In situ expression of lipase for enzymatic production of alcohol esters during fermentation

• US 8,759,044 B2
• Filed: 06/15/2012
• Issued: 06/24/2014
• Est. Priority Date: 03/23/2011
• Status: Expired due to Fees

• Current Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: DiCosimo, Robert, Kruckeberg, Arthur Leo, Van Aken, Thomas Edwin
• Primary Examiner(s): NASHED, NASHAAT T
• Application Number:
  US13/525,075
• Publication Number:
  US 20130157324A1
• Time in Patent Office: 739 Days
• Field of Search: None
• US Class Current: 435/135
• CPC Class Codes: A23K 10/10 obtained by microbiological or biochemical processes using chemicals or microorganisms for ensilaging of green fodder A23K30/15
• CPC Class Codes: A23K 10/18 of live microorganisms
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/62 Carboxylic acid esters
• CPC Class Codes: C12P 7/6436 Fatty acid esters
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

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

1. A method comprising:

(a) providing a fermentation medium comprising 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, wherein the alcohol-producing microorganism further comprises reduced or eliminated pyruvate decarboxylase activity, and wherein the microorganism ferments the fermentable carbon substrate to produce alcohol, wherein the alcohol is a C₃ to C₈ alkyl alcohol; and

(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.

2. 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.

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

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

5. The method of claim 4 wherein the alcohol esters comprise fatty acid butyl esters.

6. The method of claim 5 wherein the polypeptide having lipase activity comprises a sequence having at least 70% identity to any one of SEQ ID NOs:

249, 250, 251, 252, 253 or a fragment thereof having lipase activity.

7. The method of claim 1 wherein the polynucleotide encoding a polypeptide having lipase activity comprises a sequence with at least 95% identity to a polynucleotide having SEQ ID NO:

1, 3, 5, 7, 8, 9, 46, 48, 50, 52, 54, 255, 271 or 273.

8. The method of claim 1 wherein the polypeptide having lipase activity comprises a sequence with at least 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 a fragment thereof having lipase activity.

9. The method of claim 8 wherein the polypeptide having lipase activity does not contain a glycosylation motif.

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

11. 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.

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

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

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

15. 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.

16. 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.

17. 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.

18. 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, wherein the microorganism further comprises reduced or eliminated pyruvate decarboxylase activity;

b. contacting the engineered microorganism with

i. triglycerides, diglycerides, monoglycerides, phospholipids, free fatty acids, or a mixture thereof, wherein the secreted or displayed lipase converts at least a portion of the triglycerides, diglycerides, monoglycerides, phospholipids, or mixtures thereof into free fatty acids; and

ii. a fermentable carbon substrate, wherein the carbon substrate is selected from monosaccharides, disaccharides, oligosaccharides, polysaccharides, one carbon substrates, or mixtures thereof;

under conditions whereby the microorganism produces an alcohol, whereby the lipase activity is present in sufficient amount to convert at least a portion of the alcohol produced by the microorganism to alcohol esters, and wherein the tolerance of the microorganism to the product alcohol is increased.

19. The method of claim 18 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.

20. The method of claim 18 wherein the microorganism comprises an engineered alcohol biosynthetic pathway.

21. The method of claim 20 wherein the engineered alcohol biosynthetic pathway is an isobutanol biosynthetic pathway.

22. 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, wherein the carbon substrate is selected from monosaccharides, disaccharides, oligosaccharides, polysaccharides, one carbon substrates, or mixtures 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, and wherein said microorganism further comprises reduced or eliminated pyruvate decarboxylase activity, under conditions whereby the microorganism produces butanol, whereby the lipase activity is present in sufficient amount to convert at least a portion of the butanol produced by the microorganism to butyl esters.

23. The method of claim 22 wherein the fermentation medium further comprises one or more carboxylic acids.

24. The method of claim 22 wherein the carbon substrate is derived from biomass.