Primary alcohol producing organisms

Patent metadata

Field	Value
Publication number	US-9260729-B2
Application number	US-201113168833-A
Country	US
Kind code	B2
Filing date	Jun 24, 2011
Priority date	Mar 5, 2008
Publication date	Feb 16, 2016
Grant date	Feb 16, 2016

Abstract

The invention provides a non-naturally occurring microbial organism having at least one exogenous gene insertion and/or one or more gene disruptions that confer production of primary alcohols. A method for producing long chain alcohols includes culturing these non-naturally occurring microbial organisms.

First claim

What is claimed is:

1. A non-naturally occurring microbial organism, comprising a microbial organism having a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an acyl-reduction pathway comprising at least one exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme expressed in sufficient amounts to produce a primary alcohol, said malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase, said acyl-reduction pathway comprising an acyl-CoA reductase and an alcohol dehydrogenase, wherein said ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase converts acyl-CoA to β-ketoacyl-CoA, wherein said 3-hydroxyacyl-CoA dehydrogenase converts β-ketoacyl-CoA to β-hydroxyacyl-CoA, wherein said enoyl-CoA hydratase converts β-hydroxyacyl-CoA to trans-2-enoyl-CoA, wherein said enoyl-CoA reductase converts trans-2-enoyl-CoA to acyl-CoA, wherein said acyl-CoA reductase converts acyl-CoA to an aldehyde, wherein said alcohol dehydrogenase converts an aldehyde to said primary alcohol, wherein said microbial organism further comprises one or more gene disruptions, said one or more gene disruptions occurring in genes encoding enzymes that couple primary alcohol production to growth of said non-naturally occurring microbial organism when said gene disruption reduces an activity of said enzyme, whereby said one or more gene disruptions confers production of primary alcohols onto said non-naturally occurring microbial organism.

2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises two exogenous nucleic acid each encoding a malonyl-CoA-independent FAS pathway enzyme.

3. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises three exogenous nucleic acid each encoding a malonyl-CoA-independent FAS pathway enzyme.

4. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises four exogenous nucleic acid each encoding a malonyl-CoA-independent FAS pathway enzyme.

5. The non-naturally occurring microbial organism of claim 4 , wherein said four exogenous nucleic acids encode ketoacyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase.

6. The non-naturally occurring microbial organism of claim 4 , wherein said four exogenous nucleic acids encode ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase.

7. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises at least one exogenous nucleic acid encoding an acyl-reduction pathway enzyme.

8. The non-naturally occurring microbial organism of claim 7 , wherein said exogenous nucleic acid encoding an acyl-reduction pathway enzyme comprises an acyl-CoA reductase.

9. The non-naturally occurring microbial organism of claim 7 , wherein said exogenous nucleic acid encoding an acyl-reduction pathway enzyme comprises an alcohol dehydrogenase.

10. The non-naturally occurring microbial organism of claim 7 , wherein said exogenous nucleic acid encoding an acyl-reduction pathway enzyme comprises an enzyme having acyl-CoA reductase and alcohol dehydrogenase activity.

11. The non-naturally occurring microbial organism of claim 10 , wherein said enzyme having acyl-CoA reductase and alcohol dehydrogenase activity comprises fatty alcohol forming acyl-CoA reductase (FAR).

12. The non-naturally occurring microbial organism of claim 1 , wherein said at least one exogenous nucleic acid further comprises a heterologous encoding nucleic acid.

13. The non-naturally occurring microbial organism of claim 1 , wherein said primary alcohol is produced in amounts at least 10% greater levels compared to a microbial organism lacking said exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme.

14. The non-naturally occurring microbial organism of claim 1 , further comprising a substantially anaerobic culture medium.

15. The non-naturally occurring microbial organism of claim 1 , wherein said primary alcohol comprises an alcohol having between 4-24 carbon atoms.

16. The non-naturally occurring microbial organism of claim 15 , wherein said alcohol having between 4-24 carbon atoms is selected from butanol, hexanol, heptanol, octanol, nonanol, decanol, dodecanol, tetradecanol or hexadecanol.

17. A method for producing a primary alcohol, comprising culturing a non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce said primary alcohol.

