# US 10,119,145 B2 · Engineered organisms for production of novel lipids

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- Number: US 10,119,145 B2
- Document ID: US10119145B2
- Status: Granted
- Kind: B2
- Application number: 14/777,345
- Filed: 2014-03-14
- Published: 2016-02-25
- Granted: 2018-11-06
- Earliest claimed: 2013-03-15
- Assignees: Consejo Nacional De Investigaciones Cientificas Y Tecnicas (Conicet)
- Applicants: Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET); Inis Biotech LLC
- Inventors: Hugo Gramajo; Ana Arabolaza; Santiago Comba; Simón Menendez-Bravo; Héctor Álvarez
- Web page: https://patentgrep.com/patent/US10119145B2

## CPC

- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 9/93: {Ligases (6)}
- C12P 7/6409: Fatty acids
- C12P 7/6436: Fatty acid esters
- Y02E 50/30: Fuel from waste, e.g. synthetic alcohol or diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present disclosure provides engineered microorganisms, engineered biosynthetic pathways, methods of producing lipid compounds using genetically engineered microorganisms, and the products synthesized by those organisms. In particular, the disclosure provides genetically engineered microorganisms for the production of multi-methyl branched fatty acids (MMBFAs) and MMBFA esters (wax esters) derived from these fatty acids. In addition, the disclosure provides methods for producing acylglycerols with one of more of their acyl substituents being an MMBFA, and methods for producing alcohols derived from MMBFAs (fatty alcohols).

## Claims

### Claim 1

An engineered microorganism comprising a multi-methyl branched fatty acid (MMBFA) ester synthesis pathway comprising:
(i) an exogenous or overexpressed mycocerosic acid synthase (MAS) or active fragment thereof comprising an ACP domain;
(ii) an exogenous or overexpressed long chain fatty acid AMP ligase; and
(iii) an exogenous or overexpressed ester synthase,
wherein the MAS catalyzes the iterative conversion of methyl-malonyl-CoA to MMBFA, and the ester synthase catalyzes the transesterification of MMBFA covalently bound to the ACP domain of MAS with an acceptor alcohol to produce an MMBFA ester,
wherein said engineered microorganism further comprises:
(iv) an exogenous or overexpressed propionyl-CoA carboxylase (PCC) complex comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 81, an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 83, and an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 84; and/or
(v) an exogenous or overexpressed acyl-acyl carrier protein (ACP) thioesterase comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 89.

### Claim 2 (depends on 1)

The engineered microorganism according to claim 1, wherein the MAS or active fragment thereof comprises an acyl transferase (AT) domain, an acyl carrier protein (ACP) domain, and a ketide synthase (KS) domain.

### Claim 3 (depends on 1)

The engineered microorganism according to claim 1, wherein the MAS or active fragment thereof comprises SEQ ID NO: 3, or a homolog, fragment, or variant thereof.

### Claim 4 (depends on 1)

The engineered microorganism according to claim 1, wherein the long chain fatty acid AMP ligase is a FADD28.

### Claim 5 (depends on 4)

The engineered microorganism according to claim 4, wherein the FADD28 comprises SEQ ID NO:72, or a homolog, active fragment, or variant thereof.

### Claim 6 (depends on 1)

The engineered microorganism according to claim 1, wherein the ester synthase is a PAPA5.

### Claim 7 (depends on 6)

The engineered microorganism according to claim 6, wherein the PAPA5 ester synthase comprises SEQ ID NO:76, or a homolog, active fragment, or variant thereof.

### Claim 8 (depends on 1)

The engineered microorganism according to claim 1, wherein the MMBFA synthesis pathway further comprises the following conversions:
(vi) propionate to propionyl-CoA, wherein the enzyme that catalyzes the conversion is a propionyl-CoA synthetase comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:80;
(vii) transfer of 4′-phosphopantetheine (4′-PP) moiety from CoA to ACP, wherein the enzyme that catalyzes conversion is an exogenous or overexpressed phosphopantetheinyl transferase comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:90; or
(viii) a combination thereof.

