Microbial organism and method of producing a fatty acyl-coa derivative

专利摘要:
MICROBIAL ORGANISM AND METHOD OF PRODUCING A FATTY ACYL-CoA DERIVATIVE This invention provides microbial organisms, particularly yeasts such as Yarrowia lipolytica, which have one or more disrupted genes. The gene disruption(s) may yield better production of fatty acyl-CoA derivatives.
公开号:BR112013015797B1
申请号:R112013015797-6
申请日:2011-12-19
公开日:2021-08-31
发明作者:Douglas A. Hattendorf；Jennifer L. Shock；Louis Clark
申请人:Shell Internationale Research Maatschappij B.V.；
IPC主号:

专利说明:

CROSS REFERENCES TO RELATED ORDERS
[001] This application claims the priority benefit of provisional application US 61/427,032, filed December 23, 2010, and provisional application US 61/502,697 filed June 29, 2011, the entire contents of which are incorporated herein by reference. REFERENCE TO A “SEQUENCE LISTING”, A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE.
[002] The list of sequences written in the file 90834-824523_ST25.TXT, created on December 19, 2011, 188,336 bytes, IBM-PC machine format, MS-Windows operating system, is here incorporated by reference in its entirety and with all purposes. FIELD OF THE INVENTION
[003] This invention concerns the modified microbial organisms that exhibit better properties, especially better production of fatty acyl-CoA derivatives. FUNDAMENTALS OF THE INVENTION
[004] Microbial organisms produce fatty acyl-CoA and fatty acyl-CoA derivatives such as fatty alcohols, fatty acids, fatty aldehydes, fatty esters, fatty acetates, wax esters, alkanes and alkenes. Such fatty acyl-CoA derivatives can be used to produce a wide variety of products, including aviation and diesel fuels (eg biodiesel), chemical surfactants, polymers, nutritional supplements, pharmaceutical compounds, food additives, cosmetics and food products. personal care.
[005] Fatty acids are a major component of cell membranes and are used by organisms for energy storage. Fatty acids are metabolized by e-oxidation of fatty acyl-CoA, or conversely, fatty acids are synthesized from acetyl-CoA by multi-enzyme fatty acid synthase complexes. Fatty alcohols are the reduction products of fatty acyl-thioester substrates (eg, fatty acyl-CoA or fatty acyl-ACP), and just as fatty acids can be produced enzymatically by cultured cells. Enzymes that convert fatty acyl-thioester substrates (eg, fatty acyl-CoA or fatty acyl-ACP) to fatty alcohols are commonly referred to as “fatty-alcohol-forming acyl-CoA reductases” or “fatty acyl reductases” (“ FARs”).
[006] The commercial production and recovery of fatty alcohols from microbial organisms is a challenge, in part because fatty alcohols are not very stable in many microorganisms. Fatty alcohols (eg hexadecanol) can be used as a carbon source for the microorganism and thus can be metabolized by the microorganism prior to recovery for commercial purposes. Fatty alcohols are likely degraded by enzymes that catalyze the oxidation of alkanes to fatty acids (via fatty alcohols). Fatty acids can then be further degraded to acetyl-CoA by enzymes in the β-oxidation pathway, or converted to storage lipids by a set of acetyltransferases.
[007] Thus, there is a need for microbial organisms for the efficient production of fatty acyl-CoA derivatives. SUMMARY OF THE INVENTION
[008] This invention provides modified microbial organisms that exhibit better properties, including better production of fatty acyl-CoA derivatives. In some respects, modified microbial organisms have a disrupted gene that provides better production of fatty acyl-CoA derivatives, compared to a control organism of the same type in which the gene is not disrupted. In one modality the organism is Yarrowia lipolytica.
[009] In one aspect, the invention relates to a microbial organism in which one or more endogenous genes are disrupted, wherein the endogenous gene is YALI0C17545 or a homolog thereof, and/or YALI0E28336 or a homolog thereof, and comprising a exogenous gene encoding a functional fatty acyl reductase (FAR) protein operably linked to a promoter. In another aspect, both the endogenous gene YALI0C17545 or its homologue, and the endogenous gene YALI0E28336 or its homologue are interrupted. In another aspect, the microbial organism further comprises an interruption of one or more of endogenous gene YALI0E11099 or a homolog thereof, and endogenous gene YALI0E28534 or a homolog thereof. In another aspect, both the endogenous gene YALI0E11099 or its homologue, and the endogenous gene YALI0E28534 or its homologue are interrupted. In a further aspect, the microbial organism further comprises an interrupt one or more selected endogenous gene YALI0B10406, YALI0A19536, YALI0E32769, YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512, and homologs thereof. In another aspect, the endogenous YALI0B17512 gene is disrupted.
[0010] In another aspect, two or more of the endogenous genes are disrupted. In another aspect, three or more of the endogenous genes are disrupted. In another aspect, four or more of the endogenous genes are disrupted.
[0011] In another aspect, the microbial organism comprises: a deletion of all or a portion of the coding sequence of the endogenous gene, a mutation in the endogenous gene in such a way that the gene encodes a polypeptide with reduced activity, antisense RNA or small RNA of interference that inhibits endogenous gene expression, or a modified regulatory sequence that reduces endogenous gene expression. In one embodiment, the microbial organism comprises a deletion of all or a portion of the endogenous gene coding sequence.
[0012] In one aspect, the exogenous gene encodes a functional FAR protein comprising a polypeptide sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a Marinobacter algicola FAR protein comprising SEQ ID NO:2 . In another aspect, the exogenous gene encodes a functional FAR protein comprising a polypeptide sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a FAR protein from Marinobacter aquaeolei comprising SEQ ID NO:4. In another aspect, the exogenous gene encodes a functional FAR protein comprising a polypeptide sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a FAR protein from Oceanobacter sp. RED65 comprising SEQ ID NO:6. In one aspect, the exogenous gene includes a nucleic acid sequence with at least 80% sequence identity, often at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleic acid sequence of FAR_Maa (SEQ ID NO:1), FAR_Maq (SEQ ID NO:3), or FAR_Ocs (SEQ ID NO:5). In one embodiment, fatty acyl reductase has a gene with at least 80% sequence identity, often at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the FAR_Maa nucleic acid sequence (SEQ ID NO:1).
[0013] In one aspect, the functional FAR protein is a FAR variant that comprises one or more amino acid substitutions related to SEQ ID NO:2, 4 or 6, respectively, wherein a cell in which the FAR variant is expressed produces at least 1.5 times more fatty acyl-CoA derivatives than a corresponding cell of the same type, in which a wild-type FAR protein is expressed, from which the FAR variant is derived. In another aspect, the exogenous FAR gene encodes a FAR variant comprising from 1 to about 50, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 amino acid substitutions with respect to FAR_Maa (SEQ ID NO:2), FAR_Maq (SEQ ID NO:4), or FAR_Ocs (SEQ ID NO:6). In one embodiment, the exogenous FAR gene encodes a FAR variant comprising from 1 to about 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 amino acid substitutions with respect to FAR_Maa (SEQ ID NO:2).
[0014] In another aspect, the microbial organism has multiple copies of the endogenous gene (eg a diploid number) and more than one copy of the endogenous gene is disrupted. In another aspect, the microbial organism expresses multiple copies of the exogenous gene. In another aspect, the exogenous gene is integrated into the genome of the microbial organism.
[0015] In another aspect, the microbial organism additionally comprises a second exogenous gene encoding a fatty acid synthase (FAS), an ester synthase, an acyl-ACP thioesterase (TE), a fatty acyl-CoA synthase (FACS), an acetyl-CoA carboxylase (ACC), a xylose isomerase or an invertase.
[0016] In one aspect, the microbial organism is represented by algae, bacteria, mold, filamentous fungus or yeast, such as an oleaginous yeast. In one aspect, the microbial organism is yeast. In one aspect, the yeast is Yarrowia, Brettanomyces, Candida, Cryptococcus, Endomycopsis, Hansenula, Kluyveromyces, Lipomyces, Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Trichosporon or Trigonopsis. In one aspect, the yeast is an oleaginous yeast, such as Yarrowia lipolytica, Yarrowia paralipolytica, Candida revkauji, Candida pulcherrima, Candida tropicalis, Candida utilis, Candida curvata D, Candida curvataR, Candida diddensiae, Candida boldinii, Rhodotorula glutinous, Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorula minuta, Rhodotorula bacarum, Rhodosporidium toruloides, Cryptococcus (terricolus) albidus var. albidus, Cryptococcus laurentii, Trichosporon pullans, Trichosporon cutaneum, Trichosporon cutancum, Trichosporon pullulans, Lipomyces starkeyii, Lipomyces lipoferus, Lipomyces tetrasporus, Endomycopsis vernalis, Hansenula ciferri, or Trigonula sa. In one aspect, the yeast is Yarrowia lipolytica.
[0017] In another aspect, the microbial organism exhibits at least a 1-fold, at least 1.2-fold, at least 1.5-fold, at least 4-fold, or at least 20-fold increase in production of a fatty acyl-CoA derivative compared to a control organism of the same type (eg, an otherwise identical control microbial organism, in which the one or more genes are not disrupted).
[0018] In another aspect, the invention relates to a microbial organism comprising one or more endogenous disrupted genes, wherein at least one of the disrupted genes is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512, or homologue of any one thereof, and an exogenous gene encoding an acyl fatty functional reductase operably linked to a promoter, in which the microbial organism exhibits at least an increase. 1 time, at least 1.2 times, at least 1.5 times, at least 4 times, or at least 20 times in the production of a fatty acyl-CoA derivative compared to a control organism of the same type (eg, an otherwise identical control microbial organism, in which the one or more genes are not disrupted).
In yet another aspect, at least one of the disrupted endogenous genes is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283, YALI0E10E3, YALI0E1246 of any such homologue.
[0020] In one aspect, YALI0C17545 or a homolog thereof is stopped. In another aspect, YALI0E28336 or a homolog of it is stopped. In yet another aspect, either YALI0C17545 or a homolog of it, or YALI0E28336 or a homolog of it are interrupted.
[0021] In yet another aspect, the microbial organism further comprises a second disrupted gene which is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283, YALI15A, YALI01044 YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512 or a homolog of any of these.
[0022] In one aspect, the microbial organism comprises two disrupted endogenous genes. When two genes are disrupted, YALI0C17545 or a homologue thereof, and/or YALI0E30283 or a homolog thereof may be disrupted. In another aspect, the microbial organism comprises three disrupted endogenous genes. In yet another aspect, the microbial organism comprises four or more disrupted endogenous genes.
[0023] In another aspect, the microbial organism comprises a combination of interrupted endogenous genes or homologs thereof. The combination can be: a. YALI0C17545 and YALI0E28336; B. YALI0C17545 and YALI0B10406; ç. YALI0C17545 and YALI0E28534; d. YALI0C17545 and YALI0E30283; and. YALI0E28336 and YALI0E30283; f. YALI0E11099 and YALI0E30283; g. YALI0A19536 and YALI0E30283; H. YALI0A19536 and YALI0E28534; i. YALI0E30283 and YALI0E12463; j. YALI0B10406 and YALI0E14729; k. YALI0C17545 and YALI0E14729; l. YALI0E11099 and YALI0E14729; m. YALI0C17545, YALI0E28336 and YALI0E11099; n. YALI0C17545, YALI0E28336 and YALI0B10406; O. YALI0C17545, YALI0E28336 and YALI0A19536; for. YALI0C17545, YALI0E28336 and YALI0E28534; q. YALI0C17545, YALI0E28336 and YALI0E32769; a. YALI0C17545, YALI0E28336 and YALI0E12463; s. YALI0C17545, YALI0E11099 and YALI0B10406; t. YALI0C17545, YALI0B10406 and YALI0A19536; u. YALI0E28336, YALI0E11099 and YALI0B10406; v. YALI0E11099, YALI0B10406 and YALI0A19536; w. YALI0C17545, YALI0E28534 and YALI0B17512; x. YALI0E11099, YALI0A19536, YALI0B10406 and YALI0B17512; y. YALI0C17545, YALI0E28336, YALI0E11099 and YALI0B10406; z. YALI0C17545, YALI0E28336, YALI0E11099 and YALI0A19536; yy. YALI0C17545, YALI0E28336, YALI0E11099 and YALI0E28534; bb. YALI0C17545, YALI0E28336, YALI0E11099 and YALI0E32769; cc. YALI0C17545, YALI0E28336, YALI0B10406 and YALI0A19536; dd. YALI0C17545, YALI0E28336, YALI0B10406 and YALI0E32769; and is. YALI0C17545, YALI0E28336, YALI0A19536 and YALI0E28534; ff. YALI0C17545, YALI0E28336, YALI0E28534 and YALI0E32769; gg. YALI0C17545, YALI0E28336, YALI0E28534 and YALI0E12463; hh. YALI0E28336, YALI0E11099, YALI0B10406 and YALI0E32769; or ii. YALI0E11099, YALI0E28336, YALI0C17545 and YALI0E14729.
[0024] In one aspect, a Yarrowia lipolytica cell comprises one or more endogenous disrupted genes, wherein the at least one disrupted gene is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E28534, YALI0C175E, YALI0C175E12,02769, or a homolog of any of these, and an exogenous gene encoding a functional fatty acyl reductase operably linked to a promoter, wherein the Yarrowia lipolytica cell exhibits at least a 1-fold increase, at least a 1.2-fold, at least less than 1.5 times, at least 4 times, or at least 20 times in the production of a fatty acyl-CoA derivative compared to a control organism of the same type (eg, an otherwise identical control microbial organism, where the one or more genes are not disrupted). In one aspect, the exogenous gene includes a nucleic acid sequence with at least 80% sequence identity, often at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least at least 98%, or at least 99% sequence identity with a nucleic acid sequence of FAR_Maa (SEQ ID NO:1), FAR_Maq (SEQ ID NO:3), or FAR_Ocs (SEQ ID NO:5), or encodes a polypeptide that includes an amino acid sequence having at least 80% sequence identity, often at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a polypeptide of FAR_Maa (SEQ ID NO:2), FAR_Maq (SEQ ID NO:4), or FAR_Ocs (SEQ ID NO:6); or encodes a FAR polypeptide variant comprising from 1 to about 50, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 25, 30, or 40 amino acid substitutions with respect to FAR_Maa (SEQ ID NO:2), FAR_Maq (SEQ ID NO:4), or FAR_Ocs (SEQ ID NO:6). In one embodiment, the exogenous FAR gene encodes a FAR variant comprising from 1 to about 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 amino acid substitutions with respect to FAR_Maa (SEQ ID NO:2).
[0025] In another aspect, the invention provides a microbial organism in which one or more endogenous genes are disrupted, wherein the endogenous gene is selected from YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI3083, YALI020E3 YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F2250, YALI175, YALI1201 homologist of these. In another aspect, the endogenous YALI0B17512 gene, or homologue thereof, is disrupted. In another aspect, YALI0B17512 encodes a polypeptide comprising a cytoplasmic domain and the interruption comprises a deletion of at least a portion of the cytoplasmic domain. In another aspect, one or more of the endogenous gene YALI0C17545, or homolog thereto, and the endogenous gene YALI0E28336, or homolog thereto, is disrupted.
[0026] In another aspect, the invention provides a method for producing a fatty acyl-CoA derivative which comprises providing a microbial organism in the manner described herein; and cultivating the microbial organism under conditions in which fatty acyl-CoA derivatives are produced. The method may further include recovering (e.g. isolating) the fatty acyl-CoA derivative. In one aspect, at least 5 g/L or at least 15 g/L of fatty acyl-CoA derivatives per liter of culture medium are produced.
[0027] In another aspect, a method for producing a fatty acyl-CoA derivative may include placing a biomass containing cellulose in contact with one or more cellulases to yield fermentable sugars; and putting fermentable sugars in contact with the microbial organism. In another aspect, the method for producing a fatty acyl-CoA derivative may include contacting the fermentable sugars comprising sucrose with the microorganism in the manner described herein.
[0028] In one aspect, the fatty acyl-CoA derivative is a fatty alcohol, fatty acid, fatty aldehyde, fatty ester, fatty acetate, wax ester, alkane or alkene. In another aspect, the fatty acyl-CoA derivative is a fatty alcohol. In one aspect, the fatty acyl-CoA derivative has a carbon chain size of 8 to 24 carbon atoms, such as a fatty alcohol having 8 to 24 carbon atoms.
[0029] In another aspect, the invention provides a composition comprising the fatty acyl-CoA derivative(s) produced by a method as described herein. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 illustrates pathways for the biosynthesis of fatty acyl-CoA derivatives in Y. lipolytica. The natural pathways for the biosynthesis of fatty acyl-CoA from glucose (reactions 1-3) and for the degradation of alkanes and alkane oxidation products to fatty acyl-CoA are shown (reactions 47). Natural and exogenous pathways for the production of fatty acyl-CoA derivative products are also shown and include: acyltransferases (triacylglycerides), thioesterases (fatty acids), ester synthases (esters), acyl-CoA reductases ("FARs") (aldehydes fatty acids and fatty alcohols), and aldehyde decarbonylases (alkanes).
[0031] Figure 2 illustrates plasmid pCEN411 for the expression of FAR genes in Y. lipolytica. DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS
[0032] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In general, the nomenclature used herein and laboratory procedures in analytical chemistry, cell culture, molecular genetics, organic chemistry and nucleic acid chemistry, and hybridization described below are those well known and commonly employed in the art. It is noted that as used herein, “a”, “an”, “the” and “a” include the plural references, unless otherwise clearly indicated in the context. The term “who understands” and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates.
[0033] The techniques and procedures are generally performed according to conventional methods in the art and various general references. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed.; Ausubel, ed., 1990-2008, Current Protocols in Molecular Biology. Standard techniques, or modifications thereof, are used for nucleic acid and polypeptide synthesis, and for chemical synthesis and chemical analysis. In general, enzymatic reactions and purification steps are carried out according to the manufacturer's specifications. For techniques relating to recombinant yeast, nutrition and growth technologies, see, for example, Walker, 1998, Yeast Physiology and Biotechnology.
[0034] The term "disrupted", as applied to a gene, refers to any genetic modification that decreases or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (mRNA and/or protein). Genetic modifications include the complete or partial inactivation, deletion, deletion, interruption, blocking or downregulation of a gene. This can be accomplished, for example, by "neutralization", inactivation, gene mutation (eg insertion, deletion, point mutations or by frameshift that interrupt the expression or activity of the gene product), or by the use of RNAs inhibitory (eg sense, antisense, or RNAi RNA technology). An interruption can include all or part of a gene coding sequence.
[0035] The term "neutralized" has its conventional meaning in the art, and refers to an organism or cell in which a specific gene has been inactivated by genetic manipulation, generally by a recombination event in which all or a portion of the gene is deleted or heterologous DNA is inserted so that the cell or organism does not produce a functional product encoded by the gene. Neutralized also refers to the process of making an organism or cell with an inactivated gene, usually by replacing at least a portion of a gene's coding sequence with an artificial piece of DNA (eg, encoding a selection marker) and /or deleting at least a portion of the gene's coding sequence such that a functional gene product is not expressed in the cell or organism. In some embodiments the complete coding sequence of the gene is excised.
"Coding sequence" refers to that portion of a nucleic acid that encodes an amino acid sequence of a protein.
The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term "fatty acyl-CoA derivative" is a compound that can be metabolically derived from fatty acyl-CoA, fatty acyl-ACP, or other similar fatty acyl thioester in a microorganism. Derivatives include, but are not limited to, fatty alcohols, fatty acids, fatty aldehydes, fatty esters, fatty acetates, wax esters, alkanes and alkenes. Saturated or unsaturated fatty acyl-CoA derivatives can be described using the observation "Ca:b", where "a" is an integer that represents the total number of carbon atoms, and "b" is an integer that represents the total number of carbon atoms. refers to the number of double bonds in the carbon chain. Unsaturated fatty acyl CoA derivatives may be referred to as "cisΔx" or "transΔx", where "cis" and "trans" refer to the configuration of the carbon chain around the double bond. The “x” indicates the number of the first carbon of the double bond, where carbon 1 is, for example, the carbon of the fatty acid carboxylic acid or the carbon bond to the OH group of the fatty alcohol. With respect to the derivatives described below, “R” is a C8 to C24 saturated, unsaturated, linear, branched, or cyclic hydrocarbon (or “C7 to C23” in derivative formulas that expressly articulate the terminal carbon).
The term "fatty alcohol", as used herein, refers to an aliphatic alcohol of the formula R-OH, where "R" is as defined above. In some embodiments, a fatty alcohol produced in accordance with the methods disclosed herein is a saturated or unsaturated C8-C24 fatty alcohol (i.e., a C8, C9, C10, C11, C12, C13, C14, C15, C16, fatty alcohol, C17, C18, C19, C20, C21, C22 or C24). In some embodiments, one or more of the following fatty alcohols are produced: 1-octanol (C8:0), 1-decanol (C10:0), 1-dodecanol (C12:0), 1-tetradecanol (C14:0), 1-hexadecanol (C16:0), 1-octadecanol (C18:0), 1-icosanol (C20:0), 1-docosanol (C22:0), 1-tetracosanol (C24:0), cis Δ9-1- hexadecenol (C16:1), and cis Δ11-1-octadecenol (C18:1). It is understood that, unless otherwise specified, a reference to a “Cx fatty alcohol” includes both saturated and unsaturated fatty alcohols with “x” carbon atoms.
[0040] The term "fatty acid", as used herein, refers to a compound of the formula O

