MODIFIED NATIVE BETA-KETOACYL-ACP SYNTHASES AND ENGINEERED MICROORGANISMS

• US 20160010115A1
• Filed: 12/04/2013
• Published: 01/14/2016
• Est. Priority Date: 12/14/2012
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Clark, Louis, Osborne, Robert, Vroom, Jonathan, Valle, Fernando, Shankar, Smita, Cho, Catherine M.
• Granted Patent: US 9,512,447 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2800/10 General cosmetic use
• CPC Class Codes: A61K 8/342 Alcohols having more than seven atoms in an unbroken chain
• CPC Class Codes: A61Q 19/00 Preparations for care of the skin
• CPC Class Codes: C07C 31/125 containing five to twenty-two carbon atoms
• CPC Class Codes: C07C 33/025 with only one double bond
• CPC Class Codes: C11D 3/2013 fatty or with at least 8 carbon atoms in the alkyl chain
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12Y 203/01041 Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Genetically engineered cells and microorganisms are provided that produce fatty alcohols and fatty acids. In particular, engineered microbial cells comprise a modified native gene having β-ketoacyl-acp synthase activity.

Claims

1. A method for altering the carbon chain length of acyl-derived products produced from a bacterial microorganism, said method comprising modifying a native bacterial gene having β

-ketoacyl-ACP synthase activity.

2. The method of claim 1, wherein the native bacterial gene having β

-ketoacyl-ACP synthase activity is a native β

-ketoacyl-ACP synthase I (FabB) (EC 2.3.1.41).

3. The method of claim 1, wherein the native bacterial gene having β

-ketoacyl-ACP synthase activity is a native β

-ketoacyl-ACP synthase II (FabF) (EC 2.3.1.179).

4. The method of claim 2, wherein the native β

-ketoacyl-ACP synthase I has at least 90% sequence identity to SEQ ID NO;

2 and includes one or more amino acid residue substitutions when aligned with SEQ ID NO;

2.

5. The method of claim 1, wherein the acyl-derived product is a fatty acid or a fatty alcohol.

6. The method of claim 1, wherein the C12 carbon chain length of the acyl-derived product produced by the bacterial microorganism is increased by 10% as compared to the C12 carbon chain length of the same acyl-derived product produced by a corresponding bacterial microorganism comprising the native bacterial gene when grown under essentially the same conditions.

7. A variant FabB polypeptide, wherein the polypeptide sequence of said variant comprises at least 85% amino acid sequence identity to SEQ ID NO:

2 or a functional fragment thereof and wherein the polypeptide sequence of said variant comprises an amino acid substitution at one or more of the following positions 30, 34, 38, 40, 42, 43, 45, 51, 53, 61, 62, 63, 64, 65, 66, 95, 96, 97, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 124, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 149, 151, 161, 162, 164, 165, 191, 196, 197, 200, 201, 204, 205, 206, 209, 210, 212, 216, 217, 224, 225, 226, 229, 231, 267, 268, 270, 271, 272, 273, 275, 282, 285, 298, 300, 302, 303, 304, 305, 308, 311, 314, 315, 333, 335, 336, 360, 361, 367, 390, 391, 392, and/or 396, wherein the positions are numbered with reference to SEQ ID NO;

2.

8. The variant FabB polypeptide of claim 7, wherein said variant polypeptide comprises at least one amino acid substitution at one or more of the following positions R30, T34, E38, K40, S42, G43, R45, N51, K53, D61, R62, K63, V64, V65, R66, N95, N96, P97, G106, G107, G108, P110, R111, F112, Q113, V114, F115, G116, A117, R124, K127, A128, G130, P131, Y132, V133, V134, T135, K136, A137, M138, A139, S140, P149, K151, S161, A162, A164, T165, E191, E196, M197, E200, F201, M204, G205, A206, T209, K210, N212, E216, K217, A224, H225, R226, F229, I231, A267, D268, V270, A271, P272, S273, E275, K282, M285, H298, T300, T302, P303, V304, G305, K308, A311, R314, E315, H333, L335, G336, N360, I361, Q367, F390, G391, F392, and/or N396, wherein the positions are numbered with reference to SEQ ID NO:

2.

13. A recombinant polynucleotide encoding the variant polypeptide of claim 7.

14. The polynucleotide of claim 13, wherein the polynucleotide is codon optimized.

15. A DNA construct comprising the polynucleotide of claim 13.

16. A host cell comprising the DNA construct of claim 15.

17. The host cell of claim 16, wherein the DNA construct comprising a polynucleotide encoding the variant FabB is chromosomally integrated into the host cell.

18. The host cell of claim 16, wherein the host cell is a bacterial cell.

20. The host cell of claim 16, further comprising a heterologous polynucleotide encoding a fatty alcohol reductase (FAR) enzyme.

