Microorganisms and methods for the production of caprolactone

Patent metadata

Field	Value
Publication number	US-9267162-B2
Application number	US-201514596072-A
Country	US
Kind code	B2
Filing date	Jan 13, 2015
Priority date	Nov 2, 2011
Publication date	Feb 23, 2016
Grant date	Feb 23, 2016

Abstract

The invention provides non-naturally occurring microbial organisms containing caprolactone pathways having at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce caprolactone. The invention additionally provides methods of using such microbial organisms to produce caprolactone by culturing a non-naturally occurring microbial organism containing caprolactone pathways as described herein under conditions and for a sufficient period of time to produce caprolactone.

First claim

What is claimed is:

1. A method for producing caprolactone, said method comprising culturing a non-naturally occurring microorganism for a sufficient period of time to produce caprolactone, wherein said caprolactone is produced at an intracellular concentration of about 0.1 mM to about 200 mM, wherein said non-naturally occurring microorganism comprises a caprolactone pathway comprising at least one exogenous nucleic acid encoding a caprolactone pathway enzyme expressed in a sufficient amount to produce caprolactone and wherein said caprolactone pathway comprises a pathway selected from:
(1) 1D, 1A, 1B, and 1C;
(2) 1E, 1B, 1C and 1D;
(3) 1F, 1A, 1B, 1C and 1D;
(4) 1F, 1E, 1B, 1C and 1D;
(5) 1A, 1B and 1G;
(6) 1E, 1B and 1G;
(7) 1F, 1A, 1B and 1G;
(8) 1F, 1E, 1B and 1G;
(9) 1A, 1B, 1C, 1J and 1I;
(10) 1E, 1B, 1C, 1J and 1I;
(11) 1F, 1A, 1B, 1C, 1J and 1I;
(12) 1F, 1E, 1B, 1C, 1J and 1I;
(13) 1A, 1B, 1H and 1I;
(14) 1E, 1B, 1H and 1I;
(15) 1F, 1A, 1B, 1H and 1I;
(16) 1F, 1E, 1B, 1H and 1I;
(17) 2A, 2B, 2C, 2D, 2E and 2F;
(18) 2A, 2B, 2C, 2D and 2G;
(19) 2A, 2B, 2C, 2D, 2E, 2J and 2I;
(20) 2A, 2B, 2C, 2D, 2H and 2I;
(21) 4A, 4B, 4C, 4D and 3A; and
(22) 5A, 5B, 5C an 3A,
wherein 1A is an adipyl-CoA reductase, wherein 1B is an adipate semialdehyde reductase, wherein 1C is a 6-hydroxyhexanoyl-CoA transferase or a 6-hydroxyhexanoyl-CoA synthetase, wherein 1D is a 6-hydroxyhexanoyl-CoA cyclase or spontaneous cyclization, wherein 1E is an adipate reductase, wherein 1F is an adipyl-CoA transferase, an adipyl-CoA synthetase or an adipyl-CoA hydrolase, wherein 1G is a 6-hydroxyhexanoate cyclase, wherein 1H is a 6-hydroxyhexanoate kinase, wherein 1I is a 6-hydroxyhexanoyl phosphate cyclase or spontaneous cyclization, wherein 1J is a phosphotrans-6-hydroxyhexanoylase, wherein 2A is a 4-hydroxybutyryl-CoA:acetyl-CoA acyltransferase, wherein 2B is a 3-oxo-6-hydroxyhexanoyl-CoA reductase, wherein 2C is a 3,6-dihydroxyhexanoyl-CoA dehydratase, wherein 2D is a 6-hydroxyhex-2-enoyl-CoA reductase, wherein 2E is a 6-hydroxyhexanoyl-CoA transferase, a 6-hydroxyhexanoyl-CoA synthetase or a 6-hydroxyhexanoyl-CoA hydrolase, wherein 2F is a 6-hydroxyhexanoate cyclase, wherein 2G is a 6-hydroxyhexanoyl-CoA cyclase or spontaneous cyclization, wherein 2H is a phosphotrans-6-hydroxyhexanoylase, wherein 2I is a 6-hydroxyhexanoyl phosphate cyclase or spontaneous cyclization, wherein 2J is a 6-hydroxyhexanoate kinase, wherein 3A is a cyclohexanone monooxygenase, wherein 4A is an adipate semialdehyde dehydratase, wherein 4B is a cyclohexane-1,2-dione reductase, wherein 4C is a 2-hydroxycyclohexanone reductase, wherein 4D is a cyclohexane-1,2-diol dehydratase, wherein 5A is a 2-ketocyclohexane-1-carboxoyl-CoA hydrolase (acting on C-C), wherein 5B is a 2-ketocyclohexane-1-carboxoyl-CoA transferase, a 2-ketocyclohexane-1-carboxoyl-CoA synthetase or a 2-ketocyclohexane-1-carboxoyl-CoA hydrolase, and wherein 5C is a 2-ketocyclohexane-1-carboxylate decarboxylase.