[0041] The term "fatty aldehyde" as used herein refers to a compound of the formula jj

[0042] jj The term "fatty esters" includes compounds of the formula

where R' is a short chain hydrocarbon, for example C1 to C6, preferably C1 to C4. For example, the fatty acyl-CoA can be reacted with a short-chain alcohol (eg, methanol or ethanol) to form conventional fatty esters. In contrast, fatty alcohols can be reacted with short-chain thioesters (eg, acetyl CoA) to form esters. Both types of esters are included by the term "fatty esters".
[0043] The term "fatty acetates", as used herein, refers to a compound of formula 11

[0044] The term "wax esters", as used herein, refers to an ester derived from a long chain fatty acid and a long chain alcohol.
[0045] Reference herein to particular endogenous genes by name is for illustration and not limitation. Gene names are understood to vary from organism to organism and reference to a gene name is not intended to be limiting, but is intended to include homologues (ie, which may be endogenous to a related microbial organism) and polymorphic variants. Homologues and variants can be identified based on sequence identity and/or similar biological (e.g., enzymatic) activity. In certain embodiments, the invention includes a polynucleotide or polypeptide sequence of at least 50%, 60%, 70%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identity with the named gene or gene product.
[0046] "Identity" or "percent identity", in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or sub-sequences that are the same or have a specified percentage of nucleotides or residues of amino acids, respectively, which are the same. Percent identity can be determined by comparing two ideally aligned sequences in a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions (i.e. gaps) compared to the reference sequence ( which can also contain gaps to optimize alignment) for the alignment of the two sequences. For example, the sequence may have a percent identity of at least 50%, 60%, 70%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% with a specified region in a reference string, when compared and aligned for maximum match with a comparison window, or determined region, measured using string comparison algorithms or by manual alignment and visual inspection.
Alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the similarity method search of Pearson and Lipman, 1988, Proc. Natl. Academic Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection (see, in general, Current Protocols in Molecular Biology, FM Ausubel et al. , eds., Current Protocols, John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)).
[0048] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 33893402, respectively. Software for performing the BLAST analysis is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high similarity sequence pairs (HSPs) by identifying short words of length W in the input sequence, which both match and satisfy some positive threshold score T when aligned with a word of the same length in a sequence of data base. T is referred to as the nearest word score threshold (Altschul et al, supra). These initial next word hits act as key pieces to initiate searches and find longer HSPs containing them. The word hits are then extended in both directions along each sequence, as far as the cumulative alignment score can be greater. Cumulative scores are calculated using, relative to the nucleotide sequences, the parameters M (fidelity score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score decreases by the quantity X from its maximum achieved value; the cumulative score goes to zero or less due to the accumulation of one or more negative-scoring residue alignments; or the end of each sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as standards a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as standards a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89 :10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI), using the standard parameters provided.
[0049] The "reference sequence" refers to a defined sequence and used as a basis for a sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. In general, a reference sequence is at least 20 nucleotide or amino acid residues in size, at least 25 residues in size, at least 50 residues in size, at least 100 residues in size, or the full length of the nucleic acid or polypeptide. Since the two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two In sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides with a "comparison window" to identify and compare local regions of sequence similarity.
[0050] The "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide or amino acid residue positions, in which a sequence can be compared to a reference sequence of at least 20 nucleotides or amino acids contiguous, and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be greater than 20 contiguous residues and optionally includes 30, 40, 50, 100 or more windows.
As used herein, "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and complements thereof.
The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.
[0053] "Best production" refers to an increase in the measurable amount of fatty acyl-CoA derivatives produced by a modified microbial organism (ie, a microbial organism in which one or more endogenous genes are disrupted), compared to amount produced by a control microbial organism of the same type, in which the genes are not disrupted when grown under the same conditions. The "control organism of the same type" means an organism of the same species with a genome that is essentially identical to the genome of the modified microbial organism, except for an interrupted gene or combination of genes described herein. For example, a strain of Y. lipolytica (eg, DSMZ 1345), in which a fatty acid synthase is overexpressed, may be a “control organism of the same type” for the same strain of Y. lipolytica (eg, DSMZ 1345 ), where a fatty acid synthase is overexpressed and where the specified gene or combination of genes is disrupted. The term “otherwise identical organism” is used interchangeably with “control organism of the same type”. The best production can occur by any mechanism, for example, greater production and/or less degradation or utilization.
The term "functional", as used in referring to a polypeptide, means that the polypeptide exhibits catalytic activity in vivo. The term “functional” can be used interchangeably with the term “biologically active”.
The terms "wild type" or "natural" used in reference to a polypeptide or protein means a polypeptide or protein expressed by a microorganism found in nature. When used in reference to a microorganism, the term means a naturally occurring microorganism (not genetically modified).
[0056] A "FAR" (also known as "acyl-CoA reductase forming fatty alcohol" or "fatty acyl reductase"), as used herein, refers to an enzyme that converts fatty acyl-thioester substrates (by example, fatty acyl-CoA or fatty acyl-ACP) in fatty alcohols. “CoA” is a non-protein acyl carrier factor (or fraction) involved in fatty acid synthesis and oxidation. "ACP" is a fatty acid synthase polypeptide or protein subunit used in fatty acid synthesis.
The term "wild-type FAR", as used herein, refers to a FAR polypeptide that is produced in nature. In some embodiments, a wild-type FAR is produced by a gammaproteobacterium including, but not limited to, strains of Marinobacter, Oceanobacter, and Hahella. Naturally occurring FAR polypeptides are described, for example, in U.S. patent application 2011/0000125, incorporated herein by reference. In some embodiments, a wild-type FAR is a naturally occurring FAR polypeptide that is produced by the strain Marinobacter algicola DG893 (SEQ ID NO:2). In some embodiments, a wild type FAR is a naturally occurring FAR polypeptide that is produced by the strain Marinobacter aquaeolei VT8 (SEQ ID NO:4) In some embodiments, a wild type FAR is a naturally occurring FAR polypeptide that is produced by Oceanobacter sp. RED65 (SEQ ID NO:6).
The term "FAR variant", as used herein, refers to full-length FAR polypeptides with substitutions at one or more amino acid positions relative to a wild-type FAR polypeptide, and functional fragments thereof, wherein a cell (eg, a microbe) in which the variant is expressed, and is capable of catalyzing greater production of fatty alcohols compared to a cell in which the wild-type FAR polypeptide is expressed. In some embodiments, a FAR variant comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a FAR polypeptide of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, and also comprises one or more amino acid substitutions that give rise to increased production of the fatty acyl-CoA derivative (eg, fatty alcohol), compared to the production of the fatty acyl-CoA derivative which can be targeted with the wild-type FAR polypeptide from which it is derived. FAR variants are described, for example, in U.S. application 13/171,138, incorporated herein by reference. As used herein, except where otherwise clear in context, reference to a "FAR", "FAR protein", "FAR variant", or "FAR fragment" is intended to refer to a functional FAR protein , functional variant of FAR, or functional fragment FAR, even if not explicitly indicated.
[0059] The term "endogenous" refers to a gene or protein that is originally contained in an organism (that is, encodes a sequence found in the wild-type organism). In contrast, the terms "exogenous" or "heterologous", as used in reference to a gene, refer interchangeably to a gene that originates outside the microorganism, such as a gene from another species, or a modified or recombinant. An exogenous or heterologous gene can be introduced into the microorganism by methods known in the art.
Nucleic acid sequences can be "introduced" into a cell by transfection, transduction, transformation or any other method. A nucleic acid sequence introduced into a eukaryotic or prokaryotic cell can be integrated into a chromosome, or it can be maintained in an episome.
[0061] The terms "transform" or "transformation", as used in reference to a cell, means a cell that has an unnatural nucleic acid sequence integrated into its genome or as an episome (eg, plasmid), which it is maintained through several generations.
"Vector" refers to a DNA construct that comprises a protein DNA coding sequence. A vector may be an expression vector which comprises a protein coding sequence operably linked to a suitable control sequence (i.e. promoter) capable of effecting DNA expression in a suitable host.
"Operably linked" means that the segments of the DNA sequence are arranged in such a way that they function in combination with their intended purposes, for example, a promoter controls the transcription of a gene sequence to which it is operably linked.
The "promoter sequence" is a nucleic acid sequence that is recognized by a cell for the expression of the coding region. The control sequence may comprise an appropriate promoter sequence. The promoter sequence contains transcriptional control sequences, which mediate expression of the polypeptide. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in the cell of choice, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides either endogenous or exogenous (heterologous) to the host cell.
[0065] The term "cultivate" refers to the growth of a population of microbial cells under suitable conditions, in a liquid or solid medium. More often, a liquid medium is used. In some modalities, cultivar refers to the fermentative bioconversion of a substrate into a final product.
[0066] The term "putting in contact" refers to combining an enzyme and a substrate under conditions in which the enzyme can act on the substrate. Those skilled in the art will understand that mixing a solution containing an enzyme (eg a cellulase) with a substrate (eg a biomass containing cellulose) will affect “putting in contact”. Similarly, in the context of cultivating microorganisms, cultivating microorganisms in a medium that contains a substrate (eg a fermentable sugar) will affect “putting” the microorganism into contact with the substrate.
[0067] The term "cellulase" refers to a category of enzymes capable of interrupting the crystal structure of cellulose, and hydrolyzing cellulose (β-1,4-glucan or β-D-glycosidic bonds) into oligosaccharides, disaccharides ( eg cellobiose) and/or minor monosaccharides (eg glucose). Cellulases include endoglucanases, cellobiohydrolases and beta-glucosidases.
[0068] The terms "cellulose containing biomass", "cellulosic biomass" and "cellulosic substrate" refer to materials that include cellulose. Biomass can be derived from plants, animals or microorganisms, and can include agricultural, industrial and forestry waste, municipal solid waste, industrial waste, and land and aquatic crops grown for energy purposes. Examples of biomass include, but are not limited to, wood, wood pulp, paper pulp, corn fiber, corn grain, corn cobs, agricultural crop residues such as corn husks, corn waste, grasses, wheat, straw of wheat, barley, barley straw, hay, rice straw, fast-growing grass, waste paper, pulp and paper processing residue, woody or herbaceous plants, fruit or vegetable pulp, grain for distillation, rice husks, cotton, hemp, flax, sisal, sugarcane bagasse, sorghum, soybeans, components obtained from the milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, excrement of animal and mixtures thereof.
[0069] The "fermentable sugar" means simple sugars (monosaccharides, disaccharides and small oligosaccharides) including, but not limited to, glucose, fructose, xylose, galactose, arabinose, mannose and sucrose.
[0070] The term "recoverable fatty acyl-CoA derivative" refers to the amount of fatty acyl-CoA derivatives that can be isolated from a reaction mixture that yields the fatty acyl-CoA derivatives, according to known methods in technique. II. INTRODUCTION
[0071] It has surprisingly been found that disruption of certain endogenous genes and gene combinations in a microbial organism, eg Yarrowia lipolytica, which express a fatty acyl reductase (FAR) results in increased production of acyl-CoA derivatives fat. A FAR (also known as "fatty alcohol-forming acyl-CoA reductase" or "fatty acyl reductase") refers to an enzyme that catalyzes the reduction of a fatty acyl-CoA, a fatty acyl-ACP, or other complex of fatty acyl thioester NAD(P)+, as shown in scheme 1 below:
where "R" represents a saturated, unsaturated, straight, branched, or cyclic C7 to C23 hydrocarbon chain, and "R1" represents CoA, ACP or other fatty acyl thioester substrates. CoA is a non-protein acyl carrier group (or fraction) factor involved in fatty acid synthesis and oxidation. "ACP" is a fatty acid synthase polypeptide or protein subunit used in fatty acid synthesis. In some embodiments, a fatty aldehyde intermediate can be produced in the reaction depicted in scheme 1.
Wild-type protein FARs have been described in WO 2011/008535 (published 20 January 2011), incorporated herein by reference for all purposes. Certain FAR enzymes isolated from genera of the marine bacteria class, such as gammaproteobacteria found in seawater (and particularly FARs obtained from the Marinobacter and Oceanobacter strains, or taxonomic equivalents thereof), are capable of generating high yields of fatty alcohols when the genes that encode these enzymes are expressed in heterologous cells. As described in the examples section below, microbial organisms in which certain genes or gene combinations are disrupted, and which express a gene encoding a FAR protein, have now been found to have increased production of fatty acyl-CoA derivatives compared to otherwise identical microbial organisms, which express the exogenous gene encoding the FAR protein in which the genes have not been disrupted. Thus, in one aspect, the present invention relates to a microbial organism that exhibits increased production of fatty acyl-CoA derivatives, wherein the microbial organism comprises one or more disrupted endogenous genes and an exogenous gene encoding a FAR protein. These modified microbial organisms can be used in the commercial production of fatty acyl-CoA derivatives.
[0073] Various aspects of the invention are described in the following sections. III. INTERRUPTION OF ENDOGENOUS GENES Endogenous Genes for Interruption
[0074] In one aspect, the present invention concerns recombinant microbial organisms, such as yeasts, in which one or more endogenous genes are disrupted, and that exhibit better production of fatty acyl-CoA derivatives and methods of using such microbial organisms .
[0075] The endogenous genes described herein are named with reference to the genome of Yarrowia lipolytica. Dujon, et al., 2004, "Genome evolution in Yeasts" Nature 430:35-44. The gene's short name (eg, “C17545”) and the gene's full name (eg, “YALI0C17545”) are used interchangeably, and both include polymorphic gene variants. In some embodiments, the host cell is other than Y. lipolytica, and the endogenous gene is a homolog of the Y. lipolytica gene. As noted above, gene names vary from organism to organism and any gene name used herein is not intended to be limiting, but is intended to include homologues as well. Table 1 provides a listing of nucleotide sequences for exemplary disrupted genes from Y. lipolytica, as well as the activities of the encoded proteins. Biological activities are determined based on reference to the scientific literature, and/or based on functional and sequence characterization. Although known or predicted biological activities can be used to identify homologues, a nucleotide and/or protein sequence for use in the present invention is not limited to those nucleotide and/or protein sequences, which have previously been identified as being involved in production of the fatty acyl-CoA derivative.
[0076] In some embodiments, a microbial organism of the present invention (e.g., algae, bacteria, mold, filamentous fungus, or yeast, e.g., Yarrowia lipolytica) has one or more disrupted endogenous genes selected from the genes listed in Table 1, and counterparts of these.
[0077] Table 1. Nucleotide sequences for disrupted genes in Yarrowia lipolytica




