Methods and organisms for converting synthesis gas or other gaseous carbon sources and methanol to 1,3-butanediol

Patent metadata

Field	Value
Publication number	US-9284581-B2
Application number	US-201213597095-A
Country	US
Kind code	B2
Filing date	Aug 28, 2012
Priority date	Dec 10, 2009
Publication date	Mar 15, 2016
Grant date	Mar 15, 2016

Abstract

A non-naturally occurring microbial organism having a 1,3-butanediol (1,3-BDO) pathway includes at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme or protein expressed in a sufficient amount to produce 1,3-BDO. A method for producing 1,3-BDO that includes culturing the this non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce 1,3-BDO.

First claim

What is claimed is:

1. A non-naturally occurring microbial organism comprising:
(1) a Wood-Ljungdahl pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a Wood-Ljungdahl pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said Wood-Ljungdahl pathway enzyme is selected from the group consisting of:
a) a formate dehydrogenase that catalyzes the incorporation of CO2 into formate;
b) a formyltetrahydrofolate synthetase that ligates formate to tetrahydrofolate to form 10-formyltetrahydrofolate,
c) a methenyltetrahydrofolate cyclohydrolase that converts 10-formyltetrahydrofolate to methyltetrahydrofolate,
d) a methylenetetrahydrofolate dehydrogenase that converts methyltetrahydrofolate to methenyltetrahydrofolate,
e) a methylenetetrahydrofolate reductase that converts metheneyltetrahydrofolate to 5-methyltetrahydrofolate;
f) a methyltetrahydrofolate:corrinoid protein methyltransferase that catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to corrinoid iron sulfur protein,
g) a corrinoid iron-sulfur protein,
h) a nickel-protein assembly protein,
i) a ferredoxin,
j) a acetyl-CoA synthase that catalyzes the condensation of the methylated corrinoid iron sulfur protein, carbon monoxide and coenzyme A, yielding acetyl-CoA,
k) a carbon monoxide dehydrogenase that converts CO and water to CO2 while passing the electrons to a reduced acceptor,
l) a pyruvate ferredoxin oxidoreductase that converts the pyruvate to acetyl-CoA or pyruvate dehydrogenase that converts the pyruvate to acetyl-CoA, and
m) a pyruvate formate lyase that converts the pyruvate and CoA to acetyl-CoA; and

(2) a 1,3-butanediol pathway that converts acetyl-CoA to 1,3-butanediol, wherein the 1,3-butanediol pathway comprises at least three enzymes selected from the group consisting of:
a) an acetoacetyl-CoA thiolase that converts acetyl-CoA to acetoacetyl-CoA,
b) an acetoacetyl-CoA reductase (CoA-dependent, alcohol forming) that converts acetoacetyl-CoA to 4-hydroxy,2-butanone,
c) a 3-oxobutyraldehyde reductase (aldehyde reducing) that converts 3-oxobutyraldehyde into 4-hydroxybutanone,
d) a 4-hydroxy,2-butanone reductase that converts 4-hydroxy,2-butanone to 1,3-butanediol,
e) an acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming) that converts acetoacetyl-CoA to 3-oxobutyraldehyde,
f) a 3-oxobutyraldehyde reductase (ketone reducing) that converts 3-oxobutyraldehyde to 3-hydroxybutyraldehyde,
g) a 3-hydroxybutyraldehyde reductase that converts 3-hydroxybutyraldehyde to 1,3-BDO,
h) an acetoacetyl-CoA reductase (ketone reducing) that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA,
i) a 3-hydroxybutyryl-CoA reductase (aldehyde forming) that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde,
j) a 3-hydroxybutyryl-CoA reductase (alcohol forming) that converts 3-hydroxybutyryl-CoA to 1,3-butanediol,
k) an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or a phosphotransacetoacetylase/acetoacetate kinase that converts acetoacetyl-CoA to acetoacetate,
l) an acetoacetate reductase that converts acetoacetate to 3-oxobutyraldehyde,
m) a 3-hydroxybutyryl-CoA transferase, a 3-hydroxybutyryl-CoA hydrolase, or a 3-hydroxybutyryl-CoA synthetase that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyrate,
n) a 3-hydroxybutyrate reductase that converts 3-hydroxybutyrate to 3-hydroxybutyraldehyde; and
o) a 3-hydroxybutyrate dehydrogenase that converts acetoacetate to 3-hydroxybutyrate.

2. The non-naturally occurring microbial organism of claim 1 , wherein the organism comprises exogenous nucleic acids encoding each of the Wood-Ljungdahl pathway enzymes (a)-(m).

3. The non-naturally occurring microbial organism of claim 1 , wherein at least one of the 1,3-BDO pathway enzymes is encoded by an exogenous nucleic acid.

4. The non-naturally occurring microbial organism of claim 1 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

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

6. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism uses a carbon feedstock selected from 1) methanol and CO, 2) methanol, CO 2 , and H 2 , 3) methanol, CO, CO 2 , and H 2 , 4) methanol and synthesis gas comprising CO and H 2 , 5) methanol and synthesis gas comprising CO, CO 2 , and H 2 , 6) one or more carbohydrates, 7) methanol and one or more carbohydrates, and 8) methanol.

7. A method for producing 1,3-BDO, comprising culturing the organism of claim 1 under conditions and for a sufficient period of time to produce 1,3-BDO.

8. A culture medium comprising 1,3-BDO produced according to the method of claim 7 .

9. The culture medium of claim 8 , wherein said culture medium is separated from the non-naturally occurring microbial organism.

10. A composition comprising a biosynthesized 1,3-BDO and the non-naturally occurring microbial organism of claim 1 .

11. A biosynthesized 1,3-BDO produced according to the method of claim 7 .

Assignees

• Burgard Anthony P
• Burk Mark J
• Pharkya Priti
• Genomatica Inc

Inventors

• Burgard Anthony P
• Burk Mark J
• Pharkya Priti

Classifications

• C12P7/18 Primary

  polyhydric · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12P7/16

  Butanols · CPC title

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title