### Claim 9 (depends on 1)

The engineered microorganism according to claim 1 comprising:
(i) a MAS from Mycobacterium tuberculosis (SEQ ID NO:3);
(ii) a FADD28 long chain fatty acid AMP ligase from Mycobacterium tuberculosis (SEQ ID NO:72);
(iii) a TESA′ thioesterase from Escherichia coli (SEQ ID NO:89);
(iv) a propionyl-CoA carboxylase (PCC) complex comprising the ACCA1 (SEQ ID NO:81), PCCB (SEQ ID NO:83), and PCCE (SEQ ID NO:84) subunits from PCC from Streptomyces coelicolor; and,
(v) a PAPA5 ester synthase from Mycobacterium tuberculosis (SEQ ID NO:76),
wherein the MAS catalyzes the iterative conversion of methyl-malonyl-CoA to MMBFA, and
wherein the ester synthase catalyzes the transesterification of an MMBFA with an acceptor alcohol to produce an MMBFA ester.

### Claim 10 (depends on 1)

The engineered microorganism according to claim 1, wherein said microorganism is selected from the group consisting of bacteria, fungi, yeast, and algae.

### Claim 11 (depends on 1)

The engineered microorganism according to claim 1, wherein the MMBFA comprises
(i) a fatty acid chain length of about 10 to about 40 carbons, and at least one branching at an even position carbon;
(ii) one to four methyl branches located at carbons 2, 4, 6, or 8 of the fatty acid chain; or,
(iii) a combination thereof.

### Claim 12 (depends on 1)

The engineered microorganism according to claim 1, wherein the acceptor alcohol is ethanol, 1-butanol, 1-pentanol, 2-propanol, 2-butanol, 1-octanol, (+/−) 2-octanol, (R)-(−)-2-octanol, 1-nonanol, 1-decanol, (+/−) 2-undecanol, myristyl alcohol (1-tetradecanol), palmityl alcohol (1-hexadecanol), stearyl alcohol (1-octadecanol), 1-eicosanol, trans-2-octene-1-ol, DL-3-OH-methyl palmitate, (S)-(−)-1,2-decanediol, (R)-1,2-decanediol, (+/−) 1,2-octanediol, trimethylpropane, pentaerythrite, neopentylclycol, or a combination thereof.

### Claim 13 (depends on 1)

The engineered microorganism according to claim 1, wherein the MMBFA ester product has an ester chain length of about 2 to about 24 carbons.

### Claim 14 (depends on 1)

The engineered microorganism according to claim 1, wherein the MMBFA ester product comprises (i) octyl 2,4,6,8-tetramethyltetracosanoate; (ii) (Z)-octyl 2,4,6,8-tetramethylhexacos-17-enoate; (iii) (Z)-octyl 2,4,6,8-tetramethyltetracos-17-enoate; (iv) octyl 2,4,6,8-tetramethyltriacontonoate; (v) ethyl 2,4,6,8-tetramethyltetracosanoate; (vi) (Z)-ethyl 2,4,6,8-tetramethylhexacos-17-enoate; (vii) (Z)-ethyl 2,4,6,8-tetramethyltetracos-17-enoate; (viii) ethyl 2,4,6,8-tetramethyltriacontanoate; (ix) pentyl 2,4,6,8-tetramethyltetracosanoate; (x) (Z)-pentyl 2,4,6,8-tetramethylhexacos-17-enoate; (xi) (Z)-pentyl 2,4,6,8-tetramethyltetracos-17-enoate; (xii) pentyl 2,4,6,8-tetramethyltriacontonoate; (xiii) butyl 2,4,6,8-tetramethyltetracosanoate; (xiv) (Z)-butyl 2,4,6,8-tetramethylhexacos-17-enoate; (xv) (Z)-butyl 2,4,6,8-tetramethyltetracos-17-enoate; (xvi) butyl 2,4,6,8-tetramethyltriacontonoate; (xvii) and combinations thereof.

### Claim 15 (depends on 1)

The engineered microorganism according to claim 1, wherein at least one gene encoding one of the exogenous or overexpressed enzymes has been subjected to molecular evolution or mutated to enhance the enzymatic activity of the enzyme.