butanol.
    The last step in all the pathways described for the three products is the reduction of a more oxidized fraction of the alcohol fraction by an enzyme with butanol dehydrogenase activity.
    The methods disclosed in these publications can be used - to develop the recombinant host cells of the present invention.
    The information presented in these publications is fully incorporated by reference.
    In exemplary embodiments, the recombinant microbial host cell produces isobutanol.
    In exemplary embodiments, the recombinant microbial host cell comprises at least two heterologous polynucleotides that encode enzymes that catalyze the conversion of a substrate into product selected from the group consisting of: pyruvate to acetolactate; acetolactate for 2,3-dihydroxyisovalerate; 2,3-dihydroxyisovalerate for alpha-ketoisovalerate; alpha-ketoisovalerate - for —isobutyraldehyde, and isobutyraldehyde for isobutanol.
    In exemplary embodiments, the recombinant microbial host cell comprises at least three heterologous polynucleotides that encode enzymes that catalyze the conversion of a substrate into product selected from the group consisting of: pyruvate to acetolactate; acetolactate for 2,3-dihydroxyisovalerate; 2,3-dihydroxyisovalerate for alpha-ketoisovalerate; alpha-ketoisovalerate - for isobutyraldehyde, and isobutyraldehyde for isobutanol.
    In exemplary embodiments, the recombinant microbial host cell comprises at least four heterologous polynucleotides that encode enzymes that catalyze the conversion of a substrate into product selected from the group consisting of: pyruvate to acetolactate; acetolactate for 2,3-dihydroxyisovalerate; 2,3-dihydroxyisovalerate for alpha-ketoisovalerate; alpha-ketoisovalerate for isobutyraldehyde, and isobutyraldehyde for isobutanol.
    In exemplary embodiments, the recombinant microbial host cell comprises heterologous polynucleotides that encode enzymes that catalyze the conversion of pyruvate to acetolactate; from acetolactate to 2,3-dihydroxyisovalerate; from 2,3-dihydroxyisovalerate to alpha-ketoisovalerate; Aalpha-ketoisovalerate for isobutyraldehyde, and isobutyraldehyde for isobutanol. In exemplary embodiments, (a) the polypeptide that catalyzes a conversion of substrate into pyruvate to acetolactate product is acetolactate synthase which has the EC number 2.2.1.6, (b) the polypeptide that catalyzes the conversion of a substrate to product of acetolactate for 2,3-dihydroxyisovalerate is acetohydroxy acid isomeroreductase having the EC number 1.1.186, (c) the polypeptide that catalyzes the conversion of a substrate into 2,3-dihydroxyisovalerate product for alpha-ketoisovalerate is the acetohydroxy acid dehydratase having the EC number 4.2.1.9, and (d) the polypeptide that catalyzes the conversion of a substrate into an alpha-ketoisovalerate product to isobutyraldehyde is the branched-chain alpha-keto acid decarboxylase having the EC number
    411.72.
    In exemplary embodiments, the recombinant microbial host cell additionally comprises at least one heterologous polynucleotide that encodes an enzyme that catalyzes the conversion of a substrate into a product selected from the group consisting of: pyruvate to alpha-acetolactate, alpha-acetolactate to acetoin, acetoin to 2,3-butanediol; 2,3-butanediol to 2-butanone; and 2-butanone for 2-butanol, and wherein said microbial host cell produces 2-butanol. In exemplary embodiments, (a) the polypeptide that catalyzes the conversion of substrate into pyruvate to acetolactate product is acetolactate synthase having the EC number 2.2.1.6; (b) the polypeptide that catalyzes the conversion of substrate into acetolactate to acetoin product is acetolactate decarboxylase which has the EC number 4.1.1.5; (c) the polypeptide that catalyzes the conversion of substrate in acetoin product to 2,3-butanediol is butanediol dehydrogenase which has the number EC 1.1.1.76 or EC 1.1.1.4; (d) the polypeptide that catalyzes the conversion of substrate in butanediol product to 2-butanone is butanediol dehydratase which has the EC number 4.2.1.28. In exemplary embodiments, (e) the polypeptide that catalyzes the conversion of substrate into 2-butanone product to 2-butanol is 2-butanol dehydrogenase which has the EC number 1.1.1.1.
    In exemplary embodiments, the recombinant microbial host cell further comprises at least one heterologous polynucleotide that encodes an enzyme that catalyzes the conversion of a substrate into a product selected from the group consisting of: acetyl-CoA to acetoacetyl-CoA; acetoacetyl-CoA for 3-hydroxybutyryl-CoA; 3-hydroxybutyryl-CoA for crotonyl-CoA; crotonyl-CoA for butyryl-CoA; butyryl-CoA for butyraldehyde, and butyraldehyde for 1-butanol; and wherein said microbial host cell produces 1-butanol. In exemplary embodiments, (a) the polypeptide that catalyzes the conversion of substrate into product from acetyl-CoA to acetoacetyl-CoA is acetyl-CoA acetyltransferase which has the number EC 2.3.1.9 or EC 2.3.1.16; (b) the polypeptide that catalyzes the conversion of substrate in product from acetoaceti-CoA to 3-hydroxybutyrllCoA is 3-hydroxybutyryl-CoA-dehydrogenase which has the EC number 1.1.1.35, 1.1.1.30, 1.1.1.157, or
    1.1.1.36; (c) the polypeptide that catalyzes the conversion of substrate into a product of 3-hydroxybutyryl-CoA to crotonyl-CoA is the crotonase that has the EC number 4.2.1.17 or 4.2.1.55; (d) the polypeptide that catalyzes the conversion of substrate in a crotonyl-CoA product to butyrill-COA is butyrylCoA dehydrogenase which has the EC number 1.3.1.44 or 1.3.1.38; (e) the polypeptide that catalyzes the conversion of substrate in a product from butiri-CoA to butyrylaldehyde is butyraldehyde dehydrogenase which has the EC number 1.2.1.57. In exemplary embodiments, (f) the polypeptide that catalyzes the conversion of substrate in butyrylaldehyde product to 1-butanol is 1-butanol dehydrogenase which has the EC number 1.1.1.1.
    In some exemplary embodiments, the recombinant microbial host cell further comprises at least one modification that improves the flow of carbon into the isobutanol pathway. In some exemplary embodiments, the recombinant microbial host cell further comprises at least one modification that improves the flow of carbon into the 1-butanol pathway. In some embodiments, the recombinant microbial host cell further comprises at least one modification that improves the flow of carbon into the 2-butanol pathway.
    METHODS OF PRODUCING LOWER ALKYL ALCOHOLS Another aspect of the present invention is directed to methods for the production of lower alkyl alcohols. These methods essentially employ the recombinant host cells of the present invention. In an example of an embodiment, the method of the present invention comprises; providing a recombinant host cell as discussed above; contacting the recombinant host cell with a fermentable carbon substrate in a fermentation medium under conditions in which lower alkyl alcohol is produced; and the recovery of lower alkyl alcohol.
    Carbon substrates may include, but are not limited to; monosaccharides (such as fructose, glucose, mannose, rhamnose, xylose or galactose), oligosaccharides (such as lactose, maltose or sucrose), polysaccharides, such as starch, maltodextrin or cellulose or mixtures of these and impure mixtures of raw materials renewable sources, such as cheese whey permeate, corn steeping water, beet molasses, and barley malt. Other carbon substrates can include ethanol, lactate, succinate or glycerol.
    In addition, the carbon substrate can also be a substrate of a carbon such as carbon dioxide, or methanol so that the metabolic conversion to fundamental biochemical intermediates is demonstrated. In addition to substrates of one and two carbon (s) methylotrophic organisms are also known to use a variety of other carbon-containing compounds, such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to use carbon from methylamine to form trehalose or glycerol (Bellion et al, Microb. Growth CI Compa., [7th Int. Symp.], (1993), 415 32, Editors: Murrell , J. Collin; Kelly, Don P. iIntercept, —Andover, United Kingdom). Likewise, several species of Candida will metabolize alanine or oleic acid (Suiter et al. Arch. Microbiol. 755: 485-489 (1990)). Therefore, the possibility is contemplated that the carbon source used in the present invention may include a wide variety of substrates that contain carbon and the source used will only be limited by the “chosen organism”.
    While it is contemplated that all of the carbon substrates mentioned above and their mixtures are suitable for the present invention, the preferred carbon substrates are glucose, fructose and sucrose or mixtures of these with C5 sugars such as xylose and / or —abarabinosis for cells yeasts modified to use C5 sugars. Sucrose can be derived from renewable sources, such as sugar cane, beet, cassava, sweet sorghum, and mixtures of these. Glucose and dextrose can be derived from renewable grain sources from saccharification of starch-based raw materials, including grains such as corn, wheat, rye, - barley, oats and mixtures thereof. In addition, fermentable sugars can be derived from renewable sources of cellulosic or lignocellulosic biomass through pre-treatment and saccharification processes, as described, for example, in Published Patent Application US 2007 / 0031918A1,
    | 58 which is incorporated into the present by reference.
    Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, hemicellulose and, optionally, further comprising lignin, starch, oligosaccharides and / or monosaccharides.
    Biomass - may also include additional components, such as proteins and / or lipids.
    Biomass can be derived from a single source, or biomass can comprise a mixture of derivatives from more than one source, for example, biomass can comprise a mixture of corn cob and corn straw, or a mixture of grass and leaves .
    Biomass includes, but is not limited to, bioenergy, agricultural waste, urban solid waste, industrial solid waste, papermaking sludge, green waste, wood and forest waste.
    Examples of biomass include, but are not limited to, corn kernels, corn cobs, crop residues such as corn husks, corn husks, grasses, wheat, wheat straw, barley, barley straw, hay, straw rice, grass (Panicum virgatum), waste paper, sugarcane bagasse, sorghum, soy, components obtained from grinding grains, trees, branches, roots, leaves, wood chips, sawdust and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof.
    Carbon substrates can be supplied in any medium that is suitable for the growth and reproduction of the host cell.
    Non-limiting examples of means that can be used include; M122C, MOPS, SOB, TSY, YGM, YPD, 2XYT, LB, M17, or the M9 minimum medium. Other examples of media that can be used include solutions containing potassium phosphate and / or sodium phosphate.
    The appropriate media can be supplemented with NADH or NADPH.
    The fermentation conditions for the production of a lower alkyl alcohol may vary according to the host cell to be used.
    In one embodiment, the method for producing a lower alkyl alcohol is carried out under anaerobic conditions. In one embodiment, the method for producing a lower alkyl alcohol is performed under aerobic conditions. In one embodiment, the method for producing a lower alkyl alcohol is carried out under microaerobic conditions.
    In one example of an embodiment, the method for producing a lower alkyl alcohol results in a title of at least about 20 g / L of a lower alkyl alcohol. In another embodiment, the method for producing a lower alkyl alcohol results in a title of at least about 30 g / L of a lower alkyl alcohol. In another embodiment, the “method for producing a lower alkyl alcohol results in a titre of about 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L or 40 g / L of lower alkyl alcohol.
    Non-limiting examples of lower alkyl alcohols produced by the methods of the present invention include butanol, isobutanol, - propanol, isopropanol and ethanol. In one example, isobutane! Is Produced.
    In exemplary embodiments, isobutanol is produced. In exemplary embodiments, the method for producing isobutanol comprises: (a) providing a recombinant host cell comprising a heterologous polypeptide that catalyzes the conversion of substrate into isobutyraldehyde product to isobutanol and which has one or more of the following characteristics: () the Ky value of a lower alkylaldehyde is lower for the polypeptide when compared to that of a control polypeptide having —amino acid sequence of SEQ ID NO: 26; (ii) the K value of a lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26;
    (iii) the Kca / Km value of a lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; and (b) the contact of the host cell of item (a) with a substrate - carbon under conditions in which isobutane! Is Produced.
    In exemplary embodiments, 2-butanol is produced.
    In exemplary embodiments, the method for producing 2-butanol comprises: (a) providing a recombinant microbial host cell comprising a heterologous polypeptide that catalyzes the conversion of substrate in 2-butanone product to 2-butanol and which has one or more of the following characteristics: (i) the Kvw value for a lower alkylaldehyde is lower for the polypeptide when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; (ii) the value of a lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; and (iii) the K.a / Km value of a lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; and (b) contacting the host cell of item (a) with a carbon substrate under conditions where 2-butane! Is Produced.
    In exemplary embodiments, 1I-butanol is produced.
    In exemplary embodiments, the method for producing 1-butanol comprises: (a) providing a recombinant microbial host cell comprising a heterologous polypeptide that catalyzes the conversion of substrate in butyraldehyde product to 1-butanol and which has one or more of the following features:
    (i) the Kvw value for a lower alkylaldehyde is lower for the polypeptide when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; (ii) the value of a lower alkylaldehyde for the polypeptide is higher - when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; and (iii) the Kca / Km value of a lower alkylaldehyde for the polypeptide is greater when compared to that of a control polypeptide having the amino acid sequence of SEQ ID NO: 26; and (b) contacting the host cell of item (a) with a carbon substrate under conditions in which 1-butanol is produced.
    BIOSYNTHETIC PATHWAYS Host = production recombinant microbials expressing a 1-butanol biosynthetic pathway (Donaldson et al, US Patent Application 2008 / 0182308A1, incorporated herein by reference), a 2-butanol biosynthetic pathway (Donaldson et a /. , US Patent Application Publication 2007 / 0259410A1 and US 2007/0292927, and US 2009/0155870, all incorporated herein by reference), and an isobutanol biosynthetic pathway (Maggio-Hall et al, US Patent Application publication 2007 / 0092957, incorporated herein by reference) have been described in the state of the art. Certain suitable proteins having the ability to catalyze the conversions of substrate indicated in products are described in these publications and other suitable proteins are described in the prior art. The person skilled in the art will understand that polypeptides - which have the activity of such steps in the pathway can be isolated from a variety of sources and can be used in a recombinant host cell disclosed in the present invention. For example, US Patent Application Publications 20080261230 and US20090163376, US20100197519, and US Application No. 12/893077 describe acetohydroxy acid isomeroreductases; Publications US20070092957 and US20100081154, describe suitable dihydroxyacid dehydratases.
    Equipped with the present disclosure, a person skilled in the art will be able to use publicly available sequences to construct relevant pathways in the host cells provided by the present invention.
    In addition, one skilled in the art with the present disclosure will understand other suitable routes of isobutanol, 1-butanol or 2-butanol.
    ISOBUTANOL BIO-SYNTHETIC PATH Isobutanol can be produced from carbohydrate sources with recombinant microorganisms through the various biosynthetic pathways.
    Suitable pathways for converting pyruvate to isobutane! L include the four complete reaction pathways shown in Figure 6. A suitable isobutanol pathway (Figure 6, steps from (a) to (e)), comprises the following substrate conversions in product: a) pyruvate in acetolactate, catalyzed, for example, by the enzyme acetolactate synthase; b) acetolactate in 2,3-dihydroxyisovalerate, catalyzed, for example, by the enzyme acetohydroxy acid isomeroredutase; c) 2,3-dihydroxyisovalerate in α-ketoisovalerate, catalyzed, for example, by the enzyme acetohydroxy acid dehydratase; d) a-ketoisovalerate in isobutyraldehyde, catalyzed, for example, by the branched-chain keto acid decarboxylase enzyme; and e) isobutyraldehyde in isobutanol, catalyzed, for example, by a branched chain alcohol dehydrogenase enzyme.
    Another suitable route for converting pyruvate to isobutanol comprises the following conversions from substrate to product (Figure 6,
    steps a, b, c, f, g, e): a) pyruvate in acetolactate, catalyzed, for example, by the enzyme acetolactate synthase; b) acetolactate in 2,3-dihydroxyisovalerate, catalyzed, for example, by the enzyme acetohydroxy acid isomeroredutase; c) 2,3-dihydroxyisovalerate in α-ketoisovalerate, catalyzed, for example, by the enzyme acetohydroxy acid dehydratase; f) a-ketoisovalerate in isobutyryl-CoA, catalyzed, for example, by the branched-chain keto acid decarboxylase enzyme, g) isobutyri-CoA in isobutyraldehyde, catalyzed, for example, by an acylating aldehyde dehydrogenase enzyme, and e) isobutyraldehyde in isobutyraldehyde, in isobutyraldehyde, in isobutyraldehyde catalyzed, for example, by a branched-chain alcohol dehydrogenase enzyme.
    The first three stages of this route (a, b, c) are the same as those described above.
    Another suitable route for converting pyruvate to isobutane! comprises the following conversions from substrate to product (Figure 6, steps a, by c, h, ij, e): a) pyruvate to acetolactate, catalyzed, for example, by the enzyme — acetolactate synthase; b) acetolactate in 2,3-dihydroxyisovalerate, catalyzed, for example, by the enzyme acetohydroxy acid isomeroredutase; c) 2,3-dihydroxyisovalerate in α-ketoisovalerate, catalyzed, for example, by the enzyme acetohydroxy acid dehydratase; h) valine a-ketoisovalerate, catalyzed, for example, by the enzyme valine dehydrogenase or transaminase, i) valine in isobutylamine, catalyzed, for example, by the enzyme valine decarboxylase,
    j) isobutyralamine in isobutyraldehyde, catalyzed, for example, by the enzyme omega transaminase, and e) isobutyraldehyde in isobutanol, catalyzed, for example, by a branched chain alcohol dehydrogenase enzyme.
    The first three stages of this route (a, b, c) are the same as those described above.
    A suitable fourth isobutanol biosynthetic pathway comprises the substrate-to-product conversions shown as steps k, g, and in Figure 6.
    BIO-SYNTHETIC PATHWAY OF 1-BUUTANOL An example of a suitable biosynthetic pathway for the production of 1-butanol is that disclosed in the publication of US Patent Application 2008/0182308 A1. As disclosed in this publication, the steps in the 1-butane biosynthetic pathway! disclosed include the conversion of: aceti-CoA to acetoacetyl-CoA, catalyzed, for example, by the enzyme acetyl-CoA acetyltransferase; - acetoacetyl-CoA in 3-hydroxybutyryl-CoA, catalyzed, for example, by the enzyme 3-hydroxybutyri-CoA dehydrogenase; - 3-hydroxybutyri-CoA in crotonyl-CoA, catalyzed, for example, by the enzyme crotonase; - crotoni-CoA in butiri-CoA, catalyzed, for example, by the enzyme butyryl-CoA dehydrogenase; - butiri-CoA in butyraldehyde, catalyzed, for example, by the enzyme butyraldehyde dehydrogenase; and - butyraldehyde in 1-butanol, catalyzed, for example, by the enzyme butanol dehydrogenase. 2-BUTANOL BIO-SYNTHETIC ROUTE An example of a suitable biosynthetic pathway for the production of 2-butanol is described by Donaldson et al. in Patent Application Publications US 20070259410A1 and US 20070292927A1, and in PCT publication - WO 2007/130521, which are hereby incorporated by reference. A suitable 2-butanol biosynthetic pathway comprises the following conversions from - substrate to product: a) pyruvate to alpha-acetolactate, which can be catalyzed, for example, by acetolactate synthase; b) alpha-acetolactate for acetoin, which can be catalyzed, for example, by acetolactate decarboxylase; c) acetoin to 2,3-butanediol, which can be catalyzed, for example, by butanediol dehydrogenase; d) 2,3-butanediol to 2-butanone, which can be catalyzed, for example, by butanediol dehydratase; and e) 2-butanone to 2-butanol, which can be catalyzed, for example, by 2-butanol dehydrogenase.
