Production of fatty alcohols from engineered microorganisms

• US 9,650,655 B2
• Filed: 07/19/2013
• Issued: 05/16/2017
• Est. Priority Date: 07/20/2012
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Clark, Louis, Valle, Fernando, Vroom, Jonathan, Barak, Yoram, Karlshoej, Kristian, Chan, Kaman, Balatskaya, Svetlana, Choudhary, Trish
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US14/415,665
• Publication Number:
  US 20150176040A1
• Time in Patent Office: 1,397 Days
• CPC Class Codes: A61K 2800/85 Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
• CPC Class Codes: A61K 8/342 Alcohols having more than seven atoms in an unbroken chain
• CPC Class Codes: C11D 3/2013 fatty or with at least 8 carbon atoms in the alkyl chain
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• CPC Class Codes: C12Y 101/01 with NAD+ or NADP+ as acceptor (1.1.1)
• CPC Class Codes: C12Y 101/011 3-Oxoacyl-[acyl-carrier-protein] reductase (1.1.1.100)
• CPC Class Codes: C12Y 103/01009 Enoyl-[acyl-carrier-protein] reductase (NADH) (1.3.1.9)
• CPC Class Codes: C12Y 103/99 with other acceptors (1.3.99)
• CPC Class Codes: C12Y 201/01005 Betaine--homocysteine S-methyltransferase (2.1.1.5)
• CPC Class Codes: C12Y 203/01029 Glycine C-acetyltransferase (2.3.1.29)
• CPC Class Codes: C12Y 203/01041 Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41)
• CPC Class Codes: C12Y 203/01086 : Fatty-acyl-CoA synthase (2.3.1.86)
• CPC Class Codes: C12Y 203/0118 : Beta-ketoacyl-acyl-carrier-protein synthase III (2.3.1.180)
• CPC Class Codes: C12Y 301/02 : Thioester hydrolases (3.1.2)
• CPC Class Codes: C12Y 301/02001 : Acetyl-CoA hydrolase (3.1.2.1)
• CPC Class Codes: C12Y 301/02027 : Choloyl-CoA hydrolase (3.1.2.27)
• CPC Class Codes: C12Y 402/01059 : 3-Hydroxyacyl-[acyl-carrier-protein] dehydratase (4.2.1.59)
• CPC Class Codes: C12Y 602/01 : Acid-Thiol Ligases (6.2.1)
• CPC Class Codes: C12Y 602/01003 : Long-chain-fatty-acid-CoA ligase (6.2.1.3)

Abstract

Recombinant microorganisms are provided which have been engineered to produce fatty alcohols. Also provided are recombinant microorganisms which comprise a heterologous polynucleotide encoding a fatty alcohol reductase enzyme and an introduced polynucleotide encoding a β-ketoacyl acyl carrier protein synthase.

Claims

1. An engineered prokaryotic cell comprising two recombinant polynucleotides, the first polynucleotide encodes a heterologous fatty alcohol forming reductase (FAR) comprising the amino acid sequence of SEQ ID NO:

8, and the second polynucleotide encodes a β

-ketoacyl acyl carrier protein synthase III (FabH).

2. The engineered prokaryotic cell of claim 1, wherein the engineered prokaryotic cell is a bacterial cell.

3. The engineered prokaryotic cell of claim 2, wherein the bacterial cell is an E. coli cell.

4. The engineered prokaryotic cell of claim 1, further comprising a recombinant polynucleotide encoding a thioesterase (TE), wherein the TE comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO:

63.

5. The engineered prokaryotic cell of claim 1, wherein the TE comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO:

12.

6. The engineered prokaryotic cell of claim 1, wherein the TE comprises an amino acid sequence at least 95% sequence identity to SEQ ID NO:

12 or SEQ ID NO;

63.

7. The engineered prokaryotic cell of claim 1, further comprising one or more introduced polynucleotide encoding a FabD and/or FabG enzyme.

8. The engineered prokaryotic cell of claim 7, wherein said introduced polynucleotide encodes the FabD enzyme.

9. The engineered prokaryotic cell of claim 7, wherein the FabD enzyme comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO:

26.

10. The engineered prokaryotic cell of claim 7, wherein said introduced polynucleotide sequence encodes the FabG enzyme.

11. The engineered prokaryotic cell of claim 7, wherein the FabG enzyme comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO:

18.

