# US 10,246,725 B2 · Microorganisms and methods for producing pyruvate, ethanol, and other compounds

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- Number: US 10,246,725 B2
- Document ID: US10246725B2
- Status: Granted
- Kind: B2
- Application number: 15/815,327
- Filed: 2017-11-16
- Published: 2018-03-29
- Granted: 2019-04-02
- Earliest claimed: 2014-09-09
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Jennifer L. Reed; Xiaolin Zhang
- Web page: https://patentgrep.com/patent/US10246725B2

## CPC

- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/88: Lyases (4.)
- C12P 7/06: Ethanol, i.e. non-beverage
- C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1)
- C12Y 401/01001: Pyruvate decarboxylase (4.1.1.1)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

Microorganisms comprising modifications for producing pyruvate, ethanol, and other compounds. The microorganisms comprise modifications that reduce or ablate activity of one or more of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, phosphate acetyltransferase, acetate kinase, pyruvate oxidase, lactate dehydrogenase, cytochrome terminal oxidase, succinate dehydrogenase, 6-phosphogluconate dehydrogenase, glutamate dehydrogenase, pyruvate formate lyase, pyruvate formate lyase activating enzyme, and isocitrate lyase. The microorganisms optionally comprise modifications that enhance expression or activity of pyruvate decarboxylase and alcohol dehydrogenase. The microorganisms are optionally evolved in defined media to enhance specific production of one or more compounds. Methods of producing compounds with the microorganisms are provided.

## Claims

### Claim 1

A microorganism comprising activity-reducing or activity-ablating mutations in endogenous genes encoding pyruvate dehydrogenase, pyruvate oxidase, succinate dehydrogenase, and 6-phosphogluconate dehydrogenase.

### Claim 2 (depends on 1)

The microorganism of claim 1, further comprising an activity-reducing or activity-ablating mutation in an endogenous gene encoding an enzyme selected from the group consisting of a pyruvate formate lyase and a pyruvate formate lyase activating enzyme.

### Claim 3 (depends on 1)

The microorganism of claim 1, wherein the microorganism comprises one or more recombinant genes encoding one or more enzymes selected from the group consisting of a pyruvate decarboxylase and an alcohol dehydrogenase.

### Claim 4 (depends on 3)

The microorganism of claim 3, further comprising an activity-reducing or activity-ablating mutation in an endogenous gene encoding an enzyme selected from the group consisting of a pyruvate formate lyase and a pyruvate formate lyase activating enzyme.

### Claim 5 (depends on 1)

The microorganism of claim 1, wherein the activity-reducing or activity-ablating mutations in the endogenous genes are independently selected from the group consisting of a nucleotide substitution in the endogenous gene, a nucleotide insertion in the endogenous gene, a partial deletion of the endogenous gene, and a complete deletion of the endogenous gene.

### Claim 6 (depends on 1)

The microorganism of claim 1, wherein the microorganism is a bacterium or a yeast.

### Claim 7 (depends on 1)

The microorganism of claim 1, wherein the microorganism is a bacterium.

### Claim 8 (depends on 1)

The microorganism of claim 1, wherein the microorganism is produced by sequentially culturing a precursor microorganism in media comprising decreasing concentrations of acetate, wherein the precursor microorganism comprises the activity-reducing or activity-ablating mutations of the microorganism, and wherein the microorganism produced from sequentially culturing the precursor microorganism exhibits one or more of increased growth rate compared to the precursor microorganism and increased pyruvate production compared to the precursor microorganism.

### Claim 9 (depends on 8)

The microorganism of claim 8, wherein the concentrations of acetate in the media in which the precursor microorganism is sequentially cultured to produce the microorganism range from about 0.1 mg/L acetate to about 3 g/L acetate.

### Claim 10 (depends on 1)

A method for producing a chemical comprising culturing the microorganism as recited in claim 1.

### Claim 11 (depends on 10)

The method of claim 10, wherein the microorganism further comprises:
an activity-reducing or activity-ablating mutation in an endogenous gene encoding an enzyme selected from the group consisting of a pyruvate formate lyase and a pyruvate formate lyase activating enzyme; and
one or more recombinant genes encoding one or more enzymes selected from the group consisting of a pyruvate decarboxylase and an alcohol dehydrogenase.

### Claim 12 (depends on 10)

The method of claim 10, wherein the culturing comprises culturing the microorganism in a medium, the chemical is selected from the group consisting of pyruvate and ethanol, and the method further comprises purifying the chemical from the medium.

### Claim 13 (depends on 10)

The method of claim 10, wherein the culturing comprises culturing the microorganism in a medium comprising a biomass hydrolysate.

### Claim 14 (depends on 1)

The microorganism of claim 1, further comprising an activity-reducing or activity-ablating mutation in an endogenous gene encoding a pyruvate formate lyase.

### Claim 15 (depends on 14)

The microorganism of claim 14, further comprising recombinant genes encoding a pyruvate decarboxylase and an alcohol dehydrogenase.

### Claim 16 (depends on 1)

The microorganism of claim 1, further comprising an activity-reducing or activity-ablating mutation in an endogenous gene encoding a pyruvate formate lyase activating enzyme.

### Claim 17 (depends on 16)

The microorganism of claim 16, further comprising recombinant genes encoding a pyruvate decarboxylase and an alcohol dehydrogenase.

### Claim 18 (depends on 1)

The microorganism of claim 1, further comprising recombinant genes encoding a pyruvate decarboxylase and an alcohol dehydrogenase.

### Claim 19 (depends on 18)

The microorganism of claim 18, further comprising an activity-reducing or activity-ablating mutation in an endogenous gene encoding an enzyme selected from the group consisting of a pyruvate formate lyase and a pyruvate formate lyase activating enzyme.

### Claim 20 (depends on 10)

The method of claim 10, wherein the microorganism further comprises:
an activity-reducing or activity-ablating mutation in an endogenous gene encoding an enzyme selected from the group consisting of a pyruvate formate lyase and a pyruvate formate lyase activating enzyme; and
recombinant genes encoding a pyruvate decarboxylase and an alcohol dehydrogenase.