2. The method of claim 1 , wherein said intracellular concentration of said caprolactone is about 3 mM to about 150 mM.

3. The method of claim 1 , wherein said intracellular concentration of said caprolactone is about 8 mM to about 100 mM.

4. The method of claim 1 , wherein said caprolactone is separated from other components in said culture.

5. The method of claim 4 , wherein said separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.

6. The method of claim 1 , wherein said microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids each encoding a caprolactone pathway enzyme.

7. The method of claim 1 , wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(22).

8. The method of claim 1 , wherein said culturing is performed in a fermentation broth comprising glucose.

9. The method of claim 1 , wherein said culturing is performed in a fermentation broth comprising methanol.

10. The method of claim 1 , wherein said non-naturally occurring microbial further comprises:
(i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a citryl-CoA synthetase, a citryl-CoA, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase;
(ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or
(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof.

11. The method of claim 1 , wherein said exogenous nucleic acid is a heterologous nucleic acid.

12. The method of claim 1 , wherein said non-naturally occurring microorganism is generated in E. coli.

13. Bioderived caprolactone synthesized from the method of claim 1 having a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source.

14. A composition comprising at least 3 mM caprolactone and a viable non-naturally occurring microbial organism having a caprolactone pathway and comprising at least one exogenous nucleic acid encoding a caprolactone pathway enzyme expressed in a sufficient amount to produce caprolactone, wherein said caprolactone pathway comprises a pathway selected from:
(1) 1D, 1A, 1B, and 1C;
(2) 1E, 1B, 1C and 1D;
(3) 1F, 1A, 1B, 1C and 1D;
(4) 1F, 1E, 1B, 1C and 1D;
(5) 1A, 1B and 1G;
(6) 1E, 1B and 1G;
(7) 1F, 1A, 1B and 1G;
(8) 1F, 1E, 1B and 1G;
(9) 1A, 1B, 1C, 1J and 1I;
(10) 1E, 1B, 1C, 1J and 1I;
(11) 1F, 1A, 1B, 1C, 1J and 1I;
(12) 1F, 1E, 1B, 1C, 1J and 1I;
(13) 1A, 1B, 1H and 1I;
(14) 1E, 1B, 1H and 1I;
(15) 1F, 1A, 1B, 1H and 1I;
(16) 1F, 1E, 1B, 1H and 1I;
(17) 2A, 2B, 2C, 2D, 2E and 2F;
(18) 2A, 2B, 2C, 2D and 2G;
(19) 2A, 2B, 2C, 2D, 2E, 2J and 2I;
(20) 2A, 2B, 2C, 2D, 2H and 2I;
(21) 4A, 4B, 4C, 4D and 3A; and
(22) 5A, 5B, 5C an 3A,
wherein 1A is an adipyl-CoA reductase, wherein 1B is an adipate semialdehyde reductase, wherein 1C is a 6-hydroxyhexanoyl-CoA transferase or a 6-hydroxyhexanoyl-CoA synthetase, wherein 1D is a 6-hydroxyhexanoyl-CoA cyclase or spontaneous cyclization, wherein 1E is an adipate reductase, wherein 1F is an adipyl-CoA transferase, an adipyl-CoA synthetase or an adipyl-CoA hydrolase, wherein 1G is a 6-hydroxyhexanoate cyclase, wherein 1H is a 6-hydroxyhexanoate kinase, wherein 1I is a 6-hydroxyhexanoyl phosphate cyclase or spontaneous cyclization, wherein 1J is a phosphotrans-6-hydroxyhexanoylase, wherein 2A is a 4-hydroxybutyryl-CoA:acetyl-CoA acyltransferase, wherein 2B is a 3-oxo-6-hydroxyhexanoyl-CoA reductase, wherein 2C is a 3,6-dihydroxyhexanoyl-CoA dehydratase, wherein 2D is a 6-hydroxyhex-2-enoyl-CoA reductase, wherein 2E is a 6-hydroxyhexanoyl-CoA transferase, a 6-hydroxyhexanoyl-CoA synthetase or a 6-hydroxyhexanoyl-CoA hydrolase, wherein 2F is a 6-hydroxyhexanoate cyclase, wherein 2G is a 6-hydroxyhexanoyl-CoA cyclase or spontaneous cyclization, wherein 2H is a phosphotrans-6-hydroxyhexanoylase, wherein 2I is

Assignees

• Genomatica Inc

Inventors

• Burgard Anthony P
• Osterhout Robin E
• Pharkya Priti
• Burk Mark J

Classifications

• C12P17/08 Primary

  containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons · CPC title

• C07D313/04

  not condensed with other rings · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C08G18/4277

  Caprolactone and/or substituted caprolactone · CPC title

• C12N5/0018

  Culture media for cell or tissue culture (media for specific animal cell type C12N5/06) · CPC title