[0078] In some embodiments, the microbial organism, e.g., Yarrowia lipolytica, has one or more endogenous disrupted genes, wherein at least one of the disrupted genes is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E69,08534, YALI0E28534, YALI0E28534 YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI12F12, YALI2 or any one of these.
[0079] In some embodiments, the endogenous disrupted gene is C17545 (SEQ ID NO:7) or a homolog thereof. In some embodiments, the endogenous disrupted gene is E28336 (SEQ ID NO:8) or a homolog thereof. In some embodiments, the endogenous disrupted gene is E11099 (SEQ ID NO:9) or a homolog thereof. In some embodiments, the endogenous disrupted gene is E28534 (SEQ ID NO:12) or a homolog thereof. In some embodiments, the endogenous disrupted gene is B17512 (SEQ ID NO:54) or a homolog thereof.
[0080] In some embodiments, the microbial organism, for example, Yarrowia lipolytica, is one in which one, two, three, four, or five endogenous genes in the microbial organism are disrupted. In some embodiments, one or more, two or more, three or more, four or more, or five or more endogenous genes are disrupted. Microbial organisms with multiple disrupted endogenous genes can advantageously exhibit synergistic effects, as seen in yeast (see examples below). The present invention includes, but is not limited to, the exemplary embodiments shown in the examples section. In some modalities, the microbial organism has two, three, or four disrupted endogenous genes.
[0081] In another modality, the microbial organism presents at least two endogenous genes interrupted. In some embodiments, both the first disrupted gene and the second disrupted gene are selected from the following: YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283, YALIA14,YAL0E1206 , YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417 , YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512, or a homolog of any of these. In some modalities three, four, five, or more than five genes from this list are disrupted.
[0082] In two gene disrupted embodiments, genes particularly used for disruption include, but are not limited to, the C17545 gene (or homolog thereof), and/or the E30283 gene (or homolog thereof), and/or the E28336 gene ( or homolog thereto), and/or the E11099 gene (or homolog thereto), and/or the E28534 gene (or homolog thereto), and/or the B17512 gene (or homolog thereto). In some modalities, both the C17545 gene (or its homologue) and the E28336 gene (or its homolog) are disrupted. In some modalities, both the C17545 gene (or its homologue) and the E11099 gene (or its homolog) are disrupted. In some modalities, both the C17545 gene (or its homologue) and the E28534 gene (or its homolog) are disrupted. In some modalities, both the C17545 gene (or its homologue) and the B17512 gene (or its homolog) are disrupted. In some modalities, both the E28336 gene (or its homologue) and the E11099 gene are disrupted. In some modalities, both the E28336 gene (or its homologue) and the E28534 gene (or its homolog) are disrupted. In some modalities, both the E28336 gene (or its homologue) and the B17512 gene (or its homolog) are disrupted. In some modalities, both the E11099 gene (or its homologue) and the E28534 gene (or its homolog) are disrupted. In some modalities, both the E11099 gene (or its homologue) and the B17512 gene (or its homolog) are disrupted. In some modalities, both the E38534 gene (or its homologue) and the B17512 gene (or its homolog) are disrupted.
[0083] In one embodiment, the microbial organism, e.g., Yarrowia lipolytica, has at least one endogenous disrupted gene that is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E3275069, YALI0E3275069, YALIE3275069, YALI0E3275069, YALI0E3275069, YALI0E28534 homologue of any of these. In another modality, the microbial organism has an interrupted first gene and a second interrupted gene, both selected from this group of genes. In this modality, the microbial organism may have additional disrupted genes (eg, a third, fourth, or fifth disrupted gene also selected from this group), or it may have only two disrupted genes.
[0084] In some modalities, the microbial organism, for example, Yarrowia lipolytica, presents two interrupted genes or homologues of these. In some embodiments, microbial organisms that exhibit better production of fatty acyl-CoA derivatives comprise any of the following combinations of two disrupted endogenous genes: a. YALI0C17545 and YALI0E28336; B. YALI0C17545 and YALI0B10406; ç. YALI0C17545 and YALI0E28534; d. YALI0C17545 and YALI0E30283; and. YALI0E28336 and YALI0E30283; f. YALI0E11099 and YALI0E30283; g. YALI0A19536 and YALI0E30283; H. YALI0A19536 and YALI0E28534; i. YALI0E30283 and YALI0E12463; j. YALI0E14729 and YALI0B10406; k. YALI0E14729 and YALI0C17545; and l. YALI0E14729 and YALI0E11099; and homologues of (a) - (l).
[0085] In another modality, the microbial organism, for example, Yarrowia lipolytica, presents three or more (for example, 3) interrupted genes or homologues of these. In some embodiments, microbial organisms that exhibit better production of fatty acyl-CoA derivatives comprise any of the following combinations of three disrupted endogenous genes: m. YALI0C17545, YALI0E28336 and YALI0E11099; n. YALI0C17545, YALI0E28336 and YALI0B10406; O. YALI0C17545, YALI0E28336 and YALI0A19536; for. YALI0C17545, YALI0E28336 and YALI0E28534; q. YALI0C17545, YALI0E28336 and YALI0E32769; a. YALI0C17545, YALI0E28336 and YALI0E12463; s. YALI0C17545, YALI0E11099 and YALI0B10406; t. YALI0C17545, YALI0B10406 and YALI0A19536; u. YALI0E28336, YALI0E11099 and YALI0B10406; v. YALI0E11099, YALI0B10406 and YALI0A19536; and w. YALI0C17545, YALI0E28534 and YALI0B17512; and homologues of (m)-(w).
[0086] In some embodiments, where the microbial organism, for example, Yarrowia lipolytica, has three or more (for example, 3) disrupted genes, two or more of the disrupted genes are selected from gene C17545, gene E28336, gene E11099, from the E28534 gene, from the B17512 gene of homologues thereof. In some embodiments, the C17545 gene (or its homologue) and the E28336 gene (or its homolog) are disrupted. In some embodiments, the C17545 gene (or its homologue) and the E11099 gene (or its homolog) are disrupted. In some embodiments, the C17545 gene (or its homologue) and the E28534 gene (or its homolog) are disrupted. In some embodiments, the C17545 gene (or its homologue) and the B17512 gene (or its homolog) are disrupted. In some embodiments, the E28336 gene (or homologue thereof) and the E11099 gene are disrupted. In some embodiments, the E28336 gene (or its homologue) and the E28534 gene (or its homolog) are disrupted. In some embodiments, the E28336 gene (or its homologue) and the B17512 gene (or its homolog) are disrupted. In some embodiments, the E11099 gene (or its homologue) and the E28534 gene (or its homolog) are disrupted. In some embodiments, the E11099 gene (or its homologue) and the B17512 gene (or its homolog) are disrupted. In some embodiments, the E38534 gene (or homolog thereto) and the B17512 gene (or homolog thereto) are disrupted. In some embodiments, all three disrupted genes are selected from gene C17545, gene E28336, gene E11099, gene E28534, gene B17512, and homologues thereof. In some embodiments, the C17545 gene (or its homologue), the E28336 gene (or its homolog), and the E11099 gene (or its homolog) are disrupted. In some embodiments, the C17545 gene (or homologue thereof), the E28336 gene (or homolog thereto), and the E28534 gene (or homolog thereto) are disrupted. In some embodiments, the C17545 gene (or its homologue), the E28336 gene (or its homolog) and the B17512 gene (or its homolog) are disrupted. In some embodiments, the C17545 gene (or its homologue), the E11099 gene (or its homolog), and the B17512 gene (or its homolog) are disrupted. In some embodiments, the C17545 gene (or its homologue), the E28534 gene (or its homolog), and the B17512 gene (or its homolog) are disrupted. In some embodiments, the E28336 gene (or its homologue), the E11099 gene (or its homolog), and the E28534 gene (or its homolog) are disrupted. In some embodiments, the E28336 gene (or its homologue), the E11099 gene (or its homolog), and the B17512 gene (or its homolog) are disrupted. In some embodiments, the E28336 gene (or homologue thereof), the E28534 gene (or homolog thereto), and the B17512 gene (or homolog thereto) are disrupted. In some embodiments, the E11099 gene (or its homologue), the E28534 gene (or its homolog), and the B17512 gene (or its homolog) are disrupted.
[0087] In yet another modality, the microbial organism presents four or more (for example, 4) interrupted genes or homologues of these. In some embodiments, microbial organisms that exhibit better production of fatty acyl-CoA derivatives comprise any of the following combinations of four disrupted endogenous genes: x. YALI0C17545, YALI0E28336, YALI0E11099 and YALI0B10406; y. YALI0C17545, YALI0E28336, YALI0E11099 and YALI0A19536; z. YALI0C17545, YALI0E28336, YALI0E11099 and YALI0E28534; yy. YALI0C17545, YALI0E28336, YALI0E11099 and YALI0E32769; bb. YALI0C17545, YALI0E28336, YALI0B10406 and YALI0A19536; cc. YALI0C17545, YALI0E28336, YALI0B10406 and YALI0E32769; dd. YALI0C17545, YALI0E28336, YALI0A19536 and YALI0E28534; and is. YALI0C17545, YALI0E28336, YALI0E28534 and YALI0E32769; ff. YALI0C17545, YALI0E28336, YALI0E28534 and YALI0E12463; gg. YALI0E28336, YALI0E11099, YALI0B10406 and YALI0E32769; hh. YALI0E11099, YALI0EA19536, YALI0B10406 and YALI0B17512; and ii. YALI0E11099, YALI0E28336, YALI0C17545 and YALI0E14729; and homologues of (x)-(ii).
[0088] In some embodiments, where the microbial organism, for example Yarrowia lipolytica, has four or more (eg 4) disrupted genes, two or more of the disrupted genes are selected from gene C17545, gene E28336, gene E11099 , gene E28534, gene B17512 and homologues thereof. In some embodiments, three or more of the disrupted genes are selected from gene C17545, gene E28336, gene E11099, gene E28534, gene B17512, and homologues thereof. In some embodiments, all four disrupted genes are selected from gene C17545, gene E28336, gene E11099, gene E28534, gene B17512, and homologues thereof. In some modalities, the C17545 gene (or its homologue), the E28336 gene (or its homologue), the E11099 gene (or its homologue) and the E28534 gene (or its homologue) are interrupted. In some modalities, the C17545 gene (or homologue thereof), the E28336 gene (or homolog thereto), the E11099 gene (or homolog thereto), and the B17512 gene (or homolog thereto) are disrupted. In some modalities, the E28336 gene (or its homologue), the E11099 gene (or its homolog), the E28534 gene (or its homologue), and the B17512 gene (or its homologue) are interrupted.
[0089] In some embodiments, any of the endogenous genes or specific combinations of endogenous genes listed in Table 3 or Table 4 are disrupted in the organism. In some embodiments, the organism comprises additional disrupted genes. The genes cited in Table 3 and Table 4 are named with reference to the genome of Yarrowia lipolytica; however, those skilled in the art will understand that equivalent disruptions can be made in a microbial organism other than Y. lipolytica (eg, in algae, bacteria, mold, filamentous fungus, or yeast) by disrupting a homolog(s) of a gene listed in Table 3 or Table 4 in this microbial organism.
[0090] In addition to any of the endogenous gene disruptions described herein, one or more additional genes may optionally be disrupted (for example, by "neutralization", inactivation, mutation or inhibition in the manner described herein), introduced, and/or modified in a microbial organism of the present invention. These additional genes may be, but need not be, genes that were previously identified as those involved in the production of the fatty acyl-CoA derivative. Interrupt Methods
[0091] As described in the definitions, the term "disrupted", as applied to a gene, refers to a genetic modification that decreases or eliminates the expression of the gene and/or the biological activity of the corresponding gene product (mRNA) and/or protein) (eg, for genes listed in table 1, known or predicted biological activity listed in table 1). In some embodiments, disruption eliminates or substantially reduces expression of the gene product determined, for example, by immunoassays. "Substantially reduces" in this context means the amount of expressed protein that is reduced by at least 50%, often by at least 75%, sometimes by at least 80%, at least 90% or at least 95%, compared to the expression of uninterrupted gene. In some embodiments, a gene product (eg, protein) is expressed from the disrupted gene, but the protein is mutated (eg, a deletion of one or more amino acids, or an insertion of one or more amino acid substitutions) , in such a way that the biological activity (eg, enzymatic activity) of the protein is completely eliminated or substantially reduced. As used herein, "completely deleted" means that the gene product has no measurable activity. "Substantially reduced" in this context means that the biological activity of the protein is reduced by at least 50%, often by at least 75%, sometimes by at least 80%, at least 90% or at least 95%, compared to non-protein. mutated. The biological activity of a gene product (eg protein) can be assessed by a functional assay such as an enzymatic assay. For example, in some embodiments, the microbial organism has a deletion of all or a portion of the endogenous gene's protein coding sequence, a mutation in the endogenous gene in such a way that the gene encodes a polypeptide with no or reduced activity (for example , insertion, deletion, point or frameshift mutation), reduced expression due to antisense RNA or small interfering RNA that inhibits endogenous gene expression, or a modified or deleted regulatory sequence (eg, promoter) that reduces endogenous gene expression, any of which can result in a disrupted gene. In some embodiments, all disrupted genes in the microorganism are disrupted by deletion.
[0092] It will be appreciated that methods for gene disruption in yeast and other microorganisms are well known, and the particular method used to reduce or eliminate endogenous gene expression is not important to the invention. In one embodiment, disruption can be accomplished by homologous recombination, in which case the gene to be disrupted is disrupted (eg, by insertion of a selectable marker gene) or becomes inoperative (eg, “gene neutralized”). Methods to neutralize gene and to neutralize multiple genes are well known. See, for example, example 5 below; Rothstein, 2004, “Targeting, Interruption, Replacement, and Alelle Rescue: Integrative DNA Transformation in Yeast” In: Guthrie et al., Eds. Guide to Yeast Genetics and Molecular and Cell Biology, Part A, p. 281-301; Wach et al., 1994, “New heterologus modules for classical or PCR-based gene interruptions in Saccharomyces cerevisiae” Yeast 10:1793-1808. Methods for insertional mutagenesis are also well known. See, for example, Amberg et al., eds., 2005, Methods in Yeast Genetics, p. 95-100; Fickers et al., 2003, "New interruption cassettes for rapid interruption gene and marker rescue in the yeast Yarrowia lipolytica" Journal of Microbiological Methods 55:727-737; Akada et al., 2006, “PCR-mediated seamless gene elimination and marker recycling in Saccharomyces cerevisiae” Yeast 23:399-405; Fonzi et al., 1993, "Isogenic strain construction and gene mapping in Candida albicans" Genetics 134:717-728.
Antisense inhibition is well known in the art. Endogenous genes can be disrupted by inhibiting transcription, stability and/or translation using antisense methods. Regarding antisense technology, a strand of nucleic acid (DNA, RNA, or analogue) complementary to the mRNA of the gene is introduced into the cell. This complementary strand will bind to the gene's mRNA and thus efficiently disrupt the gene.
[0094] The disrupting method can be applied independently for each disrupted gene. So when multiple genes are disrupted, the genes need not be disrupted in the same way. For example, a microbial organism may have a gene that is disrupted or replaced by an artificial fragment of DNA ("neutralized"), a gene that is disrupted by an insertion mutation, and another gene whose promoter is altered to decrease expression . In some embodiments, two or more genes are disrupted in the same way. In some embodiments, two or more genes are disrupted by the same disruption event (eg, recombination event). In one embodiment, all disrupted genes are disrupted in the same way or by the same disruption event. In one embodiment, all disrupted genes are "killed" genes, that is, genes that are inactivated by disrupting or replacing at least a portion of the coding sequence. In another embodiment, all disrupted genes are neutralized genes that are disrupted by the same disruption event.
[0095] In one embodiment, multiple gene copies are disrupted. A "gene copy", as used herein, refers to the same target gene (eg, an endogenous gene as described herein) on a homologous chromosome in a diploid or polyploid organism. For example, a microbial organism may have multiple sets of chromosomes and thus have multiple copies of each target gene. In some embodiments, a microbial organism is diploid (that is, with two sets of chromosomes and thus two copies of each target gene). In some embodiments, a microbial organism is polyploid (that is, with more than two sets of chromosomes). In some embodiments, a microbial organism is triploid (that is, with three sets of chromosomes and thus three copies of each target gene). In some embodiments, a microbial organism is tetraploid (that is, with four sets of chromosomes and thus four copies of each target gene). In some embodiments, a microbial organism has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of a target gene. In some embodiments, the microbial organism has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more interrupted copies of an endogenous target gene. In one embodiment, all copies of the endogenous target gene are disrupted in the microbial organism.
[0096] The term "one or more gene copies" refers to the number of copies of the same target gene, while "one or more disrupted genes" refers to one or more individual genes. For example, a microbial organism may have two copies of a disrupted gene copy, while displaying only one disrupted gene.
[0097] Where two or more endogenous genes are disrupted, the number of copies to be disrupted can be independently selected for each disrupted gene. Multiple copies of a gene can be disrupted, for example, by carrying out multiple rounds of recombination with a retrievable marker. IV. TRUNCATED SEC62 EXPRESSION
[0098] In another aspect, the invention concerns recombinant microbial organisms, such as yeast, in which an endogenous gene encoding a Sec62 protein, or an allelic homologue or variant thereof, has been modified. Sec62 is a protein that is involved in protein translocation in the endoplasmic reticulum in yeast. Yarrowia Sec62 is encoded by YALI0B17512, has the amino acid sequence shown as SEQ ID NO:64, and contains a cytoplasmic domain (amino acids 207 to 396 of SEQ ID NO:64). See also GenBank accession number CAA67878.1 and Swennen et al., 1997, “Cloning the Yarrowia lipolytica homologue of the Saccharomyces cerevisiae SEC62 gene,” Curr Genet 31(2):128-132. As described in the example below, it was found that yeast cells expressing a truncated Sec62 protein, which lacks a complete cytoplasmic domain, exhibit increased production of fatty acyl-CoA derivatives compared to a control yeast cell in which the Sec62 protein is not truncated.
Thus, the invention provides a microbial organism that expresses a truncated or homologous Sec62 protein. The organism can be used by any of the methods or processes described herein, and can be combined with disrupted genes described herein and in combinations described herein.
[00100] Thus, in some embodiments, the organism, for example an alga, a bacteria, a mold, a filamentous fungus or a yeast (eg Yarrowia lipolytica), is one in which the endogenous gene encoding Sec62 (YALI0B17512 or a homolog thereof) comprises a partial deletion of the sequence encoding at least a portion of the cytoplasmic domain of the encoded Sec62 protein. In some embodiments, the partial deletion of the coding sequence comprises a deletion of the entire cytoplasmic domain of the encoded Sec62 protein.
In some embodiments, the Sec62 protein is SEQ ID NO:64 or is an allelic homologue or variant substantially identical to SEQ ID NO:64 (e.g., exhibits a sequence identity of at least one embodiment, the Sec62 protein is isolated or derived from an organism selected from the group consisting of Saccharomyces cerevisiae (Genbank accession number CAB56541.1; SEQ ID NO:77), Kluyveromyces lactis (Genbank accession number CAH00127.1; SEQ ID NO:78) and Schizosaccharomyces pombe (Genbank accession number CAB16220.1; SEQ ID NO:79).
In some embodiments, the microbial organism (eg Y. lipolytica) expresses a truncated or homologous Sec62 protein in which the entire cytoplasmic domain (corresponding to amino acids 207-396 of SEQ ID NO:64) has been deleted. In some embodiments, the microbial organism expresses a truncated or homologous Sec62 protein, in which a portion of the cytoplasmic domain is deleted, for example, from about position 210 to about position 396; from about position 250 to about position 396; from about position 300 to about position 396; from about position 330 to about position 396; from about position 210 to about position 350; from about position 210 to about position 300; from about position 250 to about position 350; or from about position 300 to about position 350, where the amino acids are numbered with reference to SEQ ID NO:64. In some embodiments, the microbial organism expresses a truncated or homologous Sec62 protein, in which a portion of the cytoplasmic domain from about position 267 to about position 396 is deleted. In some embodiments, the microbial organism expresses a truncated or homologous Sec62 protein, in which a portion of the cytoplasmic domain from about position 302 to about position 396 is deleted. In some embodiments, the microbial organism expresses a truncated or homologous Sec62 protein in which a portion of the cytoplasmic domain from about position 337 to about position 396 is deleted.
[00103] In some embodiments, a microbial organism is diploid (ie, it has two sets of chromosomes and thus two copies of the gene encoding Sec62). In some embodiments, a microbial organism is polyploid (that is, it has more than two sets of chromosomes and thus more than two copies of the gene encoding Sec62). In some embodiments, more than one copy of the Sec62 gene is modified to express a truncated Sec62 protein. In some embodiments, all copies of the Sec62 gene are modified to express a truncated Sec62 protein.
[00104] It will be understood that the particular method used to eliminate all or a portion of the cytoplasmic domain of Sec62 is not important to the invention. In some embodiments, deletion of the cytoplasmic domain or portion thereof can be accomplished by replacing the portion of the sequence encoding the cytoplasmic domain, or portion thereof, with an artificial DNA fragment (e.g., a selectable marker). In some embodiments, deletion of the cytoplasmic domain, or portion thereof, can be accomplished by removing the portion of the coding sequence that encodes the cytoplasmic domain, or portion thereof. V. EXOGENOUS FAR EXPRESSION FAR Protein
[00105] In one aspect, the modified microbial organism that exhibits better production of fatty acyl-CoA derivatives (eg, a microbial organism such as Yarrowia lipolytica, in which one, two, three, four, or more endogenous genes are disrupted as described herein), expresses or overexpresses a FAR. As described in the examples section, microbial organisms in which certain endogenous genes or gene combinations are disrupted, and which express an exogenous gene encoding a FAR protein, show greater production of fatty acyl-CoA derivatives compared to control microbial organisms (eg, otherwise identical microbial organisms), which express the exogenous gene encoding the FAR protein in which the corresponding endogenous genes have not been disrupted.
[00106] In some embodiments, the organism, for example, an alga, a bacteria, a mold, a filamentous fungus, or a yeast (eg, Yarrowia lipolytica), expresses an exogenous FAR protein (ie, an unexpressed FAR normally in the body, such as a protein derived from a different species). In some embodiments, the exogenous FAR protein is a wild-type FAR protein. In some embodiments, the exogenous FAR protein is selected or genetically modified for increased activity or yield of fatty acyl-CoA derivatives, e.g., fatty alcohols (i.e., a FAR variant as described herein). In some embodiments, the FAR protein is a FAR protein or variant as described in US patent application 2011/0000125, or US patent application 13/171,138, filed June 28, 2011, the entire contents of which are hereby incorporated by reference.
[00107] In one embodiment, the exogenous FAR protein is from a genus of marine bacteria such as gammaproteobacteria (eg, Marinobacter and Oceanobacter). In one embodiment, the exogenous FAR protein is from a species of the genus Marinobacter including, but not limited to, M. aquaeolei, M. arcticus, M. actinobacterium, and M. lipolyticus. In one embodiment, the exogenous FAR protein is from M. algicola (also referred to herein as "FAR_Maa"). In one embodiment, the exogenous FAR protein is from M. aquaeolei (also referred to herein as "FAR_Maq"). In another embodiment, the exogenous FAR protein is from a species of the genus Oceanobacter including, but not limited to, Oceanobacter sp. Red65 (renamed Bermanella marisrubi) (also referred to herein as “FAR_Ocs”), Oceanobacter strain WH099 and O. kriegii. In another embodiment, the exogenous FAR protein is from Hahella including, but not limited to, H. chejuensis and equivalent species thereof.
[00108] In one embodiment, the exogenous FAR gene is FAR_Maa (wild type FAR from Marinobacter algicola strain DG893, SEQ ID NO:1), FAR_Maq (wild type FAR from Marinobacter aquaeolei, SEQ ID NO:3), FAR_Ocs (FAR wild type of Oceanobacter sp. RED65, SEQ ID NO:5), or a fragment encoding a functional FAR enzyme. In one embodiment, the FAR gene exhibits a DNA sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with any of SEQ ID NOs: 1, 3 or 5. In one embodiment, the FAR gene exhibits sequence identity of DNA at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with SEQ ID NO:1.
[00109] In another embodiment, the exogenous FAR protein exhibits a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with any of SEQ ID NOs:2, 4 or 6, which correspond to FAR-like polypeptide sequences wild_Maa, wild_Maq_FAR, and wild_Ocs_FAR, respectively. In one embodiment, the FAR protein has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with SEQ ID NO:2.
[00110] In other embodiments, the FAR enzyme is FAR_Hch (Hahella chejuensis KCTC 2396, GenBank number YP_436183.1, SEQ ID NO:65), FAR_Mac (from marine actinobacterium strain PHSC20C1, SEQ ID NO:66), FAR_JVC ( JCVI_ORF_1096697648832, GenBank No. EDD40059.1, SEQ ID NO:67), FAR_Fer (JCVI_SCAF_1101670217388, SEQ ID NO:68), FAR_Key (JCVI_SCAF_1097205236585, SEQ ID NO:69), FAR_Gal (167 or SEQ ID NO:68F) a functional variant or fragment thereof. Table 2 provides the approximate amino acid sequence identity of this bacterial protein FARs for FAR_Maa (SEQ ID NO:2) and FAR_Ocs (SEQ ID NO:6).
[00111] Table 2. Amino acid sequence identity of homologs with respect to FAR_Maa and FAR_Ocs