    ADDITIONAL MODIFICATIONS Additional modifications that may be useful for the cells provided in the present invention include modifications to reduce the activity of pyruvate decarboxylase and / or glycerol-3-phosphate dehydrogenase, as described in US Patent Application Publication 20090305363 (incorporated herein by reference) , modifications to a host cell that provides an increased carbon flow through an Entner-Doudoroff pathway or a reduction in the balance of equivalents, as described in US Patent Application Publication 20100120105 (incorporated herein by reference). Yeast strains with increased activity of heterologous proteins that require the binding of an Fe-S center for their activity are described in US Patent Application Publication 20100081179 (incorporated herein by reference). Other modifications include modifications to an endogenous polynucleotide that encodes a polypeptide having a dual role in hexokinase activity, described in Provisional Application US 61/290639, and the integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a biosynthetic pathway that uses the pyruvate described in Provisional Application US 61/380563 (both fully incorporated into the present invention by reference). Additional modifications that may be suitable for the exemplary embodiments of the present invention are described in US Application 12/893089. In addition, host cells comprising at least one deletion, mutation and / or replacement of an endogenous gene that encodes a polypeptide that affects the biosynthesis of Fe-S centers are described in Provisional Patent Application US 61/305333 (incorporated herein by reference), and host cells comprising a heterologous polynucleotide encoding a polypeptide with phosphocetolase activity and host cells comprising a heterologous polynucleotide encoding a polypeptide with phosphotransacetylase activity are described in Provisional Patent Application US 61/356379. IDENTIFICATION AND ISOLATION OF ADH ENZYMES WITH HIGH ACTIVITY The present invention is directed to the elaboration of a strategy to identify several ADH enzymes with superior properties in relation to the conversion of isobutyraldehyde into isobutanol in a host organism that was developed by genetic engineering for the production of isobutanol.
    The process of selecting the candidate ADH involves researching among naturally occurring enzymes.
    Enzymes are identified based on their natural propensity to use aldehydes as powdered substrates and convert them to the respective alcohols with a reasonably high Kcat and / or low Kyw values for the corresponding aldehyde substrates, as documented by examples in the literature.
    Once a set of candidates is identified, the strategy involves using this set to isolate closely related counterparts through bioinformatics analysis. Therefore, in one embodiment, the method of screening the invention 'comprises conducting a search using bioinformatics or literature for candidate ADH enzymes. In one example, the search for bioinformatics uses a phylogenetic analysis.
    The DNA sequences encoding protein from the candidate genes are either amplified directly from host organisms or acquired as synthetic codon-optimized genes for expression in a host cell, such as E. coli. The various candidate ADH enzymes used in the present invention are listed in Table 3. TABLE 3 SEQ ID NO of | SEQ ID NO of Gene; ; :; polynucleotide: | polypeptide: ADH6 of liver of ADH6 of Sacch: cerevisiae ADH7 of Saccharomyces 3 23 cerevisiae BdhA of Clostridium 4 24 acetobutylicum BdhB of Clostridium 5 25 acetobutylicum SadB of Achromobacter 26 xylosoxidans ARD of Bos taurus ADHB of Rana perzez | = - = - = 8 | 28 | ADH of Clostridium 29 beijerinckii ADH1 of Entamoeba 10 30 histolytica
    Gene SEQ ID NO of | Polynucleotide SEQ ID NO: | polypeptide: Refills HA nonvegiois pH oe Themus * ”I love 'Phenylobacterium 14 ADH 34 zucineum HLK1 ADH of Methyloceclla 1 silvestris BL2 ADH of Acinetobacter 16 baumannii YE = | ss' ADH of Geobacillus sp.
    ADH of Vanderwaltozyma 18 38 polyspora DSM 70294 ADH of HDI of Mucor 19 39 circinelloides ADH of Rhodococeus 20 to erythropolis PR4 The present invention is not limited to the ADH enzymes listed in Table 3. Additional candidate enzymes can be identified based on the sequence homology for these candidates, or candidate enzymes, can be derived from these sequences through —mutagenesis and / or protein evolution.
    Suitable ADH enzymes include ADH enzymes having at least about 95% identity with the sequences provided in the present invention.
    Tables 4 and 5 provide polynucleotide sequences (codon-optimized for expression in E. coli except for SEQ ID NOS. 2,3,4,5e6) and polypeptide sequences of the candidate ADH enzymes shown in Table 3, respectively.
    TABLE 4 [DEFOLINUCLEOTIDE SEQUENCE SEGMENT | | atgtcaacagccggtaaagttattaagtgtaaageggcagttttataggaagagaaaaag cegtttagcatagaagaagtagaagtagegecaccaaaageacacgaggttagaatca agatgagttgccaceggaatetgtagatcegacgaccatgtggtgagtggcactctagttact coetttgccagtaategegggacacgaggctgceggaategttgaatccataggtaaaggta ttaccactgttcgtectagtgataaagtgatccecactatticactccteaatatagtaagtataga gtctgcaaacatcctgaggagtaatttctaccttaaaaatgatttatcetatgcctagagagtactat gcaggatggtacaagcagatttacatgcagagggaaacctatacaccatttccttagtactt ctacattttcccaatacacagtggtggacgagatatctategctaaaategatgcagettcac 1 cactggaaaaagtttacttgatagggtacagattttecacegattacgagtticegcagttaaag ttgcaaaggttacacagggttegacttgtgcagtatteggtttaggaggagtaggactaage gttattatagagtgtaaagctgcaggegcagegaggattataggtatagacatcaataago acaaatttgcaaaagctaaggaggteggggctactgaatgtattaaccctceaagattataa gaaaccaatacaagaagtccttactgaaatgtcaaacagtggagttgatttctetitigaagtt ataggccgtettgatactatggtaactgcagttgtectactateaagaggcatatagagtcagt gtgatcgtaggtattectectgattcacaaaatttategatgaatcctatactattgctaagegg tcgtacatggaagggagctatatttagegattttaag agcaaggatagtgattccaaaacttat tgcegactttatagegaagaagtttactcttgatcctttaattacacatgatattgccatticgaga aaatcaatgaagggtttaatttattaagaagtggtaaatctattcgatacaattttaactttttaa atgatcttatoctgagaaatttaaaggtatcgctaticaatcacacgaagattagaaaaacec aaagaagacaaagtatgacccaaaaccattttacgatcatgacattgacattaagatega agcatgatggtatetacgagtagtgatattcattatacagetagteattggageaatatgaagatg cegcetagtegttiggtcatgaaatecgttggtaaagttateaagetagggcccaagtcaaacag tgggttgaaagteggteaacatattggtatagatactcaagtettttcatgcttggaatgtaace gttgtaagaatgataatgaaccatactgcaccaagtttattaccacatacagtcagccttato aagacggctatatgtegcaggagtagetatgcaaactacgtcagagttcatgaacattttgta gtgcctatcccagagaatattccateacatttagetactecactattatatagtagtttaactata tactctecattggttegtaacggttgcggtecagataaaaaagttggtatagttggtcttagtag 2 ttatcggcagtatgggtacattgatttccaaagecataggggcagagacgtatattatttetegt tcttegagaaaaagagaagatgcaatgaagatgggcgcegatcactacattgctacatta gaagaaggtgattagggtgaaaagtactttgacaccttegacctgaattgtagtctatacttcet cocttacegacattgacttcaacattataccaaaggctatgaagattggtagtagaa ttgtcte aatctctataccagaacaacacgaaatgttategctaaagecatatggcttaaaggctatect ccatttcttacagtactttaggticcatcaaagaattigaaccaactctigaaattagtctetgaa aaagatatcaaaatttaggtggaaacattacctgattggtaaagecggegtecatgaagectt cgaaaggatggaaaaggagigacgttagatatagatttaccttagteggctacgacaaaga attttcagactag atgctttacccagaaaaattteaggacatcegatatitecaacgcaaaggattagaageatec 3 taaattagtgagttttyacccaaaaccctitagegatcatgacgttgatatigaaattgaagec tatagtatctacgagatoetgatttteatatagcegttagtaatiggggtecagteccagaaaate
    [sEaDNO SEQUENCIA DEFOLINUCKEOTIDEOS = | aaatccttggacatgaaataatiggcegegtagtyaagattggatccaagigecacactgg ggtaaaaatcagigaccatattggtattggtacccaagecttggeagtattttyagtgtgaacat tgcaaaagigacaacgagcaatactgtaccaatgaccacgttttgactatgtagactectta caaggacggctacattttcacaaggaggctttgceteccacgatgaggacttcatgaacacttta ctattcaaataccagaaaatattccaagtcegetagecgcetecattattgtatagtagtattac agttttetetecactactaagaaatggctatagtecaggtaagaggagtagatattagttggeatc ggtgagtattaggcatatagggattctattggctaaagetataggagecgagatttatgcatttto gcgaggccactecaageggagaggattctatgaaactegatactgatcactatattgctatatt ggaggataaaggctggacagaacaatactctaacgctttagaccttcttategtttacteate atctttategaaagttaattttgacagtatcgttaagattatgaagattggaggctecategttte aattgctgctcectgaagttaatgaaaagcttgttttaaaacegttgggcctaataggagtatca atctcaagcagtgctatcggatcetaggaaggaaatcegaacaactattgaaattagtttccga aaagaatgtcaaaatatagatagaaaaacttcegatcagegaagaaggegteagecatg cctttacaaggatggaaageggagacgtcraaatacagatttactttagtcegattatgataag aaattccataaatag atgctaagttttgattaticaataccaactaaagttttttttyggaaaaggaaaaatagacgtaat tggagaag aaattaagaaatatggctcaagagtocttatagtttatagcggaggaagtata aaaaggaacggtatatatgatagagcaacagctatattaaaagaaaacaatatagotttct atgaactttcaggagtagagccaaatcctaggataacaacagtaaaaaaaggcatagaa atatgtagagaaaataatgtagatttagtattagcaatagggagaggaaagitgcaatagact gttctaaggtaattgcagctagagttitattataatagegatacatgggacatagttaaagatco atctaaaataactaaagttcttecaattgcaagtatacttactctttecagcaacagggtetgaa atggatcaaattgcagtaattticaaatatagagactaatgaaaagcttiggagtaggacatg atgatatgagacctaaattttcagtattagatcctacatatacttttacagtacctaaaaatcaa
    4 acagcagcgggaacagctgacattatgagtcacacctitgaatcttactttagtggtatigaa gatacttatatacaggacgagtatacgagaagcaatcttaagaacatgtataaagtatagaa aaatagcaatggagaagactgatgattacgaggctagagctaatttgatgtaggcttcaag tttagctataaatggtctattatcactiggtaaggatagaaaatagagttatcatectatagaac acgagttaagtgcatattatgatataacacatgagtataggactigcaattttaacacctaattg gatggaatatattctaaatgacgatacacttcataaatttgtttettatagaataaatgtttaggg aatagacaagaacaaagataactatgaaatagcacgagaggctattaaaaatacgaga gaatactttaattcattgggtattccticaaagettagagaagttggaataggaaaagataaa ctagaactaatggcaaagcaagctattagaaattctagaggaacaataggaagtttaaga ccaataaatgcagaggatgttcttgagatatttaaaaaatcttattaa atggttgatttegaatattcaataccaactagaattttttteggtaaagataagataaatgtactt ggaagagagcitaaaaaatatggttctaaagtacttatagtttatagtagaggaagtataaa gagaaatggaatatatgataaagctgtaagtatactigaaaaaaacagtattaaattttatga acttgcaggagtagagccaaatccaagagtaactacagttgaaaaaggagttaaaatato tagagaaaatggagttgaagtagtactagctataggtagaggaagtgcaatagatigege aaaggttatagcagcagcatgtgaatatgatagaaatccatgggatattgtattagatage t caaaaataaaaagggtgacttectatagctagtatattiaaccattgctacaacaggatcaga
    [SEGIDNO [SEQUENCE DEPOLINUCLEOTIDEOS - aatggatacgtgggcagtaataaataatataggatacaaacgaaaaactaatigcggcaca tccagatatggctectaagttttetatattagatecaacgtatacgtatacegtacctaccaate aaacagcagcaggaacagctgatattatgagtcatatatttgaggtgatattttagtaatacaa aaacagcatatttgcaggatagaatggcagaagcgttattaagaacttgtattaaatataga ggaatagctcttyagaagceggatgattataaggcaagagecaatctaatgtaggacticaa gtcttgcgataaatggacittitaacatatagtaaagacactaattggagtgtacacttaataga acatgaattaagtgcttattacgacataacacacggcatagggcttacaattttaacacctaa ttagatggagtatatittaaataatgatacagtgtacaagtttattgaatatggtataaatoattta gggaatagacaaagaaaaaaatcactatgacatagcacatcaagcaatacaaaaaac aagagattactttgtaaatgtactaggtttaccatctagactgagagatgatiggaatigaaga agaaaaattggacataatggcaaaggaatcagtaaagettacaggaggaaccatagga aacctaagaccagtaaacgcctecgaagitectacaaatattcaaaaaatctotataa atgaaagctctggtttateacggtgaccacaagatctegettyaagacaageccaagece acccettrcaaaageccacgagatgtagtagtacggattittuaagaccacgatctgcggeacg gatecteggcatcetacaaaggcaagaatccagaggtegecgacgggcgcatcetaggeca tgaaggggtaggc gtcategaggaagtaggcgagagtateacgcagttraagaaagge gacaaggtcctgatttectgcgteacticttacggctegtgegactactgcaagaagcagcttt actcccattgcegegacggegagtagatcectagattacatgategatagegtacaggeca aatacgtcegcatecegeatgcegacaacagectetacaagatececcagacaattigacga acgaaatcgccgtectgctgagegacatectacecaceggecacgaaateggcgtecagt atgggaatgtecagecgggegatgcagtagetatigteggegegggececgteggcatat cegtactgtigaccgeccagttetactececctegaccatcategtgategacatagacgag aategcectecagetegecaaggageteggggeaacgcacaccateaactecggeacgg agaacgttgtegaagcegtacataggattgcggcagagggagtegatgatigegatrIagga cggtgggcataccggcgacttgggacatctgccaggagategtrcaageceggegegeas atcgccaacgteggegtacatggegteaagattgacttcgagattcagaagetetggatea agaacctgacgatcaccacgggactagtgaacacgaacacgacgcccatactgatgaa ggtegectegacegacaagcettccgttgaagaagatgattacccategettegagetages gagatcgagcacgcctatcaggtattccteaatagegecaaggagaaggcegatgaagat catcctetegaacgcaggegctacetga atggcggegagctgacattttgctgcacacceggtcaaaagatgcegetgateggtetaggca cctggaaatctgacccaggateaagtgaaggcgagcaattaagtatgcgctgagecg tegatta tcgtcacattgactgcgeggcaatcetacggcaatgaaacegagattiggegaggcgtigaa agagaacgtcggtecgaggataagetagtecegegtgaagaactatttateacgagcaaget gtggaataccaagcaccacceggggga
    7 gatctgcagttggagtacttggatctgatatttaatacactggcegtatgcgtttuaacgeggta actctecgttecegaagaacgeegacggcaccateegttacgacagcactcattataaag aaacctggegtacactagaggacactagttacaaaagatetagtacatacectagatttaag caattttaattctegtcagatcgacgatgttctgagegtggectetatacatecggetatatige aggtcegagtateacccttatetagegeaaaacgagetgategcteattgteaagegegtaat ctggaagtgaccgcegtactececgetaggatagcagegacegegectagegtgatcegga
    [SEaiDNo JsEauêNCIA DEFOLINUELEOTÍDEOS MN agaacctattctactaaaagaaceggtegtgetagegetagetgaaaageacagtegeag cecagegceagatettgctycatiggcaagtteagegcaaagtttettacatecegaaatetat cacgcegagoccgtattetagagaacattcaagttttegacttcacetitagccecggaagaaa tgaagcagctggacgcccetgaacaagaatoetgacagtittattgtacegatatigaccatagac ggcaagegegttecgegtgacgegggteacecgttgtatecatttaacgatcegtactaatg the atgtgcacegecggtaaagatattacgtgtaaageggeggtegettgggagecgacataaa cegcetgtecetagaaacgatcacgagtigcacotecaaaagegcatgaggtacatattaaa atcctggegtetggeatetacagtagegacagcagegttctigaaagagatcatecegage aagttcccggtgyattctagateataaggeggtagacatagttaagagcateggtacaggeg ttacgtgcgtgaaacegggtgacaaggatgatccegetattegtacegcaatatagttcttate goegcatgtaaaagcagcaatagcaacttctgtaagaagaatgatataggegegaaaacg gatttgatagcagacatgaccagcecgttttacgtacegtagtaagecgatttataatctagta ggcaccagcacctttacggagtacacgagttgtagecgatategeggtegeaaagategac ccaaaagcececeegetggagagcetacetgateggttgtagttttacgacagattatagtacage g gattaacacggccaaagttacecetageageacetatacagtatttagectaggacagtatta ttteagegctattgtiggttigtaaagcagctggegceateccecegtattattagegttagtacteata aggataagttccegaaggcaategaactaggegcaactgagigcetgaatcegaaggac tatgacaaaccgatctatgaggttatttacgagaaaaccaatggeggtatagattacgeggt cgagtgtgcgagategtatigaaactatgatgaacgcattgcagtegacctattacagttetag cattactattatattigggtetagegagecegaacgagegtetagcegetagacecgttgtiget gctgacgggccgttcectgaaagatagcgtatttagegactttaaagatagaagaagttage cgtctggtagatgactacatgaagaagaagatcaatgttaatitectgagigagcaccaaact gacgctggatcagatcaacaaagegttegaattgctgagcageggteaaggcgtticatag cattatgatctactaatga atgaaaggtttcgctatattagatattaataagctgggtiggatigagaaagagegtecggte gcaggcagctatgatgcaategttegtecatiggcegttagecogtgcacgagegacattca tacggtattegagagtgcactaggatgacegtaagaacatgatcctagggtcatgagaccagtt ggtgaagttgategaagteggtagegaagtcaaagattttaaaccgggegacegtateateg ttecatgcacgacgccagattggegtagectagaggatacaggcagatttecagcageata gcaatggcatgctagctagetagaaattetetaatttraaggatggtatattcggtgaatattte cacgtgaacgacgctgacatgaacctggctatcctacegaaggatatgcegetagagaa cgcgagtgatgatcacagatatg atgactacgggtittecatagtacgagagctggeggacatee aaatgggtagcagcgtagitcgtcateggacateggcgctatagatetgatagacatigcagg cgcaaaactgcgegatgcagategtatcateggtatagatageegecctatetacatagag gecggcgaagttttacggtacgactgacattctgaactataagaacggtcacattgttgatca agtgatgaagctgaccaacgagtaaagacatagatcgacattateatagegagtagtagtteg gaaacgctgagccaggcagttagcatggtcaagecgggatagcattatcagcaatattaatt accacggtagcgagtgatacgactactgatcccacgatategagtgaggattgtagtatageaca caagaccattaaaggcggtetatacecaggtagtegtitacatacgagaaatgactgcgatgata tagttgtctataaccgtatigacctaagcaagetagtgacgcacatetatcacgagettigace
