# US 10,370,686 B2 · Yeast cell modified to overproduce fatty acid and fatty acid-derived compounds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,686 B2 - Document ID: US10370686B2 - Status: Granted - Kind: B2 - Application number: 15/005,719 - Filed: 2016-01-25 - Published: 2016-07-28 - Granted: 2019-08-06 - Earliest claimed: 2013-07-25 - Assignees: The Regents of the University of California - Applicants: The Regents of the University of California - Inventors: Weerawat Runguphan; Jay D. Keasling - Web page: https://patentgrep.com/patent/US10370686B2 ## CPC - C12P 7/6409: Fatty acids - C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)} - C12N 9/93: {Ligases (6)} - C12N 15/81: for yeasts - C12P 7/04: acyclic - C12P 7/6458: by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis - C12P 7/6463: obtained from glyceride producing microorganisms, e.g. single cell oil - C12P 7/649: Biodiesel, i.e. fatty acid alkyl esters - C12Y 203/01085: Fatty-acid synthase (2.3.1.85) - C12Y 604/01002: Acetyl-CoA carboxylase (6.4.1.2) - Y02E 50/10: Biofuels, e.g. bio-diesel - 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) - C12Y: ENZYMES - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract The present invention provides for a genetically modified yeast host cell capable of producing one or more fatty acids, or fatty acid-derived compounds, or a mixture thereof, comprising: (a) increased expression of acetyl-CoA carboxylase (such as ACC1), (b) increased expression of one or more fatty acid synthases (such as FAS1 and FAS2), and (c) optionally reduced expression of one or more enzymes involved in or in the β-oxidation pathway (such as peroxisomal transporters PXA1 and PXA2, and β-oxidation enzymes POX1, POX2, and POX3). ## Claims ### Claim 1 A genetically modified Saccharomyces cerevisiae host cell capable of producing one or more fatty acids, or fatty acid-derived compounds, or a mixture thereof, comprising: (a) increased expression of acetyl-CoA carboxylase, wherein the endogenous promoter of the ACC1 gene is replaced with promoter PTEF1; (b) increased expression of one or more fatty acid synthases, wherein the endogenous promoter of the FAS1 and FAS2 genes are replaced with promoter PTEF1; (c) optionally reduced expression of one or more enzymes in the β-oxidation pathway, wherein the endogenous POX1 and/or PXA2 genes are deleted; (d) a plasmid encoding an Escherichia coli TesA, which lacks a membrane signal peptide at a N-terminal end of TesA, operatively linked to promoter PGAL10; and (e) reduced expression of fatty-acyl-CoA synthetases, wherein the endogenous FAA1 and/or FAA4 genes are deleted; wherein the host cell does not comprise the ΔSnf2 mutation and the host cell is capable of producing at least 162 mg of free fatty acids when cultured in one liter of culture medium. ### Claim 2 (depends on 1) The host cell of claim 1, wherein the one or more enzymes involved in or in the β-oxidation pathway are peroxisomal transporters PXA1 and PXA2, and β-oxidation enzymes PDX1, PDX2, and PDX3. ### Claim 3 (depends on 1) The host cell of claim 1, wherein the fatty acid-derived compound is a fatty acyl ethyl ester (FAEE), a fatty alcohol, a triacylglycerol (TAG), or a mixture thereof. ### Claim 4 (depends on 1) The host cell of claim 1, wherein the fatty acid is a C12:0, C14:0, C16:0, C16:1, C18:0, or C18:1, or mixture thereof, and the fatty acid-derived compound is a fatty acid methyl and ethyl esters (FAEE), fatty alcohol, or triacylglycerol (TAG) derived from one or more the following fatty acids: C12:0, C14:0, C16:0, C16:1, C18:0, and C18:1. ### Claim 5 (depends on 1) The host cell of claim 1, wherein the host cell is capable of producing at least 201 mg of free fatty acids when cultured in one liter of culture medium. ### Claim 6 (depends on 5) The host cell of claim 5, wherein the host cell is capable of producing at least 400 mg of free fatty acids when cultured in one liter of culture medium. ### Claim 7 (depends on 1) The host cell of claim 1, wherein the host cell comprises the following genotype: Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0. ### Claim 8 A method of constructing a genetically modified Saccharomyces cerevisiae host cell of the claimed invention, comprising: (a) replacing the endogenous promoter of the ACC1 gene with promoter PTEF1 and the endogenous promoter of the FAS1 and FAS2 genes with promoter PTEF1; (b) optionally the deleting the endogenous POX1 and/or PXA2 genes; (c) introducing a plasmid encoding an Escherichia coli TesA, which lacks a membrane signal peptide at a N-terminal end of TesA, operatively linked to promoter PGAL10 into the host cell; (d) deleting the endogenous FAA1 and/or FAA4 genes; and (e) culturing the host cell in a cell culture such that the cell culture produces at least 162 mg of free fatty acids per one liter of culture medium; wherein the host cell does not comprise the ΔSnf2 mutation. ### Claim 9 (depends on 8) The method of claim 8, wherein the host cell comprises the following genotype: Matα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0. ### Claim 10 (depends on 1) A method of producing one or more fatty acids, or fatty acid-derived compounds, or a mixture thereof, in a genetically modified yeast host cell of claim 1, comprising culturing the genetically modified yeast host cell of claim 1 under a suitable condition such that the culturing results in the genetically modified yeast host cell producing the one or more fatty acids, or fatty acid-derived compounds, or a mixture thereof. ### Claim 11 (depends on 10) The method of claim 10, further comprising: recovering the one or more fatty acids, or fatty acid-derived compounds, or a mixture thereof, and optionally chemically treating the recovered compound to produce a second compound, wherein the second compound when combusted, can produce more energy that the combustion of the recovered compound.