[00112] In other embodiments, the FAR enzyme or functional fragment is isolated or derived from an organism selected from the group consisting of Vitis vinifera (GenBank accession number CAO22305.1, SEQ ID NO:71; or CAO67776.1, SEQ ID NO:72), Desulfatibacillum alkenivorans (GenBank accession number NZ_ABII01000018.1), Stigmatella aurantiaca (NZ_AAMD01000005.1, SEQ ID NO:73) and Phytophthora ramorum (GenBank accession number: AAQX01001105.1). FAR variants
[00113] In some embodiments, variants of FAR enzymes are used, such as functional fragments, and variants are selected using the technology of molecular evolution. A "functional fragment", as used herein, refers to a polypeptide with an amino-terminal and/or carboxy-terminal deletion, and/or internal deletion, but in which the remaining amino acid sequence is identical or substantially identical to the corresponding positions. in the sequence to which it is being compared (eg, a full-length wild-type FAR protein or a full-length FAR protein variant), and which retains substantially all (eg, retains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more) full-length polypeptide activity (for example, the full-length wild-type FAR protein or the full-length FAR protein variant). Functional fragments may comprise up to 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the full-length FAR protein. Thus, a functional fragment in this context is a fragment of a naturally occurring FAR polypeptide, or variant thereof, that exhibits catalytic activity. In some embodiments, the functional fragment has at least 50% of the activity of the corresponding full-length wild-type FAR from which it is derived (eg, FAR_Maa, FAR_Maq, or FAR_Ocs).
[00114] In some embodiments, a FAR variant comprises one or more mutations (e.g., substitutions) compared to a wild-type FAR, such that the resulting FAR polypeptide variant exhibits better characteristics and/or properties compared to the FAR-type wild type, for example, such as increased fatty alcohol production when the FAR variant is expressed in a host cell. In some embodiments, a FAR protein variant may have from 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 25, 30, 35, 40, 45 or more amino acid substitutions with respect to a natural (wild-type) FAR protein, such as FAR_Maa (SEQ ID NO:2), FAR_Maq (SEQ ID NO:4) or FAR_Ocs (SEQ ID NO:6). In some embodiments, a variant of the FAR protein may have from 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 25, 30, 35, 40, 45 or more amino acid substitutions relative to the natural FAR protein of SEQ ID NO:2. In some embodiments, a variant of the FAR protein may have from 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 25, 30, 35, 40, 45 or more amino acid substitutions relative to the natural FAR protein of SEQ ID NO:4. In some embodiments, a FAR protein variant may have from 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 25, 30, 35, 40 45, or more amino acid substitutions relative to the natural FAR protein of SEQ ID NO:6.
[00115] In some embodiments, a FAR variant comprises at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to a wild-type FAR (e.g., a FAR polypeptide of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6), and further comprises one or more amino acid substitutions (for example, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 25, 30, 35, 40, 45 or more amino acid substitutions) relative to wild-type FAR, and is capable of producing at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least ce rca 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times more fatty alcohol than wild-type FAR, from which it is derived when tested under the same conditions.
In certain embodiments, the microbial organism does not express an endogenous FAR (ie, the wild-type organism's genome does not encode a FAR). In some embodiments, the microbial organism is an organism that expresses an endogenous FAR protein. In certain embodiments, the microbial organism is an organism that does not express an exogenous FAR protein. In some embodiments, the microbial organism is an organism that does not express an endogenous FAR protein or an exogenous FAR protein. In some embodiments, the microbial organism expresses both the endogenous FAR(s) and the exogenous FAR(s).
[00117] Methods for introducing exogenous genes (eg, genes encoding FAR) into a host organism and expressing an exogenous protein are known in the art. See section VII below. SAW. MICROBIAL ORGANISMS Host cells
[00118] The microbial organism in which one or more endogenous genes are disrupted, and which exhibits increased production of fatty acyl-CoA derivatives, can be any "host cell" that produces fatty acyl-CoA derivatives. Suitable host cells include, but are not limited to, algae, bacteria, mold, filamentous fungus, and yeast, including oleaginous yeast (e.g., Yarrowia lipolytica). In some modalities, the microbial organism is an oleaginous organism, for example, an organism that tends to store its energy source in the form of oil. The host cell can be either eukaryotic or prokaryote.
[00119] In one embodiment, the microbial organism is a fungus. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti. Particularly preferred fungal host cells are yeast cells and filamentous fungus cells.
[00120] In one embodiment, the microbial organism is a yeast. In one embodiment, the yeast is of one of the genera: Yarrowia, Brettanomyces, Candida, Cryptococcus, Endomycopsis, Hansenula, Kluyveromyces, Lipomyces, Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Trichosporon, or Trichosporon. In one embodiment, the yeast is of the Yarrowia genus. In some embodiments of the invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichiaaccharomyces finlandica, Pichiaeph. Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans and Yarrowia lipolytica.
[00121] In one embodiment, the microbial organism is an oleaginous yeast. Oil yeasts accumulate lipids such as tri-acyl glycerols. Examples of oleaginous yeast include, but are not limited to, Yarrowia lipolytica, Yarrowia paralipolytica, Candida revkauji, Candida pulcherrima, Candida tropicalis, Candida utilis, Candida curvata D, Candida curvataR, Candida diddensiae, Candida boldinii, Rhodotorula glutinous, Rhodotorula graminis, Rhodotorula mucilaginos , Rhodotorula minuta, Rhodotorula bacarum, Rhodosporidium toruloides, Cryptococcus (terricolus) albidus var. albidus, Cryptococcus laurentii, Trichosporon pullans, Trichosporon cutaneum, Trichosporon cutancum, Trichosporon pullulans, Lipomyces starkeyii, Lipomyces lipoferus, Lipomyces tetrasporus, Endomycopsis vernalis, Hansenula ciferri, and Trichosporon pullulans.
[00122] In one embodiment, the yeast is Yarrowia lipolytica. Exemplary strains of Yarrowia lipolytica include, but are not limited to, DSMZ 1345, DSMZ 3286, DSMZ 8218, DSMZ 70561, DSMZ 70562, DSMZ 21175, available from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, as well as strains available from the Agricultural Research Service ( NRRL) such as, but not limited to, NRRL YB-421, NRRL YB-423, NRRL YB-423-12 and NRRL YB-423-3.
[00123] In one embodiment, the host cell is a filamentous fungus. The filamentous fungus host cells of the present invention include all filamentous forms of the Eumycotina and Oomycota subdivision (Hawksworth et al., 1995, in Ainsworth and Bisby's Dictionary of Fungi, 8th ed.). Filamentous fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose and other polysaccharide complexes. As used herein, the filamentous fungus host cells of the present invention are morphologically distinct from yeast. Exemplary filamentous fungus cells include, but are not limited to, the species of Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprindous, Dithis, Corynascus , Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Pyromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thillma, Trac teleomorphs, anamorphs, synonyms, basionyms, and taxonomic equivalents thereof.
[00124] In some embodiments, the host cell is an algae cell such as Chlamydomonas (eg C. Reinhardtii) and Phormidium (P. sp. ATCC29409).
[00125] Suitable prokaryote cells include Gram positive, Gram negative and Gram variable bacterial cells. Suitable eukaryotic host cells include, but are not limited to, Agrobacterium species, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizoriane, P. Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigellasy, Thermoyanaerococcus asthma, Xanthomonas, Xylella, Yersinia and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Lactococcus, Pantococcus, Salmonlomonas, Pseudomona , Streptococcus, Streptomyces or Zymomonas. Cell transformation and culture
[00126] In another embodiment, the invention provides a method comprising providing a microbial organism in the manner described herein, and culturing the microbial organism under conditions in which the fatty acyl-CoA derivatives are produced. In some modalities, the microbial organism with one or more disrupted endogenous genes is capable of better production in the manner described above, for example, with at least a 1-fold increase in the production of fatty acyl-CoA derivatives compared to a control organism. same type (eg, an otherwise identical control microbial organism, in which the one or more genes are not disrupted).
In some embodiments, a polynucleotide encoding a FAR polypeptide (eg, a wild-type FAR polypeptide or a variant FAR polypeptide) is introduced into the microbial organism for expression of the wild-type FAR polypeptide or variant FAR polypeptide. The polynucleotide can be introduced into the cell as a self-replicating episome (eg, expression vector) or it can be stably integrated into the host cell's DNA.
[00128] Methods, reagents and tools for transforming the microbial organisms described herein, such as bacteria, yeast (including oleaginous yeast) and filamentous fungi are known in the art. General methods, reagents and tools for transforming, for example, bacteria can be found, for example, in Sambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York. Methods, reagents and tools for transforming yeast are described in "Guide to Yeast Genetics and Molecular Biology," C. Guthrie and G. Fink, Eds., Methods in Enzymology 350 (Academic Press, San Diego, 2002). Methods, reagents, and tools for transforming, cultivating, and manipulating Y. lipolytica are found in "Yarrowia lipolytica" C. Madzak, J.M. Nicaud and C. Gaillardin in "Production of Recombinant Proteins. Novel Microbial and Eucaryotic Expression Systems”, G. Gellissen, Ed. 2005, which are incorporated herein by reference for all purposes. In some embodiments, introduction of the DNA construct or vector of the present invention into a host cell can be accomplished by calcium phosphate transfection, DEAE-Dextran-mediated transfection, PEG-mediated transformation, electroporation, or other common techniques (See Davis et al. al., 1986, Basic Methods in Molecular Biology, which is incorporated herein by reference).
[00129] Microbial organisms can be cultured in conventional nutrient media, modified in the appropriate manner to activate promoters, select transformants, or amplify the FAR polynucleotide. Cultivation conditions, such as temperature, pH and the like, will be apparent to those skilled in the art. As noted, many references are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archaebacteria origin. See, for example, Sambrook, Ausubel, and Berger (all supra), as well as Freshney (1994) Culture of Animal Cells, The Manual of Basic Technique, third edition, Wiley-Liss, New York and its cited references; Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, fourth edition W.H. Freeman and Company; and Ricciardelli, et al., (1989) In Vitro Cell Dev. Biol. 25:1016-1024, all of which are incorporated herein by reference. Regarding plant cell culture and regeneration, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); Jones, ed. (1984) Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, New Jersey and Plant Molecular Biology (1993) RRDCroy, Ed. Bios Scientific Publishers, Oxford, UK ISBN 0 12 198370 6, all of which are incorporated herein by reference . Cell culture media are generally presented in Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL, which is incorporated herein by reference. Additional information for cell culture is found in available commercial literature, such as the Life Science Research Cell Culture Catalog (1998) from Sigma-Aldrich, Inc (St Louis, MO) ("Sigma-LSRCCC") and, for example , The Plant Culture Catalog and Supplement (1997) also from Sigma-Aldrich, Inc (St Louis, MO) ("Sigma-PCCS"), all of which are incorporated herein by reference. VII. ADDITIONAL METABOLIC ENGINEERING
[00130] In one embodiment, the modified microbial organism that exhibits better production of fatty acyl-CoA derivatives contains an exogenous gene operably linked to a promoter that is functional in the microbial organism. Incorporation of an exogenous gene (eg, a FAR gene as described above) can be accomplished by techniques well known in the art.
[00131] In some embodiments, the microbial organism can be modified to express or overexpress one or more genes that encode enzymes, other than FAR, that are involved in the biosynthesis of fatty acyl-CoA derivative. See Figure 1. In particular embodiments, the gene encodes a fatty acid synthase (FAS), an ester synthase, an acyl-ACP thioesterase (TE), a fatty acyl-CoA synthase (FACS), or an acetyl-CoA carboxylase ( ACC). For example, in one embodiment, the microbial organism can be modified to express an ester synthase and produce fatty esters. Similarly, in another modality, the microbial organism can be modified to express thioesterase and produce fatty acids. Any of these exemplary genes can be used in place of, or in addition to, FAR. When multiple exogenous genes are expressed, in some embodiments, the expression vector encoding a first enzyme (eg, FAR) and the expression vector encoding a second enzyme (eg, a FAS, ester synthase, TE, FACS, or ACC) are separate nucleic acids. In other embodiments, the first enzyme and second enzyme are encoded on the same expression vector, and the expression of each enzyme is independently regulated by a different promoter.
[00132] In the manner shown in figure 1, the various fatty acyl-CoA derivatives can be produced by the microbial organism. When recovery of a particular derivative is desired, the expression or activity of one or more of the polypeptides involved in this metabolic pathway can be altered to preferentially yield the desired derivative. For example, one can modify the expression or activity of one or more of acetyl-CoA carboxylase, pyruvate decarboxylase, isocitrate dehydrogenase, ATP-citrate lyase, malic enzyme, AMP-deaminase, glucose-6-phosphate dehydrogenase, 6-phosphoglyconate dehydrogenase , fructose 1,6 bisphosphatase, NADH kinase, transhydrogenase, acyl-CoA:diacylglycerol acyltransferase, phospholipid:diacylglycerol acyltransferase, acyl-CoA:cholesterol acyltransferase, triglyceride lipase, and acyl-coenzyme A oxidase.
[00133] As another example, the microbial organism can be modified to utilize particular desired substrates. For example, although wild-type Y. lipolytica preferably does not use xylose as a substrate, it can be genetically modified in order to accomplish this. See, for example, Brat et al., 2009, "Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae" Applied and Environmental Microbiology 75:2304-11; Ho et al., 1998, "Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose" Applied and Environmental Microbiology 64:1852-59. Similarly, Y. lipolytica can also be genetically modified to utilize sucrose. See, for example, Nicaud et al., 1989, "Expression of invertase activity in Yarrowia lipolytica and its use as a selectable marker" Current Genetics 16:253-260. It may be advantageous to genetically modify microbial organisms to be adjusted to the conditions of a particular environment, for example to use raw material obtained from a cellulosic or lignocellulosic biomass where the raw material can be brought into contact with cellulase enzymes to provide fermentable sugars including , but not limited to, glucose, fructose, xylose and sucrose.
[00134] In some embodiments, a microbial organism as described herein (e.g., a microbial organism comprising one or more endogenous disrupted genes selected from YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI3087E, YALI20E3 , YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502 , YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0E32417, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121 , YALI0F212121 gene, YALI0F25003, a homolog additionally comprises an exogenous gene, YALI0F25003, a homolog of these gene(s) which further encodes an exogenous gene YALI0F25003 exogenous that encodes an enzyme that catalyzes the hi drolysis of a fermentable sugar (eg, sucrose, arabinose, or mannose). Examples of enzymes that catalyze the hydrolysis of a fermentable sugar include, but are not limited to, sucrase and invertases. Thus, in some modalities, the exogenous gene encodes a sucrase or an invertase. In some embodiments, the exogenous gene is a SUC2 gene that encodes invertase. Invertases (EC 3.2.1.26) catalyze the hydrolysis of sucrose which results in a mixture of glucose and fructose. Sucroses are related to invertases, but they catalyze the hydrolysis of sucrose by a different mechanism.
[00135] The exogenous gene encoding the enzyme that catalyzes the hydrolysis of a fermentable sugar can be derived from any suitable microbial organism, for example, from algae, bacteria, mold, filamentous fungus or yeast. In some embodiments, the microbial organism that comprises one or more endogenous disrupted genes is Y. lipolytica, and the exogenous gene encoding an enzyme that catalyzes the hydrolysis of sucrose is from Saccharomyces cerevisiae. In some embodiments, the exogenous gene is SUC2 invertase from Saccharomyces cerevisiae. Targeted Integration of an Exogenous Gene
[00136] In some embodiments, the expression of an exogenous gene in the microbial organism is accomplished by introducing the exogenous gene into the organism in an episomal plasmid. In some modalities, exogenous gene expression is accomplished by integrating the gene into the genome of the microbial organism. Integration of the exogenous gene into the genome of the microbial organism has several advantages with the use of plasmids including, but not limited to, less variation in protein expression, better flexibility in the choice of fermentation media, and the potential for high expression levels by introducing multiple copies of a single gene.
[00137] Thus, in some embodiments, a microbial organism with one or more endogenous genes disrupted, as described herein, further comprises an exogenous gene encoding an enzyme that is involved in the biosynthesis of the fatty acyl-CoA derivative (eg, a FAR enzyme), in which the exogenous gene is integrated into the genome of the microbial organism. In some embodiments, the microbial organism comprises an exogenous gene that encodes a FAR protein (e.g., a wild-type FAR protein that is identical or substantially identical to the FAR polypeptide of any of SEQ ID NOs:2,4, or 6, or a FAR protein variant in the manner described herein) which is integrated into the genome of the microbial organism.
[00138] In some embodiments, the microbial organism comprises a copy of the exogenous gene. In some embodiments, the microbial organism comprises two, three, four, five or more copies of the exogenous gene. In some embodiments, multiple copies of the exogenous gene (eg, two, three, four, five or more copies) are integrated into the microbial organism's genome in a direct repeating structure, or an inverted repeating structure.
[00139] In some embodiments, integration of the exogenous gene into the genome of the microbial organism may be targeted at one or more particular regions of the microbial genome. The microbial organism's genome can be mapped to identify regions where integration of an exogenous gene results in better gene expression, or a better property (eg, better fatty alcohol production) with respect to exogenous gene expression in an organism control of the same type (eg, an otherwise identical organism) by a plasmid (also called “stirring spots” of expression). As follows in the examples, after the integration of an exogenous gene encoding the FAR protein into a strain of Y. lipolytica, the strains that were identified showed particularly good improvement in fatty alcohol production relative to a strain of Y. lipolytica , which expressed FAR through the plasmid. These churning points of expression integration, once mapped, can also be targeted for subsequent integration of an exogenous gene via homologous recombination.
[00140] Thus, in some modalities, the exogenous gene is integrated into a chromosomal site in the genome of the microbial organism, which is a stirring point of expression. In some embodiments, where the microbial organism is Y. lipolytica, the exogenous gene is integrated into the microbial organism's genome at one or more of the chromosomal sites described herein, for example, in example 1.
[00141] Targeted integration of an exogenous gene into the genome of a microbial organism of the present invention can also be performed by means of "continuous" marker recycling. As described in the examples section below, in continuous marker recycling a bifunctional selectable marker is introduced at a specific genomic site, either to disrupt a natural gene or to introduce an exogenous gene. Integrants are identified using the selectable marker (positive selection, for example, using a marker that confers antibiotic resistance). The marker is then excised, or "recycled", through homologous recombination between two flanking repeats, and organisms that satisfactorily display the recycled marker are identified by counter-selection (negative selection, for example, using a marker that induces toxicity) . The selectable marker can then be used again to introduce further modifications into the organism's genome. This method is advantageous in that it allows a theoretically unlimited number of targeted modifications (for example, targeted exogenous gene deletions or targeted exogenous gene integration) to be performed in an organism's genome, thus facilitating the development of the strain.
[00142] Thus, in some embodiments, an exogenous gene (eg a gene encoding an enzyme that is involved in the biosynthesis of the fatty acyl-CoA derivative, eg a FAR enzyme) is integrated into the genome of a microbial organism of the present invention (e.g., a microbial organism with one or more endogenous genes disrupted as described herein), using a bifunctional recyclable selectable marker with a positive selectable marker and a negative selectable marker, wherein the integration of the exogenous gene into the genome is identified using the positive selectable marker, and wherein subsequent recycling of the bifunctional marker is identified using the negative selectable marker. In some embodiments, the bifunctional selectable marker has a positive selectable marker for hygromycin and a negative selectable marker for thymidine kinase. Vectors
[00143] Expression vectors can be used to transform a microbial organism of the present invention (for example, a microbial organism that has one or more endogenous genes disrupted in the manner described herein) with a gene encoding a FAR enzyme, and/or a gene encoding an enzyme other than FAR that is involved in the biosynthesis of the fatty acyl-CoA derivative, and/or a gene encoding an enzyme that catalyzes the hydrolysis of a fermentable sugar. A recombinant expression vector can be any vector, for example a plasmid or a virus, which can be manipulated by recombinant DNA techniques to facilitate expression of the exogenous gene in the microbial organism. In some embodiments, the expression vector is stably integrated into the chromosome of the microbial organism. In other embodiments, the expression vector is a replicative extrachromosomal DNA molecule, eg, a linear or closed circular plasmid, which is found in either a small copy number (eg, from about 1 to about 10 copies per equivalent genome) or high copy number (eg, more than about 10 copies per equivalent genome).
Expression vectors for expressing the one or more exogenous genes are commercially available, for example, from Sigma-Aldrich Chemicals, St. Louis, MO and Stratagene, LaJolla, CA. In some embodiments, examples of suitable expression vectors are plasmids that are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193- 201).
[00145] In some embodiments, an expression vector optionally contains a ribosome binding site (RBS) for translation initiation and a transcription terminator, such as PinII. The vector also optionally includes appropriate sequences to amplify expression, for example an enhancer.
In particular embodiments, the present disclosure provides an autonomously replicating plasmid for the expression of exogenous genes in Yarrowia, and particularly in Y. lipolytica. An exemplary plasmid is shown in Figure 2 and described in the examples. Such a plasmid can be further modified for the expression of exogenous genes used for the production of fatty acyl-CoA derivative in yeast, inter alia, Y. lipolytica.
[00147] In some embodiments, where more than one exogenous gene must be expressed in the microbial organism (eg, a first exogenous gene encoding a wild-type FAR polypeptide or a variant FAR polypeptide, and a second exogenous gene encoding a enzyme other than FAR that is involved in the biosynthesis of the fatty acyl-CoA derivative or an enzyme that catalyzes the hydrolysis of a fermentable sugar), the expression vector encoding the FAR polypeptide and the expression vector encoding the second enzyme are acids separate nucleic acids. In other embodiments, the FAR polypeptide and the second enzyme are encoded on the same expression vector, and the expression of each enzyme is independently regulated by a different promoter. Promoters