    [SEaiDNo [sequence DEPOLÍNUCLEÓTIDEOS atatcgaagaggcgttactactgatgaaggataaaccgaaggacctgattaaagegateg atgaagggcctggegatgctggatatcggtegtattggttggatigaaaagaaaatcccgg agtgeggcecactagatgcgttggtecgtecegetggegetggeceegigcaceagegaca cecacacegtgtagactagegcaateggaegacegicacgacatgattcigggtracgaag cggteggteagategtgaaggtagattecctagigaagegtetgaagattggegataaggt gatecgteceggegattacteeggactagggtaaagaagaaagecaacgtagttacccgat gcatageggtagtatactaggacagetagaagttctecaatttraaggacgagtatettttecga gatattecacgtgaacgaggeggatactaacctggcactgctacegegtgatatiaaacct gaagatgcggtcatgctgagegacatagtgaccacegactttcacggtacegaatiggeg aatattaaactgggtgataccgtatacattattagtateggeccagtgggtetgataagegta gcetggtgcgaateacetgagatacegategeatettegeggttggtagecgeaaacactatia tgatatcgctctagaatacggcgcgactgatattatcaattacaagaatagegacattatag agcaaattttygaaggcgacegatggtaaaggcatigacaagattattattgcagatagegat gttcatacgtttacacaagcggtcaagatgattaaaccgggtagecgatattggtaacgtgaa ttatctgggtgaaggcgataacattigacattcegegtagegaatagagtataggcatagate ataaacacatccacggt agtttaactectageggtegtatecgcatagaaaagttggetteg ctgattagcaccggcaaactggacaccagcaaactgattactcategtttegagggceetag agaaggtggaagatgccttigatgctgatyaagaacaagecggcagatctgattaagecg gttgtecgtattcactatgacgatgaagatacgttacactaatga atgaaagcactagtttaccgtggecctggecaaaagetagtagaagaacgtraaaagec ggagctgaaagagccaggegacgcgattgtgaaagtcaccaaaacgaccatctatagta cggacttgcacattctaaagggegatgtagegacgatataagecgggtegegtactagatca cgaaggtgtaggtattatigaaagegttagcagegacgttacegegttceaacegagtgato gcgtectgatctettatatttetagctatagcaagtgcagcettttategecgtagcatgtttagec actgtaccactagcgagctagattctgggtaatgagatigacggtacgcaggcagagtacat tegtagtecegeatacegacacctetetatategtaticcagegggtacagacgaagaggeg ctggtgatgctgagegatatcctgcegacegatttegagtgtagtatectgaatggtaaggtt "gegcectggcagcagegttgcgategttagegeaggccctategagtttagecacattgactgac ggcacagttctactetecagcagagattatcatgattgatetagacgacaacegectagges tggcgaagcaattecggegcaacgcegtacegttaatagcaceggtagtaacgcageagea gaggtcaagactetagacggagggccetyaggtattgacacggctatigaggctatiggcatce cggccaccttegagetatgcca gaacattgtagetecgggtageactatigegaatgtegge gttcacggttegaaagiggatctacatctagaatctetatagagccataatgtgactateacg acgcgtetggtagacacggcaacgacgcegatactactgaaaaccgtacaatctcataa actggaccegagcecegtetgateacecategttttagectagaccaaatectagatgcgtaca aaacgtttagtcaggeccgcaageacecaggegcetgaagattattateagcatagaggcgt aatga atgagcacecgcaggtaaagigattaaatgcaaagcagcagttetataggaacegcataa 12 accgtttaccattgaagatatigaagttgcacctcegaaagcacatgaagtgcgcattaaaa tagttacaaccggtatttategttctaatgatcatacagttageggatagcctatttacacegeta
    [SEGIDNO SEQUENCE DEFOLINUCLEOTIDEOS cetgcagttetagateatgaaggtacagatattatigaaagcattggtaaaggtattacctata ttaaaccgggtgataaagtgattccgctattttetecgacagtgtagtaaatategcatttgcaa acatccggaaagcaatcetatattgccagaccaaaaatctgacccagecgaaaggtacac tgctggatggcaccagocgttttagectategtagtaaaccgattcatcattttattageacocag cacctttagccagtataccgtggttgatgatatigccgtagcaaaaattigatgcageageac cgctggataaagtttgtctgattagittatagttttagcaceggttatagtagegcagttcaggtta caaaagttacaccgggytagcacctgtacagtttttagtetagatagtattagtctaagegttatt attggttgtaaaaccgcaggcgcageaaaaattatigccgatagatattaataaagataaattt gccaaagccaaagaactagatgcaaccgattgtattaatcegcaggattataccaaaceg attcaggaagttctacaggaaatgacegatggtagtatagattttagoctttaaagtgattggtc gtctggataccatgaccagegceactactgagctateatagegcatatagtattagegitattgt tagtattcctecgagegeacagagectgagegttaatecgatgagcctactactagategta cctggaaaggtacaatttttggtagetttaaaagcaaagatgcegttcegaaactagttaca gattttatagccaaaaaattteegetagaacegetgattaccecatgttctagcegtttyaaaaaa ttaatgaagcctttgatetactacatacaggataaaagcattegtaccgtact gaccttttaataa atgcgtgcagttgtatttyaaaacaaagaacgcgtagecattaaagaagttaacgeaceg cgtctgcagcatccgctggatgcactagttcgtaticatctagcagagtatttatagtagegatot gcatctgtatcatggtaaaattcegattctacctagtagcagttctaggatcatgaatttatiggtca gatigaagcagttagtgaagatattcaggatctacagectagtaattggattatiggtecattte atattgcatgtggcacctatecgtattategtegtcatcagtataatctatataaacgtagtggt gtttatagttatagtecgatatttagtaatetacagggtgcacaggcagaaattetagcgtattice gtttagcaatgtgaatctgegtaaactacctecgaatetatetecggaacgatacaatitttaco gatgaatattctygagcacegectatggtagtetgattcagggteagctgcgtectagtaatage
    13 gttgacagttattagtacagatecgagtigatetgatggeaattigaagttacacaggttctaggta caagcaaaattctggccattgatcegtatticoeggaacatetagaacgtacageaagectaga tgcaattcegattaatacegaacaggaaaatccggttegtcacattcatagegaaaccaat gatgaaggtccggatctggttetagaagecgtiggtggtacagcaaccctgagectageac tggaaatggttegtectggtagtegtattagegecagttagtattgataatacaccgagetttec gtttecgctagcaagegagtetagttaaagatctgacgtttegtatiggtetagcaaatgtacate tatatattgatacagttctagcactactagecageggtegtetacagecggaacatatigttag ccattatetgcegetagaagaageacctegeggttacgaactatitaategcaaagaagea ctgaaagttctactagttgtacatagttaataa atgaaagcactgagtttatggtagtccegggtcagaaaagectagaagategtcecgaaaceg gaactgcaggcaccgggtaatgcaattgttegtattgtaaaaaccaccatttatagcacega tctgcatattctaaaaggtgatatigcaacctatacacceggagtegtattctagatcatgaagat gttagtattatigatagecgttggtacagcagttaccgcatttegtecgggtgatcatgttctgatt
    14 agctgtattagcgcctatagtaaatatgattattgccgtegtagtatgtatagccattgtacaac cggtagatggattctggataatgaaatigatagcacccaggcagaatatgttcgtacaceg catgcagataccagcctgtatceggtteceggcaggegcagatgaagaggcactagttatg ctgagcgatattctacegacegattttgaatatagtatactigaatgagtaaagttgcaccgggt ggacaccgttgcaattgttagtacaggtecgattggtetagcagcactactgacegeacagttt
    [SEGIDNO SEQUENCE DEFOLINUCLEOTÍDEOS MM tattctccggcagaaattattatgattigatctagatgataatcgtetagatattgacacgteagttt ggtgcaacccagaccattaatageggtgatagtcatacagcagaaacegttaaageacta accggtggtegtagtattgataccgcaatigaagcagttggtattccggcaacctitaaacta tagtcaggatctagttagtcctagtagtattatigcaaatattggtatacatagtcataaagttgat ctgcatctggategtetatagagccagaatattgcaattaccaccegtetagttaatacegtta gcacccegatgctgctgaaaacegttcagagecgtaaactagaccegagecagetgatta cecategttttegectagatgaaattctggcagcectatgataccttitacacgtgcagcagata cccaggcactgaaagttattattgcagcectaataa atgaaagcactggtttatcatagtcegggtragaaagcactggaagaacgtcegaaace gcagattgaagcaageggtgatgccattgttaaaatigtgaaaaccaccatttatageace gatctacatattctgaaagataatatigcaacctatacaceggagtegtattctagateatgaa gatatagggtattattgatagcgttggtacegatattacegcattteagectagtaatecagtatteta attagctgtattagcagctgtggcaaatgtgattattgtegtegtggtetatatagccattataca accggtgagttagattctaggataatgaaattgatagcacccaggcagaatatgttegtacace gcatgcagataccagcctatategtatticeggcaggegcagatgaagaggcactagttata ctgagcgatattctac egacceggttttyaatatagtatactgaatggtaaagttaaacegggt agcaccgttgcaattgtiggtacaggatcecgattggtetageagcactagctgacegeacagttt tatgcaccgggtgatattattatgattgatctagatgataatcgtetagatatigcacategtttta gtgcaacccataccattaatageggtgatggtaaagcagcagaagcagttaaagcactg accggtagtattggtatigataccgcaatigaagccgttagtattceggcaacctttctactata tgaagatattgttgcaccgggtagtattattacaaatatiggtatacatagtattaaagttgatct gcatctggaacgtetatagacacataatattaccattaccaccegtetagttigatacegttac cacccegatgctgctgaaaacegticagagcaaaaaactggaccegetacagoetgattac, ccategttttaccctagatcatattetagatgcctatgatacctttagecgtgcageagatace aaagccctgaaagttattgtaagegcctaataa atggaaaatattatgaaagcaatggtgatattatagegatcatgatattcgttttyaagaacge aaaaaaccggaactgattgatcegacegatgccattattaaaatgaccaaaaccaccattt gtggcacegatctagatatttataaaggcaaaaatccggaaattigaacagaaagaacag gaaaaaaacggcagctttaatagtcatattictagatcatgaagatatiggtattatagagca 16 gattggtagcagcgtgaaaaacattaaagtgggcgataaagttattgttagctacgattagec gttataggcacctgtgaaaattgtgccaaacagctatatagccattategtaatgatagtggtta gattatgggct atatgatigataggcacccaggcagaatatgattegtacecegtttacagatac cagcctgatatattetaceggaaggtetgaatgaagatgattgcagttctactatetgatgcacta cegacegcacatgaaattggtattcagaatggcgatattaaacegggtgatacegtta caattattagtgcaggtecggtiggatataagegeactgctgacegceteagttttatagecega gccagattattatgattgatatagatgaaaategtetageaatageaaaagaactaggtaca accgataccattaatagcggcaccgaagatgcaatigcacgtattatagaactgaccaatc 17 agcgtagtattgattatacaatigaagecattggtatigaaccgacctgggatatttateagaa tattgtgaaagaaggtagtcatetageaaatattagtaticatageaaaagegtgaattttag cctggaaaaactatagattaaaaatctgaccattaccaccggtetagttaatagcaaatacca cecaggtatgctgctgaaaagetattatageggtaaactgcegatggaaaaactggeaaceo
    [SaioNo [SEQUENCE OF POLINUCLEOTIPEOS atcattttaaatitaatgaaatigaaaaggcctatgatgtatttattaatgcagccaaagaaaa agccatgaaagtgattattgatttttaataaatgaaagcactgacctatctaggtecgagataa aaaagaagtgatggaaaaaccgaaaccgaaaatigaaaaagaaaccgatgccattgta aaaattaccaaaaccaccatttgtagcaccgatctacatattctaagcggtaataticcgac cgttgaagaaggtcgtattctaggateataaaggtatagatattatigaagaagttagetetag cgttaaaaattttaaaaaaggegategcgttctgattagctatattaccagctgtagcaaatg cgaaaatigcaaaaaaggcctatatgcecatigtaaagatggtagttggattctgggccate tgattgatggcacccaggcagaatatattcgtattcegcatgcagataatagccetatateega ttecggaaggtattgatgaagaggcactgagttatactgagegatattctacegaccagtttta aaattggtgtactgaatagtaaagttcagectagtragaccgttacaattattagtacaggte cagttggtatagcagcactgctgacegceacagttttattetecggcagaaattattatagtaga tctagatgataategtetagaagtggecaaaaaatttagtgcaacccagattattaatageg cagatggtaaagccgtggaaaaaattatagaactgaccggtggcaaaggetatagatatta caatggaagcagttggtattceggtgacctttgatatttaccaggaaattgttaaacctageg gttatattgcaaatattggcgatacatagtaaaagegtagaattteatatigaaaaactatagat you gcaacattaccectgaccaceggtetagttaataccaccetetacecegatgctgctgaaaa cegttragageaaaaaactgaaaceggaacagctgattacccategttttacetitacegat attatgaaagcctatgaagtatttagtaatacagccaaagaaaaagecctaaaagitgattat tagcaatgattaataa atgagctatccggaaaaattteagggtattggcattaccaategegaagattggaaacatc cgaaaaaagtgaccttitygaaccgaaacagtttaatgataaagatgtagatattaaaattga agcctgcaggtatttatagttctgatattcattgtacagcaagecattggggtecggttacagaa aaacaggttgtaggccatgaaattattagtcgtatactgaaagttagtccgaaatgtaccac cggtattaaagttagtgatcgatatiggtattggtacacaggcatgagtcttatetagaatgtagec gttgcaaaagegataatgaaagctattgtecgaaaagegtttggacctatagcattcegtat attgatggttatattagccagggtagttatacaagecatatticgcctacatgaacattttacaat ttccgattceggataaactgagcaatgaactggcagcacegoetactatatagtagtattacceg
    18 tttattetccgctactacataatagttatagtccegggtaaaaaagttagtattgtaggacattiggta gtattggtcacatgggtctactatttacaaaaggtatagataccgaagtttatacatttagecg cacccatagcaaagaggcagacgccaaaaaactagggtagcegatoeattttattgcaacect ggaagataaagattggaccaccaaatattttgataccctagatctactagttatitatacaag cagcctgaccgatattaatittigatgaactgaccaaaattatagaaagtgaataccaaaattat tagcattagcgcaccggcagcagatgaagttctgaccetgaaaccgtttagtetaatiggtat gaccattggtaatagcgcaattggtagcegtegtgaaattigaacatctactgaattttgtage cgaaaaagatattaaaccgataggattgaaaccctacegattagtgaagecaggtattaatgaa gcatttaaacgcatggataaagatgatgtgaaatatcgtittaccctagtagattttgataaag aatttagcaattaataa atgagcgaagaaacctttaccgcatgggcatgtaaaagcaaaagegcacegetggaac
    19 cgatggaaatgaccttttaccattigggatgatgatatggttcagatggatgttatitatigtagto tttatagcacegatectgcataccgttgatgaaggatiggggatecgacegaatttecgatatatatta ggccatgaaagattggatata
    [EGiDNo JssauENCIADEPOLÍNUCLEOTIDEOS | tgatcgttatagtatiggttateagagegcaagcetatagtaaatgacgatitttacaaaaaagge atggaaaatctgtgtagcacccatgcagtttagacctitaatgatcgctatgataatagccace aaagataaaacctatggtggcttitacaaaaaaatggcgtaggcaatcaggattttattattcat gtgcegatggatttttectecggaagttgcagcaagctttetatatagtagtattaccacctatge accgctgaaacgttatagtgattggtaaaggtagcaaagttgcagttctagatetagatagtct gagccattttagtaticagtagacaaaagcaatgggtacagaagttattgcctttaacatgatt cceggataaagtagatgatgccaaaaaactaggctatgatgattatattctaatacagaaag aagagcagatggaaccgcattataatacctttacccatattctagecaccaaaattgtgaat aaatgctgggatcagtattttaaaatgctgaaaaataatggcatttttatactatacgatattico ggaagttccgctgagegatatgagegceatttattatagcaggtaaacagetgaccattgca gacacctitattagtagccegagegttaticaggaatatctagattitacagccaagcataatg ttegtacctagattaatacctttecgatggaaaaaattaatgaagcctttgaatttattcgteag gcaaaaccgcattategtacegtigtaatgaattaataa atgtttaccgttaatacacgtagcaccagegeacegggtacaccgtttaaagcagttattatt gaacgtcgtgatecgggtecagataatgttgttatigatattgcctttageggtatttateatace gatattagecegtgcacgtagegaatttageaccacecattatecac tagttecgggteatga aattgceggtattattagcaaagttagittecgatattaccaaatttacagttagtgatcgatatigg tattggttatattgtigatagctacegtaaatatgattattategtgcaggtetagaacegtattgt cgtaaagatcatgtgcgcacctataatagcatgggtcatgatagtcatattaccctagatagt tatagcgaaaaaatigtggtagatgaagattatattctacatattceggatacaattcegetag atcaggcagcaccgctactatatacaggtattaccatgtattictecgctacatcattagaaag caggtcegggtagccegtattacaattgttggttttagtagtcetagatcatattagtatigcaatta cacgtgcactgggtacacataccaccgtttttyatctgacgatggataaacatgatgatgca attcgtetaggtacagatgattatcegtetgagcacegatgcaggcatttttaaagaatttigaag gtgcctttgaactgattattagcacegttecggcaaatetagattatgacctatitetaaaaatg ctggcactagatagcacctttgttcagetaggtattcegcataatccggttagectagatattttt agcctgttttataategtegtagectagcaggcaccectagttggytagtattagtgaaacccag gaaatgctagatttitigcgcagaacatagcattgttgcegaaatigaaaccgttiggtacegat gaaattgatagecgcctatgategtatigcageeggtaatattcgttategtatagttctagatatt ggcaccctaggcaacecagegttaataa
    TABLES [SEGIDNo | SEQUENCIADEPOLIPEPTIDEO - | MSTAGKVIKCKAAVLWEEKKPFSIEEVEVAPPKAHEVRIKMVATG ICRSDDHVVSGTLVTPLPVIAGHEAAGIVESIGEGVTTVRPGDKVI
    PLFTPQCGKCRVCKHPEGNFCLKNDLSMPRGTMQDGTSRFTC 21 RGKPIHHFLGTSTFSQYTVVDEISVAKIDAASPLEKVCLIGCGFST GYGSAVKVAKVTAGSTCAVFGLGGVGLSVIMGCKAAGAARIIGV DINKDKFAKAKEVGATECVNPQDYKKPIQEVLTEMSNGGVDFSF
    EVIGRLDTMVTALSCCQEAYGVSVIVGVPPDSQNLSMNPMLLLS GRTWKGAIFGGFKSKDSVPKLVADFMAKKFALDPLITHVLPFEKI |
    [SEQIDNo [SEGUENCIADEFOLFERTDES MM [NEGEDELRSGESIRTETE MSYPEKFEGIAIOASHEDWKNPKKTKYDPKPFYDHDIDIKIEACGV CGSDIHCAAGHWGNMKMPLVVGHEIVGKVVKLGPKSNSGLKVG QRVGVGAQVFSCLECDRCKNDNEPYCTKFVTTYSQPYEDGYVS
    QGGYANYVRVHEHFVVPIPENIPSHLAAPLLCGGLTVYSPLVRN 22 GCGPGKKVGIVGLGGIGSMGTLISKAMGAETYVISRSSRKREDA MKMGADHYIATLEEGDWGEKYFDTFDLIVVCASSLTDIDFNIMPK AMKVGGRIVSISIPEQHEMLSLKPYGLKAVSISYSALGSIKELNQL LKLVSEKDIKIWVETLPVGEAGVHEAFERMEKGDVRYRFTLVGY DKEFSD MLYPEKFQGIGISNAKDWKHPKLVSFDPKPFGDHDVDVEIEACG
    ICGSDFHIAVGNWGPVPENOQILGHEIIGRVVKVGSKCHTGVKIGD RVGVGAQALACFECERCKSDNEQYCTNDHVLTMWTPYKDGY |
    SQGGFASHVRLHEHFAIQIPENIPSPLAAPLLCGGITVFSPLLRNG 23 CGPGKRVGIVGIGGIGHMGILLAKAMGAEVYAFSRGHSKREDSM KLGADHYIAMLEDKGWTEQYSNALDLLVVCSSSLSKVNFDSIVKI MKIGGSIVSIAAPEVNEKLVLKPLGLMGVSISSSAIGSRKEIEQLLK LVSEKNVKIWVEKLPISEEGVSHAFTRMESGDVKYRFTLVDYDK
    KFHK MLSFDYSIPTKVFFGKGKIDVIGEEIKKYGSRVLIVYGGGSIKRNGI | YDRATAILKENNIAFYELSGVEPNPRITTVKKGIEICRENNVDLVLA IGGGSAIDCSKVIAAGVYYDGDTWDMVKDPSKITKVLPIASILTLS
    ATGSEMDOIAVISNMETNEKLGVGHDDMRPKFSVLDPTYTFTVP 24 KNQTAAGTADIMSHTFESYFSGVEGAYVQDGIREAILRTCIKYGK IAMEKTDDYEARANLMWASSLAINGLLSLGKDRKWSCHPMEHE LSAYYDITHGVGLAILTPNWMEYILNDDTLHKFVSYGINVWGIDK NKDNYEIAREAIKNTREYFNSLGIPSKLREVGIGKDKLELMAKQA VRNSGGTIGSLRPINAEDVLEIFKKSY MVDFEYSIPTRIFFGKDKINVLGRELKKYGSKVLIVYGGGSIKRNG IYDKAVSILEKNSIKFYELAGVEPNPRVTTVEKGVKICRENGVEVWV LAIGGGSAIDCAKVIAAACEY DGNPWDIVLDGSKIKRVLPIASILTI AATGSEMDTWAVINNMDTNEKLIAAHPDMAPKFSILDPTYTYTVP TNQTAAGTADIMSHIFEVYFSNTKTAYLQODRMAEALLRTCIKYGG IALEKPDDYEARANLMWASSLAINGLLTYGKDTNWSVHLMEHEL SAYYDITHGVGLAILTPNWMEYILNNDTVYKFVEYGVNVWGIDKE KNHYDIAHQAIAKTRDYFVNVLGLPSRLRDVGIEEEKLDIMAKES VKLTGGTIGNLRPVNASEVLQIFKKSV