12. The engineered prokaryotic cell of claim 7, wherein the introduced polynucleotide encode the FabD and FabG enzymes, wherein the FabD enzyme has an amino acid sequence at least 90% sequence identity to SEQ ID NO:

26, and wherein the FabG enzyme has an amino acid sequence at least 90% sequence identity to SEQ ID NO;

18.

13. The engineered prokaryotic cell of claim 1, further comprising an introduced polynucleotide encoding a Fab enzyme selected from a FabI and a FabZ enzyme.

14. The engineered prokaryotic cell of claim 13, wherein the FabI enzyme has an amino acid sequence at least 90% sequence identity to SEQ ID NO:

20.

15. The engineered prokaryotic cell of claim 13, wherein the FabZ enzyme has an amino acid sequence at least 90% sequence identity to SEQ ID NO:

22.

16. The engineered prokaryotic cell of claim 13, wherein the introduced polynucleotide encodes the FabI enzyme and the FabZ enzyme.

17. The engineered prokaryotic cell of claim 1, further comprising the expression of at least one endogenous genes selected from fadE, fadR, fadD, fabB, fabH and fabF, is attenuated.

18. The engineered prokaryotic cell of claim 1, further comprising a recombinant acyl-CoA synthase (FadD).

19. A cell culture comprising the engineered prokaryotic cell of claim 1.

20. A method of producing a fatty alcohol composition comprising

a) providing the engineered prokaryotic cell of claim 1;

b) culturing the engineered prokaryotic cell of claim 1 under suitable culture conditions in the presence of a carbon source;

c) producing fatty alcohols; and

d) optionally recovering the fatty alcohols from the engineered cell of claim 1 or from a culture medium thereof.

21. The method of claim 20, wherein the carbon source comprises a fermentable sugar.

22. The method of claim 21, wherein the fermentable sugar comprises glucose.

23. The method of claim 21, wherein the fermentable sugar is obtained from a cellulosic feedstock.

24. The method of claim 23, wherein the cellulosic feedstock is derived from a biomass selected from grain (e.g. corn), corn stover, corn cobs, wheat straw, bagasse and beet pulp.

25. The method of claim 24, wherein the biomass has been pretreated.

26. The method of claim 20, wherein at least 1 g/L of fatty alcohols are produced by the engineered prokaryotic cell.

27. The method of claim 20, wherein the total fatty alcohol composition produced comprises at least 60% C12 to C14 fatty alcohols.

28. The method of claim 27, wherein the total fatty alcohol composition produced comprises at least 70% C12 to C14 fatty alcohols.

29. A vector comprising a first polynucleotide encoding a FAR comprising the amino acid sequence of SEQ ID NO:

8, and a second polynucleotide encoding a FabH, wherein the first and second polynucleotides are operably linked to a promoter that is functional in a prokaryotic host cell.

30. A recombinant bacterial culture that produces a composition of fatty alcohols comprising at least 60% C12, C14 and C16 fatty alcohols, wherein the recombinant bacterial culture comprises an engineered bacterial microorganism comprising a gene encoding a heterologous FAR comprising the amino acid sequence of SEQ ID NO:

8 and a gene encoding a heterologous FabH comprising an amino acid sequence at least 90% sequence identity to SEQ ID NO;

10.

31. The recombinant bacterial culture of claim 30, wherein the engineered bacterial microorganism is an E. coli strain.

32. The recombinant bacterial culture of claim 30, wherein the fatty alcohol composition produced comprises at least 80% of C12, C14 and C16 fatty alcohols, or comprises at least 60% of the combination of C12 and C14 fatty alcohols.

33. A method of shifting the C12, and C16 fatty alcohols carbon chain length profile in a fatty alcohol composition produced by an engineered bacterial cell comprising a polynucleotide encoding a heterologous FAR having the amino acid sequence of SEQ ID NO:

8, said method comprises introducing a polynucleotide encoding a FabH having at least 90% sequence identity to SEQ ID NO;

10 into the engineered bacterial cell to produce newly engineered bacterial cell, wherein the composition of C12 fatty alcohols produced by the newly engineered bacterial cell has been increased by 25% and the composition of C16 fatty alcohols has been decreased by at least 10% as compared to the engineered bacterial cell.