The promoter sequence is a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide, such as a polynucleotide containing the coding region. In general, the promoter sequence contains transcriptional control sequences, which mediate the expression of the polynucleotide. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides, either homologous or heterologous to the host cell. Methods for the isolation, identification and manipulation of promoters of varying concentrations are available or easily adapted from the art. See, for example, Nevoigt et al. (2006) Appl. Environ. Microbiol. 72:5266-5273, the disclosure of which is incorporated herein by reference in its entirety.
In a host yeast, promoters used include, but are not limited to, those donate genes for enolase (ENO-1) from Saccharomyces cerevisiae, galactokinase (GAL1) from Saccharomyces cerevisiae, alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2 /GAP) from Saccharomyces cerevisiae and 3-phosphoglycerate kinase from Saccharomyces cerevisiae. Exemplary Y. lipolytica promoters include, but are not limited to, TEF1, RPS7 (Müller et al., 1998, “Comparison of expression systems in the yeast Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of twolytica. novel promoters from Yarrowia lipolytica” Yeast 14:1267-1283), GPD, GPM (US 7259255), GPAT (US 7264949), FBA1 (US 7202356), the Leu2 promoter and variants thereof (US 5786212), the EF1alpha protein promoter ( WO 97/44470), Xpr2 (US4937189), Tefl, Caml (YALI0C24420g), YALI0DI6467g, Tef4 (YALI0BI2562g), Yef3 (YALI0E13277g), Pox2, Yat1 (US 2005/0130280), the promoters disclosed in US 2004/0146975 and US 5952195, CYP52A2A (US 2002/0034788); gene sequences from fungi (eg C. tropicalis) such as catalase, citrate synthase, 3-ketoacyl-CoA thiolase A, citrate synthase, O-acetylhornserine sulfhydrylase, protease, camitine O-acetyltransferase, hydratase dehydrogenase, epimerase; Pox4 genes (U.S. 2004/0265980); and Met2, Met3, Met6, Met25 and YALI0DI2903 genes. See also WO 2008/042338. Other promoters used for host cells of the type are described by Romanos et al., 1992, “Foreign gene expression in yeast: a review” Yeast 8:423-488.
With respect to bacterial host cells, suitable promoters include, but are not limited to, promoters obtained from E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), xylA and xylB genes of Bacillus subtilis, Bacillus megaterium promoters, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731(1978)), as well as the tac promoter (DeBoer et al., ., Proc. Natl Acad. Sci. USA 80: 21-25(1993)). Additional promoters include the trp promoter, the phage lambda PL, the T7 promoter and the like. Promoters suitable for use in the invention are described in Gilbert et al., 1980, "Useful proteins from recombinant bacteria" Sci Am 242:74-94, and Sambrook et al., supra.
With respect to filamentous fungal host cells, suitable promoters include, but are not limited to, promoters obtained from the genes for TAKA amylase from Aspergillus oryzae, Rhizomucor miehei aspartic proteinase, neutral alpha-amylase from Aspergillus niger, alpha- Acid-stable amylase from Aspergillus niger, glucoamylase (glaA) from Aspergillus niger or Aspergillus awamori, lipase from Rhizomucor miehei, alkaline protease from Aspergillus oryzae, triose phosphate isomerase from Aspergillus oryzae, acetamidase from Aspergillus and trypsnianss porgillus trypsni-like protease from Aspergillus WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters of the genes for neutral alpha-amylase from Aspergillus niger and triose phosphate isomerase from Aspergillus oryzae).
[00152] The promoter may be any of the promoters listed in U.S. patent application 13/330,324. In particular, the promoter may be a promoter region from a portion of the Y. lipolytica YALI0E12683 gene, a promoter region from a portion of the Y. lipolytica YALI0E19206 gene, or a promoter region from a portion of the Y. lipolytica YALI0E34749 gene. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:74 (a 0.25 kb sequence from YALI0E12683), SEQ ID NO:75 (a 0.25 kb sequence from YALI0E19206), or SEQ ID NO :76 (a 0.25 kb sequence of YALI0E34749). In some embodiments, the promoter is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the nucleotide sequence of SEQ ID NO :74, SEQ ID NO:75 or SEQ ID NO:76. Other regulatory elements
[00153] Exogenous gene expression can be improved also by incorporating transcription terminators, core sequences, polyadenylation sequences, secretory signals, pro-peptide coding regions, regulatory sequences, and/or selectable markers that are evident to those skilled in the art. The choice of appropriate control sequences for use in the polynucleotide constructs of the present disclosure is made by those skilled in the art and, in various embodiments, is dependent on the recombinant host cell used and the desired method of recovering the fatty alcohol compositions produced.
The regulatory sequences used for Yarrowia include, but are not limited to, fragments of the Xpr2 promoter (U.S. 6083717). Terminator sequences used include, but are not limited to, the terminator sequences Xpr2 (U.S. 4,937,189) and Pox2 (YALIOFI 0857g) from Y. lipolytica.
[00155] In various embodiments, the expression vector includes one or more selectable markers that allow easy selection of transformed cells. Selectable markers for use in a host organism in the manner described herein include, but are not limited to, genes that confer antibiotic resistance (e.g., resistance to ampicillin, kanamycin, chloramphenicol, hygromycin, or tetracycline) to the recombinant host organism that comprises the vector. VIII. BETTER PRODUCTION OF FATTY ACIL- COA DERIVATIVES
[00156] The modified microbial organisms described here exhibit better production of fatty acyl-CoA derivatives. The yield of fatty acyl-CoA derivatives of the modified microbial organism of the invention can be compared to a control organism of the same type (eg, an otherwise identical control microbial organism in which the endogenous gene has not been disrupted). In an embodiment, microbial modified organism has at least one endogenous disrupted gene is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175 , YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0B17512, or a homologue of any of these, and a gene exogenous protein that encodes a functional fatty acyl reductase operably linked to a promoter. In some embodiments, the organism exhibits at least a 1.2-fold increase in production of fatty acyl-CoA derivatives compared to a control organism of the same type (eg, an otherwise identical control microbial organism, where the one or more endogenous genes are not disrupted). In other modes, the best production is at least 1 time, at least 1.2 times, at least 1.5 times, at least 2.5 times, at least 4 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or at least 60 times compared to the control microbial organism. In some embodiments, the exogenous gene encoding a fatty acyl reductase is a gene with at least 80% sequence identity with the nucleotide sequence of FAR_Maa (SEQ ID NO:1), FAR_Maq (SEQ ID NO:3), or FAR_Ocs (SEQ ID NO:5). In some embodiments, the exogenous gene encodes a FAR polypeptide with at least 80% sequence identity to wild-type_Maa FAR (SEQ ID NO:2), wild-type_Maq FAR (SEQ ID NO:4), or wild-type_Ocs FAR (SEQ ID NO:6). In some embodiments, the exogenous gene encodes a FAR variant derived from FAR_Maa (SEQ ID NO:2), FAR_Maq (SEQ ID NO:4), or FAR_Ocs (SEQ ID NO:6).
[00157] In some embodiments, the invention provides a microbial organism (eg, an alga, a bacteria, a mold, a filamentous fungus, a yeast, or an oleaginous yeast) comprising one, two, three, four, or more disrupted endogenous genes, in which at least one of the disrupted endogenous genes is selected from gene C17545, gene E28336, gene E11099, gene E28534 and homologs thereof, and an exogenous gene encoding a functional fatty acyl reductase gene operably linked to a promoter, wherein the microbial organism exhibits at least a 1-fold, at least a 1.2-fold, at least a 1.5-fold, at least 2.5-fold, at least 4-fold, at least 10 times, at least 15 times, or at least 20 times in the production of fatty acyl-CoA derivatives compared to a control microbial organism (eg, an otherwise identical control microbial organism, where the one or more genes are not interrupted).
[00158] In one embodiment, the invention provides a Yarrowia lipolytica cell that comprises at least one endogenous disrupted gene that is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769, YALI0E32769, YALI0E28336, YALI0E110E or one homolog of either of these, and an exogenous gene encoding a functional fatty acyl reductase gene operably linked to a promoter, in which the Yarrowia lipolytica cell exhibits at least a 1-fold increase in production of fatty acyl-CoA derivatives compared to a control microbial organism (eg, an otherwise identical control microbial organism, in which the one or more genes are not disrupted). In certain embodiments, the invention provides a Yarrowia lipolytica cell comprising a disrupted gene or combination of disrupted genes shown in Table 3 or Table 4. In certain embodiments, the invention provides a yeast cell comprising a disrupted gene that is a homologous, or a combination of disrupted genes that are homologous, to the genes shown in Table 3 or Table 4.
The control microbial organism can be, for example, Y. lipolytica DSMZ 1345 (wild-type) or the Y. lipolytica CY-201 strain (a variant of Y. lipolytica DSMZ 1345 shows little growth in hexadecane media such as only source of carbon). In some embodiments, the control microbial organism is a recombinant organism that has exogenous genes incorporated in an identical manner to the microbial organism with the gene(s) interrupted. For example, both the microbial organism that has one or more endogenous disrupted genes and the control microbial organism may contain an exogenous FAR gene.
[00160] When comparing the microbial organism that has one or more endogenous disrupted genes with the control microbial organism, the organisms can be cultured under essentially identical conditions, and the fatty acyl-CoA derivatives can be evaluated or recovered using essentially identical procedures . Fatty alcohol production
[00161] In some embodiments, the fatty acyl-CoA derivative that is produced is a fatty alcohol. Thus, in some embodiments, the invention provides a modified microbial organism that exhibits at least a 1-fold, at least 1.2-fold, at least 1.5-fold, at least 2.5-fold, at least 2.5-fold increase. 4 times, at least 10 times, at least 15 times, or at least 20 times in fatty alcohol production compared to a control microbial organism, where the one or more genes are not disrupted.
[00162] Fatty alcohol production can be evaluated by methods described in the examples section (eg examples 3 and 6) and/or using any other methods known in the art. Alcohol production by an organism of the present invention (eg, a microbial organism that has an endogenous disrupted gene) can be described as an absolute amount (eg, mols/liter of culture), or as a times increase in improvement. in production by a control organism (eg, a microbial organism in which the endogenous gene has not been disrupted). The production of fatty alcohol by a microbial organism of the present invention can be evaluated, for example, using gas chromatography. In general, the microbes are cultivated, total or secreted fatty alcohols are isolated, and the amount and/or content of fatty alcohol is evaluated.
[00163] Any assay number can be used to determine whether a microbial organism comprising at least one endogenous disrupted gene, in the manner described herein, produces a greater amount of fatty alcohols (eg, at least 1 times more fatty alcohols) compared to to a control microbial organism, in which the one or more genes are not disrupted, including exemplary assays described herein. In an exemplary trial, fatty alcohols produced by productive Y. lipolytica strains are collected by extraction from cell cultures using 1 mL of isopropanol:hexane (4:6 ratio). The extraction mixture is centrifuged and the upper organic phase is transferred to a 96-well plate, and analyzed by gas chromatography (GC) equipped with a flame ionization detector (FID) and HP-5 column (30 m size, ID 0.32 mm, 0.25 µm film), starting at 100 °C, and increasing the temperature at a rate of 25 °C/min to 246 °C, and then holding for 1.96 minutes. IX. METHODS OF PRODUCING FATTY ACYLCOA DERIVATIVES
[00164] The present disclosure also provides methods of producing fatty acyl-CoA derivatives using the microbial organisms in the manner described herein, as well as the resulting fatty acyl-CoA derivative compositions produced by said methods. Fermentation
[00165] The host cell fermentation is carried out under suitable conditions and for a sufficient time to produce fatty acyl-CoA derivatives. Conditions for culturing and producing cells, including filamentous fungal, bacterial and yeast cells, are readily available. Cell culture media are generally presented in Atlas and Parks, eds., 1993, The Handbook of Microbiological Media. The individual components of such media are available from commercial sources, for example, from the trademarks DIFCO™ and BBL™. In some embodiments, the aqueous nutrient medium is a "rich medium" comprising complex sources of nitrogen, salts and carbon, such as the YP medium, which comprises 10 g/L of peptone and 10 g/L of yeast extract in a kinda like this. In other embodiments, the aqueous nutrient medium is nitrogen-based yeast (DIFCO™) supplemented with an appropriate mixture of amino acids, eg SC medium. In particular embodiments, the amino acid mixture lacks one or more amino acids, thereby imposing a selective pressure to maintain an expression vector in the recombinant host cell.
[00166] The culture medium may contain an assimilable source of carbon. Assimilable carbon sources are available in many forms, and include renewable carbon sources and the cellulosic feedstock and starch substrates obtained from them. Suitable assimilable carbon sources include, but are not limited to, monosaccharides, disaccharides, oligosaccharides, saturated and unsaturated fatty acids, succinate, acetate and mixtures thereof. Additional carbon sources include, but are not limited to, glucose, galactose, sucrose, xylose, fructose, glycerol, arabinose, mannose, raffinose, lactose, maltose, and mixtures thereof. Culture media can include, for example, raw material from a cellulose-containing biomass, a lignocellulosic biomass, or a sucrose-containing biomass.
[00167] In some embodiments, "fermentable sugars" are used as the assimilable source of carbon. The “fermentable sugar” means simple sugars (monosaccharides, disaccharides, and short oligosaccharides) including, but not limited to, glucose, fructose, xylose, galactose, arabinose, mannose, and sucrose. In one modality, fermentation is carried out with a mixture of glucose and galactose as the assimilable source of carbon. In another modality, fermentation is carried out with glucose alone to accumulate biomass, after which the glucose is substantially removed and replaced by an inducer, for example, galactose to induce expression of one or more exogenous genes involved in derivative production of fatty acyl-CoA. In yet another modality, fermentation is carried out with an assimilable carbon source that does not mediate glucose repression, eg raffinose, to accumulate biomass, after which the inducer, eg galactose, is added to induce the expression of one or more exogenous genes involved in the production of the fatty acyl-CoA derivative. In some embodiments, the assimilable carbon source is cellulosic feedstock and starch derived from, but not limited to, wood, wood pulp, paper pulp, grain, corn residue, corn fiber, rice, wood processing residue. paper and pulp, woody or herbaceous plants, fruit or vegetable pulp, grains for distillation, grasses, rice husks, wheat straw, cotton, hemp, flax, sisal, corn cobs, sugarcane bagasse, growing grass fast, and mixtures of these.
[00168] In one embodiment, the method of preparing the fatty acyl-CoA derivatives further includes the steps of placing a biomass containing cellulose in contact with one or more cellulases to yield a fermentable sugar raw material, and placing the fermentable sugars in contact with a microbial organism as described herein. In one embodiment, the microbial organism is Y. lipolytica, and the fermentable sugars are glucose, sucrose, and/or fructose.
Microorganisms can be grown under batch, fed batch or continuous fermentation conditions, which are all known in the art. Classic batch fermentation is a closed system, in which the compositions of the medium are adjusted at the start of fermentation and are not subjected to artificial alternatives during fermentation. A variation of the batch system is a fed batch fermentation, where the substrate is added in increments like the fermentation processes. Fed-batch systems are used when catabolite repression is likely to inhibit cell metabolism, and where it is desirable to present limited amounts of substrate in the medium. Continuous fermentation is an open system where a defined fermentation medium is continuously added to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains cultures at a constant high density, where cells are mostly in the log growth phase. Continuous fermentation systems strive to keep growing conditions at the stable stage. Methods to modulate nutrients and growth factors for continuous fermentation processes, as well as techniques to maximize the rate of product formation, are well known in the art of industrial microbiology.
[00170] In some embodiments, fermentations are carried out at a temperature of about 10°C to about 60°C, about 15°C to about 50°C, about 20°C to about 45°C about 20°C to about 40°C, about 20°C to about 35°C, or about 25°C to about 45°C. In one embodiment, fermentation is carried out at a temperature of about 28°C and/or about 30°C. It will be understood that, in certain embodiments where thermostable host cells are used, fermentations can be carried out at higher temperatures.
[00171] In some embodiments, fermentation is carried out for a period of time from about 8 hours to 240 hours, about 8 hours to about 168 hours, about 8 hours to 144 hours, about 16 hours to about 120 hours, or about 24 hours to about 72 hours. In some embodiments, the fermentation will be carried out at a pH of about 3 to about 8, about 4.5 to about 7.5, about 5 to about 7, or about 5.5 to about 6 .5.
[00172] In one embodiment, the method of producing fatty acyl-CoA derivatives comprises: a) providing a microbial organism (eg, a Yarrowia lipolytica cell) that has one or more disrupted endogenous genes, in which at least one gene interrupted is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14720, YALI0B17512, or homologous to either of these, and an exogenous gene encoding a functional fatty acyl reductase, op. reliably linked to a promoter; and b) culturing the microbial organism (eg the Yarrowia cell) to allow for the production of a fatty acyl-CoA derivative, where the growing conditions include a temperature from about 20°C to about 40°C, a time period of about 16 to about 120 hours, and a culture medium containing fermentable sugars obtained from a cellulosic raw material.
[00173] In another embodiment, the above method is modified to include a culture medium containing sucrose. In some embodiments, where the culture medium contains sucrose, the microbial organism (eg, the Yarrowia cell) additionally comprises an exogenous gene encoding an invertase (eg, invertase from Saccharomyces cerevisiae SUC2).
[00174] In some embodiments, the method of producing fatty acyl-CoA derivatives yields at least 0.5 g/L of fatty acyl-CoA derivatives in the manner described below. production levels
The methods described herein produce fatty acyl-CoA derivatives in high yield. Routine cultivation conditions, eg yeast cultivation such as for Yarrowia lipolytica, may yield about 0.5 g to about 35 g fatty acyl-CoA derivatives, eg fatty alcohols, per liter of medium. culture (eg nutrient medium), depending on the disrupted gene(s). In some embodiments, the amount of fatty acyl-CoA derivatives, e.g., fatty alcohols, produced by the methods described herein is at least 0.5 g/L, at least 1 g/L, at least 1.5 g/L , at least 2 g/L, at least 2.5 g/L, at least 3 g/L, at least 3.5 g/L, at least 4 g/L, at least 4.5 g/L, at least at least about 5 g/L, or at least 10 g/L, at least 20 g/L, at least 30 g/L, at least 40 g/L, or at least 50 g/L of culture medium.
[00176] In some embodiments, the amount of fatty acyl-CoA derivatives, e.g., fatty alcohols, produced by the methods described herein is about 40 mg/g about 1 g/g, about 40 mg/g about 5 g/g, about 100 mg/g, about 1 g/g, about 100 mg/g, about 5 g/g, about 500 mg/g, about 2 g/g, about 1 g/g about 4 g/g, or about 2 g/g to about 3 g/g dry cell weight by modification of routine culture conditions.
In certain embodiments, the amount of fatty acyl-CoA derivatives, e.g., fatty alcohols, produced by the methods described herein is about 4% to about 20%, about 10% to about 20%, about from 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, or about 70% to about 80% cell dry weight by modification of routine culture conditions. Recovery of fatty acyl-CoA derivatives
The methods can further include a recovery step, e.g. isolating the fatty acyl-CoA derivatives to yield the fatty acyl-CoA derivative compositions. The recovery or isolation of the produced fatty acyl-CoA derivatives refers to the separation of at least a portion of the fatty acyl-CoA derivatives from the other components of the culture medium or fermentation process. Suitable protocols for recovering or isolating fatty acyl-CoA derivatives from recombinant host cells and/or culture medium (e.g., distillation, chromatography) are known to those skilled in the art. In certain embodiments, the derivatives are purified (e.g., substantially free of organic compounds other than the derivative(s)). Derivatives can be purified using purification methods well known in the art.
[00179] In some embodiments, recombinant host microorganisms secrete the fatty acyl-CoA derivatives into the nutrient medium. In this case, the fatty acyl-CoA derivatives can be isolated by solvent extraction from the aqueous nutrient medium with a water-immiscible solvent. Phase separation after solvent removal provides the fatty acyl-CoA derivative, which can then be further purified and fractionated using methods and equipment known in the art. In other embodiments, the secreted fatty acyl-CoA derivatives coalesce to form a water-immiscible phase, which can be separated directly from the aqueous nutrient medium both during fermentation and after fermentation.
[00180] In some embodiments, the fatty acyl-CoA derivatives, for example, fatty alcohols, are isolated by separating the cells from the aqueous nutrient medium, for example, by centrifugation, resuspension and extraction of the fatty acyl-CoA derivatives from the recombinant host cells using an organic solvent or solvent mixture.
[00181] Regarding host microorganisms that do not secrete the fatty acyl-CoA derivatives into the nutrient medium, the fatty acyl-CoA derivatives can be recovered by first lysing the cells to release the fatty acyl-CoA derivatives, and extracting the derivatives of fatty acyl-CoA from the lysate using conventional medium. See Clontech Laboratories, Inc., 2009, Yeast Protocols Handbook, 100:9156-9161. X. FATTY ACIL-COA DERIVATIVES
[00182] As described above, fatty acyl-CoA derivatives include several compounds produced enzymatically by cellular metabolic pathways, as shown in Figure 1. Genetic modification of the enzymes involved in these pathways may preferentially yield the particular derivatives, by example, fatty alcohols. See section 0 above. Additionally, or alternatively, particular fatty acyl-CoA derivatives can be chemically modified (in culture or after recovery) to yield a different derivative.
Fatty acyl-CoA derivative compositions may include saturated (eg monounsaturated), unsaturated and branched fatty acyl-CoA derivatives, eg fatty alcohols. In some modalities, the amount of unsaturated fatty acyl-CoA derivatives (eg fatty alcohols) may be less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% of the total fatty acyl-CoA derivative composition. In some modalities, the amount of saturated fatty acyl-CoA derivatives may be less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1 % of total fatty acyl-CoA derivative composition. In some modalities, the amount of branched fatty acyl-CoA derivatives may be less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1 % of total fatty acyl-CoA derivative composition.
[00184] In some embodiments, fatty acyl-CoA derivatives (eg, fatty alcohols, fatty esters, alkanes, alkenes, etc.) that have a carbon chain size of C8 to C20, C10 to C18, C14 to C18, or C16 to C18 comprise at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% by weight of the total derivative composition of fatty acyl-CoA. In some embodiments, fatty alcohols having a carbon chain size of C8 to C20, C10 to C18, C14 to C18, or C16 to C18 comprise at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% by weight of a total fatty alcohol composition. In some embodiments, fatty acyl-CoA derivatives (eg, fatty alcohols) have a carbon chain size of C16 to C18. Such C16 to C18 fatty acyl-CoA derivatives, for example fatty alcohols, can be saturated, unsaturated or a mixture of saturated and unsaturated derivatives. When the derivative is an alkane or alkene, it is observed that alkanes and/or alkenes having particular carbon chain sizes can be isolated from longer and/or shorter alkanes and/or alkenes, for example, by HPLC.
