# US 7,465,564 B2 · Production of γ-linolenic acid in oleaginous yeast > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,465,564 B2 - Document ID: US7465564B2 - Status: Granted - Kind: B2 - Application number: 11/198,975 - Filed: 2005-08-08 - Published: 2006-02-16 - Granted: 2008-12-16 - Earliest claimed: 2004-08-10 - Assignees: E.I. du Pont de Nemours and Company - Inventors: Quinn Qun Zhu; Dana M. Walters Pollak - Web page: https://patentgrep.com/patent/US7465564B2 ## CPC - C12P 7/6472: containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone - C12N 9/001: {acting on the CH-CH group of donors (1.3)} - C12P 7/6427: Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to methods for the production of γ-linolenic acid (GLA) in oleaginous yeast. Thus, Δ12 and Δ6 desaturases able to catalyze the conversion of oleic acid to GLA have been introduced into the genome of Yarrowia, using zeta-directed integration. Transformed strains produced over 25% GLA in the total lipids, as opposed to wildtype Yarrowia that is unable to synthesize any GLA. Metabolic engineering and fermentation methods are provided to further enhance GLA productivity in oleaginous yeast. ## Claims ### Claim 1 A method for producing γ-linolenic acid comprising: a) providing an oleaginous Yarrowia sp. comprising: i) the nucleic acid sequence of SEQ ID NO:9 encoding a peptide having Δ6 desaturase activity; ii) the nucleic acid sequence of SEQ ID NO:39 encoding a peptide having Δ12 desaturase activity; and iii) a source of desaturase substrate selected from the group consisting of oleic acid and linoleic acid; b) growing the yeast of step (a) in the presence of a fermentable carbon source whereby at least 15% γ-linolenic acid in the total lipids is produced; and c) optionally, recovering the γ-linolenic acid. ### Claim 2 (depends on 1) A method according to claim 1 wherein the at least one genetic construct encoding the polypeptide having Δ6 desaturase activity is in multicopy in the oleaginous yeast. ### Claim 3 (depends on 1) A method according to claim 1 wherein the at least one genetic construct encoding the polypeptide having Δ12 desaturase activity is in multicopy in the oleaginous yeast. ### Claim 4 (depends on 1) A method according to claim 1 wherein the at least one genetic construct encoding the polypeptide having Δ12 desaturase activity is integrated into at least one genetic loci selected from the group consisting of the zeta loci, the Ura3 loci, the Leu 2 loci, the Lys5 loci, the Aco2 loci, the Pox3 loci, the Lip1 loci and the Lip2 loci. ### Claim 5 (depends on 1) A method according to claim 1 wherein the at least one genetic construct encoding the polypeptide having Δ6 desaturase activity is integrated into at least one genetic loci selected from the group consisting of the zeta loci, the Ura3 loci, the Leu 2 loci, the Lys5 loci, the Aco2 loci, the Pox3 loci, the Lip1 loci and the Lip2 loci. ### Claim 6 (depends on 1) A method according to claim 1 wherein the at least one genetic construct encoding the polypeptide having Δ6 desaturase activity is under the control of a strong promoter isolated from genes selected from the group consisting of: alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase, phosphoglycerate mutase, fructose-bisphosphate aldolase, glycerol-3-phosphate o-acyltransferase, phosphoglucose isomerase, phosphoglycerate kinase, acid phosphatase, lactase, metallothionein and glucoamylase. ### Claim 7 (depends on 1) A method according to claim 1 wherein the at least one genetic construct encoding the polypeptide having Δ12 desaturase activity is under the control of a strong promoter isolated from genes selected from the group consisting of: alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase, phosphoglycerate mutase, fructose-bisphosphate aldolase, glycerol-3-phosphate o-acyltransferase, phosphoglucose isomerase, phosphoglycerate kinase, acid phosphatase, lactase, metallothionein and glucoamylase. ### Claim 8 (depends on 1) A method according to claim 1 wherein at least about 20% γ-linolenic acid in the total lipids is produced. ### Claim 9 (depends on 1) A method according to claim 1 wherein the source of desaturase substrate is endogenous to the oleaginous yeast. ### Claim 10 A production host cell for the production of γ-linolenic acid comprising: a) a background Yarrowia sp. comprising: i) the nucleic acid sequence of SEQ ID NO:9 encoding a peptide having Δ6 desaturase activity; ii) the nucleic acid sequence of SEQ ID NO:39 encoding a peptide having Δ12 desaturase activity, wherein at least one of said genetic constructs of (i) or (ii) is over-expressed and whereby at least 15% γ-linolenic acid in the total lipids is produced. ### Claim 11 (depends on 10) A production host cell according to claim 10 wherein the background Yarrowia sp. is devoid of any native gene encoding a polypeptide having Δ12 desaturase activity. ### Claim 12 (depends on 10) A production host cell according to claim 10 wherein at least one of said genetic constructs is integrated into the Yarrowia sp. genome at a gene locus selected from the group consisting of: zeta, Ura3, Leu2, Lys5, Aco2, Pox3, Lip1 and the Lip2 gene locus.