    MKALVYHGDHKISLEDKPKPTLOKPTDVVVRVLKTTICGTDLGIY 26 KGKNPEVADGRILGHEGVGVIEEVGESVTQFKKGDKVLISCVTS CGSCDYCKKQLYSHCRDGGWILGYMIDGVQAEYVRIPHADNSL YKIPQTIDDEIAVLLSDILPTGHEIGVQYGNVQPGDAVAIVGAGPV
    EGIDNo SEQUENCESPOLFERTIDEO GMSVLLTAQFYSPSTIIVIDMDENRLQLAKELGATHTINSGTENVV EAVHRIAAEGVDVAIEAVGIPATWDICQEIVKPGAHIANVGVHGV KVDFEIQKLWIKNLTITTGLVNTNTTPMLMKVASTDKLPLKKMITH RFELAEIEHAYQVFLNGAKEKAMKIILSNAGAA MAASCILLHTGQKMPLIGLGTWKSDPGQVKAAIKYALSVGYRHID CAAIYGNETEIGEALKENVGPGKLVPREELFVTSKLWNTKHHPE
    DVEPALRKTLADLQLEYLDLYLMHWPYAFERGDSPFPKNADGTI 27 RYDSTHYKETWRALEALVAKGLVRALGLSNFNSRQIDDVLSVAS VRPAVLQVECHPYLAQNELIAHCQARNLEVTAYSPLGSSDRAW RDPEEPVLLKEPVVLALAEKHGRSPAQILLRWQVQRKVSCIPKS VTPSRILENIQVFDFTFSPEEMKQLDALNKNLRFIVPMLTVDGKR
    VPRDAGHPLYPFNDPY MCTAGKDITCKAAVAWEPHKPLSLETITVAPPKAHEVRIKILASGI | CGSDSSVLKEIIPSKFPVILGHEAVGVVESIGAGVTCVKPGDKVIP LFVPQCGSCRACKSSNSNFCEKNDMGAKTGLMADMTSRFTCR
    GKPIYNLVGTSTFTEYTVVADIAVAKIDPKAPLESCLIGCGFATGY 28 GAAVNTAKVTPGSTCAVFGLGGVGFSAIVGCKAAGASRIIGVGT HKDKFPKAIELGATECLNPKDYDKPIYEVICEKTNGGVDYAVECA GRIETMMNALQSTYCGSGVTVVLGLASPNERLPLDPLLLLTGRS LKGSVFGGFKGEEVSRLVDDYMKKKINVNFLVSTKLTLDQINKAF ELLSSGQGVRSIMIY MKGFAMLGINKLGWIEKERPVAGSYDAIVRPLAVSPCTSDIHTVF EGALGDRKNMILGHEAVGEVVEVGSEVKDFKPGDRVIVPCTTPD
    WRSLEVQAGFOQHSNGMLAGWKFSNFKDGVFGEYFHVNDAD 29 MNLAILPKDMPLENAVMITDMMTTGFHGAELADIQMGSSVVWVIGI GAVGLMGIAGAKLRGAGRIIGVGSRPICVEAAKFYGATDILNYKN GHIVDQVMKLTNGKGVDRVIMAGGGSETLSQAVSMVKPGGIISN INYHGSGDALLIPRVEWGCGMAHKTIKGGLCPGGRLRAEMLRD MVVYNRVDLSKLVTHVYHGFDHIEEALLLMKDKPKDLIKAVVIL MKGLAMLGIGRIGWIEKKIPECGPLDALVRPLALAPCTSDTHTVW AGAIGDRHDMILGHEAVGQIVKVGSLVKRLKVGDKVIVPAITPDW GEEESQRGYPMHSGGMLGGWKFSNFKDGVFSEVFHVNEADA NLALLPRDIKPEDAVMLSDMVTTGFHGAELANIKLGDTVCVIGIG PVGLMSVAGANHLGAGRIFAVGSRKHCCDIALEYGATDIINYKNG DIVEQILKATDGKGVDKVVIAGGDVHTFAQAVKMIKPGSDIGNVN YLGEGDNIDIPRSEWGVGMGHKHIHGGLTPGGRVRMEKLASLIS TGKLDTSKLITHRFEGLEKVEDALMLMKNKPADLIKPVVRIHYDD EDTLH
    MKALVYRGPGQKLVEERQKPELKEPGDAIVKVTKTTICGTDLHIL 31 KGDVATCKPGRVLGHEGVGVIESVGSGVTAFQPGDRVLISCISS CGKCSFCRRGMFSHCTTGGWILGNEIDGTQAEYVRVPHADTSL
    SEGIDNo JSEQUENCIADEFOLFERTIDES = | YRIPAGADEEALVMLSDILPTGFECGVLNGKVAPGSSVAIVGAGP VGLAALLTAQFYSPAEIIMIDLDDNRLGLAKQFGATRTVNSTGGN AAAEVKALTEGLGVDTAIEAVGIPATFELCOQNIVAPGGTIANVGVH GSKVDLHLESLWSHNVTITTRLVDTATTPMLLKTVOSHKLDPSRL ITHRFSLDQILDAYETFGQAASTQALKVIISMEA MSTAGKVIKCKAAVLWEPHKPFTIEDIEVAPPKAHEVRIKMVATG VCRSDDHAVSGSLFTPLPAVLGHEGAGIVESIGEGVTCVKPGDK VIPLFSPQCGKCRICKHPESNLCCQTKNLTQPKGALLDGTSRFS
    CRGKPIHHFISTSTFSQYTVVDDIAVAKIDAAAPLDKVCLIGCGFS 32 TGYGSAVQVAKVTPGSTCAVFGLGGVGLSVVIGCKTAGAAKIIAV DINKDKFAKAKELGATDCINPQDYTKPIQEVLQEMTDGGVDFSF EVIGRLDTMTSALLSCHSACGVSVIVGVPPSAQSLSVNPMSLLLG RTWKGAIFGGFKSKDAVPKLVADFMAKKFPLEPLITHVLPFEKTN EAFDLLRAGKSIRTVLTF MRAVVFENKERVAVKEVNAPRLOQHPLDALVRVHLAGICGSDLHL YHGKIPVLPGSVLGHEFVGQVEAVGEGIQDLOPGDWVVGPFHIA
    CGTCPYCRRHQOYNLCERGGVYGYGPMFGNLQGAQAEILRVPF 33 SNVNLRKLPPNLSPERAIFAGDILSTAYGGLIQGQALRPGDSVAVI GAGPVGLMAIEVAQVLGASKILAIDRIPERLERAASLGAIPINAEQ ENPVRRVRSETNDEGPDLVLEAVGGAATLSLALEMVRPGGRVS AVGVDNAPSFPFPLASGLVKDLTFRIGLANVHLYIDAVLALLASG RLQPERIVSHYLPLEEAPRGYELFDRKEALKVLLVVRG MKALVYGGPGQKSLEDRPKPELQAPGDAIVRIVKTTICGTDLHIL KGDVATCAPGRILGHEGVGIVDSVGAAVTAFRPGDHVLISCISAC
    GKCDYCRRGMYSHCTTGGWILGNEIDGTQAEYVRTPHADTSLY 34 PVPAGADEEALVMLSDILPTGFECGVLNGKVAPGGTVAIVGAGP | GLAALLTAQFYSPAEIIMIDLDDNRLGIARQFGATQOTINSGDGRAA ETVKALTGGRGVDTAIEAVGVPATFELCQDLVGPGGVIANIGVH GRKVDLHLDRLWSQNIAITTRLVDTVSTPMLLKTVOSRKLDPSQL ITHRFRLDEILAAYDTFARAADTQALKVIIAA MKALVYHGPGQKALEERPKPQIEASGDAIVKIVKTTICGTDLHILK GDVATCAPGRILGHEGVGIIDSVGAGVTAFQPGDRVLISCISSCG KCDYCRRGLYSHCTTGGWILGNEIDGTQAEYVRTPHADTSLYRI PAGADEEALVMLSDILPTGFECGVLNGKVEPGSTVAIVGAGPIGL AALLTAQFYAPGDIIMIDLDDNRLDVARRFGATHTINSGDGKAAE AVKALTGGIGVDTAIEAVGIPATFLLCEDIVAPGGVIANVGVHGVK VDLHLERLWAHNITITTRLVDTVTTPMLLKTVOQSKKLDPLQLITHR FTLDHILDAYDTFSRAADTKALKVIVSA
    MENIMKAMVYYGDHDIRFEERKKPELIDPTDAIIKMTKTTICGTDL 36 GIYKGKNPEIEQKEQEKNGSFNGRILGHEGIGIVEQIGSSVKNIKV GDKVIVSCVSRCGTCENCAKQLYSHCRNDGGWIMGYMIDGTOA
    [sEaiDNo [SEQUENCIADEFOLIPERTIDEO - - .. | EYVRTPFADTSLYVLPEGLNEDVAVLLSDALPTAHEIGVQNGDIK PGDTVAIVGAGPVGMSALLTAQFYSPSQIIMIDWDENRLAMAKEL GATDTINSGTEDAIARVMELTNQRGVDCAIEAVGIEPTWDICQNI VKEGGHLANVGVHGKSVNFSLEKLWIKNLTITTGLVNANTTGML LKSCCSGKLPMEKLATHHFKFNEIEKAYDVFINAAKEKAMKVIIDF MKALTYLGPGKKEVMEKPKPKIEKETDAIVKITKTTICGTDLHILS GDVPTVEEGRILGHEGVGIIEEVGSGVKNFKKGDRVLISCITSCG
    KCENCKKGLYAHCEDGGWILGHLIDGTQAEYVRIPHADNSLYPI 37 PEGVDEEALVMLSDILPTGFEIGVLNGKVQPGQTVAIIGAGPVGM AALLTAQFYSPAEIIMVDLDDNRLEVAKKFGATQVVNSADGKAV EKIMELTGGKGVDVAMEAVGIPVTFDICQEIVKPGGYIANIGVHG KSVEFHIEKLWIRNITLTTGLVNTTSTPMLLKTVOSKKLKPEQLIT HRFAFADIMKAYEVFGNAAKEKALKVIISND MSYPEKFQGIGITNREDWKHPKKVTFEPKQFNDKDVDIKIEACG VCGSDVHCAASHWGPVAEKQVVGHEIIGRVLKVGPKCTTGIKV GDRVGVGAQAWSCLECSRCKSDNESYCPKSVWTYSIPYIDGYV
    SQGGYASHIRLHEHFAIPIPDKLSNELAAPLLCGGITVYSPLLRNG 38 CGPGKKVGIVGIGGIGHMGLLFAKGMGAEVYAFSRTHSKEADAK KLGADHFIATLEDKDWTTKYFDTLDLLVICASSLTDINFDELTKIM KVNTKIISISAPAADEVLTLKPFGLIGVTIGNSAIGSRREIEHLLNFV AEKDIKPWVETLPVGEAGVNEAFERMDKGDVKYRFTLVDFDKE FGN AND CGTDLHTVDEGWGPTEFPCVVGHEIIGNVTKVGKNVTRIKVGDR CGVGCASASCGKCDFCKKGMENLCSTHAVWTFNDRYDNATKD
    KTYGGFAKKWRGNQDFVVHVPMDFSPEVAASFLCGGVTTYAPL 39 KRYGVGKGSKVAVLGLGGLGHFGVQWAKAMGAEVVAFDVIPD KVDDAKKLGCDDYVLMQKEEQMEPHYNTFTHILATKIVNKCWD QYFKMLKNNGIFMLCDIPEVPLSGMSAFVMAGKQLTIAGTFIGSP SVIQECLDFAAKHNVRTWVNTFPMEKINEAFEFVRQAKPRYRAV VMN MFTVNARSTSAPGAPFEAVVIERRDPGPGDVVIDIAFSGICHTDV SRARSEFGTTHYPLVPGHEIAGVVSKVGSDVTKFAVGDRVGVG
    CIVDSCRECDYCRAGLEPYCRKDHVRTYNSMGRDGRITLGGYS 40 EKIVVDEGYVLRIPDAIPLDQAAPLLCAGITMYSPLRHWKAGPGS RIAIVGFGGLGHVGVAIARALGAHTTVFDLTMDKHDDAIRLGADD YRLSTDAGIFKEFEGAFELIVSTVPANLDYDLFLKMLALDGTFVQL GVPHNPVSLDVFSLFYNRRSLAGTLVGGIGETQEMLDFCAEHS!
    VAEIETVGADEIDSAYDRVAAGDVRYRMVLDVGTLATOQR In one embodiment, the method for screening polypeptides - candidates - possessing - alcohol dehydrogenase activity comprises:
    (a) measuring the rate of oxidation of the cofactor by a lower alkylaldehyde for candidate polypeptides in the presence or absence of a lower alkyl alcohol, and
    (b) selecting only the candidate polypeptides that oxidize a cofactor more quickly compared to a control polypeptide in the presence or absence of a lower alkyl alcohol.
    In one embodiment, (b) comprises selecting only those candidate polypeptides that oxidize a cofactor more quickly compared to a
    —Control polypeptide both in the presence and in the absence of a lower alkyl alcohol.
    In one example, the cofactor is NADH.
    In another example, the cofactor is NADPH.
    In yet another embodiment, the control polypeptide is HLADH having the amino acid sequence of SEQ ID NO: 21. In another embodiment, the
    —Control polypeptide is the SadB of Achromobacter xylosoxidans having the amino acid sequence of SEQ ID NO: 26. In another embodiment, step (a) comprises monitoring a change in Asaonm-
    In another embodiment, the method for screening candidate polypeptides having alcohol dehydrogenase activity comprises:
    (a) measuring one or more of the following values for candidate polypeptides:
    (1) the Ky value for lower alkylaldehyde;
    (ii) the K value, for a lower alkyl alcohol; and
    (ii) KalKmje
    (b) selecting only candidate polypeptides that have one or more of the following characteristics:
    (i) the Kv value for a lower alkylaldehyde which is lower when compared to that of a control polypeptide; (ii) the K value, for a lower alkyl alcohol that is higher when compared to a control polypeptide; and (iii) the K.a / Km value for lower alkylaldehyde which is higher when compared to that of a control polypeptide.
    In another embodiment, the control polypeptide is the SadB of Achromobacter xylosoxidans having the amino acid sequence of SEQ ID NO: 26. In another embodiment, the selected candidate polypeptides have two or more of the above characteristics. In another example, the selected candidate polypeptides have three or more of the above characteristics. In another example, the selected candidate polypeptides preferentially use NADH as a cofactor.
    In an example of an embodiment of the invention, the polynucleotide sequence suitable for use in the screening methods of the present invention comprises nucleotide sequences that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 , SEQIDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO : 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In another example of an embodiment of the invention, the polynucleotide sequence - suitable for use in the screening methods of the present invention can be selected from the group consisting of: SEQ ID NO : 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 , SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:
    13, SEQ ID NO 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or a variant, fragment or active derivative of this. In one embodiment, the polynucleotides were codon-optimized for expression in a specific host cell.
    In an example of an embodiment of the invention, candidate polypeptides suitable for use in the screening methods of the present invention have amino acid sequences that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40. In another example of an embodiment of the invention, a candidate polypeptide suitable for use in the screening methods of the present invention has an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ I D NO: 33, SEQ ID NO 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, or a variant , fragment or active derivative thereof. In one embodiment, candidate polypeptides suitable for use in the screening methods of the present invention were codon-optimized for expression in a specific host cell.
    In an example of an embodiment of the invention, the polynucleotide sequence suitable for use in the screening methods of the present invention has a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
    95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, candidate polypeptides for use in the screening methods of the present invention comprise an amino acid sequence having at least 80%>, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:
    22. In another example, the candidate polypeptide comprises the amino acid sequence of SEQ ID NO: 22 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the polynucleotide sequence suitable for use in screening methods has a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 3. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the candidate polypeptides for use in the screening methods of the present invention comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:
    23. In another example, the candidate polypeptide comprises the amino acid sequence of SEQ ID NO: 23 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the polynucleotide sequence for use in screening methods has a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 11. In another an exemplary embodiment, the -polyucleotide comprises the nucleotide sequence of SEQ ID NO: 11 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the candidate polypeptides for use in the screening methods of the present invention comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:
    31. In another embodiment, the candidate polypeptide comprises the amino acid sequence of SEQ ID NO: 31 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the polynucleotide sequence for use in screening methods has a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 9. In another an exemplary embodiment, the -pollucleotide comprises the nucleotide sequence of SEQ ID NO: 9 or a variant, fragment or active derivative thereof.
    In an example of an embodiment of the invention, the candidate polypeptides for use in the screening methods of the present invention comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:
    29. In another embodiment, the candidate polypeptide comprises the amino acid sequence of SEQ ID NO: 29 or a variant, fragment or active derivative thereof.
    In another example, the method for screening candidate polypeptides results in selected candidate polypeptides that are able to catalyze the conversion of an aldehyde to an alcohol at a temperature of up to about 70 ° C. In another example, the screening method results in the selection of candidate polypeptides that are capable of catalyzing the conversion of an aldehyde to an alcohol at a temperature of about 10 ºC, 15 ºC, 20 ºC, 25 ºC, 30 ºC , 35 ºC, 40 ºC, 45 ºC, 50 ºC, 55 ºC, 60 ºC, 65 ºC, or 70 ºC. In another example, the screening method results in selected candidate polypeptides that are able to catalyze the conversion of an aldehyde to an alcohol at a temperature of about 30 ° C. In another embodiment, the method for screening candidate polypeptides results in selected candidate polypeptides that - are able to catalyze the conversion of an aldehyde to an alcohol at a pH of about 4 to about 9. In another embodiment , the screening method results in selected candidate polypeptides that are capable of catalyzing the conversion of an aldehyde to an alcohol at a pH of about 5 to about 8. In another example, the screening method results in selected candidate polypeptides that are able to catalyze the conversion of an aldehyde to an alcohol at a pH of about 6 to about 7. In another example, the screening method results in selected candidate polypeptides that are capable of catalyzing the conversion of an aldehyde in an alcohol at a pH of about 6.5 to about 7. In another example, the method for screening results in the selection of candidate polypeptides that are capable of catalyzing the conve ratio of an aldehyde to an alcohol at a pH of about 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5 or 9. In another embodiment, the screening method results in selected candidate polypeptides that are able to catalyze the conversion of an aldehyde to an alcohol at a pH of approximately 7. In another embodiment, the screening for candidate polypeptides results in selected candidate polypeptides - which can catalyze the conversion of an aldehyde to an alcohol in the presence of a lower alkyl alcohol, at a concentration of up to about 50 g / L.
    In another example, the method for screening results in the selection of candidate polypeptides that are capable of catalyzing the conversion of an aldehyde to an alcohol at a concentration of about 10g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L or 50 g / L.
