# US 12,760,339 B2 · Production of odd chain fatty acid derivatives in recombinant microbial cells

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- Number: US 12,760,339 B2
- Document ID: US12760339B2
- Status: Granted
- Kind: B2
- Application number: 17/169,845
- Filed: 2021-02-08
- Published: 2021-10-21
- Granted: 2026-10-13
- Earliest claimed: 2011-09-14
- Assignees: Genomatica, Inc.
- Applicants: Genomatica, Inc.
- Inventors: Grace J. Lee; John Haliburton; Zhihao Hu; Andreas W. Schirmer; Kevin Holden
- Web page: https://patentgrep.com/patent/US12760339B2

## CPC

- C12P 7/6409: Fatty acids
- C07C 53/126: Acids containing more than four carbon atoms
- C07C 57/03: Monocarboxylic acids
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 9/1018: {Carboxy- and carbamoyl transferases (2.1.3)}
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 9/1217: {Phosphotransferases with a carboxyl group as acceptor (2.7.2)}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/18: Carboxylic ester hydrolases {(3.1.1)}
- C12N 9/88: Lyases (4.)
- C12N 9/90: Isomerases (5.)
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1)
- C12Y 101/01003: Homoserine dehydrogenase (1.1.1.3)
- C12Y 101/01085: 3-Isopropylmalate dehydrogenase (1.1.1.85)
- C12Y 102/0105: Long-chain-fatty-acyl-CoA reductase (1.2.1.50)
- C12Y 102/0108: Long-chain acyl-[acyl-carrier-protein] reductase (1.2.1.80)
- C12Y 201/03001: Methylmalonyl-CoA carboxytransferase (2.1.3.1)
- C12Y 203/01041: Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41)
- C12Y 203/01182: (R)-Citramalate synthase (2.3.1.182)
- C12Y 207/01039: Homoserine kinase (2.7.1.39)
- C12Y 207/02004: Aspartate kinase (2.7.2.4)
- C12Y 301/01067: Fatty-acyl-ethyl-ester synthase (3.1.1.67)
- C12Y 301/02001: Acetyl-CoA hydrolase (3.1.2.1)
- C12Y 401/01041: Methylmalonyl-CoA decarboxylase (4.1.1.41)
- C12Y 401/03022: Citramalate lyase (4.1.3.22)
- C12Y 402/01033: 3-Isopropylmalate dehydratase (4.2.1.33)
- C12Y 402/03001: Threonine synthase (4.2.3.1)
- C12Y 403/01019: Threonine ammonia-lyase (4.3.1.19)
- C12Y 504/99002: Methylmalonyl-CoA mutase (5.4.99.2)
- 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)}
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Recombinant microbial cells are provided which have been engineered to produce fatty acid derivatives having linear chains containing an odd number of carbon atoms by the fatty acid biosynthetic pathway. Also provided are methods of making odd chain fatty acid derivatives using the recombinant microbial cells, and compositions comprising odd chain fatty acid derivatives produced by such methods.

## Claims

### Claim 1

A recombinant microbial cell comprising:
(a) polynucleotides encoding polypeptides having enzymatic activity effective to produce an increased amount of propionyl-CoA in the recombinant microbial cell relative to the amount of propionyl-CoA produced in a parental microbial cell lacking or having a reduced amount of said enzymatic activity, wherein the polynucleotides encode
(i) one or more of a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 40, 41, 42, or 43, wherein the polypeptide has (R)-citramalate synthase activity; a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 44 or 46 and 45 or 47, wherein the polypeptide has isopropylmalate isomerase activity; and a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 48, 49, or 50, wherein the polypeptide has beta-isopropylmalate dehydrogenase activity, and/or
(ii) one or more of a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 20, 21, 22, 23, or 24, wherein the polypeptide has aspartokinase activity; a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 20, 21, 25, 26, or 27, wherein the polypeptide has homoserine dehydrogenase activity; a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 28, 29, 30, or 31, wherein the polypeptide has homoserine kinase activity; a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 32, 33, or 34, wherein the polypeptide has threonine synthase activity; and a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 35, 36, 37, 38, or 39, wherein the polypeptide has threonine deaminase activity,
(b) a polynucleotide encoding a polypeptide having β-ketoacyl-ACP synthase activity that utilizes propionyl-CoA as a substrate, wherein the polypeptide having β-ketoacyl-ACP synthase activity comprises one or more sequence motif selected from SEQ ID NO: 14, 15, 16, 17, 18, or 19, and
(c) a polynucleotide encoding a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 64, 65, 66, 67, 68, 69, 70, 71, or 72, wherein the polypeptide has thioesterase enzyme activity,
wherein the recombinant microbial cell produces a fatty acid derivative composition comprising odd chain and even chain fatty acid derivatives when cultured in the presence of a carbon source under conditions effective to express the polynucleotides according to (a), (b), and (c), and
wherein at least 10% of the fatty acid derivatives in the fatty acid derivative composition are odd chain fatty acid derivatives.

