# US 2014/0155567 A1 · Microorganisms and methods for the biosynthesis of butadiene

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- Number: US 2014/0155567 A1
- Document ID: US20140155567A1
- Status: Published application
- Kind: A1
- Application number: 14/059,131
- Filed: 2013-10-21
- Published: 2014-06-05
- Earliest claimed: 2010-05-05
- Assignees: Genomatica, Inc.
- Applicants: Genomatica, Inc.
- Inventors: Mark J. Burk; Anthony P. Burgard; Jun Sun; Robin E. Osterhout; Priti Pharkya
- Web page: https://patentgrep.com/patent/US20140155567A1

## CPC

- C12P 5/026: {Unsaturated compounds, i.e. alkenes, alkynes or allenes}
- C07C 11/16: with four carbon atoms
- C08F 136/06: Butadiene
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides non-naturally occurring microbial organisms having a butadiene pathway. The invention additionally provides methods of using such organisms to produce butadiene.

## Claims

### Claim 1

A non-naturally occurring microbial organism, said microbial organism having a butadiene pathway and comprising at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, said butadiene pathway comprising converting crotonyl-CoA to crotyl-alcohol using one or more butadiene pathway enzymes, wherein said butadiene pathway enzymes are selected from the group consisting of a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming) a crotonyl-CoA hydrolase, synthetase, or transferase, a crotonate reductase, and a crotonyl-CoA reductase (alcohol forming).

### Claim 2 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises two exogenous nucleic acids each encoding a butadiene pathway enzyme.

### Claim at position 3

3-4. (canceled)

### Claim 5 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway enzymes comprise a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming).

### Claim 6 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway enzymes comprise a crotonyl-CoA reductase (alcohol forming).

### Claim 7

(canceled)

### Claim 8 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway enzymes comprise a crotonyl-CoA hydrolase, synthetase or transferase and a crotonate reductase.

### Claim 9 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway further comprises converting crotyl alcohol to butadiene using butadiene pathway enzymes, wherein said butadiene pathway enzymes are a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase.

### Claim 10 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway further comprises converting crotyl alcohol to butadiene using butadiene pathway enzymes, wherein said butadiene pathway enzymes are a butadiene synthase and a crotyl alcohol diphosphokinase.

### Claim 11 (depends on 5)

The non-naturally occurring microbial organism of claim 5, wherein said butadiene pathway further comprises converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase.

### Claim 12 (depends on 6)

The non-naturally occurring microbial organism of claim 6, wherein said butadiene pathway further comprises converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase.

### Claim 13 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway further comprises converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase.

### Claim at position 13

14-16. (canceled)

### Claim 17 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway further comprises converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase.

### Claim at position 15

18-22. (canceled)

### Claim 23 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway further comprises converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase.

### Claim at position 17

24-28. (canceled)

### Claim 29 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said butadiene pathway further comprises converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase.

### Claim at position 19

30-34. (canceled)

### Claim 35 (depends on 1)

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

### Claim 36 (depends on 1)

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

### Claim at position 22

37-57. (canceled)

### Claim 58 (depends on 1)

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

### Claim 59 (depends on 58)

The method of claim 58, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

### Claim 60 (depends on 1)

The non-naturally occurring microbial organism of claim 1, wherein said microbial organism is a bacteria, a yeast, or a fungus.

### Claim 61 (depends on 60)

The non-naturally occurring microbial organism of claim 60, wherein said a bacteria is Escherichia coli.

### Claim 62 (depends on 60)

The non-naturally occurring microbial organism of claim 60, wherein said a yeast is Saccharomyces cerevisiae.

### Claim 63 (depends on 58)

Culture medium comprising butadiene, wherein said butadiene was produced by the non-naturally occurring microbial organism of claim 58.

### Claim 64

Biosynthesized butadiene.

### Claim 65 (depends on 64)

A composition comprising the biosynthesized butadiene of claim 64.

### Claim 66 (depends on 65)

The composition of claim 65, wherein said composition is a polymer.

### Claim 67

A process for producing the polymer of 66 comprising chemical or enzymatic conversion of biosynthesized butadiene to produce said polymer.