    In another embodiment, the screening method results in selected candidate polypeptides that are capable of catalyzing the conversion of an aldehyde to an alcohol at a concentration of about 20 g / L.
    Non-limiting examples of lower alkyl alcohols that can be used in the methods of the present invention include butanol, isobutanol, propanol, isopropanol and ethanol.
    In one embodiment, the lower alkyl alcohol used in the screening method is isobutanol.
    Unless otherwise defined, the technical and scientific terms used herein have the same meaning as is commonly understood by a skilled person in the field to which this invention belongs.
    In the event of a conflict, the meanings in this application including definitions will prevail.
    In addition, unless otherwise required by the context, singular terms must also include plural forms and plural terms must include singular forms.
    All publications, patents, and other references mentioned in the present invention are incorporated herein by reference for all purposes.
    EXAMPLES The present invention is further defined in the following Examples. It should be understood that these examples, while indicating examples of embodiments of the present invention, are given by way of illustration only. From the above discussion and these Examples, a person skilled in the art can determine the essential features of the present invention, and without departing from the spirit and scope of the present invention, can make several changes and modifications to the invention to adapt it to the diverse uses and conditions.
    GENERAL METHODS Recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described in more detail in Sambrook, et al. (Sambrook, J., Fritsch, EF and Maniatis, T. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, referred to herein as Maniatis) and by Ausubel et al. (Ausubel et al. ., Current Protocols in Molecular Biology, Greene Publishing Assoc. & Wiley-Interscience, 1987). Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following Examples can be found as set forth in: Manual of Methods for General Bacteriology (Phillipp Gerhardt, et al. Eds., American Society for Microbiology, Washington, DC, 1994) or by Thomas D. Brock in: Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition, Sinauer Associates, Inc., Sunderland, MA, (1989) All reagents, restriction enzymes and materials used for the cultivation and maintenance of bacterial cells were obtained from Sigma-Aldrich Chemicals (St. Louis, MO), BD Diagn ostic Systems (Sparks, MD), Invitrogen (Carlsbad, CA), HiMedia (Mumbai, India), SD Fine Chemicals (India), or Takara Bio Inc.
    (Shiga, Japan), unless otherwise stated. The abbreviations mean: “s” means second (s), “min” means minute (s), “h” means hour (s); “Nm” means nanometers, “ul” means microliter (s), “mL” means milliliter (s), “mg / mL” means milligram per milliliter, “L” means liter (s) “nm” means nanometers, “mM ”Means millimolar,“ M ”means molar,“ mmol ”means millimoles (s),“ umol ”means micromole (s),“ kg ”means kilogram (s),“ 9 ”means gram (s),“ ug ”means micrograms (s), and "ng" means nanogram (s), "PCR" means polymerase chain reaction, "OD" means optical density, "OD600" means the optical density measured at the 600 nm wavelength, " kDa ”means kilodaltons,“ g ”can also mean the gravitational constant,“ bp ”means base pairs,“ kbp ”means kilo base pairs,“ kb ”means kilobase,“% ”means percent,“% w / v "means percent by weight / volume,"% vv "means percent by volume / volume," HPLC "means high performance liquid chromatography," g / L "means gram per liter," (vg / L "means microgram per liter, “ng / ul . ”Means nanogram per microliter,“ pmol / uL ”means picomol per microliter,“ RPM ”means revolutions per minute,“ (umol / min / mg ”means micromole per minute per milligram,“ w / v ”means weight per volume, “V / v” means volume for volume. EXAMPLE 1 SELECTION OF POTENTIAL ISOBUTYRALDEHYDE DEHYDROGENASES FOR
    TRACKING This example describes the basis for the selection of several candidate ADH enzymes for the identification of efficient isobutyraldehyde dehydrogenases. Butanol Dehydrogenases A and B (BdhA and BdhB) from Clostridium acetobutylicum were chosen for analysis based on evidence in the literature. Achromobacter xylosoxidans was selected by enriching a sample obtained from environmental mud in a medium containing
    1-butanol. The organism was then cultured and used to purify the protein fraction that contained butanol dehydrogenase activity, then the gene corresponding to secondary alcohol dehydrogenase B (SadB) was cloned as described in US Patent Application publication 20090269823A1. The - horse liver ADH enzyme (HLADH) is commercially available and has been described as having isobutanol oxidation activity by Green et al. in J. Biol. Chem. 268: 7792 (1993). Desirable properties of an ideal candidate isobutyraldehyde dehydrogenase for the isobutanol production pathway have been described above.
    An extensive search in the literature identified candidate ADH enzymes with high Kcat and / or low Kw values for isobutyraldehyde or other closely related aldehydes, or with a low Kcat & / or high Kvy value for isobutanol or other closely related alcohols. The BLAST Protein searches against the protein sequence database - non-redundant (nr) in the NCBI were performed using horse liver ADH, Achromobacter xylosoxidans SadB and Saccharomyces cerevisiae ADH6 as query strings, respectively . All hits found in BLAST were collected and combined, from which sequences with more than 95% sequence identity to each other were removed. The multiple sequence alignment (MSA) was created from the set of 95% remaining non-redundant sequences and a phylogenetic tree was generated from the MSA using the neighbor joining method. Likewise, the MSA and the phylogenetic tree were generated separately for a number of selected ADH enzymes to identify - closely related homologues of each enzyme where the BLAST alignment consisted only of the found alignments (hits) obtained using the target enzyme as the query. These enzymes included SadB from Achromobacter xylosoxidans, ADHG6 from Saccharomyces cerevisiae, and ADH7 from Saccharomyces cerevisiae. Based on these analyzes, several candidates were selected (Table 3) for performance evaluation. EXAMPLE 2 PROTEIN CLONING, EXPRESSION AND PURIFICATION, AND SCREENING FOR A
    ISOBUTYRALDEHYDE ADEQUATE DEHYDROGENASE This example describes the preparation of constructs with the ADH gene for overexpression / purification and measurement of enzymatic activities using a time curve assay. Horse liver ADH (HLADH; A-6128) was purchased from Sigma. The SadB of Achromobacter xylosoxidans (SadB), ADH6 (ScADH6) and ADH7 (ScADH7) of Saccharomyces cerevisiae, ADH1 of Entamoeba histolytica (EnADH1), Aldehyde Reductase of Bos Taurus (BtARD), ADH of Beijerinckia ATHI Indicates subsp.Indica ), Clostridium beijerinckii ADH (CDADH), Rana perezi ADH8 (RPBADH8), Rattus norvegicus ADH1 (RNADHI), Thermus sp.ATN1 ADH (TADH), Phenylobacterium zucineum ADH HLK1 (PZADH) (MSsADH)) Acinetobacter baumannii AHE (ALADH) ADH, Geobacillus sp.WCH70 (GLADH) ADH, Vanderwaltozyma polyspora DSM 70294 (VPpADH) AD, Muúucor circinelloides (McADH) ADH and Rhodocephalus ADH were the candidates for which subclones were prepared for protein expression and purification. - CONSTRUCTION OF PLASMIDIAL CONSTRUCTIONS EXPRESSING ADH CANDIDATES The coding regions of the EhADHI, BIARD, CLbADH, BIiADH, and RpADH8 genes were synthesized by DNA 2.0 (Menlo Park, CA) and the coding regions of the RnADHI, TADH, PZADH, ALADH, MSADH, MSADH, MSADH, MSADH, MSADH, MSADH, MSADH, MSADH, PADHAD, MSADH, MSADH, MSADH, MSADH, MSADH, MSADH, MSADH, MSADH, MSADH, MsADH genes. , GLbADH, VpADH, McADH, and ReADH were synthesized by GENEART AG (Germany) after optimizing the codons for expression in Escherichia coli. The amino acid sequences for these candidates were obtained from the Genbank protein database and supplied to DNA 2.0 or Geneart AG for codon optimization. Each coding region was flanked by Xhol and Kpnl sites at the 5 'and 3' ends of the coding sequence, respectively.
    These constructs were cloned and supplied in the DNA 2.0 vector pJ201 or Geneart's PMA vector.
    The plasmids were transformed into chemically competent TOP 10 cells (invitrogen) and amplified by growing the transformants in liquid LB medium containing 25 mg / ml kanamycin or 100 mg / ml ampicillin.
    Plasmids that were purified from overnight cultures (grown at 37 ° C), were digested with restriction enzymes Xhol (NEB; R0146) and Kpnl (NEB; RO0142) and ligated at the corresponding locations on the reading board with a N-terminal hexahystidine marker in the vector pBADHISA (invitrogen; V43001), using the “DNA ligation kit” version 2.1 from Takara Bio Inc. (6022). The ligation products were transformed into chemically competent TOP 10 cells (invitrogen; C4040-50). The transformed cells were seeded on a plate containing LB medium plus 100 mg / ml ampicillin.
    The clones containing the ADH inserts were confirmed by digestion with the restriction enzymes Xhol / Kpnl.
    Plasmids with the correct insertion contained the expected band of 1.2 kbp in each case.
    The cloned sequence was confirmed by DNA sequencing.
    The resulting clones were named pBADHIsA :: EhADHI, pBADHIsSA :: BtARD, PBADHIsA :: CbADH, PBADHIsSA :: BiADH, PBADHIsSA :: RpADH8, PBADHIsSA :: RnADHI, PBADHIsSA :: TADH, PBADHIsSA :: PzADHISSA :: PzADH , PBADHIsSA :: ADADH, PBADHIsA :: GbADH, PBADHISA :: VpADH, PBADHISA :: McADH, and PBADHISA :: ReADH, respectively.
    SadB, an enzyme that was previously analyzed, was amplified by PCR with the enzyme KOD polymerase (Novagen), according to the procedure described in the product manual, from pTrc99a :: SadB using the primers SadBXhol-f (CCATGGAATCTCGAGATGAAAGCTCTGGTTTACC, SEQ ID NO : 41) and SadBKpnl-r (GATCOCCCGGGTACCGAGCTCGAATTC, SEQ ID NO: 42) to introduce Xhol and Kpnl sites at the 5 'and 3' ends, - respectively.
    After confirming the PCR product by means of agarose gel electrophoresis, the 1.2 kb PCR product was digested with the enzymes Xhol and Kpnl and cloned into pBADHISA as described above for the other candidate genes.
    The genes for ScCADH6 and ScADH7 were each amplified from 100 ng genomic DNA from the wild type yeast strain BY4741 (ATCC 201388) using the ADH6 Xhol f primers (CAAGAAATCTCGAGATCATGTCTTATCCTGAG, SEQ ID NO: 43) and ADH6 Kpnl rp GAGCTTGGTACCCTAGTCTGAAAATTCTTTG, SEQ ID NO: 44) for ScADH6 and ADH7 Xhol f (CTGAAAAACTCGAGAAAAAAATGCTTTACCC, SEQ I | D NO: 45) and ADH7 Kpnl for (GAMSAMATATGGTACTATTATGATTACHTATTATGATTACTGGTACT). The PCR strategy and conditions were identical to those used for SadB amplification.
    The genes were then cloned into the Xhol and Kpnl sites of pBADHISA, according to the procedure described above.
    The plasmids containing SadB, ScADH6 and ScADH7 were named as —pBADHIsSA :: SadB, PBADHIsSA :: ScADH6 and PBADHIsSA :: ScADH7, respectively.
    EXPRESSION OF RECOMBINANT ADHs IN E.
    COLI For the data shown, BL21-CodonPlus (invitrogen; 230,240) or an E strain was used. coli for the overexpression of ADH enzymes.
    However, commercially available strains, such as BL271-codon plus, are believed to be suitable for overexpression of ADH enzymes.
    Expression plasmids (pBADHISA plasmids) containing the ADH genes were prepared from 3-mL of Top 10 transformant cultures incubated overnight using the 'Qiaprep spin miniprep' kit (Qiagen, Valencia CA; 27106) following the instructions of the manufacturer. One ng of each plasmid was transformed into electrocompetent BL21- — CodonPlus or E. coli cells using an electroporator “Bio Rad Gene Pulser II” (Bio-Rad Laboratories Inc, Hercules, CA), following the manufacturer's instructions. The transformed cells were seeded on agar plates containing LB medium plus 100 pg / ml ampicillus and spectinomycin. The plates were incubated overnight at 37 ° C. The colonies of these plates were inoculated in 3.0 mL of LB medium containing 100 Vvg / mL of ampicillin and spectinomycin, at 37 ºC, under agitation at 250 rpm. The cells from these initial cultures (grown overnight) were used to inoculate 1 L of medium at a 1: 1000 dilution. The cells were induced with 0.02% Arabinose, after the culture reached an OD of - 0.8. The induction was carried out at 37 ºC, under agitation at 250 rpm during the night. The cells were then collected by centrifugation at 4000 g for 10 min at 4 ºC. The cells were lysed by treatment with 40 ml of the “BugBuster master mix” mixture (Novagen; 71.456-4), in the presence of EDTA-free protease inhibitor cocktail tablets (Roche; 11873580001) and 1 mg / ml of lysozyme, by placing on a shaker at 4 ºC for 30 min. Cell debris was removed by centrifugation at 16,000 g for 20 min at 4 ºC.
    The total protein concentration in the samples was measured by the Bradfords assay using a concentrated Bradford dye (Bio-Rad). The samples and protein standards (serum bovine albumin, BSA) were - configured in individual cuvettes (1 mL reactions) or in a 96-well microplate following the manufacturer's protocol. Protein concentrations were calculated from the absorbance values at 595 nm, measured using a visible / UV light spectrophotometer “Cary 100 Bio” (Varian, Inc.)
    or a Spectramax card reader (Molecular Devices Corporation, Sunnyvale, CA).
    PURIFICATION AND TESTS OF ADH ENZYME ACTIVITY Extracts without cells prepared from 1 liter cultures according to the procedure described above, were used directly to purify the various ADH enzymes expressed by means of IMAC affinity chromatography (metal affinity chromatography) immobilized) in 5 mL HisTrap FF columns (GE Healthcare Life Sciences; 175255-01). The entire procedure was performed using an AKTAexplorer 10 S FPLC system (GE Healthcare Life Sciences; 18-1145-05). The extracts were mixed with 30 mM imidazole and loaded onto HisTrap columns. After loading, the column was washed with 50 mM sodium phosphate buffer, pH 8.0, containing 30 mM Imidazole (approximately -—- 10-20 column volumes) to get rid of unbound proteins and not specifically connected. The ADH protein was then eluted with a gradient from 30 mM to 500 mM Imidazole over 20 column volumes. The peak fractions were electrophoresed on Bis-Tris 10% SDS-PAGE gels (invitrogen; NPO301) using the “XCell Invitrogen from Invitrogen's SureLock Mini-Gef (EIO0001). After coomassie staining and discoloration, it can be seen that the fractions were more than 95% pure and contained only the ADH protein. Activity tests were performed to ensure that the purified proteins were active. As a routine practice, crude extracts and purified proteins were evaluated for the oxidation activity of butanol, in order to ensure that the recombinant proteins remained active throughout the purification process. In the reductive direction, isobutyraldehyde reduction assays were performed with NADH or NADPH as the cofactor and an excess of isobutyraldehyde substrate (40 mM). In each case, the enzymatic activity was measured for 1 min at 30 ºC in 1 ml reactions following the decrease or increase in absorbance at 340nm using a spectrophotometer “Cary Bio 100 UV-Visible” (Varian Inc.), depending on whether the NADH / NADPH is being consumed (absorbance is decreased) or generated (absorbance is increased) in the reaction.
    The oxidation activities of the alcohol were carried out in 50 MM sodium phosphate buffer at pH 8.8 and the aldehyde reduction reactions were evaluated in 100 mM potassium phosphate buffer at pH 7.0. Depending on the nature of the reaction to be performed, the enzyme and cofactor stock solutions were diluted in the reaction buffers at the respective pHs.
    The buffer or cell extract prepared from the coliproperty E strain (without the ADH plasmid) was used as a negative control for assays with purified protein and cell-free extracts,
    respectively.
    In the initial experiments, there was an insufficient level of protein expression with EhADHI and RpADH8. Subsequently, activity assays failed to detect ADH activity in extracts from cells that expressed these enzymes.
    Likewise, initially, although BtARD exhibited good levels of protein expression and the protein was purified to homogeneity, there was no detectable activity under the conditions used for the assay.
    It is believed that a technician skilled in the subject could further optimize the conditions of expression and testing for these candidates.
    Sufficient amounts of active protein could be purified with all other enzymes for which the data are presented.
    The specificities of cofactors were measured with all these enzymes in isobutyraldehyde reduction reactions (as in the proc mentioned above), using both NADH and NADPH as cofactors.
    In each case, a difference of at least 10 times was observed in the activity numbers when NADH or NADPH were used as a cofactor, against the corresponding number for the other form of cofactor. THE
    Table 6 summarizes the cofactor preferences for some of the ADH enzymes. TABLE 6 ADH Candidate Cofactor of Preference ADH of horse liver NADH ADHS of Saccharomyces NADPH cerevisiae ADH7 ofSaccharomyces NADPH cerevisiae SadB of Achromobacter NADH xylosoxidans ADH of Beijerickia NADHHHRD of MuscleDadHusky RADH1 of NADH1 RADH of NADHHH RADH of norms of NERHHHHH RADH of NERHHHHHHHHHH! ATN1 NADH ADH from Phenylobacterium NADH zucineum HLK1 ADH from ADMethylocella silvestris NADH BL2 ADH from Acinetobacter baumannii ADH from Geobacillus sp. WCH70 NADPH ADH by Mucor circinelloides NADH SELECTING PURIFIED CANDIDATE ADHS USING A SEMIFYSIOLOGICAL TIME CURVE TEST.
    The ideal way to characterize and compare several ADH candidates would be to calculate and compare the complete set of kinetic constants, that is, K.at values for aldehyde reduction and alcohol oxidation, Ky values for isobutyraldehyde, isobutanol, NAD (P) and NAD (P) H, and values for isobutyraldehyde and isobutanol. Detailed characterization for several candidate enzymes would require a considerable expenditure of time, effort and money. Thus, a qualitative essay was developed to allow quick and efficient comparison of the various candidates. A semi-physiological assay was designed to compare the performance of various enzymes. The tests involve the initiation of all reactions with a constant amount of each enzyme. In this case, 1 µg of each enzyme was used to initiate the reactions that contained isobutyraldehyde and NADH in concentrations of 1 mM and 200 UM, respectively. Each reaction time curve was followed for 10 min by measuring the decrease in absorbance at 340nm, until the reaction proceeded to equilibrium. An enzyme with a High Kkcat would lead the reaction to equilibrium more quickly than an enzyme with a KkKca: lower. A parallel test was also carried out under identical conditions, but with the inclusion of 321 mM isobutane! (24 g / L) in the reaction. An enzyme that is not relatively inhibited by this concentration of isobutanol would have a time curve that mimics the time curve in the absence of isobutanol. Figure 1 compares the time curves displayed for the candidate ADH enzymes in these assays.
    Based on the results presented in Figure 1, it appears that Beijerickia indica ADH is likely to have Kat Maior for the isobutyraldehyde reduction reaction and ADH6 is probably the enzyme that is least inhibited by isobutane! in the reaction.
    EXAMPLE 3 BEIJERINCKIA ADH IDENTIFICATION INDICATES WITH HIGH KCAR AND A LOW KM VALUE FOR ISOBUTYRALDEHYDE The kinetic constants of ADH enzymes were calculated and compared to identify those candidate ADH enzymes with the most desirable properties for converting isobutyraldehyde into isobutanol stage of the road developed for the production of isobutanol. The tests for determining the kinetic constants were performed using the initial rates from the tests described above. Reductions in NADH can be correlated with consumption of aldehyde (Biochemistry by Voet and Voet, John Wiley & Sons, Inc.). However, the amount of a given enzyme used in the reaction was in the range of 0.1 to 5 µg. The concentration of a given enzyme was such that it was favorable for measuring initial velocities over a 1 min time course.