[00185] In some embodiments, the fatty acyl-CoA derivative is a fatty alcohol. The fatty alcohol can be one or more of 1-octanol (C8:0), 1-decanol (C10:0), 1-dodecanol (C12:0), 1-tetradecanol (C14:0), 1-hexadecanol (C16 :0), 1-octadecanol (C18:0), 1-icosanol (C20:0), 1-docosanol, 1-tetracosanol, hexadecenol (C16:1) and octadecenol (C18:1). Alkane and/or alkene compositions
[00186] In some embodiments, the fatty acyl-CoA derivative is an alkane and/or alkene. Alkanes and/or alkenes can be isolated from the reaction mixture (which may contain unreduced fatty alcohols) to yield a composition that comprises substantially all of the alkanes and/or alkenes. Alternatively, unreduced alkanes/alkenes and fatty alcohols can be isolated from the reaction mixture to yield a composition comprising alkanes and/or alkenes and fatty alcohols. In some embodiments, the fatty acyl-CoA derivative compositions comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% alkanes and/or alkenes by weight of the composition after reduction.
In some embodiments, the alkane is octane, decane, dodecane, tetradecane, hexadecane, octadecane, icosane, docosane, tetracosane, or mixtures thereof. In some embodiments, the alkene is octene, decene, dodecene, tetradecene, hexadecene, octadecene, icosene, docosene, tetracosene, or mixtures thereof.
[00188] In some embodiments, fatty alcohols produced according to the methods described herein can be reduced to yield alkanes and/or alkenes, which have the same carbon chain size as the fatty alcohol starting materials. Without being bound by any particular theory, the hydroxyl group of an alcohol is a weak leaving group and therefore, in principle, a chemical moiety that binds to the oxygen atom of the hydroxyl group to make it a better leaving group can be used to reduce the fatty alcohols described herein.
[00189] Any method known in the art can be used to reduce fatty alcohols. In some embodiments, the reduction of fatty alcohols can be carried out chemically, for example, by a Barton deoxygenation (or Barton-McCombie deoxygenation), a two-step reaction in which the alcohol is first converted to a methyl xanthate or thioimidazoyl carbamate , and the xanthate or thioimidazoyl carbamate is reduced with a tin hydride or trialkylsilane reagent under radical conditions to produce the alkane and/or alkene. See Li et al., 2007, Modern Organic Synthesis in the Laboratory, p. 81-83.
[00190] In another modality, alkanes can be produced by hydrogenation of fatty alcohols or fatty acids. Ester Compositions
[00191] In other embodiments, fatty alcohols are reacted with a carboxylic acid to form acid esters. Esterification reactions of fatty alcohols are well known in the art. In certain embodiments, the transesterification reaction is carried out in the presence of a strong catalyst, for example, a strong alkali such as sodium hydroxide. In other embodiments, an esterification reaction is carried out enzymatically using an enzyme that catalyzes the conversion of fatty alcohols to acid esters, such as lipoprotein lipase. See, for example, Tsujita et al., 1999, "Fatty Acid Alcohol Ester-Synthesizing Activity of Lipoprotein Lipase" J. Biochem. 126:1074-1079. XI. EXEMPLARY COMPOSITIONS INCLUDING FATTY ACIL-COA DERIVATIVES
[00192] In yet another aspect, the present invention relates to the use of microbial organisms, as described herein, for the production of various compositions including, but not limited to, fuel compositions (e.g., biodiesel and petrodiesels), compositions detergents (eg laundry detergents in liquid or powder form, hard surface cleaning agents, dishwashing liquids and the like); industrial compositions (eg lubricants, solvents and industrial cleaning agents); and personal care compositions (eg soaps, cosmetics, shampoos and gels). Fuel Compositions
[00193] In certain embodiments, the fatty acyl-CoA derivative compositions described herein can be used as components of fuel compositions. In certain embodiments, the fatty acyl-CoA derivatives produced by the methods described above can be used directly in fuel compositions. Fuel compositions containing fatty acyl-CoA derivatives produced by the methods of the present invention include any of the compositions used in energy combustion machines including, but not limited to, biodiesel fuels and petrodiesel fuels (e.g. aviation and rocket fuels).
[00194] In some modalities, the fuel composition is diesel fuel. Diesel fuel is any fuel used in diesel engines and includes both petrodiesel and biodiesel. Petrodiesel is a specific fractional distillate of fossil fuel oil. It is comprised of about 75% saturated hydrocarbons and 25% aromatic hydrocarbons. Biodiesel is not derived from petroleum, but from vegetable oil or animal fats, and contains long-chain alkyl esters. Biodiesel is prepared from transesterification of lipids (eg, vegetable oil consumed from frying or seed oils) with an alcohol and burns cleaner than petrodiesel. Biodiesel can be used alone or mixed with petrodiesel in any amount for use in modern machines.
[00195] In some modalities, the fuel composition is kerosene. Kerosene is a fuel hydrocarbon that is also a specific fractional distillate of fossil fuel, and contains hydrocarbons that have 6 to 16 carbon atoms. Kerosene has a combustion heat comparable to that of petrodiesel, and is widely used in jet fuel for starting aircraft engines and for heating in certain countries. Kerosene-based fuels can also be burned with liquid oxygen and used as rocket fuel (eg RP-1).
[00196] In particular embodiments, fatty esters are used as components of biodiesel fuel composition. In various embodiments, fatty acid esters are used as biodiesel fuel without being mixed with other components. In certain embodiments, fatty acid esters are mixed with other components, such as petrodiesel fuel. In other embodiments, alkanes and/or alkenes (eg, C10 to C14) are used as components of jet fuel compositions. In other embodiments, alkanes and/or alkenes are used as rocket fuel components. In yet other embodiments, alkanes and/or alkenes (eg, C16 to C24) are used as components in a petrodiesel-type fuel composition.
[00197] In some embodiments, the fuel composition comprises an alkane and/or alkene. In certain embodiments, alkanes and/or alkenes are from 6 to 16 carbons, and the fuel composition is a kerosene type fuel composition. In various embodiments, kerosene-type fuel compositions are included in aviation fuel compositions. In particular embodiments, kerosene-type fuel compositions are included in various grades of jet fuel including, but not limited to, Avtur grades, Jet A, Jet A-1, Jet B, JP-4, JP-5, JP- 7 and JP-8. In other embodiments, kerosene-type fuel compositions are included in heating fuel compositions. In yet other embodiments, kerosene-type fuel compositions are burned with liquid oxygen to provide rocket fuel. In particular embodiments, kerosene-type fuel compositions are used in RP-1 rocket fuel.
[00198] In some embodiments, alkanes and/or alkenes are used in fuel compositions that are similar to petrodiesel fuel compositions, for example, fuels that contain saturated and aromatic hydrocarbons. In certain embodiments, the fuel compositions comprise only alkanes and/or alkenes. In other embodiments, fuel compositions comprise alkanes and/or alkenes mixed with other components, such as petrodiesel fuel.
[00199] In certain embodiments, fatty alcohols, fatty esters, alkanes, and/or alkenes are combined with other fuels or fuel additives to produce compositions that exhibit the desired properties for their intended use. Exemplary fuels and fuel additives for particular applications are well known in the art. Exemplary fuels that can be combined with the compositions described herein include, but are not limited to, traditional fuels such as ethanol and petroleum based fuels. Exemplary fuel additives that can be combined with the compositions described herein include, but are not limited to, low cloud point additives, surface active agents, antioxidants, metal deactivators, corrosion inhibitors, anti-freeze additives, anti-wear additives, deposit modification additives and octane enhancers. Detergent Compositions
In some embodiments, the fatty acyl-CoA derivative compositions described herein, and the compounds derived therefrom, can be used as components of detergent compositions. Detergent compositions containing fatty acyl-CoA derivatives produced by the methods of the present invention include compositions used in cleaning applications including, but not limited to, laundry detergents, manual cleaning agents, dishwashing detergents, detergents that assist. in washing, household detergents and household cleaners, in liquid, gel, granular, powder or tablet form. In some embodiments, fatty acyl-CoA derivatives (eg, fatty alcohols) produced by the methods described above can be used directly in detergent compositions. In some embodiments, fatty acyl-CoA derivatives (eg, fatty alcohols) can be reacted with a sulfonic acid group to produce sulfate derivatives, which can be used as components of detergent compositions. Detergent compositions that can be generated using the fatty acyl-CoA derivatives produced by the methods of the present invention include, but are not limited to, hair shampoos and conditioners, carpet shampoos, fast acting household cleaners, fast acting household detergents fast-acting household cleaners and long-acting household detergents. Detergent compositions in general include, in addition to the fatty acyl-CoA derivatives, one or more building agents (eg, sodium carbonate, complexing agents, soap and zeolites), enzymes (eg, a protease, a lipase and an amylase); carboxymethyl cellulose, optical brighteners, fabric softeners, dyes and perfumes (eg cyclohexyl salicylate).
[00201] In some embodiments, sulfate derivatives (eg, C12-15) from fatty acyl-CoA derivatives are used in products such as hair shampoos, carpet shampoos, fast-acting household cleaners and household detergents. fast action. In some embodiments, sulfate derivatives (eg, C16-C18) from fatty acyl-CoA derivatives are used in products such as shampoos and hair conditioners. In some embodiments, sulfate derivatives (eg, C16-18) from fatty acyl-CoA derivatives are used in products such as long-acting household cleaners and longer-acting household detergents. Personal Care Compositions
In some embodiments, fatty acyl-CoA derivative compositions as described herein, and compounds derived therefrom, can be used as components of personal use compositions. In some embodiments, the fatty acyl-CoA derivatives produced by the methods described above can be used directly in personal use compositions. Personal use compositions containing fatty acyl-CoA derivatives produced by the methods of the present invention include compositions used for application to the body (e.g., for application to the skin, hair, nails, or oral cavity) for the purposes of care for the appearance, cleanse, beautify, or care for the body, including, but not limited to, lotions, plasters, creams, gels, serums, cleansers, paints, masks, sunscreens, soaps, shampoos, conditioners, shower gels, hair care products. finishing and cosmetic compositions (eg makeup in liquid, cream, solid, anhydrous or pencil form). In some embodiments, fatty acyl-CoA derivatives (eg fatty alcohols) can be reacted with a sulfonic acid group to produce sulfate derivatives that can be used as components of said compositions.
In some embodiments, fatty acyl-CoA derivative compositions (eg, C12) produced by the methods described herein are used in products such as lubricating oils, pharmaceuticals, and as an emollient in cosmetics. In some embodiments, fatty acyl-CoA derivative compositions (eg, C14) produced by the methods described herein are used in products such as cosmetics (eg, cold creams) with their emollient properties. In some embodiments, fatty acyl-CoA derivative compositions (e.g., C16), produced by the methods described herein, are used in products such as cosmetics (e.g., skin creams and lotions) as an emollient, emulsifier, agent. of thickening. In some embodiments, fatty acyl-CoA derivative compositions (e.g., C18), produced by the methods described herein, are used in products such as lubricants, resins, perfumes, and cosmetics, e.g., as an emollient, emulsifier, or agent. of thickening. In some embodiments, sulfate derivatives (e.g., C12-14) derived from the fatty acyl-CoA derivative compositions produced by the methods described herein are used in products such as toothpastes. Other compositions
[00204] In some embodiments, fatty acyl-CoA derivatives (eg fatty alcohols, especially cetyl alcohol, stearyl alcohol and myristyl alcohol) can be used as food additives (eg, adjuvants and production aids). XII. EXAMPLES
[00205] The following examples are offered to illustrate but not to limit the claimed invention. Example 1: Expression of wild-type M. algicola DG893 FAR in Y. lipolytica strains
Wild-type M. algicola FAR (FAR_Maa) was expressed in Y. lipolytica strains. The codon optimized sequence of the M. algicola DG893 FAR gene corresponds to SEQ ID NO:1, and the corresponding polypeptide sequence is determined to SEQ ID NO:2. An autonomous replicating plasmid, pCEN354, was constructed for the expression of the FAR gene from M. algicola DG893 FAR in Y. lipolytica strains. The replicating plasmid was genetically modified with two antibiotic selection marker cassettes for resistance to hygromycin and phleomycin (HygB(R) or Ble(R), respectively). The expression of each cassette is independently regulated by a strong and constitutive promoter isolated from Y. lipolytica: pTEF1 for the expression of Ble(R), and pRPS7 for the expression of HygB(R). Plasmid pCEN354 was used to assemble the Y. lipolytica expression plasmids. Using the “unrestricted cloning” methodology, the Ble(R) gene was replaced by the FAR gene from M. algicola DG893 to provide plasmid pCEN411 (figure 2). In pCEN411, the expression of the FAR gene is in control of the constitutive TEF1 promoter, and the HygB(R) gene allows selection in media containing hygromycin. Ars18 is an autonomous replicating sequence isolated from the genomic DNA of Y. lipolytica. The resulting plasmid was transformed into Y. lipolytica strains using routine transformation methods. See, for example, Chen et al., 1997, "One-step transformation of the dimorphic yeast Yarrowia lipolytica" Appl Microbiol Biotechnol 48:232-235.
[00207] FAR was also expressed by integrating an expression cassette at a specific site in the genome of Y. lipolytica. In this case, the DNA to be integrated contained a M. algicola FAR FAR expression cassette, and a second expression cassette that encoded hygromycin resistance. The DNA encoding these expression cassettes was flanked on both sides by ~1 kb Y. lipolytica DNA, which acted to target this DNA to a specific intergenic site on the E chromosome. This site has been identified as a “type” expression. stirred point” by random integration of a FAR expression cassette, followed by mapping of the integration sites of most active transformants. Integration constructs were amplified by PCR and transformed into Y. lipolytica using routine transformation methods.
[00208] Through the random integration of an M. algicola FAR expression cassette into the genome of Y. lipolytica, numerous strains were identified with better fatty alcohol titers compared to strains with plasmid-based FAR expression. Integration sites in five of the best random integrant strains were determined using the “vectoret” PCR method. In each of these strains, there are two copies of the FAR gene in both the direct and inverted repeat structure.
One copy of an M. algicola FAR FAR expression cassette was introduced by targeted integration into both the positive strand and the smallest of one of the five shake dots identified in the genome of the Y. lipolytica strain CY-201. The tFARi-1 integration site was located on the E chromosome between 1433493 bp and 1433495 bp on the negative strand. The tFARi-2 integration site was located on the C chromosome between 2526105 bp and 2526114 bp on the positive strand. The tFARi-3 integration site was located on chromosome B between 2431420 bp and 2431425 bp on the positive strand. The tFARi-4 integration site was located on the D chromosome between 1669582 bp and 1669588 bp on the positive strand. The tFARi-5 integration site was located on the D chromosome between 518746 bp and 518789 bp on the positive strand. The tFARi-6 integration site was located on chromosome B between 2431420 bp and 231425 bp on the negative strand. The tFARi-7 integration site was located on the D chromosome between 1669582 bp and 1669588 bp on the negative strand. The tFARi-8 integration site was located on the D chromosome between 518746 bp and 518789 bp on the negative strand. Example 2: In vivo activity of exogenous M. algicola FAR in recombinant strains of Y. lipolytica
Two strains of Y. lipolytica were used to build neutralized genes: 1) Y. lipolytica DSMZ 1345, obtained from the German Resource Center for Biological Material (DSMZ), and 2) Y. lipolytica CY-201, a better host production obtained by UV mutagenesis of Y. lipolytica DSMZ 1345 and deficient in growth on media with hexadecane as the only carbon source. When transformed with pCEN411, Y. lipolytica CY-201 produced 7 to 10 times more fatty alcohols compared to Y. lipolytica DSMZ 1345, and also significantly reduced the degradation rate of exogenous 1-hexadecanol in YPD media containing 8% glucose and 500 µg/ml of hygromycin. The expression of alternative and variant FAR genes in modified strains of Y. lipolytica can be evaluated using similar methodology. Example 3: Analysis of fatty alcohol production in Y. lipolytica strains containing exogenous FAR
Y. lipolytica strains comprising a plasmid containing an exogenous gene, encoding M. algicola DG893 FAR, were grown in Axygen 96-well plates containing 250 μL of YPD supplemented with 2% glucose and 500 μg /ml hygromycin. The plates were incubated in a Kuhner shaking incubator for approximately 40-48 hours at 30°C, 200 rpm and 85% relative humidity. Cell cultures were diluted by transferring 50 µL of overnight-grown cultures into Axygen 96-well plates containing 250 µL of YPD supplemented with 2% glucose and 500 µg/mL of hygromycin. Plates were incubated for approximately 24-28 hours in a Kuhner shaking incubator under the same conditions. 20 μL of the cell cultures were transferred to 96-deep well plates containing 380 μL of YPD, supplemented with 8% glucose and 500 μg/mL hygromycin. Plates were incubated for approximately 22-26 hours under the same conditions. Cells were collected by centrifugation for 10 minutes at 3,500 rpm. Cell pellets were resuspended in 400 μL of nitrogen limiting media (1.7 g/L yeast nitrogen base, 1.4 g/L (NH4)2SO4, 30 g/L glucose), containing 500 μg/mL of hygromycin, and were incubated for 22-26 hours in a Kuhner shaking incubator at 30°C, 200 rpm and 85% relative humidity. Cell cultures were extracted with 1 mL of isopropanol:hexane (4:6 ratio) for 2 hours. The extracts were centrifuged, and the upper organic phase was transferred to 96-well polypropylene plates. Samples were analyzed using the GC-FID method below.
[00212] A 1μL sample was analyzed by GC-FID with a separation ratio of 1:10 using the following conditions: GC-6890N from Agilent Technologies equipped with FID detection and HP-5 column (size 30 m, ID 0, 32 mm, 0.25 µm film). GC method: start at 100°C, increase temperature at a rate of 25°C/min to 246°C, and this was held for 1.96 min. Total running time, 7.8 minutes. In the above GC compositions, the approximate retention times (minutes) of the alcohols and fatty acids produced are as follows: 5.74, C16:1-OH; 5.93, C16:0-OH; 6.11, C16:0-OOMe (internal standard); 6.16, C16:1-OOH; 6.29, C16:0-OOH; 6.80, C18:1-OH; 6.90, C18:0-OH; 7.3, C18:0- and C18:1-OOH. Identification of individual fatty alcohols was performed by comparison with commercial standards (Sigma Chemical Company). Under the conditions tested, the expression of FAR of M. algicola DG893 in the parental strains of Y. lipolytica DSMZ 1345 and CY-201 resulted in production of 5-20 mg/L and 100-200 mg/L of fatty alcohols, respectively. Fatty alcohols were produced: 70-80% C16:0 (1-hexadecanol), 10-15% 18:0 (1-octadecanol) and 10-15% C18:1 (cis Δ9-1-octadecenol) . Example 4: Identification of Y. lipolytica target genes for disruption
[00213] The genes selected for the interruption have been identified in several ways. Some genes were selected based on their roles in hydrocarbon assimilation pathways in yeast using alkane. Since the fatty acyl-CoA is an intermediate in these pathways, which result from the oxidation of alkanes, the stability of fatty acyl-CoA derivatives can be improved by disrupting the genes responsible for the use of alkane. Other genes for disruption were selected based on their homology to those genes. These include genes whose sequence determined they could function as alcohol dehydrogenases or acyltransferases involved in lipid biosynthesis.
Additional genes for disruption include those encoding proteins involved in the import of newly synthesized proteins into the endoplasmic reticulum. These include the trimeric protein-carrying channel subunits (Sec61, Ssh1, Sbh1, and Sss1), the Sec62/Sec63 tetrameric complex (Sec62, Sec63, Sec66, and Sec72), and other proteins found in the endoplasmic reticulum (Kar2 and Sls1) (Boisrame A. et al., "Interaction of Kar2p and Sls1p IS Required for Efficient Co-translational Translocation of Secreted Proteins in the yeast Yarrowia lipolytica," J. Biol. Chem. (1998) 273: 30903).
[00215] Other genes for disruption have been identified by comparing global gene expression in glucose and glycerol-based media. In particular, genes whose expression is repressed by glycerol were selected, since the use of alkane is repressed in media containing glycerol. Glycerol repressed genes were identified by microarray analysis, using RNA prepared from Y. lipolytica DSMZ 1345 cultivated both in rich medium and in medium with lipid accumulation. Example 5: Construction and analysis of strains that have disrupted genes
Neutralized strains can be constructed by transforming Y. lipolytica with a DNA construct determined to replace most or all of the open reading frame of interest with a selectable marker by homologous recombination. As such, the DNA constructs can comprise a selectable marker flanked by ~1 kb sequences immediately upstream and downstream of the gene of interest, which are necessary for homologous recombination to occur. These DNA constructs are contained in plasmids assembled using standard methods for plasmid construction. For transformation, the DNA construct of interest is amplified from the corresponding plasmid using PCR to generate ~1 µg of linear DNA. This DNA is transformed into Y. lipolytica using the method described in Madzak et al., 2003, "Yarrowia lipolytica." In Gellissen, ed. Production of Recombinant Proteins Novel Microbial and Eukaryotic Expression Systems, p. 163-189. Strains in which the gene of interest is replaced by the selectable marker are identified by their ability to grow on selective media, and by PCR genotyping. Typical selective markers are familiar to those skilled in the art (see, for example, Fickers et al., 2003, "New interruption cassettes for rapid gene interruption and marker rescue in the yeast Yarrowia lipolytica" Journal of Microbiological Methods 55:727-737) .
[00217] In a second step, the selectable marker is excised from the chromosome using methods that are familiar to those skilled in the art. See, for example, Fickers et al., supra; Akada et al., 2006, “PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae” Yeast 23:399-405; Fonzi et al., 1993, "Isogenic strain construction and gene mapping in Candida albicans" Genetics 134:717-728. Strains with excised markers can easily be identified by growth on counter-selection media if the selectable marker used is bifunctional, that is, if it encodes an enzyme whose product(s) is(are) essential for the growth on positive selection media, and toxic on another selection medium. Such bifunctional markers are familiar to those skilled in the art.
[00218] For the construction of strains with multiple gene disruptions, Y. lipolytica can be sequentially transformed with a series of DNA constructs determined to neutralize the genes of interest. Each transformation can be performed by the method described above, such that the selectable marker is excised after each interruption step. Thus, any combination of neutralized ones can be created in a given strain using the collection of plasmids that carry the DNA constructs described above. Example 6: Analysis of fatty alcohol production in modified strains of Y. lipolytica
[00219] A collection of ~233 strains comprising strains with single gene gaps, and strains with 2 or more gene gaps, has been created for both the earlier strains DSMZ1345 and CY-201. These strains were transformed with plasmid pCEN411 for expression of wild-type M. algicola DG893 FAR (see Figure 2), and were selected for fatty alcohol production in the manner described above. Tables 3 and 4 below provide the relative fatty alcohol production for targeted gene disruption of Y. lipolytica DSMZ 1345 and CY-201 strains, which express the wild-type M. algicola DG893 FAR gene, with respect to the strain counterpart of Y. lipolytica without any targeted gene deletion and expressing the wild-type M. algicola DG893 FAR gene. The fatty alcohols produced were: 70-80% C16:0 (1-hexadecanol), 10-15% 18:0 (1-octadecanol), and 10-15% C18:1 (cis Δ9-1-octadecenol ).