21. The host cell of claim 20, wherein the FAR has at least 80% amino acid sequence identity to SEQ ID NO:

4 or SEQ ID NO;

6.

22. The host cell of claim 21, further comprising a variant FAR having at least 80% sequence identity to SEQ ID NO:

4 or SEQ ID NO;

6 and having a substitution at one or more positions corresponding to residues N134, E138, P188, P405, Q418 and/or A511, wherein the positions are numbered with reference to SEQ ID NO;

4.

23. The host cell of claim 22, further comprising a variant FAR having a substitution at one or more positions corresponding to residues Q7, Q18, R65, V104, N128, E138, N177, K224, L226, E227, M365, G401, G410, S433, S458, G487, L502, R508, and/or K509 when aligned with SEQ ID NO:

4.

24. An engineered microorganism comprising at least one polynucleotide sequence encoding at least one variant FabB of claim 7.

25. The engineered microorganism of claim 24, further comprising a heterologous polynucleotide encoding a fatty alcohol reductase (FAR), said FAR comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

4 or SEQ ID NO;

6.

26. The engineered microorganism of claim 24, wherein the endogenous polynucleotide encoding a FabB having at least 85% sequence identity to SEQ ID NO:

2 is replaced with a polynucleotide encoding a variant FabB.

27. The engineered microorganism of claim 24, further comprising one or more heterologous polynucleotides encoding FabI, FabZ, FabH, FabD, and/or FabG.

28. The engineered microorganism of claim 24, further comprising a heterologous polynucleotide encoding a thioesterase.

29. The engineered microorganism of claim 24, wherein when said microorganism is cultured in the presence of a carbon source and is capable of producing fatty alcohols, fatty acids and/or fatty aldehydes.

30. The engineered microorganism of claim 29, wherein said microorganism is capable of producing a fatty alcohol composition comprising at least 25% (30%, 35%, 40%, 45%, and 50%) of C12 to C14 fatty alcohols.

31. The engineered microorganism of claim 30, wherein said microorganism is capable of producing a fatty alcohol composition comprising at least 20% C12 fatty alcohols.

32. The engineered microorganism of claim 24, wherein said microorganism is a bacteria or yeast.

41. A method of producing a fatty alcohol composition comprising culturing the engineered cell of claim 20, with a carbon substrate under suitable culture conditions to allow expression of the variant FabB and heterologous FAR, and allowing production of fatty alcohols, wherein the fatty alcohol composition comprises at least 20% C12 and C14 fatty alcohols.

42. The method of claim 41, further comprising recovering the fatty alcohol composition.

43. The method of claim 41, wherein at least about 1.0 g/L of fatty alcohols are produced.

44. The method of claim 41, wherein at least about 80% of the produced fatty alcohols have a carbon chain length of C10 to C16.

45. The method of claim 41, wherein at least about 60% of the produced fatty alcohols have a carbon chain length of C12 to C16.

46. The method of claim 41, wherein at least about 30% of the produced fatty alcohols have a carbon chain length of C12 to C14.

49. The fatty alcohol composition produced according to the method of claim 41, wherein said composition is used in a detergent composition or a personal care composition.

9-12. -12. (canceled)

19. (canceled)

33. (canceled)

34. (canceled)

35. A recombinant cell culture comprising engineered bacterial cells, said engineered bacterial cells comprising (a) a modified native gene having β

-ketoacyl-ACP synthase activity and (b) one or more heterologous polynucleotides encoding a fatty alcohol reductase (EC1.1.1.*) and/or a thioesterase (EC3.1.2.- or EC 3.1.1.5).

37. The recombinant cell culture of claim 35, wherein the engineered bacterial cells comprise a polynucleotide encoding a heterologous fatty alcohol reductase comprising at least 80% sequence identity to SEQ ID NO:

4 or 6.

38. The recombinant cell culture of claim 37, wherein the engineered cells produce a fatty alcohol composition selected from:

a) at least 60% C12 to C16 fatty alcohols relative to the total fatty alcohols produced by the engineered microbial cells;

b) at least 25% C12 to C14 fatty alcohols relative to the total fatty alcohols produced by the engineered microbial cells;

c) at least 10% C12 fatty alcohols relative to the total fatty alcohols produced by the engineered microbial cells;

d) at least 20% C14 fatty alcohols relative to the total fatty alcohols produced by the engineered microbial cells; and

e) less than 5% C18 fatty alcohols relative to the total fatty alcohols produced by the engineered cells.

39. The recombinant cell culture of claim 38, wherein the fatty alcohols have been secreted from said engineered microbial cells and are present in the culture medium.

36. (canceled)

40. (canceled)

47. (canceled)

48. (canceled)

50. (canceled)