    For each enzyme, Michaelis-Menten plots were generated with a wide range of substrate concentrations.
    Crude estimates of Ky values were obtained based on tests that were redesigned in order to use substrate concentrations in the range of 0.5 to 10 times the Km value, to be able to obtain adequate kinetic constants.
    The isobutyraldehyde (isobutanal) reduction reactions were carried out at 30 ºC in 100 mM potassium phosphate buffer, pH 7.0, containing 200 µM of NADH.
    When calculating for isobutanol, the same reactions were carried out in the presence of various concentrations of isobutanol (usually 0-535 mM) in the reaction (see Figure 7, for example). The reactions with isobutanol substrate were carried out at 30 ºC in 50 mM sodium phosphate buffer, pH 8.8, containing 7.5 mM NAD.
    The “enzyme kinetics” module (Version 1.3) of the SigmaPlot 11 program (Systat Software, Inc.) was used to adjust the data to the Michaelis-Menten equations and calculate the kinetic constants.
    The kinetic constants obtained for the indicated ADH enzymes are given in Table 7. The kKca / Km is derived from the individual numbers of K.at and Kvw, and is not the value determined experimentally.
    The proportions of Km, Ki, and Kca / Km for each candidate enzyme, compared to the same parameter for SadB, are given in Table 9. TABLE 7 Ku K Other properties
    (isobututanol) | (isobutanol) | k.a / Km | enzymatic and cofactor of
    (mM) (mM) preference [oxidation of Isobutanol:
    HLADH * 0.1 2 82 Kcat = 5 sec ”;
    Km = 0.4mM]
    Km (NADH) = 0.02mM
    SadB * 109 1 180 105 [oxidation of Isobutane !: kcai = 2 sec ”; Ku = 24mM]
    «Ku K Other properties (sec (isobutane!) | (Isobutane!) | Kca / Knm | enzymatic and 9 (mM) cofactor (mM) preference ScADH6 | 47 | o6êe |] 4170 specific NADPH SaADHT” specific NADPH Km (NADH ) = 0.06 mM; [oxidation of Isobutane !: BIiADH | 283 2 3 252 ia 8. 6 | 1252 | a = 9 sec; Ku = 4.7 mM] Bound | 123 | 15 | No | & | NADPA specific TADA | 15 | 13 | ND | 71 | NOTHING specific RnADH1 <0003 | ND Jj-1667] specific NADH For those enzymes marked with an asterisk in Table 7, at least 3 assays were performed with separate enzyme preparations. are values from one assay or are they “averages of 2 assays performed on the same enzyme sample.
    The ADH value of Beijerickia indica (BIADH) shows the highest number for kK.at and a reasonably high Kca / Km value, and is the enzyme pdita. The RnADH1 enzyme appears to have a low Ky for isobutyraldehyde and, consequently, can have a high catalytic efficiency. However, the low Kvy value prevents accurate determination of its Kyw value using spectrophotometric methods. However, the performance of the enzyme in the isobutanol-producing host may be further limited by K.at if the intracellular steady-state levels of isobutyraldehyde are above its Ku value. Comparing with BIADH with SadB, the efficiency —catalytic of the first enzyme for reducing isobutyraldehyde is — 12 times that of the last enzyme, although it is more sensitive to isobutanol than SadB. With respect to the nucleotide cofactor, SadB has a lower Km value for NADH, when compared to the BIADH value. The ScADH6 enzyme has a high value for isobutanol, indicating that this enzyme is susceptible to functioning in vivo, without being affected by the presence of isobutanol, in concentrations that are expected in an isobutanol production host.
    Among the candidates analyzed so far, SadBé has the least catalytic efficiency for the oxidation of isobutanol (Kca / Km = 0.083), followed by BIADH (1.91) and HLADH (12.5). EXAMPLE 4 Seven additional candidate ADH enzymes were synthesized, expressed, and evaluated according to methods described in Example 2. The kinetic constants obtained for the indicated ADH enzymes (Phenylobacterium zucineum HLK1 ADH (PZADH), Methylocella silvestris BL2 ADH (MSsADH) , ADH of Acinetobacter baumannii AYE (ALADH), ADH of Geobacillus sp.WCH70 (GLbADH), and ADH of Mucor circinelloides (McADH)) are provided in Table 8. A comparison of Knu, Ki, and Kkca / Km for each candidate enzyme compared to the same parameter for SadB it is given in Table 9 as a percentage of the values determined (Table 7) for SadB.
    Percentages below 100 indicate a lower value than that determined for SadB; percentages greater than 100 indicate a higher value than that determined for SadB.
    There was no expression of Rhodococcus erythropolis PR4 ADH (ReADH) and there was no detectable ADH activity of Vanderwaltozyma polyspora DSM 70294 (VPpADH) in these assays.
    TABLE 8 Kea Ku K, Other properties Enzymes A (isobututane! L) | (isobutanol) | KaKm | enzymes and (sec An (mM) (mM) cofactor preference specific NADH Conversion of isobutane! PZADH so 18 321 to isobutyraldehyde not measurable Specific NADH Conversion of isobutane! MSADH | 33 1st Are to non-measurable isobutyraldehyde k. Ku K, Others properties Enzymes A a (isobutane!) | (isobutane!) | Ka / Km | enzymatic and cofactor (sec AA. (mM) (mM) preference specific NADH Conversion of isobutane! ADADH 10 sos 10 into isobutyraldehyde NADPH specific GLADH | 32 13 72 Conversion of isobutane! Into non-measurable isobutyraldehyde MCADH specific NADH | 151 30 79 Conversion of isobutanol to non-measurable isobutyraldehyde TABLE 9 Parameter indicated as a percentage of the same parameter determined for SadB Without] sm De Ds
    FIA 100% 100% 100% 100% EXAMPLE 5 CONSTRUCTION OF S. CEREVISIAE CEPA PNY 2211 The PNY2211 was built in several stages from S.
    cerevisiae cepa PNY1507 as described in Order US 61/380563, filed on September 7, 2010, and in the following paragraphs. The strain was first modified to contain a phosphocetolase gene. The construction of cassettes with the phosphocetolase gene and its integration into the strains was - previously described in order US 61/356379 deposited on June 18,
    2010. Then, an acetolactate synthase gene (a / sS) was added to the strain, using an integration vector described earlier in application US 61/308563. Finally, homologous recombination was used to remove the phosphocetolase gene and integration vector sequences, resulting in a Scarless insertion of the alsS gene into the intergenic region between pdclA :: ilvD (a deletion / insertion previously described in the ORF PDC1 in the US patient) 61/308563) and the TRX1 gene native to chromosome XII. The genotype resulting from the PNY2211 strain is: MATa uradA: loxP his3A pdc6A pdce1Aa :: P [PDC1] - DHADI / ilvD Sm-PDC1t-P [FBA1] -ALS | alsS Bs-CYC1t pdc5A :: P [PDC5] - ADHisadB Ax -PDC5t gpd24 :: lo0xP fra2dá —adhiA: UAS (PGK1) P [FBA1] - kivD Ll (y) -ADH1t. A cassette with the phosphocetolase gene was introduced into PNY1507 by homologous recombination. The integration construction was generated as follows. Plasmid pRS423 :: CUP1-alsS + FBA-budA (as described in US Publication 2009/0305363 A1) was digested with Notl and Xmal to remove the 1.8 kb FBA-budA sequence, and the vector was rewired after treatment with Klenow fragments. Then, the CUP1 promoter was replaced with a variant TEF1 promoter (M4 variant described by Nevoigt et al. Appl. Environ. Microbiol. 72 (8): 5266-5273 (2006)), through DNA synthesis and construction of the vector from DNA 2.0 (Menlo Park, CA). The resulting plasmid, pRS423 :: TEF (M4) -alsS was cut with the restriction enzymes Stul and Mlul (which remove a 1.6 kb fraction containing part of the alsS gene and the terminator CYC1), and combined with the product from 4 kb PCR generated from pRS426 :: GPD-xpkIl + -ADH-eutD (SEQ ID NO: 81; the plasmid is described in Order US 61/356379) with primers N1176 and N1177 (SEQ ID NOs: 47 and 48 , respectively) and a 0.8 kb PCR product DNA generated from the yeast genomic DNA (ENO1 promoter region) with primers N822 and N1178 (SEQ ID NOs: 49 and 50, respectively) and transformed into S. cerevisiaãee strain BY4741 (ATCC * 201388; by gap-repair cloning methodology, see Ma and Botstein). The transformants were obtained by plating cells in a complete synthetic medium without histidine.
    The correct assembly of the expected plasmid (pRS423 :: TEF (M4) -xpk1 + ENO1-eutD, SEQ ID NO: 51) was confirmed by PCR using primers and N821 and N1115 (SEQ ID NOs: 52 and 53, respectively) and digestion with the restriction enzyme (Bg / l). Two clones were subsequently sequenced.
    The 3.1 kb TEF (M4) -xpk1 gene was isolated by digestion with Sacl and Notl and cloned into the vector puC19-URA3 :: ilvD-TRX1 described in Order US 61/356379 (Clone A, cut with the Afill enzyme) . The cloning fragments were treated with Klenow fragment to generate blunt ends for ligation.
    The ligation reactions were transformed into E. coli Stbl3 cells, selecting them for resistance to ampulla.
    The insertion of TEF (M4) -xpk1 was confirmed by PCR using primers N1110 and N1114 (SEQID NOs: 54 and 55, respectively). The vector was linearized with Afill and treated with Klenow fragment.
    The 1.8 kb Kpnl-Hindl geneticin resistance cassette described in US Application 61/356379 was cloned by ligation after treatment with Klenow fragment.
    The binding reactions were transformed into E. coli Stbl3 cells, selecting them for ampicillin resistance.
    The insertion of the geneticin cassette was confirmed by PCR using primers NI6O0SegF5 and BK468 (SEQ ID NOs: 56 and 57, respectively). The plasmid sequence is provided as SEQ ID NO:
    58 (pUC19-URA3 :: pde1 :: TEF (M4) -xpk1 :: kan).
    The resulting integration cassette (pdc1t :: TEF (M4) - xpk1 :: KanMX :: TRX1) was isolated (digestion with Ascl and Nael generated a 5.3 kb band that was purified on gel) and transformed into PNY 1507 using the yeast transformation kit 'Zymo Research Frozen-EZ Yeast Transformation Kit' (Cat. No. T2001). The transformants were selected by plating in YPE medium with the addition of 50 µg / ml of G418. The insertion of the geneticin cassette was confirmed by PCR using primers N160SegF5 and BK468 (SEQ ID NOs: 59 and 60, respectively). Then, plasmid pRS423 :: GAL1p-Cre, which encodes a Cre recombinase, was used to remove the KanMX cassette flanked by loxP (vector and methods described in Order US 61/308563). Adequate removal of the cassette was confirmed by PCR using the primers oBP512 and N1I60SeqgF5 (SEQ ID NOs: 61 and 62, respectively). Finally, the a / sS integration plasmid described in Order US 61/308563 (pUC19-kan :: pdc1 :: FBA-alsS :: TRX1, clone A) was transformed into this strain using the included geneticin selection marker. Two members were tested for acetolactate synthase activity by transformation with plasmids pYZ090AalsS and pBP915 (plasmids described in Order US 61/308563, transformed using the% 2 protocol in 2005 in Methods in Yeast Genetics; Amberg, Burke and Strathern) and evaluation of growth and production of isobutanol in glucose-containing medium (the methods of growth and measurement of isobutanol are described in US Application 61/308563 and US Patent Application Publication 2007 / 0092957A1) .One of the two clones was positive and was named PNY2218 An isolate of PNY2218 containing plasmids pYZ090AalsS and pBP915 was designated PNY2209.
    PNY2218 was treated with Cre recombinase and the resulting clones were screened for the loss of the xpk1 gene and the sequences of the pUC19 integration vector by PCR using primers N886 and N160SegR5 (SEQ ID NOs: 59 and 56, respectively). This left only the alsS gene integrated in the pde1-TRX1 intergenic region after recombination of the DNA upstream of xpk1 and the homologous DNA introduced during the insertion of the integration vector (a “scarless” insertion since the vector sequences, marker gene and loxP are lost, see Figure 9). Although this recombination may have occurred at any point, the integration of the vector appeared to be stable, even without geneticin selection and the recombination event was only observed after the introduction of Cre recombinase. One clone was designated PNY2211. EXAMPLE 6 CONSTRUCTION OF SACCHAROMYCES CEREVISIAE CEPA PNY 1540 The purpose of this example is to describe the construction of the Saccharomyces cerevisiae strain PNY1540 from strain PNY2211. This strain was obtained from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center, Netherlands) and is described in Example 5 above. PNY 1540 contains a deletion of the sadB gene, from —Achromobacter xylosoxidans, which had been integrated into the PDC5 locus in PNY2211. The deletion, which completely removed the entire coding sequence, was created by homologous recombination with a PCR fragment containing the homologous regions upstream and downstream of the target gene and a URA3 gene for selection of transformants. The URAS3 gene was removed by homologous recombination to create a Scarless deletion.
    The Scarless deletion procedure was adapted from Akada et al. 2006 Yeast v23 page 399. The PCR cassette for the Scarless deletion was made by combining four fragments, A-B-U-C, and by overlapping PCR. The PCR cassette contained a selectable marker / marker — against-selectable, URA3 (U fragment), which consists of the native URAS3 gene of the CEN.PK 113-7D, along with the promoter (250bp upstream of the URAS3 gene) and terminator ( 150bp downstream of the URA3 gene). Fragments A and C, each 500 bp in length, corresponding to 500 bp immediately upstream of the target gene (fragment A) and 500 bp 3 'from the target gene (fragment C). Fragments A and C were used to integrate the cassette into the chromosome by homologous recombination. Fragment B (254 bp in length) corresponded to the sequence immediately downstream of the target gene and was used to excise the URA3 marker and fragment C from the chromosome by homologous recombination, thus a direct repetition of the sequence corresponding to fragment B was created by integrating the cassette into the chromosome. Using the PCR product from the ABUC cassette, the URAS3 marker was first integrated and then excised from the chromosome - by homologous recombination. The initial integration deleted the gene, excluding 500 bp in the 3 'direction. After excision, the 3 '500 bp region of the gene was also deleted.
    SADB DELECTION The four fragments for the PCR cassette for the Scarless deletion of sadB were amplified using the “Phusion High Fidelity PCR Master Mix” (New England Biolabs; Ipswich, MA) and the CEN.PK 113-7D genomic DNA as template DNA for Fragment U and the genomic DNA of PNY1503 as template DNA for fragments A, B, and C. The genomic DNA was prepared with the “Gentra Puregene Yeast / Bact” kit (Qiagen; Valencia, CA). The sadB fragment A was amplified with the primer oBP540 (SEQ ID NO: 63) and the primer oBP835 (SEQ ID NO: 64), containing a 5th tail homologous to the 5 'end of sadB fragment B. The sadB fragment B was amplified with primer oBP836 (SEQ ID NO: 65), containing a 5 'tail with homology to the 3' end of sadB fragment A, and primer oBP837 (SEQ ID NO: 66), containing a 5 'tail with homology to 5' end of sadB fragment U. The sadB fragment U was amplified with primer oBP838 (SEQ ID NO: 67), containing a 5 'tail homology to the 3' end of sadB fragment B, and primer oBP839 (SEQ ID NO: 68) , containing a 5 'tail with homology to the 5' end of sadB fragment C.
    The sadB fragment C was amplified with the primer oBP840 (SEQ ID NO: 69) containing a 5 'tail with homology to the 3' end of sadB fragment U, and the primer oBP841 (SEQ IDNO: 70). PCR products were purified with the 'PCR Purification kit' (Qiagen) purification kit. The sadB Fragment AB was created by overlapping PCR mixing sadB fragment A and sadB fragment B and amplifying with primers oBP540 (SEQ ID NO: 63) and oBP837 (SEQ ID NO: 66). The sadB Fragimento UC was created by overlapping PCR mixing the sadB fragment U and sadB fragment C and amplifying them with primers oBP838 (SEQ ID NO: 67) and oBP841 (SEQ ID NO: 70). The resulting PCR products were purified on an agarose gel followed by purification with the “Gel Extration” kit (Qiagen). The ABB sadB cassette was created by overlapping PCR by mixing sadB Fragment AB and sadB Fragment UC, by amplification with the primers oBP540 (SEQ ID NO: 63) and oBP841 (SEQ ID NO: 70) The PCR product was purified with
    The purification kit "PCR Purification kit" (Qiagen). Competent PNY2211 cells were made and transformed with the ABB sadB PCR cassette using a "Frozen-EZ Yeast Transformation Il kit" transformation kit (Zymo Research; Orange, CA ). The mixtures of the transformants were seeded in a complete synthetic medium without uracil supplemented with 1% ethanol at 30 ºC.
    Transformants with a sadB knockout were screened by PCR with the ura3-end primers (SEQ ID NO: 71) and oBP541 (SEQ ID NO: 72). A correct transformant was cultured in YPE medium (1% ethanol) and plated in complete synthetic medium containing 5-fluoro-orotic acid (0.1%) at 30 ºC to select the isolates that lost the URA3 marker. The deletion and removal of the marker was confirmed by PCR with the
    | 110 primers oBP540 (SEQ ID NO: 63) and oBP541 (SEQ ID NO: 72) using genomic DNA prepared with a genomic DNA kit “YeaStar Genomic DNA Kit” (Zymo Research). The absence of the sadB gene from the isolate was demonstrated by a negative PCR result using the primers - specific for the sadB coding sequence that was deleted, oPP530 (SEQ ID NO: 73) and oPP531 (SEQ ID NO: 74). The correct isolate was selected as PNY 1540 strain (BP 1746) EXAMPLE 7 CONSTRUCTION OF A TRANSPORT VECTOR (SHUTTLE) FOR YEAST CARRYING A GENE THAT CODES THE ADH OF B. INDICA AND A VECTOR
    NEGATIVE CONTROL Plasmid pLH468 (SEQ ID NO: 75), as described in US Patent Application Publication 2009/0305363 A1, is a yeast / E shuttle vector. coli c that carries 3 chimeric genes that encode enzymes that make up part of an isobutanol production pathway (dihydroxy acid dehydratase, aKIV decarboxylase and isobutanol dehydrogenase). The existing isobutanol dehydrogenase gene has been replaced by B. indica's ADH using the gap-repair cloning methodology. The ADH coding region of BB indicates with the appropriate 5 'and 3' flanking sequences it was first obtained by DNA synthesis (DNA2.0, Menlo Park, CA) with the codon optimization for yeast. The sequence is provided as SEQ ID NO: 76. The vector pLH468 was linearized with Bsu36! / And transformed together with the B. indica ADH (released from the supplier's cloning vector with EcoRI and BamHI) in the yeast strain BY4741 . The transformants were plated on a complete synthetic medium without histidine (Teknova Cat. No. C3020). Plasmids were prepared from the various transformants using a “Zymoprep" ”Yeast Plasmid kit
    | 111 Miniprep "(Zymo Research. Cat No.: D2004). PCR (primers N1092 and N1093 and SEQ ID NOs: 77 and 78) and digestion with restriction enzymes (Kpnl) were used to confirm the incorporation of BiADH in the desired location This plasmid is said to be pl H468 :: BiADH.
    A second vector was constructed, in which most of the original isobutanol dehydrogenase (hADH) gene was eliminated from PLHA468. This was done by releasing an 808 bp fragment by digesting it with Bsu36 / and Pacl, filling the ends of the DNA with Klenow fragment and rewiring the vector. The ligation reaction was transformed into E. coli Stbli3 cells. The loss of the hADH gene was confirmed by EcoRI digestion of isolated plasmid clones. A successful clone was selected for the experiment described in Example 8, below. This plasmid is said to be pLH468AhADH.
