# US 9,434,964 B2 · Microorganisms for the production of 1,4-butanediol and related methods

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- Number: US 9,434,964 B2
- Document ID: US9434964B2
- Status: Granted
- Kind: B2
- Application number: 13/361,799
- Filed: 2012-01-30
- Published: 2012-09-06
- Granted: 2016-09-06
- Earliest claimed: 2009-06-04
- Assignees: Genomatica, Inc.
- Applicants: Stephen J. Van Dien; Anthony P. Burgard; Robert Haselbeck; Catherine J. Pujol-Baxley; Wei Niu; John D. Trawick; Harry Yim; Mark J. Burk; Robin E. Osterhout; Jun Sun
- Inventors: Stephen J. Van Dien; Anthony P. Burgard; Robert Haselbeck; Catherine J. Pujol-Baxley; Wei Niu; John D. Trawick; Harry Yim; Mark J. Burk; Robin E. Osterhout; Jun Sun
- Web page: https://patentgrep.com/patent/US9434964B2

## CPC

- C12P 7/18: polyhydric
- C07D 207/267: with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to the ring nitrogen atom
- C07D 307/08: Preparation of tetrahydrofuran
- C07D 307/33: in position 2, the oxygen atom being in its keto or unsubstituted enol form
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 9/001: {acting on the CH-CH group of donors (1.3)}
- C12N 9/13: {transferring sulfur containing groups (2.8)}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/88: Lyases (4.)
- C12N 9/93: {Ligases (6)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 17/04: containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C}
- C12P 17/10: Nitrogen as only ring hetero atom
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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)}
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention provides non-naturally occurring microbial organisms comprising a 1,4-butanediol (BDO) pathway comprising at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO and further optimized for expression of BDO. The invention additionally provides methods of using such microbial organisms to produce BDO.

## Claims

### Claim 1

A non-naturally occurring Escherichia coli, comprising a 1,4-butanediol (BDO) pathway comprising one or more heterologous polynucleotides encoding BDO pathway enzymes expressed in a sufficient amount to produce BDO, wherein said E. coli:
(A) comprises a BDO pathway comprising:
(a) alpha-ketoglutarate decarboxylase; or alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase; or glutamate:succinate semialdehyde transaminase and glutamate decarboxylase;
(b) 4-hydroxybutyrate dehydrogenase;
(c) 4-hydroxybutyryl-CoA transferase; or 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and
(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase; or aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol; and
(B) comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system; or expresses an exogenous NADH insensitive citrate synthase.

### Claim 2 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said E. coli comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system.

### Claim 3 (depends on 2)

The non-naturally occurring E. coli of claim 2, wherein said gene encoding the protein in the aerobic respiratory control regulatory system is an arcA gene.

### Claim 4 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said E. coli expresses an exogenous NADH insensitive citrate synthase.

### Claim 5 (depends on 4)

The non-naturally occurring E. coli of claim 4, wherein said NADH insensitive citrate synthase is encoded by a gltA gene or a mutant gltA gene encoding an R163L mutant NADH insensitive citrate synthase.

### Claim 6 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said E. coli further expresses an exogenous phosphoenolpyruvate carboxykinase.

### Claim 7 (depends on 1)

The non-naturally occurring E. coli of claim 1, further comprising disruption of a gene encoding malate dehydrogenase.

### Claim 8 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein one or more of said one or more heterologous nucleotides encoding BDO pathway enzymes are integrated into the fimD locus of the E. coli.

### Claim 9 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said E. coli further expresses an exogenous non-phosphotransferase sucrose uptake system.

### Claim 10 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said E. coli further comprises disruption of endogenous lactate dehydrogenase, endogenous alcohol dehydrogenase, and endogenous pyruvate formate lyase.

### Claim 11 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said E. coli further expresses an exogenous pyruvate dehydrogenase.

### Claim 12 (depends on 11)

The non-naturally occurring E. coli of claim 11, wherein one or more genes encoding pyruvate dehydrogenase subunits is under the control of a pyruvate formate lyase promoter.

### Claim 13 (depends on 11)

The non-naturally occurring E. coli of claim 11, wherein said exogenous pyruvate dehydrogenase is NADH insensitive.

### Claim 14 (depends on 11)

The non-naturally occurring E. coli of claim 11, wherein said exogenous pyruvate dehydrogenase is encoded by the Klebsiella pneumonia lpdA gene.

### Claim 15 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase and 4-hydroxybutyryl-CoA/acetyl-CoA transferase are encoded by Porphyromonas gingivalis W83 genes.

### Claim 16 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said 4-hydroxybutyryl-CoA reductase is encoded by the Clostridium beijerinckii ald gene.

### Claim 17 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said E. coli further expresses an exogenous succinyl-CoA synthetase.

### Claim 18 (depends on 17)

The non-naturally occurring E. coli of claim 17, wherein said succinyl-CoA synthetase is encoded by the Escherichia coli sucCD genes.

### Claim 19 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said alpha-ketoglutarate decarboxylase is encoded by the Mycobacterium bovis sucA gene.

### Claim 20 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said 4-hydroxybutyrate kinase and said phosphotrans-4-hydroxybutyrylase are encoded by the Clostridium acetobutylicum buk1 and ptb genes.

### Claim 21 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said 4-hydroxybutyryl-CoA reductase is encoded by the Clostridium beijerinckii ald gene.

### Claim 22 (depends on 1)

The non-naturally occurring E. coli of claim 1, wherein said 4-hydroxybutyraldehyde reductase is encoded by the Geobacillus thermoglucosidasius adh1 gene.

### Claim 23 (depends on 6)

The non-naturally occurring E. coli of claim 6, wherein said phosphoenolpyruvate carboxykinase is encoded by the Haemophilus influenza phosphoenolpyruvate carboxykinase gene.