### Claim 2 (depends on 1)

The recombinant microbial cell of claim 1, wherein at least 20% of the fatty acid derivatives in the fatty acid derivative composition are odd chain fatty acid derivatives.

### Claim 3 (depends on 1)

The recombinant microbial cell of claim 1, wherein the cell produces at least 100 mg/L of odd chain fatty acid derivatives.

### Claim 4 (depends on 1)

The recombinant microbial cell of claim 1, wherein expression of the at least one polynucleotide according to (a) is modulated by overexpression of the polynucleotide in the recombinant microbial cell.

### Claim 5 (depends on 1)

The recombinant microbial cell of claim 1, wherein the polypeptide having β-ketoacyl-ACP synthase activity that utilizes propionyl-CoA as a substrate is exogenous to the recombinant microbial cell, and expression of a polypeptide having beta-ketoacyl-ACP synthase activity endogenous to the recombinant microbial cell is attenuated.

### Claim 6 (depends on 1)

The recombinant microbial cell of claim 1, wherein the fatty acid derivative enzyme activity comprises thioesterase activity and the recombinant microbial cell produces a fatty acid composition comprising odd chain fatty acids, wherein at least 10% of the fatty acids in the composition are odd chain fatty acids.

### Claim 7 (depends on 1)

The recombinant microbial cell of claim 1, wherein the fatty acid derivative enzyme activity comprises ester synthase activity and the recombinant microbial cell produces a fatty ester composition comprising odd chain fatty esters, wherein at least 10% of the fatty esters in the composition are odd chain fatty esters.

### Claim 8 (depends on 1)

The recombinant microbial cell of claim 1, wherein the fatty acid derivative enzyme activity comprises fatty aldehyde biosynthesis activity and the recombinant microbial cell produces a fatty aldehyde composition comprising odd chain fatty aldehydes, wherein at least 10% of the fatty aldehydes in the composition are odd chain fatty aldehydes.

### Claim 9 (depends on 1)

The recombinant microbial cell of claim 1, wherein the fatty acid derivative enzyme activity comprises fatty alcohol biosynthesis activity and the recombinant microbial cell produces a fatty alcohol composition comprising odd chain fatty alcohols, wherein at least 10% of the fatty alcohols in the composition are odd chain fatty alcohols.

### Claim 10 (depends on 1)

The recombinant microbial cell of claim 1, wherein the fatty acid derivative enzyme activity comprises hydrocarbon biosynthesis activity and the recombinant microbial cell produces a hydrocarbon composition comprising even chain hydrocarbons, wherein at least 10% of the hydrocarbons in the composition are even chain hydrocarbons.

### Claim 11 (depends on 1)

A cell culture comprising the recombinant microbial cell of claim 1.

### Claim 12 (depends on 1)

A method of making a fatty acid derivative composition comprising odd chain fatty acid derivatives, the method comprising:
culturing the recombinant microbial cell of claim 1 in a culture medium containing a carbon source under conditions effective to express the polynucleotides according to (a), (b), and (c) and to produce a fatty acid derivative composition comprising odd chain fatty acid derivatives, wherein at least 10% of the fatty acid derivatives in the composition are odd chain fatty acid derivatives, and
optionally recovering the odd chain fatty acid derivative composition from the culture medium.

### Claim 13 (depends on 12)

The method of claim 12, wherein the recombinant microbial cell further expresses one or more polynucleotides encoding:
(1) a polypeptide having thioesterase activity;
(2) a polypeptide having decarboxylase activity;
(3) a polypeptide having carboxylic acid reductase activity;
(4) a polypeptide having alcohol dehydrogenase activity;
(5) a polypeptide having aldehyde decarbonylase activity;
(6) a polypeptide having acyl-CoA reductase activity;
(7) a polypeptide having acyl-ACP reductase activity;
(8) a polypeptide having ester synthase activity;
(9) a polypeptide having OleA activity; or
(10) a polypeptide having OleCD or OleBCD activity;
wherein the recombinant microbial cell produces one or more of odd chain fatty acids, odd chain fatty esters, odd chain fatty aldehydes, odd chain fatty alcohols, even chain alkanes, even chain alkenes, even chain terminal olefins, even chain internal olefins, or even chain ketones.

