# US 2013/0157324 A1 · In situ expression of lipase for enzymatic production of alcohol esters during fermentation

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- Number: US 2013/0157324 A1
- Document ID: US20130157324A1
- Status: Published application
- Kind: A1
- Application number: 13/525,075
- Filed: 2012-06-15
- Published: 2013-06-20
- Earliest claimed: 2011-03-23
- Assignees: Butamax(Tm) Advanced Biofuels LLC
- Inventors: Robert DICOSIMO; Arthur Leo Kruckeberg; Thomas Edwin Van Aken
- Web page: https://patentgrep.com/patent/US20130157324A1

## CPC

- C12N 15/81: for yeasts
- A23K 10/10: obtained by microbiological or biochemical processes (using chemicals or microorganisms for ensilaging of green fodder A23K30/15)
- A23K 10/18: of live microorganisms
- C12N 9/20: Triglyceride splitting, e.g. by means of lipase
- C12P 7/06: Ethanol, i.e. non-beverage
- C12P 7/16: Butanols
- C12P 7/62: Carboxylic acid esters
- C12P 7/6436: Fatty acid esters
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23K: FODDER
- 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

Disclosed herein are methods of producing alcohol esters during a fermentation by providing alcohol-producing microorganisms which further comprise an engineered polynucleotide encoding a polypeptide having lipase activity.

## Claims

### Claim 1

A method comprising:
a) providing a fermentation medium comprising fermentable carbon substrate derived from a biomass feedstock, alcohol produced from a fermentable carbon substrate derived from a biomass feedstock, and an alcohol-producing yeast microorganism wherein the alcohol-producing microorganism comprises an engineered polynucleotide encoding a polypeptide having lipase activity and the microorganism expresses and displays or secretes said polypeptide such that the lipase activity is present in the fermentation medium;
b) contacting the fermentation medium with a carboxylic acid
wherein the lipase activity is present in the fermentation medium in sufficient amount to convert at least a portion of the alcohol produced by the microorganism to alcohol esters extracellularly

### Claim 2 (depends on 1)

The method of claim 1 further comprising contacting the fermentation medium with an extractant to form a two-phase mixture comprising an aqueous phase and an organic phase.

### Claim 3 (depends on 2)

The method of claim 2 wherein the extractant comprises the carboxylic acid.

### Claim 4 (depends on 1)

The method of claim 1 wherein the product alcohol is a C2 to C8 alkyl alcohol.

### Claim 5 (depends on 1)

The method of claim 1 wherein the product alcohol is ethanol.

### Claim 6 (depends on 5)

The method of claim 5 wherein the alcohol esters comprise fatty acid ethyl esters.

### Claim 7 (depends on 1)

The method of claim 1 wherein the product alcohol is butanol.

### Claim 8 (depends on 7)

The method of claim 7 wherein the alcohol esters comprise fatty acid butyl esters.

### Claim 9

(canceled)

### Claim 10

(canceled)

### Claim 11 (depends on 1)

The method of claim 1 wherein the polypeptide having lipase activity comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 249, 250, 251, 252, 253 or a fragment thereof.

### Claim 12 (depends on 1)

The method of claim 1 wherein the polynucleotide encoding a polypeptide having lipase activity comprises a sequence with at least about 70% identity to a polynucleotide having SEQ ID NO: 1, 3, 5, 7, 8, 9, 46, 48, 50, 52, 54, 255, 271 or 273.

### Claim 13 (depends on 1)

The method of claim 1 wherein the polypeptide having lipase activity comprises a sequence with at least about 70% identity to a polypeptide having SEQ ID NO: 2, 4, 6, 256, 47, 49, 51, 53, 55, 241, 242, 243, 244, 245, 246, 247, 248, 272, or 274 or an active fragment thereof.

### Claim 14 (depends on 13)

The method of claim 13 wherein the polypeptide having lipase activity does not contain a glycosylation motif.

### Claim 15 (depends on 1)

The method of claim 1 wherein the polypeptide having lipase activity is not glycosylated.

### Claim 16 (depends on 1)

The method of claim 1 wherein the carboxylic acid comprises free fatty acids derived from corn oil, canola oil, palm oil, linseed oil, jatropha oil, or soybean oil.

### Claim 17 (depends on 1)

The method, of claim 1 wherein the carboxylic acid is derived from the same biomass feedstock as the fermentable carbon substrate.

### Claim 18 (depends on 1)

The method of claim 1 wherein the carboxylic acid comprises carboxylic acids having C12 to C22 linear or branched aliphatic chains.

### Claim 19 (depends on 3)

The method of claim 3 wherein the contacting with extractant and the contacting with carboxylic acid occur contemporaneously.

### Claim 20 (depends on 1)

The method of claim 1 wherein at least about 60% of the effective titer of alcohol produced by the microorganism is converted to alcohol esters.

### Claim 21 (depends on 1)

The method of claim 1 wherein the fermentation medium further comprises triglycerides, diglycerides, monoglycerides, and phospholipids, or combinations thereof and wherein the lipase activity hydrolyzes at least a portion of the triglycerides, diglycerides, monoglycerides, and phospholipids, or combinations thereof to form free fatty acids.

