Microorganisms and methods for producing alkenes

Patent metadata

Field	Value
Publication number	US-11932893-B2
Application number	US-201815971977-A
Country	US
Kind code	B2
Filing date	May 4, 2018
Priority date	Sep 16, 2011
Publication date	Mar 19, 2024
Grant date	Mar 19, 2024

Abstract

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

First claim

What is claimed is:

1. A method for producing an alkene comprising culturing a non-naturally occurring microbial organism having an alkene pathway under conditions and for a sufficient period of time to produce said alkene, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a alkene pathway enzyme expressed in sufficient amount to convert an alcohol to said alkene, wherein said alkene pathway comprises alkene pathway enzymes selected from:
(1) an alcohol kinase that transfers a phosphate group to a hydroxyl group, and a phosphate lyase that converts an alkyl-phosphate to an alkene, wherein the alcohol kinase is selected from enzymes having an E.C. number selected from the group consisting of 2.7.1.30 (glycerol kinase), 2.7.1.36 (mevalonate kinase), or 2.7.1.39 (homoserine kinase);
(2) a diphosphokinase that transfers a di-phosphate group to a hydroxyl group and a diphosphate lyase that converts an alkyl di-phosphate to an alkene, wherein the diphosphokinase is selected from enzymes having an E.C. number selected from the group consisting of, 2.7.6.1 (ribose-phosphate diphosphokinase) or 2.7.6.2 (thiamine diphosphokinase); and
(3) an alcohol kinase that transfers a phosphate group to a hydroxyl group, an alkyl phosphate kinase that transfers a phosphate group to a phosphate group of an alkyl-phosphate, and a diphosphate lyase that converts an alkyl di-phosphate to an alkene, wherein:
(i) the alcohol kinase is selected from enzymes having an E.C. number selected from the group consisting of 2.7.1.30 (glycerol kinase), 2.7.1.36 (mevalonate kinase), or 2.7.1.39 (homoserine kinase);
(ii) the alkyl phosphate kinase is selected from enzymes having an E.C. number selected from the group consisting of 2.7.4.2 (phosphomevalonate kinase), 2.7.4.18 (farnesyl-diphosphate kinase); and
(iii) the diphosphate lyase is selected from enzymes having an E.C. number selected from the group consisting of 4.2.3.5 (Chorismate synthase), 4.2.3.15 (Myrcene synthase), 4.2.3.36 (Terpentriene synthase), 4.2.3.46 ((E, E)-alpha-Farnesene synthase), or 4.2.3.47 (Beta-Farnesene synthase);

wherein said alcohol is a compound of Formula (I)

wherein said alkene is a compound of Formula (II)

wherein,
(a) R 1 , R 2 , R 3 , and R 4 of Formula I and R 1 , R 2 , R 3 , and R 4 of Formula II are the same and are independently hydrogen, methyl, ethyl, or ethenyl, and
(b) wherein R 1 , R 2 , R 3 , and R 4 are selected such that the compound of Formula (II) is a C 4 alkene.

2. The method of claim 1 , wherein said alkene is selected from, But-1-ene, Isobutylene, but-2-ene, and 1,3-Butadiene.

3. The method of claim 1 , wherein the non-naturally occurring microbial organism comprises two exogenous nucleic acids each encoding an alkene pathway enzyme when the microbial organism comprises an alkene pathway selected from (1) or (2) and the microbial organism comprises two or three exogenous nucleic acids each encoding an alkene pathway enzyme when the microbial organism comprises an alkene pathway selected from (3).

4. The method of claim 3 , wherein said two exogenous nucleic acids encode an alcohol kinase and a phosphate lyase.

5. The method of claim 3 , wherein said two exogenous nucleic acids encode a diphosphokinase and a diphosphate lyase.

6. The method of claim 3 , wherein said three exogenous nucleic acids encode an alcohol kinase, an alkyl phosphate kinase and a diphosphate lyase.

7. The method of claim 1 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

8. The method of claim 1 , wherein said non-naturally occurring microbial organism is cultured in a substantially anaerobic culture medium.

9. The method of claim 1 , wherein said microbial organism converts n-butanol to but-1-ene, isobutanol to isobutylene, tert-butanol to isobutylene, butan-2-ol to but-1-ene or but-2-ene, but-3-en-1-ol to 1,3-butadiene, but-3-en-2-ol to 1,3-butadiene, or but-2-en-1-ol to 1,3-butadiene.

10. The method of claim 1 , wherein the alcohol kinase is:
(1) a mevalonate kinase from Saccharomyces cerevisiae, Methanocaldococcus jannaschi, Homo sapiens, Arabidopsis thaliana coli, Methanosarcina mazei or Streptococcus pneumonia;
(2) a glycerol kinase from Escherichia coli, Saccharomyces cerevisiae , or Thermotoga maritime; or
(3) a homoserine kinase from Escherichia coli, Saccharomyces cerevisiae or Streptomyces sp. ACT-1.

11. The method of claim 1 , wherein the phosphate lyase is:
(1) a chorismate synthase from Escherichia coli, Streptococcus pneumoniae, Neurospora crassa , or Saccharomyces cerevisiae;
(2) a myrcene synthase from Solanum lvcopersicum, Picea abies, Abies grandis , or Arabidopsis thaliana ; or
(3) a farnesyl diphosphate from Arabidopsis thaliana, Picea abies, Cucumis sativus, Matus x domestica , or Zea mays.

12. The method of claim 1 , wherein the diphosphate lyase is:
(1) a chorismate synthase from Escherichia coli, Streptococcus pneumoniae, Neurospora crassa , or Saccharomyces cerevisiae;
(2) a myrcene synthase from Solanum lvcopersicum, Picea abies, Abies Grandis , or Arabidopsis thaliana ; or
(3) a farnesyl diphosphate from Arabidopsis thaliana, Picea abies, Cucumis sativus, Matus x domestica , or Zea mays.

13. The method of claim 1 , wherein the alkyl phosphate kinase is:
(1) a phosphomevalonate kinase from Saccharomyces cerevisiae, Staphylococcus aureus, Streptococcus pneumoniae , or Enterococcus faecalis ; or
(2) a farnesyl monophosphate kinase from Nicotiana tabacum.

14. The method of claim 1 , wherein the diphosphokinase is
(1) a ribose-phosphate diphosphokinase from Escherichia coli and Mycoplasma pneumoniae M129; or
(2) a thiamine diphosphokinase from Arabidopsis thaliana.

Assignees

• Genomatica Inc

Inventors

• Burk Mark J
• Osterhout Robin E

Classifications

• C12P5/026 Primary

  Unsaturated compounds, i.e. alkenes, alkynes or allenes · CPC title

• C12N9/1205

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title

• C12N9/1229

  Phosphotransferases with a phosphate group as acceptor (2.7.4) · CPC title

• C12N9/1235

  Diphosphotransferases (2.7.6) · CPC title

• C12N9/88

  Lyases (4.) · CPC title