    EXAMPLE 8 ISOBUTANOLOGICAL STRUCTURES LOADING BI'ADH EXHIBIT BETTER DEPENDENT GROWTH OF GLUCOSE, GREATER CONSUMPTION OF GLUCOSE AND GREATER TITLE AND YIELD OF ISOBUTANOL THAN CONTROL STRUCTURES. Plasmids plH468 :: BiIADH and pLH468AhADH were each transformed together with a second plasmid with an isobutanol pathway (pYZ090AalsS, Order US 61/380563) in PNY1540. The transformants were plated in a complete synthetic medium without histidine and uracil, containing 1% ethanol as a carbon source. Several transformants were plated on fresh plates. After 48 hours, the plates (3 from each strain) were used to inoculate complete synthetic medium (without histidine and uracil) containing 0.3% glucose and 0.3% ethanol as carbon sources. After 24 hours, growth in this medium was similar for all replicates of both strains. The cultures were then subcultured in complete synthetic medium (without histidine and uracil) containing 2% glucose and 0.05% ethanol as carbon sources.
    The cultures (starting at an optical density (OD) of 600 nm were 0.2; the culture volume was 20 ml in tightly capped 125 ml flasks) were incubated for 48 hours.
    The samples were collected for analysis by HPLC, at the time of subculture and again after 48 hours.
    Final ODs were also determined.
    The 48h average OD for the BiADH strain was 3.3 (+0.1) compared to 2.37 (+0.07) for the control strain without ADH.
    Thus, the inclusion of BIADH increased DO by 39% under these conditions.
    Likewise, glucose consumption (assessed by HPLC compared to samples collected immediately after subculture) increased by 69% (81 + 1 MM vs 47.9 + 0.6 mM). Isobutanol titers were 4 times higher and molar yields (ie, isobutanol yield per mole of glucose consumed) were twice as high, as shown in the table below.
    In the control strain without ADH, a significant amount of carbon from the isobutanol pathway accumulated as isobutyrate, indicating that aldehyde dehydrogenases were acting on isobutyraldehyde.
    TABLE 10 (mM) (mM) (mM) IPNY1540 / pLH468 :: BIADH 0.401 (20.006) 0.135 (= 0.005)] - - ND - |
    权利要求:
    Claims (24)
    [1]
    1. RECOMBINANT MICROBIAL HOSTING CELL, characterized by the fact that it comprises: a biosynthetic pathway for the production of a lower alkyl alcohol, the biosynthetic pathway comprising a substrate for product conversion catalyzed by a polypeptide with alcohol dehydrogenase activity and one or more of the following characteristics: (a) the Kvw value for isobutyraldehyde is lower for said polypeptide when compared to a control polypeptide having the amino acid sequence of SEQ ID NO: 26; (b) the K value for isobutanol for said polypeptide is higher when compared to a control polypeptide having the amino acid sequence of SEQ ID NO: 26; and (c) the K.a / Km value for isobutyraldehyde for said polypeptide is higher when compared to a control polypeptide having the amino acid sequence of SEQ ID NO: 26.
    [2]
    2. HOSTING CELL according to claim 1, characterized by the fact that the biosynthetic pathway for the production of a lower alkyl alcohol is a biosynthetic pathway of butanol, propanol, isopropanol or ethanol.
    [3]
    3. HOSTING CELL according to claim 1, characterized by the fact that the polypeptide with alcohol dehydrogenase activity has at least 95% identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 31, 32, 34, 35, 36, 37 or 38.
    [4]
    4. HOSTING CELL, according to claim 1, characterized by the fact that the polypeptide with alcohol dehydrogenase activity has the amino acid sequence of SEQ ID NO: 31.
    [5]
    5. HOSTING CELL according to claim 1,
    i 2 characterized by the fact that the polypeptide with alcohol dehydrogenase activity is encoded by a polynucleotide having at least 85% identity with a nucleotide sequence of SEQ ID NO: 1,2,3,4,5, 6, 11, 12 , 14, 15, 16 or 17.
    [6]
    6. HOSTING CELL, according to claim 1, characterized by the fact that the polypeptide with alcohol dehydrogenase activity preferably uses NADH as a cofactor.
    [7]
    7. HOSTING CELL according to claim 1, characterized by the fact that said polypeptide having alcohol dehydrogenase activity catalyzes the conversion of isobutyraldehyde to isobutanol in the presence of isobutanol at a concentration of at least about 15 g / L.
    [8]
    8. HOSTING CELL according to claim 1, characterized by the fact that the biosynthetic pathway for the production of a lower alkyl alcohol is a butanol biosynthetic pathway.
    [9]
    9. HOSTING CELL according to claim 1, characterized by the fact that the biosynthetic pathway for the production of a lower alkyl alcohol is an isobutanol biosynthetic pathway comprising heterologous polynucleotides that encode polypeptides that catalyze the conversion of substrate into product for each step of the following steps: (a) pyruvate in acetolactate; (b) acetolactate in 2,3-dihydroxyisovalerate; (c) 2,3-dihydroxyisovalerate in α-ketoisovalerate; (d) a-ketoisovalerate in isobutyraldehyde; and (e) isobutyraldehyde in isobutanol; and wherein said microbial host cell produces isobutanol.
    [10]
    10. HOSTING CELL according to claim 1, characterized by the fact that the biosynthetic pathway for the production of a lower alkyl alcohol is an isobutanol biosynthetic pathway comprising
    | 3 heterologous polynucleotides that encode polypeptides that catalyze substrate-to-product conversions for each step of the following steps: (a) pyruvate to acetolactate; (b) acetolactate in 2,3-dihydroxyisovalerate; (c) 2,3-dihydroxyisovalerate in α-ketoisovalerate; (d) a-ketoisovalerate in isobutyryl-CoA; (e) isobutyryl-CoA in isobutyraldehyde; and (f) isobutyraldehyde in isobutane! 1; and wherein said microbial host cell produces isobutanol.
    [11]
    11. HOSTING CELL, according to claim 1, characterized by the fact that the biosynthetic route for the production of an alcohol! lower alkyl is an isobutanol biosynthetic pathway comprising heterologous polynucleotides that encode polypeptides that catalyze substrate-to-product conversions for each step of the following steps: (a) pyruvate to acetolactate; (b) acetolactate in 2,3-dihydroxyisovalerate; (c) 2,3-dihydroxyisovalerate in α-ketoisovalerate; (d) a-ketoisovalerate in valine; (e) valine in isobutylamine; (f) isobutylamine in isobutyraldehyde; and (g) isobutyraldehyde in isobutanol; and wherein said microbial host cell produces isobutanol.
    [12]
    12. RECOMBINANT MICROBIAL HOST CELL, characterized by the fact that it comprises a biosynthetic pathway for the production of a lower alkyl alcohol and a heterologous polynucleotide that encodes a polypeptide with alcohol dehydrogenase activity having at least 85% identity with the SEQ amino acid sequence ID NO: 21,22, 23, 24, 25, 31, 32, 34, 35, 36, 37 or 38.
    | 4
    [13]
    13. HOSTING CELL according to claim 12, characterized by the fact that the biosynthetic pathway for the production of a lower alkyl alcohol is a 2-butanol biosynthetic pathway comprising heterologous polynucleotides that encode polypeptides that catalyze substrate conversions in product for each of the following steps: (a) pyruvate in a-acetolactate; (b) a-acetolactate in acetoin; (c) acetoin in 2,3-butanediol; (d) 2,3-butanediol in 2-butanone; and (e) 2-butanone in 2-butanol; and wherein said microbial host cell produces 2-butanol.
    [14]
    14. - HOSTING CELL, according to claim 12, characterized by the fact that the biosynthetic pathway for the production of a lower alkyl alcohol is a 1-butanol biosynthetic pathway comprising - heterologous polynucleotides that encode polypeptides that catalyze the conversion of product substrate for each of the following steps: (a) acetyl-CoA in acetoacetyl-CoA; (b) acetoacetyl-CoA in 3-hydroxybutyryl-CoA; (c) 3-hydroxybutyryl-CoA to crotonyl-CoA; (d) crotonyl-CoA in butyryl-CoA; (e) butiri-CoA in butyraldehyde; and (f) butyraldehyde in 1-butanol; and wherein said microbial host cell produces 1-butanol.
    [15]
    15. HOSTING CELL, according to claim 12, characterized by the fact that said polypeptide having alcohol dehydrogenase activity comprises an amino acid sequence with at least 95% identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 36, 37, or 38.
    [16]
    16. HOSTING CELL according to claim 12, characterized in that said polypeptide having alcohol dehydrogenase activity comprises an amino acid sequence with at least 95% identity with the amino acid sequence of SEQ ID NO: 31.
    [17]
    5 17. HOSTING CELL, according to claim 1 or 12, characterized by the fact that the genus of said host cell is selected from the group consisting of: Saccharomyces, Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zygosaccharomyces, Galactomyces, Schizosaccharomyces, Torulaspora, Debayomyces, Williopsis, Dekkera, Kloeckera, Metschnikowia, Issatchenkia and Candida.
    [18]
    18. METHOD FOR THE PRODUCTION OF ISOBUTANOL, characterized by the fact that it comprises: (a) the supply of a recombinant microbial host cell comprising an isobutanol biosynthetic pathway, the pathway comprising a heterologous polypeptide that catalyzes the conversion of substrate into isobutyraldehyde product for isobutanol, where the polypeptide has at least 90% identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 36, 37 or3d8e (b ) the contact of the host cell of (a) with a carbon substrate under conditions in which isobutanol is produced.
    [19]
    19. METHOD, according to claim 18, characterized by the fact that the heterologous polypeptide that catalyzes the conversion of substrate into isobutyraldehyde product to isobutane! it has at least 95% identity with the amino acid sequence of SEQ ID NO: 31.
    [20]
    20. METHOD, according to claim 18,
    | 6 characterized by the fact that the heterologous polypeptide that catalyzes the conversion of substrate in isobutyraldehyde product to isobutanol has the amino acid sequence of SEQ ID NO: 31.
    [21]
    21. METHOD FOR THE PRODUCTION OF 2-BUTANOL, characterized by the fact that it comprises: (a) the supply of a recombinant microbial host cell comprising a 2-butanol biosynthetic pathway, the pathway comprising a heterologous polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27,31,32,34,35,36,37 or 38, and (b) contact of the host cell of (a) with a carbon substrate under conditions in which 2-butanol is produced.
    [22]
    22. METHOD, according to claim 21, characterized by the fact that the heterologous polypeptide has at least 95% identity with the amino acid sequence of SEQ ID NO: 31.
    [23]
    23. METHOD FOR THE PRODUCTION OF 1-BUTANOL, characterized by the fact that it comprises: (a) the supply of a recombinant microbial host cell comprising a biosynthetic 1-butanol pathway, the pathway comprising a heterologous polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 36, 37 or 38, and (b) the contact of the host cell of (a) with a carbon substrate under conditions in which 1-butanol is produced.
    [24]
    24. METHOD, according to claim 23, characterized by the fact that the heterologous polypeptide has at least 95% identity with the amino acid sequence of SEQ ID NO: 31.
    类似技术:
    公开号 | 公开日 | 专利标题
    BR112012016042A2|2020-09-15|&#34;recombinant microbial host cell, method for the production of isobutanol, method for the production of 2-butanol and method for the production of 14-butanol
    US9404117B2|2016-08-02|Increased production of isobutanol in yeast with reduced mitochondrial amino acid biosynthesis
    JP6321631B2|2018-05-09|Ketolate reduct isomerase enzyme and method of use
    US9689004B2|2017-06-27|Acetate supplemention of medium for butanologens
    JP6099623B2|2017-03-22|Host cells and methods for isobutanol production
    AU2009345976B8|2015-02-05|Enhanced pyruvate to acetolactate conversion in yeast
    AU2010300653B2|2016-01-28|Improved yeast production host cells
    JP2013515507A|2013-05-09|Expression of hexose kinase in recombinant host cells
    CA2737112A1|2010-06-03|Carbon pathway optimized production hosts for the production of isobutanol
    WO2014031831A1|2014-02-27|Production of fermentation products
    同族专利:
    公开号 | 公开日
    CN102762722A|2012-10-31|
    WO2011090753A3|2011-10-13|
    ZA201204375B|2014-08-27|
    US20140377824A1|2014-12-25|
    EP2519628A2|2012-11-07|
    KR20120115349A|2012-10-17|
    US9410166B2|2016-08-09|
    US20110269199A1|2011-11-03|
    CA2784903A1|2011-07-28|
    JP2013515506A|2013-05-09|
    WO2011090753A2|2011-07-28|
    NZ600509A|2014-08-29|
    JP5947219B2|2016-07-06|
    AU2010343048A1|2012-06-28|
    MX2012007583A|2012-07-30|
    US8765433B2|2014-07-01|
    CN102762722B|2015-05-20|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    JPS54129189U|1978-02-28|1979-09-07|
    JPS5724752B2|1978-03-31|1982-05-26|
    US4266034A|1978-04-14|1981-05-05|Exxon Research And Engineering Company|Method for producing microbial cells and use thereof to produce oxidation products|
    CS197080B1|1978-06-06|1980-04-30|Ladislav Skursky|Mixture for the photometric determination of activities of the alcoholdehydrogenasis in the biologic material|
    US4241184A|1979-03-27|1980-12-23|Exxon Research & Engineering Co.|Secondary alcohol dehydrogenase enzyme and use thereof|
    CA1189807A|1982-06-28|1985-07-02|Ching-Tsang Hou|Microbiological process for oxidation of alkanes,vinyl compounds and secondary alcohols|
    CA1211728A|1982-06-28|1986-09-23|Exxon Research And Engineering Company|Primary and secondary alcohol dehydrogenase enzymesand use thereof|
    SE454330B|1984-03-28|1988-04-25|Santrade Ltd|REQUEST FOR TEAM DISPOSAL PROCESSING|
    US5162516A|1988-05-31|1992-11-10|University Of Florida|Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis|
    US5028539A|1988-08-31|1991-07-02|The University Of Florida|Ethanol production using engineered mutant E. coli|
    US5000000A|1988-08-31|1991-03-19|University Of Florida|Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes|
    US5821093A|1988-08-31|1998-10-13|University Of Florida Research Foundation, Inc.|Recombinant cells that highly express chromosomally-integrated heterologous genes|
    US7192772B1|1988-08-31|2007-03-20|The University Of Florida Research Foundations, Inc.|Recombinant cells that highly express chromosomally-integrated heterologous gene|
    US5487989A|1988-08-31|1996-01-30|Bioenergy International, L.C.|Ethanol production by recombinant hosts|
    US5385833A|1992-02-26|1995-01-31|The Scripps Research Institute|Pseudomonas sp. ATCC No. 49794 alcohol dehydrogenase|
    JPH08504326A|1992-12-15|1996-05-14|コモンウェルスサイエンティフィックアンドインダストリアルリサーチオーガナイゼーション|DNA molecule encoding an inducible plant promoter and tomato ADH2 enzyme|
    US6011199A|1992-12-15|2000-01-04|Commonwealth Scientific|Method for producing fruiting plants with improved fruit flavour|
    FR2706906B1|1993-06-21|1995-12-08|Ifremer|
    JP3574682B2|1993-09-24|2004-10-06|ダイセル化学工業株式会社|Novel enzymes, methods for producing the enzymes, DNAs encoding the enzymes, transformants containing the DNAs, methods for producing optically active alcohols and the like using the enzymes|
    US5855881A|1996-02-22|1999-01-05|Loike; John D.|Mammalian alcohol dehydrogenase and aldehyde dehydrogenase production in plants|
    WO1998017788A1|1996-10-22|1998-04-30|Daicel Chemical Industries, Ltd.|Novel secondary alcohol dehydrogenase, process for preparing said enzyme, and process for preparing alcohols and ketones using said enzyme|
    JPH11127883A|1997-10-28|1999-05-18|Meidensha Corp|Biological production of propane|
    US6737257B2|1998-05-18|2004-05-18|Board Of Regents Of The University Of Nebraska|Hyperthermophilic enzymes for industrial chemical redox reactions: a method for biofuel ethanol production|
    JP2000078969A|1998-09-04|2000-03-21|Unitika Ltd|Alcohol dehydrogenase and its production|
    US6432688B1|1999-01-18|2002-08-13|Daicel Chemical Industries, Ltd.|Amino alcohol dehydrogenase converts keto alcohol to amino alcohol and amino alcohol to keto alcohol|
    TWI275645B|2000-02-16|2007-03-11|Daicel Chemical Industries Ltd.|-2-octanol dehydrogenases, methods for producing the enzymes, DNA encoding the enzymes, and methods for producing alcohols using the enzymes|
    AU6274201A|2000-06-16|2001-12-24|Res Inst Innovative Tech Earth|Process for producing ethanol by using recombinant coryneform bacterium|
    GB0024554D0|2000-10-06|2000-11-22|Agrol Ltd|Ethanol production|
    US6440711B1|2000-12-08|2002-08-27|Board Of Trustees Southern Illinois University, The|Dehydrogenase enzymatic synthesis of methanol|
    DE10112401A1|2001-03-13|2002-09-19|Degussa|Alcohol dehydrogenase and its use|
    DE10119274A1|2001-04-20|2002-10-31|Juelich Enzyme Products Gmbh|Enzymatic process for the enantioselective reduction of keto compounds|
    JP4630486B2|2001-05-28|2011-02-09|ダイセル化学工業株式会社|Novel -2,3-butanediol dehydrogenase, method for producing the same, and method for producing optically active alcohol using the same|
    AU2003214111A1|2002-03-18|2003-09-29|Ciba Specialty Chemicals Holding Inc.|Alcohol dehydrogenases with high solvent and temperature stability|
    DE10218689A1|2002-04-26|2003-11-20|Degussa|ADH from Rhodococcus erythropolis|
    JP4160417B2|2003-02-25|2008-10-01|株式会社カネカ|Secondary alcohol dehydrogenase and production method thereof|
    CN100376677C|2003-03-12|2008-03-26|株式会社味滋集团公司|Alcohol dehydrogenase gene of acetic acid bacterium|
    KR100532627B1|2003-05-27|2005-12-02|엔자이텍 주식회사|Alcohol dehydrogenase -producing microorganisms and the method of making optically active 2-phenyl-1-propanol by these microorganisms|
    KR20040104076A|2003-06-03|2004-12-10|엔자이텍 주식회사|Alcohol dehydrogenase-producing microorganisms for the synthesis of optically active ethyl 3-hydroxy-3-phenylpropionate from ethyl benzoylacetate|
    EP1664286A1|2003-09-18|2006-06-07|Ciba SC Holding AG|Alcohol dehydrogenases with increased solvent and temperature stability|
    JP2005102511A|2003-09-26|2005-04-21|Kaneka Corp|New alcohol dehydrogenase and its gene|
    SE526429C2|2003-10-24|2005-09-13|Swedish Biofuels Ab|Intensifying fermentation of carbohydrate substrate for, e.g. producing one to five carbon alcohols, involves using amino acid leucine, isoleucine, and/or valine as source of nitrogen|