### Claim 14 (depends on 1)

A method of making the recombinant microbial cell of claim 1, which produces a higher titer or higher proportion of odd chain fatty acid derivatives than produced by a parental microbial cell, the method comprising:
obtaining a parental microbial cell comprising a polynucleotide encoding a polypeptide having β-ketoacyl-ACP synthase activity that utilizes propionyl-CoA as a substrate and a polynucleotide encoding a polypeptide having fatty acid derivative enzyme activity, and
engineering the parental microbial cell to obtain the recombinant microbial cell of claim 1, which produces or is capable of producing a greater amount of propionyl-CoA than the amount of propionyl-CoA produced by the parental microbial cell when cultured under the same conditions, wherein the step of engineering the parental microbial cell comprises:
engineering the parental microbial cell to express polynucleotides encoding:
(a) polypeptides:
(i) having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity, and threonine deaminase activity;
and/or
(ii) polypeptides having (R)-citramalate synthase activity, isopropylmalate isomerase activity, and beta-isopropylmalate dehydrogenase activity; and
(b) a polypeptide having β-ketoacyl-ACP synthase activity that utilizes propionyl-CoA as a substrate; and
(c) a polypeptide having fatty acid derivative enzyme activity;
wherein the recombinant microbial cell of claim 1 produces a higher titer or higher proportion of odd chain fatty acid derivatives when cultured in the presence of a carbon source under conditions effective to express the polynucleotides, relative to the titer or proportion of odd chain fatty acid derivatives produced by the parental microbial cell cultured under the same conditions.

### Claim 15 (depends on 14)

The method of claim 14, wherein the recombinant microbial cell is engineered to express an exogenous polynucleotide encoding a polypeptide having β-ketoacyl-ACP synthase activity that utilizes propionyl-CoA as a substrate, and expression of an endogenous polynucleotide encoding a polypeptide having β-ketoacyl-ACP synthase activity is attenuated.

### Claim 16 (depends on 1)

A method of increasing the titer or the proportion of odd chain fatty acid derivatives produced by the microbial cell of claim 1, the method comprising:
obtaining a parental microbial cell which produces fatty acid derivatives, and engineering the parental microbial cell to obtain the recombinant microbial cell of claim 1, which produces or is capable of producing a greater amount of propionyl-CoA than the amount of propionyl-CoA produced by the parental microbial cell when cultured under the same conditions, wherein the step of engineering the parental microbial cell comprises:
engineering the parental microbial cell to express polynucleotides encoding:
(a) polypeptides:
(i) having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity, and threonine deaminase activity;
and/or
(ii) polypeptides having (R)-citramalate synthase activity, isopropylmalate isomerase activity, and beta-isopropylmalate dehydrogenase activity; and
(b) a polypeptide having β-ketoacyl-ACP synthase activity that utilizes propionyl-CoA as a substrate; and
(c) a polypeptide having fatty acid derivative enzyme activity;
wherein the recombinant microbial cell of claim 1 produces a higher titer or higher proportion of odd chain fatty acid derivatives when cultured in the presence of a carbon source under conditions effective to produce propionyl-CoA and fatty acid derivatives in the recombinant microbial cell, relative to the titer or proportion of odd chain fatty acid derivatives produced by the parental microbial cell cultured under the same conditions.

### Claim 17 (depends on 12)

A fatty acid derivative composition produced by the method of claim 12.

### Claim 18 (depends on 1)

The recombinant microbial cell of claim 1, further comprising polynucleotides according to (a) encoding:
(iii) one or more of a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, or 58, wherein the polypeptide has methylmalonyl-CoA mutase activity; a polypeptide comprising at least 85% sequence identity to SEQ ID NOs: 59, 60 or 61, wherein the polypeptide has methylmalonyl-CoA decarboxylase activity a polypeptide comprising at least 85% sequence identity to SEQ ID NO: 62, wherein the polypeptide has methylmalonyl-CoA carboxyltransferase activity; and a polypeptide comprising at least 85% sequence identity to SEQ ID NO: 63, wherein the polypeptide has methylmalonyl-CoA epimerase activity.

### Claim 19 (depends on 1)

The recombinant microbial cell of claim 1, comprising one or more polypeptides according to (i) and one or more polypeptides according to (ii).

### Claim 20 (depends on 1)

The recombinant microbial cell of claim 1, wherein the recombinant microbial cell expresses or overexpresses one or more polypeptides having fatty acid derivative enzyme activity selected from:
(1) a polypeptide having thioesterase activity;
(2) a polypeptide having decarboxylase activity;
(3) a polypeptide having carboxylic acid reductase activity;
(4) a polypeptide having alcohol dehydrogenase activity;
(5) a polypeptide having aldehyde decarbonylase activity;
(6) a polypeptide having acyl-CoA reductase activity;
(7) a polypeptide having acyl-ACP reductase activity;
(8) a polypeptide having ester synthase activity;
(9) a polypeptide having OleA activity; and
(10) a polypeptide having OleCD or OleBCD activity;
wherein the recombinant microbial cell produces a composition comprising odd chain fatty acids, odd chain fatty esters, odd chain wax esters, odd chain fatty aldehydes, odd chain fatty alcohols, even chain alkanes, even chain alkenes, even chain internal olefins, even chain terminal olefins, or even chain ketones.