+ = 0.0 to 0.99 times improvement Example 7: Fatty alcohol production in fermentation with a modified strain of Y. lipolytica
A derivative of the CY-201 strain which comprises deletions of YALI0E11099g, YALI0E28336g, YALI0C17545, and YALI0E14729 and which bears two integrated copies of M. algicola FAR ("the CY-202 strain") was used to produce fatty alcohol in a stirred tank fermenter. Fermentation followed a two-stage protocol, in which cells are propagated in a nutrient-rich medium and then transferred to a nutrient-limited medium for the production of fatty alcohol. Regarding the first stage, an inoculation culture was prepared by growing the CY-202 strain in YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose) in a shake flask grooved on the bottom at 30°C for 24 hours. This culture was used to inoculate a fermenter containing 10 L of propagation medium (6.7 g/L of nitrogen-free yeast base without amino acids, 20.9 g/L of Bis Tris buffer, 80 g/L of glucose, 10 g /L of corn cinnamon, and 0.22 mL/L of antifoam (a 1:1 mixture of poly(propylene glycol) and antifoam B), adjusted to pH 6.5 with KOH. This seed culture was grown at 30°C in a batch process with controlled oxygen transfer rate (15-20 mM O2/hour), at a final OD600 of 12-18. For the second stage, cells in propagation medium were collected by centrifugation, then they were resuspended in 1.1L of fatty alcohol production medium (200 g/L of glucose, 1 g/L of KH2PO4, 5 g/L of (NH4)2SO4. 2.5 mg/L of MgSO4 *7H20 1 mg/L of FeSO4*7H20, 0.5 mg/L of H3BO3, 0.5 mg/L of MnSO4-H20, 0.5 mg/L of Na2MoO4-2H20, 0.5 mg/L of ZnSO4 *7H20, 0.5 mg/L of CoCl2*H20, 0.1 mg/L of KI, 0.1 mg/L of CuCl2*2H20, 50 mg/L of thiamine HCl, and 50 mg/L of inositol and 0.8 mL/L of anti-foam ). The cell resuspension volume was adjusted to provide an initial cell density for the second stage of 20 g/L (dry cell weight), then the resuspension was placed in a stirred tank fermenter. Fermentation was carried out in a batch process at 30°C with dissolved oxygen control (30% dO2). The pH was controlled to 3.5 by the addition of KOH. Glucose was added as needed to prevent glucose depletion (35 g/L as the fermentation progressed).
[00221] Samples were collected at 24 hours, 48 hours and 72 hours after inoculation of the culture in the production stage. The fatty alcohol titration was analyzed by GC-FID essentially as described in example 3. After 24 hours, a fatty alcohol titration of 9 g/L was observed. After 48 hours, a fatty alcohol titre of 16 g/L was observed. After 72 hours, a fatty alcohol titer of 21 g/L was observed. Example 8: Partial deletion of Sec62 gene
Strains with a partial deletion of the Sec62 gene (YALI0B17512g; SEQ ID NO:54) were constructed by transforming Y. lipolytica with a determined DNA construct (1) to mutate codon Trp235 into a stop codon and (2) to replace codons 236-396 with a selectable marker by homologous recombination. Thirty nucleotides from the 3' untranslated region immediately following the Sec62 coding sequence were also deleted. This partial deletion corresponds to a deletion of the cytoplasmic domain of Sec62, which starts immediately after the predicted transmembrane domain in Leu206, and continues at the end of the protein in Glu396. As shown in Table 3, this strain (identified in the table as “B17512”) provided ~10 times greater production of fatty alcohol than the corresponding DSMZ 1345 strain, without a partial deletion of the Sec62 gene.
[00223] Three other partial deletions of the Sec62 gene (YALI0B17512g) were performed by transforming Y. lipolytica with a DNA construct that (1) mutated both the codon encoding Glu267, the codon encoding Ala302, and the codon encoding Ile337 into a stop codon and (2) replaced subsequent codons with a selectable marker using homologous recombination. These strains provided ~1.5 to 2-fold higher production of fatty alcohol than the corresponding DSMZ 1345 strain without a partial deletion of the Sec62 gene.
[00224] The methods used for the construction and transformation of DNA are described in example 4 and are familiar to those skilled in the art. Briefly, the transforming DNA construct comprised a bifunctional selectable marker flanked by ~1 kb of genomic sequences immediately upstream and downstream of the nucleotides to be deleted. After transformation, strains with the desired modification were selected by growth on positive selective media. The selectable marker was then excised from the genome, and strains that showed marker loss were identified by growth on counter-selection media. PCR genotyping was used to confirm that the strains had the desired modification.
[00225] It is understood that the examples and modalities described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested by those skilled in the art, and shall be included in the spirit and competence of this application and the scope of the appended claims . All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety and for all purposes.