18. The non-naturally occurring microbial organism of claim 1 , wherein said one or more gene disruptions comprise a deletion of a negative regulatory effector of one or more malonyl-CoA-independent FAS pathway enzymes.

19. The non-naturally occurring microbial organism of claim 1 , wherein said one or more gene disruptions comprises a deletion of said one or more genes.

20. The non-naturally occurring microbial organism of claim 1 , wherein said one or more gene disruptions encode enzymes selected from the group consisting of an acetaldehyde-CoA dehydrogenase and a lactate dehydrogenase.

21. The non-naturally occurring microbial organism of claim 20 , wherein said one or more gene disruptions further comprise encoded enzymes selected from the group consisting of a pyruvate formate lyase, a phosphotransacetylase, an acetate kinase, a fumarate reductase, a fumarase, and a malate dehydrogenase.

22. The non-naturally occurring microbial organism of claim 21 , wherein said one or more gene disruptions further comprise encoded enzymes selected from the group consisting of a glutamate dehydrogenase, a NAD(P) transhydrogenase, and an ATP synthase.

23. The non-naturally occurring microbial organism of claim 1 , wherein said one or more gene disruptions comprise:
(1) acetylaldehyde-CoA dehydrogenase (ADHEr), lactate dehydrogenase (LDH_D), L-aspartase (ASPT), malate dehydrogenase (MDH), pyruvate formate lyase (PFLi), phosphogluconate dehydratase (PGDHY), pyruvate kinase (PYK,) and dihydroxyacetone phosphotransferase (DHAPT);
(2) ADHEr, LDH_D, ASPT, MDH, PFLi, 6-phosphogluconolactonase (PGL), PYK, and DHAPT;
(3) ADHEr, LDH_D, ASPT, glucose 6-phosphate dehydrogenase (G6PDHy), MDH, PFLi, PYK, and DHAPT;
(4) ADHEr, LDH_D, ASPT, 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA), MDH, PFLi, PYK, and DHAPT;
(5) ADHEr, LDH_D, D-glucose transport via PEP:Pyr PTS (GLCpts), glutamate dehydrogenase (GLUDy), phosphogluconate dehydrogenase (PGDH), glucose-6-phosphate isomerase (PGI), and phosphotransacetylase (PTAr);
(6) ADHEr, LDH_D, acetate kinase (ACKr), GLCpts, GLUDy, PGDH, and PGI;
(7) ADHEr, LDH_D, GLCpts, GLUDy, PGI, PTAr, and transaldolase (TAL);
(8) ADHEr, LDH_D, GLCpts, GLUDy, PGI, PTAr, and transketolase (TKT1);
(9) ADHEr, LDH_D, ACKr, GLCpts, GLUDy, PGI, TKT1;
(10) ADHEr, LDH_D, ACKr, GLCpts, GLUDy, PGI, and TAL;
(11) ADHEr, LDH_D, fructose-bisphosphate aldolase (FBA), GLCpts, GLUDy, and PTAr;
(12) ADHEr, LDH_D, GLCpts, GLUDy, PTAr, and triose-phosphate isomerase (TPI);TPI;
(13) ADHEr, LDH_D, ACKr, GLCpts, GLUDy, and TPI;
(14) ADHEr, LDH_D, GLCpts, GLUDy, phosphofructokinase (PFK), and PTAr;
(15) ADHEr, LDH_D, ACKr, FBA, GLCpts, and GLUDy;
(16) ADHEr, LDH_D, ACKr, GLCpts, GLUDy, and PFK;
(17) ADHEr, LDH_D, ACKr, GLCpts, GLUDy, PGI, and ribulose

Assignees

• Sun Jun
• Pharkya Priti
• Burgard Anthony P
• Genomatica Inc

Inventors

• Sun Jun
• Pharkya Priti
• Burgard Anthony P

Classifications

• C12Y203/01016

  Acetyl-CoA C-acyltransferase (2.3.1.16) · CPC title

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12Y402/01017

  Enoyl-CoA hydratase (4.2.1.17), i.e. crotonase · CPC title

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12Y203/01009

  Acetyl-CoA C-acetyltransferase (2.3.1.9) · CPC title