    KR100543115B1|2003-11-04|2006-01-20|주식회사한국야쿠르트|Lactic acid bacteria having high alcohol dehydrogenase and acetaldehyde dehydrogenase activity, and metabolize ethanol and acetaldehyde|
    AT413541B|2004-05-10|2006-03-15|Iep Gmbh|PROCESS FOR THE PREPARATION OF 2-BUTANOL|
    DE102004038054A1|2004-08-05|2006-03-16|Maxens Gmbh|Process for the preparation of primary alcohols|
    WO2006016432A1|2004-08-12|2006-02-16|Marine Biotechnology Institute Co., Ltd.|Novel aromatic dehydrogenase gene and process for producing aromatic carboxylic acid|
    AT501928B1|2004-10-27|2010-09-15|Iep Gmbh|PROCESS FOR THE PREPARATION OF CHIRAL ALCOHOLS|
    DE102004055508A1|2004-11-17|2006-06-01|Basf Ag|Process for the preparation of optically active alcohols|
    EP1869197A2|2005-04-12|2007-12-26|E.I. Dupont De Nemours And Company|Treatment of biomass to obtain ethanol|
    JP4704814B2|2005-06-15|2011-06-22|神戸天然物化学株式会社|Novel aromatic alcohol oxidase gene and method for producing aromatic carboxylic acid|
    EP1896573A1|2005-06-24|2008-03-12|Novozymes A/S|Mutant cells suitable for recombinant polypeptide production|
    WO2007005646A2|2005-07-01|2007-01-11|The University Of Florida Research Foundation, Inc.|Recombinant host cells and media for ethanol production|
    US9297028B2|2005-09-29|2016-03-29|Butamax Advanced Biofuels Llc|Fermentive production of four carbon alcohols|
    JP5276986B2|2005-10-26|2013-08-28|ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー|Fermentative production of four-carbon alcohol|
    US9303225B2|2005-10-26|2016-04-05|Butamax Advanced Biofuels Llc|Method for the production of isobutanol by recombinant yeast|
    US8273558B2|2005-10-26|2012-09-25|Butamax Advanced Biofuels Llc|Fermentive production of four carbon alcohols|
    DE102006009744A1|2006-03-02|2007-09-06|Wacker Chemie Ag|Preparation of -2-butanol by oxidative resolution|
    DE102006010994A1|2006-03-09|2007-09-13|Wacker Chemie Ag|Process for the enzymatic preparation of chiral alcohols|
    US8962298B2|2006-05-02|2015-02-24|Butamax Advanced Biofuels Llc|Recombinant host cell comprising a diol dehydratase|
    US8828704B2|2006-05-02|2014-09-09|Butamax Advanced Biofuels Llc|Fermentive production of four carbon alcohols|
    US7659104B2|2006-05-05|2010-02-09|E.I. Du Pont De Nemours And Company|Solvent tolerant microorganisms and methods of isolation|
    US7541173B2|2006-06-15|2009-06-02|E.I. Du Pont De Nemours And Company|Solvent tolerant microorganisms and methods of isolation|
    WO2008006038A2|2006-07-06|2008-01-10|Integrated Genomics, Inc.|Methods and compositions for butanol production|
    US20080293101A1|2006-07-27|2008-11-27|Peters Matthew W|Engineered microorganisms for increasing product yield in biotransformations, related methods and systems|
    WO2008013949A2|2006-07-28|2008-01-31|Michigan State University|Molecular design of thermostable alcohol dehydrogenase for synthesis for chiral aromatic alcohols|
    US20080293086A1|2006-09-18|2008-11-27|Cobalt Technologies, Inc. A Delaware Corporation|Real time monitoring of microbial enzymatic pathways|
    WO2008035187A2|2006-09-21|2008-03-27|Dow Global Technologies Inc.|Alcohol dehydrogenase from agromyces sp. and a method of producing a chiral secondary alcohol using same|
    US7973214B2|2006-09-25|2011-07-05|Ut-Battelle, Llc|Designer organisms for photosynthetic production of ethanol from carbon dioxide and water|
    WO2008052991A2|2006-10-31|2008-05-08|Dsm Ip Assets B.V.|Butanol production in a eukaryotic cell|
    CN101528936A|2006-10-31|2009-09-09|代谢探索者公司|Process for the biological production of 1,3-propanediol from glycerol with high yield|
    JP5308163B2|2006-11-29|2013-10-09|株式会社カネカ|Novel alcohol dehydrogenase, its gene, vector, transformant, and method for producing optically active alcohol using them|
    DE102006056526A1|2006-11-30|2008-06-05|Archimica Gmbh|Process for the stereoselective synthesis of chiral epoxides by ADH reduction of alpha-leaving group-substituted ketones and cyclization|
    US8017364B2|2006-12-12|2011-09-13|Butamax Advanced Biofuels Llc|Solvent tolerant microorganisms|
    EP2102351A4|2006-12-15|2010-01-06|Biofuelchem Co Ltd|Method for preparing butanol through butyryl-coa as an intermediate using bacteria|
    WO2008074794A1|2006-12-19|2008-06-26|Dsm Ip Assets B.V.|Butanol production in a prokaryotic cell|
    WO2008080124A2|2006-12-21|2008-07-03|Gevo, Inc.|Butanol production by metabolically engineered yeast|
    KR20090117739A|2007-02-09|2009-11-12|더 리전트 오브 더 유니버시티 오브 캘리포니아|Biofuel production by recombinant microorganisms|
    KR101573775B1|2007-04-18|2015-12-04|부타맥스 어드밴스드 바이오퓨얼스 엘엘씨|- fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes|
    US20080274526A1|2007-05-02|2008-11-06|Bramucci Michael G|Method for the production of isobutanol|
    US8945899B2|2007-12-20|2015-02-03|Butamax Advanced Biofuels Llc|Ketol-acid reductoisomerase using NADH|
    US8129162B2|2007-12-20|2012-03-06|Butamax Advanced Biofuels Llc|Ketol-acid reductoisomerase using NADH|
    US8518678B2|2007-12-21|2013-08-27|Butamax Advanced Biofuels Llc|Strain comprising increased expression of a CFA coding region for butanol production|
    US8372612B2|2007-12-21|2013-02-12|Butamax Advanced Biofuels Llc|Production of four carbon alcohols using improved strain|
    US8017375B2|2007-12-23|2011-09-13|Gevo, Inc.|Yeast organism producing isobutanol at a high yield|
    EP2446043A4|2009-06-22|2013-02-13|Gevo Inc|Yeast organisms for the production of isobutanol|
    US8188250B2|2008-04-28|2012-05-29|Butamax Advanced Biofuels Llc|Butanol dehydrogenase enzyme from the bacterium Achromobacter xylosoxidans|
    US8389252B2|2008-05-12|2013-03-05|Butamax Advanced Biofuels Llc|Yeast strain for production of four carbon alcohols|
    US8906666B2|2008-05-22|2014-12-09|Butamax Advanced Biofuels Llc|Engineering resistance to aliphatic alcohols|
    US20120058541A1|2009-05-22|2012-03-08|Butamax Advanced Biofuels Llc|Engineering resistance to aliphatic alcohols|
    BRPI0909989A2|2008-06-05|2021-06-22|Butamax Advanced Biofuels Llc|recombinant yeast cell and method for producing a product|
    US20110281314A1|2008-08-04|2011-11-17|Lynch Michael D|Methods, systems and compositions related to microbial bio-production of butanol and/or isobutanol|
    US8455224B2|2008-09-29|2013-06-04|Butamax Advanced Biofuels Llc|Enhanced pyruvate to 2,3-butanediol conversion in lactic acid bacteria|
    CN102186983A|2008-09-29|2011-09-14|布特马斯先进生物燃料有限责任公司|Increased heterologous Fe-S enzyme actiivty in yeast|
    EP2821484B1|2008-09-29|2016-06-29|Butamax Advanced Biofuels LLC|Identification and use of Bacterial [2Fe-2S] dihydroxy-acid dehydratases|
    BRPI0913680A2|2008-09-29|2019-09-24|Butamax Advanced Biofuels Llc|"Lactic acid bacterial cell, method for increasing the activity of a heterologous dihydroxy acid dehydratase polypeptide in a lactic acid bacterial cell and insobutanol production method"|
    WO2010037105A1|2008-09-29|2010-04-01|Butamax™ Advanced Biofuels LLC|Enhanced dihydroxy-acid dehydratase activity in lactic acid bacteria|
    WO2010062597A1|2008-10-27|2010-06-03|Butamax™ Advanced Biofuels LLC|Carbon pathway optimized production hosts for the production of isobutanol|
    CA2779262C|2008-10-31|2021-09-07|Gevo, Inc.|Engineered microorganisms capable of converting pyruvate to isobutanol under anaerobic conditions|
    US8465964B2|2008-11-13|2013-06-18|Butamax Advanced Biofules LLC|Increased production of isobutanol in yeast with reduced mitochondrial amino acid biosynthesis|
    US8828694B2|2008-11-13|2014-09-09|Butamax Advanced Biofuels Llc|Production of isobutanol in yeast mitochondria|
    US8652823B2|2008-12-03|2014-02-18|Butamax Advanced Biofuels Llc|Strain for butanol production with increased membrane unsaturated trans fatty acids|
    US8557562B2|2008-12-29|2013-10-15|Butamax Advanced Biofuels Llc|Yeast with increased butanol tolerance involving filamentous growth response|
    US8455225B2|2008-12-29|2013-06-04|Butamax Advanced Biofuels Llc|Yeast with increased butanol tolerance involving high osmolarity/glycerol response pathway|
    US8795992B2|2008-12-29|2014-08-05|Butamax Advanced Biofuels Llc|Yeast with increased butanol tolerance involving cell wall integrity pathway|
    US8614085B2|2009-02-27|2013-12-24|Butamax Advanced Biofuels Llc|Yeast with increased butanol tolerance involving a multidrug efflux pump gene|
    CA2776151A1|2009-09-29|2011-04-07|Butamax Advanced Biofuels Llc|Improved flux to acetolactate-derived products in lactic acid bacteria|
    CA2775893A1|2009-09-29|2011-04-07|Butamax Advanced Biofuels Llc|Improved yeast production host cells|
    IN2012DN02227A|2009-09-29|2015-08-21|Butamax Tm Advanced Biofuels|
    US20110195505A1|2009-10-08|2011-08-11|Butamax Advanced Biofuels Llc|Bacterial strains for butanol production|
    KR20120099509A|2009-12-29|2012-09-10|부타맥스 어드밴스드 바이오퓨얼스 엘엘씨|Expression of hexose kinase in recombinant host cells|
    WO2011090753A2|2009-12-29|2011-07-28|Butamax Advanced Biofuels Llc|Alcohol dehydrogenases useful for fermentive production of lower alkyl alcohols|
    MX330710B|2010-02-12|2015-06-11|
    CA2788842C|2010-02-17|2019-03-19|Butamax Advanced Biofuels Llc|Improving activity of fe-s cluster requiring proteins|
    NZ603546A|2010-06-17|2014-10-31|Butamax Tm Advanced Biofuels|Yeast production culture for the production of butanol|
    US8871488B2|2010-06-18|2014-10-28|Butamax Advanced Biofuels Llc|Recombinant host cells comprising phosphoketolases|
    MX346408B|2010-09-07|2017-03-17|Butamax Advanced Biofuels Llc|Integration of a polynucleotide encoding a polypeptide that catalyzes pyruvate to acetolactate conversion.|
    BR112013024328A2|2011-03-24|2020-08-25|Butamax Advanced Biofuels Llc.|Recombinant host cells, methods for producing isobutanol, composition, methods for producing a recombinant host cell, to reduce the conversion of isobutiraldehyde, polypeptides, polynucleqtide, paraconverter acetolactate method, recombinant yeasts, methods for producing butanol, to increase the activity and KARL ENZYME PRODUCTIVITY|
    US20120258873A1|2011-04-06|2012-10-11|Butamax Advanced Biofuels Llc|Reduction of 2,3-dihydroxy-2-methyl butyrate in butanol production|
    CA2838519A1|2011-06-17|2012-12-20|Butamax Advanced Biofuels Llc|Lignocellulosic hydrolysates as feedstocks for isobutanol fermentation|
    US9238828B2|2011-07-28|2016-01-19|Butamax Advanced Biofuels Llc|Keto-isovalerate decarboxylase enzymes and methods of use thereof|
    EP2798072A2|2011-12-30|2014-11-05|Butamax Advanced Biofuels LLC|Fermentative production of alcohols|
    US9181566B2|2011-12-30|2015-11-10|Butamax Advanced Biofuels Llc|Genetic switches for butanol production|
    JP2015510774A|2012-03-23|2015-04-13|ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー|Acetic acid supplementation of medium for butanol-producing bacteria|
    CA2873109A1|2012-05-11|2013-11-28|Butamax Advanced Biofuels Llc|Ketol-acid reductoisomerase enzymes and methods of use|
    EP2900811B1|2012-09-26|2018-12-19|Butamax Advanced Biofuels LLC|Polypeptides with ketol-acid reductoisomerase activity|
    US9273330B2|2012-10-03|2016-03-01|Butamax Advanced Biofuels Llc|Butanol tolerance in microorganisms|
    US20140186911A1|2012-12-28|2014-07-03|Butamax Advanced Biofuels Llc|Recombinant host cells and methods for producing butanol|
    WO2014106107A2|2012-12-28|2014-07-03|Butamax Advanced Biofuels Llc|Dhad variants for butanol production|
    EP2970863B1|2013-03-14|2019-04-24|E. I. du Pont de Nemours and Company|Glycerol 3- phosphate dehydrogenase for butanol production|
    WO2014151190A1|2013-03-15|2014-09-25|Butamax Advanced Biofuels Llc|Dhad variants and methods of screening|US9303225B2|2005-10-26|2016-04-05|Butamax Advanced Biofuels Llc|Method for the production of isobutanol by recombinant yeast|
    KR101573775B1|2007-04-18|2015-12-04|부타맥스 어드밴스드 바이오퓨얼스 엘엘씨|- fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes|
    US8945899B2|2007-12-20|2015-02-03|Butamax Advanced Biofuels Llc|Ketol-acid reductoisomerase using NADH|
    US8188250B2|2008-04-28|2012-05-29|Butamax Advanced Biofuels Llc|Butanol dehydrogenase enzyme from the bacterium Achromobacter xylosoxidans|
    CN102186983A|2008-09-29|2011-09-14|布特马斯先进生物燃料有限责任公司|Increased heterologous Fe-S enzyme actiivty in yeast|
    CA2775893A1|2009-09-29|2011-04-07|Butamax Advanced Biofuels Llc|Improved yeast production host cells|
    WO2011090753A2|2009-12-29|2011-07-28|Butamax Advanced Biofuels Llc|Alcohol dehydrogenasesuseful for fermentive production of lower alkyl alcohols|
    CA2788842C|2010-02-17|2019-03-19|Butamax Advanced Biofuels Llc|Improving activity of fe-s cluster requiring proteins|
    WO2011140386A2|2010-05-05|2011-11-10|Mascoma Corporation|Detoxification of biomass derived acetate via metabolic conversion to ethanol, acetone, isopropanol, or ethyl acetate|
    US8871488B2|2010-06-18|2014-10-28|Butamax Advanced Biofuels Llc|Recombinant host cells comprising phosphoketolases|
    MX346408B|2010-09-07|2017-03-17|Butamax Advanced Biofuels Llc|Integration of a polynucleotide encoding a polypeptide that catalyzes pyruvate to acetolactate conversion.|
    US8765425B2|2011-03-23|2014-07-01|Butamax Advanced Biofuels Llc|In situ expression of lipase for enzymatic production of alcohol esters during fermentation|
    US8759044B2|2011-03-23|2014-06-24|Butamax Advanced Biofuels Llc|In situ expression of lipase for enzymatic production of alcohol esters during fermentation|
    BR112013024328A2|2011-03-24|2020-08-25|ButamaxAdvanced Biofuels Llc.|Recombinant host cells, methods for producing isobutanol, composition, methods for producing a recombinant host cell, to reduce the conversion of isobutiraldehyde, polypeptides, polynucleqtide, paraconverter acetolactate method, recombinant yeasts, methods for producing butanol, to increase the activity and KARL ENZYME PRODUCTIVITY|
    WO2012173660A2|2011-06-17|2012-12-20|ButamaxAdvanced Biofuels Llc|Co-products from biofuel production processes and methods of making|
    US9238828B2|2011-07-28|2016-01-19|Butamax Advanced Biofuels Llc|Keto-isovalerate decarboxylase enzymes and methods of use thereof|
    BR112014013741A2|2011-12-09|2017-06-13|Butamax Advanced Biofuels Llc|alcohol removal methods, alcohol recovery system, compositions and food|
    US9181566B2|2011-12-30|2015-11-10|Butamax Advanced Biofuels Llc|Genetic switches for butanol production|
    EP2798072A2|2011-12-30|2014-11-05|Butamax Advanced Biofuels LLC|Fermentative production of alcohols|
    WO2013102554A1|2012-01-06|2013-07-11|Firmenich Sa|Genetically engineered yeast cells|
    JP2015510774A|2012-03-23|2015-04-13|ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー|Acetic acid supplementation of medium for butanol-producing bacteria|
    CA2873109A1|2012-05-11|2013-11-28|Butamax Advanced Biofuels Llc|Ketol-acid reductoisomerase enzymes and methods of use|
    CA2883627A1|2012-09-12|2014-03-20|Butamax Advanced Biofuels Llc|Processes and systems for the production of fermentation products|
    AU2013329024A1|2012-10-11|2015-03-12|Butamax Advanced Biofuels Llc|Processes and systems for the production of fermentation products|
    US20140024064A1|2012-07-23|2014-01-23|Butamax Advanced Biofuels Llc|Processes and systems for the production of fermentative alcohols|
    BR112015003701A2|2012-08-22|2017-12-12|Butamax Advanced Biofuels Llc|recombinant host cells, method for enhancement, process for producing an alcohol, isolated polynucleotide, expression cassette and composition|
    US9840724B2|2012-09-21|2017-12-12|Butamax Advanced Biofuels Llc|Production of renewable hydrocarbon compositions|
    EP2900811B1|2012-09-26|2018-12-19|Butamax Advanced Biofuels LLC|Polypeptides with ketol-acid reductoisomerase activity|
    AU2013323396B2|2012-09-28|2017-04-20|Butamax Advanced Biofuels Llc|Production of fermentation products|
    US9273330B2|2012-10-03|2016-03-01|Butamax Advanced Biofuels Llc|Butanol tolerance in microorganisms|
    WO2014106107A2|2012-12-28|2014-07-03|ButamaxAdvanced Biofuels Llc|Dhad variants for butanol production|
    WO2014105840A1|2012-12-31|2014-07-03|Butamax Advanced Biofuels Llc|Fermentative production of alcohols|
    EP2970863B1|2013-03-14|2019-04-24|E. I. du Pont de Nemours and Company|Glycerol 3- phosphate dehydrogenase for butanol production|
    CN105229156A|2013-03-15|2016-01-06|布特马斯先进生物燃料有限责任公司|For utilizing the method extracting fermentative production butanols|
    WO2014144210A2|2013-03-15|2014-09-18|Butamax Advanced Biofuels Llc|Competitive growth and/or production advantage for butanologen microorganism|
    WO2014144643A1|2013-03-15|2014-09-18|Butamax Advanced Biofuels Llc|Method for producing butanol using extractive fermentation|
    WO2014151645A1|2013-03-15|2014-09-25|Butamax Advanced Biofuels Llc|Process for maximizing biomass growth and butanol yield by feedback control|
    WO2014151190A1|2013-03-15|2014-09-25|Butamax Advanced Biofuels Llc|Dhad variants and methods of screening|
    CN105073991B|2013-03-28|2019-04-05|株式会社钟化|Change type carbonyl reductase and its gene|
    WO2015002913A1|2013-07-03|2015-01-08|Butamax Advanced Biofuels Llc|Partial adaptation for butanol production|
    DE102013113719A1|2013-12-09|2015-06-11|Oxea Gmbh|Process for the preparation of pentane derivatives and derivatives of α, β-unsaturated decenals|
    WO2016025425A1|2014-08-11|2016-02-18|Butamax Advanced Biofuels Llc|Yeast preparations and methods of making the same|
    法律状态:
    2020-10-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2020-10-27| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 9A ANUIDADE. |
    2021-02-17| B08K| Patent lapsed as no evidence of payment of the annual fee has been furnished to inpi [chapter 8.11 patent gazette]|Free format text: EM VIRTUDE DO ARQUIVAMENTO PUBLICADO NA RPI 2599 DE 27-10-2020 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDO O ARQUIVAMENTO DO PEDIDO DE PATENTE, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |
    2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    US29063609P| true| 2009-12-29|2009-12-29|
    US61/290,636|2009-12-29|
    PCT/US2010/062390|WO2011090753A2|2009-12-29|2010-12-29|Alcohol dehydrogenasesuseful for fermentive production of lower alkyl alcohols|
    [返回顶部]