权利要求:
Claims (13)
[0001]
1. Microbial organism, characterized by the fact that one or more endogenous genes are disrupted, in which the endogenous gene is YAL1OC17545 or a homolog thereof, and/or YAL1OE28336 or a homolog thereof, and comprising an exogenous gene encoding an acyl protein fatty reductase (FAR) operably linked to a promoter, in which said microbial organism is an alga, a bacterium or a yeast.
[0002]
2. Microbial organism according to claim 1, characterized in that one or more of the endogenous gene YAL1OE11099 or a homolog thereof, and the endogenous gene YAL1OE28534 or a homolog thereof, is interrupted.
[0003]
3. Microbial organism according to claim 1 or 2, characterized in that it further comprises one or more endogenous genes selected from YALI0B10406, YALI0A19536, YALI0E32769, YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014, YALI079 , YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, YALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417 , YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512, and homologues thereof, interrupted.
[0004]
4. Microbial organism according to claim 3, characterized in that the endogenous YAL1OB17512 gene is disrupted.
[0005]
5. Microbial organism according to any one of claims 1 to 4, characterized in that it additionally comprises a second exogenous gene encoding a fatty acid synthase (FAS), an ester synthase, an acyl-ACP thioesterase (TE), a fatty acyl-CoA synthase (FACS), an acetyl-CoA carboxylase (ACC), a xylose isomerase, or an invertase.
[0006]
6. Microbial organism according to claim 1, characterized in that one or more endogenous genes are disrupted and comprises an exogenous gene encoding a functional fatty acyl reductase (FAR) protein operably linked to a promoter, in which one or more endogenous genes are selected from: (a) YALI0G17545 and YALI0E28336; (b) YALI0G17545 and YALI0B10406; (c) YALI0G17545 and YALI0E28534; (d) YALI0G17545 and YALI0E30283; (e) YALI0E28336 and YALI0E30283; (f) YALI0E11099 and YALI0E30283; (g) YALI0A19536 and YALI0E30283; (h) YALI0A19536 and YALI0E28534; (i) YALI0E30283 and YALI0E12463; (j) YALI0B10406 and YALI0E14729; (k) YALI0C17545 and YALI0E14729; (l) YALI0E11099 and YALI0E14729; (m) YALI0C17545, YALI0E28336 and YALI0E11099; (n) YALI0C17545, YALI0E28336 and YALI0B10406; (o) YALI0C17545, YALI0E28336 and YALI0A19536; (p) YALI0C17545, YALI0E28336 and YALI0E28534; (q) YALI0C17545, YALI0E28336 and YALI0E32769; (r) YALI0C17545, YALI0E28336 and YALI0E12463; (s) YALI0C17545, YALI0E11099 and YALI0B10406; (t) YALI0C17545, YALI0B10406 and YALI0A19536; (u) YALI0E28336, YALI0E11099 and YALI0B10406; (v) YALI0E11099, YALI0B10406 and YALI0A19536; (w) YALI0C17545, YALI0E28534 and YALI0B17512; (x) YALI0E11099, YALI0A19536, YALI0B10406 and YALI0B17512; (y) YALI0C17545, YALI0E28336, YALI0E11099 and YALI0B10406; (z) YALI0C17545, YALI0E28336, YALI0E11099 and YALI0A19536; (aa) YALI0C17545, YALI0E28336, YALI0E11099 and YALI0E28534; (bb) YALI0C17545, YALI0E28336, YALI0E11099 and YALI0E32769; (cc) YALI0C17545, YALI0E28336, YALI0B10406 and YALI0A19536; (dd) YALI0C17545, YALI0E28336, YALI0B10406 and YAL1I0E32769; (ee) YALI0C17545, YALI0E28336, YALI0A19536 and YALI0E28534; (ff) YALI0C17545, YALI0E28336, YALI0E28534 and YALI0E32769; (gg) YALI0C17545, YALI0E28336, YALI0E28534 and YALI0E12463; (hh) YALI0E28336, YALI0E11099, YALI0B10406 and YALI0E32769; and (ii) YALI0E11099, YALI0E28336, YALI0G17545 and YALI0E14729.
[0007]
7. Microbial organism according to claim 1 characterized in that one or more endogenous genes is disrupted in which the endogenous gene is selected from YALI0G17545 YALI0F25003, YALI0E14729, YALI0B17512, and homologs thereof.
[0008]
8. Microbial organism according to claim 7, characterized in that the endogenous YAL10B17512 gene, or homologue thereof, is disrupted, and wherein YAL10B17512 optionally encodes a polypeptide comprising a cytoplasmic domain, and wherein the disruption comprises a elimination of at least a portion of the cytoplasmic domain.
[0009]
9. Method of producing a fatty acyl-CoA derivative, characterized in that the method comprises cultivating the microbial organism as defined in any one of claims 1 to 8 under conditions in which the fatty acyl-CoA derivative is produced.
[0010]
10. Method according to claim 9, characterized in that the fatty acyl-CoA derivative is a fatty alcohol, fatty acid, fatty aldehyde, fatty ester, fatty acetate, wax ester, alkane or alkene.
[0011]
11. Method according to claim 10, characterized in that the fatty acyl-CoA derivative is a fatty alcohol.
[0012]
12. Method according to any one of claims 9 to 11, characterized in that it comprises: contacting a biomass containing cellulose with one or more cellulases to produce fermentable sugars; and contacting said microbial organism with the fermentable sugars under conditions in which the fatty acyl-CoA derivative is produced.
[0013]
13. Method according to claim 12, characterized in that fermentable sugars comprise sucrose.

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法律状态:
2020-08-11| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-09-08| B25A| Requested transfer of rights approved|Owner name: SHELL INTERNATIONALE RESEARCH MAATSCHAPPIJ B.V. (NL) |

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2020-11-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-02| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-31| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 19/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201061427032P| true| 2010-12-23|2010-12-23|

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US61/427,032|2010-12-23|

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US61/427032|2010-12-23|

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US201161502697P| true| 2011-06-29|2011-06-29|

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US61/502,697|2011-06-29|

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US61/502697|2011-06-29|

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PCT/US2011/065899|WO2012087964A1|2010-12-23|2011-12-19|Gene disruptants producing fatty acyl-coa derivatives|

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