### Claim 22 (depends on 1)

The method of claim 1 wherein the effective titer or the effective rate of alcohol produced during a fermentation is greater than that produced during a fermentation by an alcohol-producing microorganism that does not comprise a polynucleotide encoding a polypeptide having lipase activity and the microorganism expresses and secretes or displays said polypeptide such that the lipase activity is present in the fermentation medium.

### Claim 23

(canceled)

### Claim 24

A recombinant host cell comprising:
(a) an engineered alcohol production pathway; and
(b) an engineered polynucleotide encoding a polypeptide having lipase activity.

### Claim 25 (depends on 24)

The recombinant host cell of claim 24 wherein the polypeptide having lipase activity comprises a sequence having at least about 70% identity to SEQ ID NO: 2, 4, 6, 256, 47, 49, 51, 53, 55, 241, 242, 243, 244, 245, 246, 247, 248, 272, or 274 or an active fragment thereof.

### Claim 26 (depends on 24)

The recombinant host cell of claim 24 wherein the polypeptide having lipase activity comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 249, 250, 251, 252, 253 or a fragment thereof.

### Claim 27 (depends on 26)

The recombinant host cell of claim 26 wherein the polypeptide having lipase activity does not contain a glycosylation motif.

### Claim 28 (depends on 24)

The recombinant host cell of claim 24 wherein the polypeptide having lipase activity is not glycosylated.

### Claim 29 (depends on 24)

The recombinant host cell of claim 24 wherein the engineered polynucleotide encoding a polypeptide having lipase activity comprises a sequence having at least about 70% identity to SEQ ID NO: 1, 3, 5, 7, 8, 9, 46, 48, 50, 52, 54, 255, 271 or 273.

### Claim 30

A recombinant host cell comprising:
(a) an alcohol production pathway; and
(b) an engineered polynucleotide encoding a polypeptide having lipase activity wherein the polypeptide having lipase activity comprises a sequence having at least about 70% identity to SEQ ID NO: 2, 4, 6, 256, 47, 49, 51, 53, 55, 241, 242, 243, 244, 245, 246, 247, 248, 272, or 274 or an active fragment thereof.

### Claim 31 (depends on 30)

The recombinant host cell of claim 30 wherein the polypeptide having lipase activity further comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 249, 250, 251, 252, 253 or a fragment thereof.

### Claim 32 (depends on 24)

The recombinant host cell of claim 24 wherein the alcohol production pathway is a butanol production pathway.

### Claim 33 (depends on 32)

The recombinant host cell of claim 32 wherein the butanol production pathway is an isobutanol production pathway

### Claim 34 (depends on 24)

The recombinant host cell of claim 24 wherein the host cell further comprises reduced or eliminated pyruvate decarboxylase activity.

### Claim 35

A method of increasing tolerance of an alcohol-producing microorganism to the produced alcohol, the method comprising:
(a) engineering a microorganism to express and secrete or display a polypeptide having lipase activity;
(b) contacting the engineered microorganism with
i. triglycerides, diglycerides, monoglycerides, phospholipids, free fatty acids, or a mixture thereof; and
ii. a carbon substrate;
under conditions whereby the microorganism produces an alcohol.

### Claim 36 (depends on 35)

The method of claim 35 wherein the engineered microorganism is contacted with triglycerides, diglycerides, monoglycerides, and phospholipids, or combinations thereof and wherein the secreted or displayed lipase converts at least a portion of the triglycerides, diglycerides, monoglycerides, and phospholipids, or combinations thereof into free fatty acids.

### Claim 37 (depends on 35)

The method of claim 35 wherein the lipase catalyzes the formation of alcohol esters.

### Claim 38 (depends on 35)

The method of claim 35 wherein the microorganism produces alcohol at an effective titer greater than that produced by a microorganism that has not been engineered to express and secrete a polypeptide with lipase activity.

### Claim 39 (depends on 35)

The method of claim 35 wherein the alcohol biosynthetic pathway is an engineered alcohol biosynthetic pathway.

### Claim 40 (depends on 39)

The method of claim 39 wherein the engineered alcohol biosynthetic pathway is an isobutanol biosynthetic pathway.

### Claim 41

(canceled)

### Claim 42

A method of producing butyl esters during a fermentation comprising
(a) providing a fermentation medium comprising a carbon substrate and triglycerides, diglycerides, monoglycerides, and phospholipids, or a mixture thereof; and
(b) contacting the fermentation medium with an alcohol-producing microorganism comprising a butanol biosynthetic pathway wherein said microorganism further comprises an engineered polynucleotide encoding a polypeptide having lipase activity and which expresses and secretes or displays the polypeptide such that the lipase activity is present in the fermentation medium.

### Claim 43 (depends on 42)

The method of claim 42 wherein the fermentation medium further comprises one or more carboxylic acids.

### Claim 44 (depends on 42)

The method of claim 42 wherein the carbon substrate is derived from biomass.

### Claim 45

(canceled)

### Claim 46

(canceled)

### Claim 47

A fermentation medium comprising an alcohol-producing microorganism comprising a butanol biosynthetic pathway and further comprising an engineered polynucleotide encoding a polypeptide having lipase activity which is expressed and secreted or displayed, butyl esters, and butanol.

### Claim 48 (depends on 24)

An animal feed product comprising a microorganism of claim 24.