# US 8,541,212 B2 · Genetic modification for production of 3-hydroxypropionic acid

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- Number: US 8,541,212 B2
- Document ID: US8541212B2
- Status: Granted
- Kind: B2
- Application number: 13/368,953
- Filed: 2012-02-08
- Published: 2012-12-27
- Granted: 2013-09-24
- Earliest claimed: 2011-06-27
- Assignees: Samsung Electronics Co., Ltd.
- Applicants: Jae Young Kim; Jae Chan Park; Sung Min Park; Hyun Min Koo; Byung Jo Yu; Hwa Young Cho; Young kyoung Park
- Inventors: Jae Young Kim; Jae Chan Park; Sung Min Park; Hyun Min Koo; Byung Jo Yu; Hwa Young Cho; Young kyoung Park
- Web page: https://patentgrep.com/patent/US8541212B2

## CPC

- C12P 7/42: Hydroxy-carboxylic acids
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1)
- C12Y 101/01027: L-Lactate dehydrogenase (1.1.1.27)
- C12Y 203/01008: Phosphate acetyltransferase (2.3.1.8)
- 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

## Abstract

A method of increasing 3-HP production efficiency by inhibiting expression of a lactate dehydrogenase, a phosphotransacetylase, and an alcohol dehydrogenase in production of 3-HP using a malonic semialdehyde reduction pathway to prevent metabolite leak and increase a malonyl-CoA pool is disclosed.

## Claims

### Claim 1

A recombinant microorganism for producing 3-hydroxypropionic acid (3-HP), wherein the recombinant microorganism comprises a metabolic pathway in which metabolites are sequentially produced in a sequence of pyruvate, acetyl-CoA, malonyl-CoA and malonic semialdehyde, and comprises a gene encoding an enzyme that reduces malonyl-CoA into malonic semialdehyde and a gene encoding an enzyme that reduces malonic semialdehyde into 3-HP; and wherein the recombinant microorganism has undergone deletion or knockout of a gene encoding at least one selected from the group consisting of lactate dehydrogenase, phosphotransacetylase, and alcohol dehydrogenase.

### Claim 2 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the recombinant microorganism belongs to a genus selected from Escherichia, Saccharomyces, and Kluyveromyces.

### Claim 3 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the recombinant microorganism is Escherichia coli.

### Claim 4 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the gene encoding the lactate dehydrogenase is a ldhA or a homolog or variant thereof.

### Claim 5 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the gene encoding the phosphotransacetylase is a pta or a homolog or variant thereof.

### Claim 6 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the alcohol dehydrogenase is an ethanol dehydrogenase.

### Claim 7 (depends on 6)

The recombinant microorganism for producing 3-HP of claim 6, wherein the gene encoding the ethanol dehydrogenase is adhE or a homolog or variant thereof.

### Claim 8 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the recombinant microorganism has undergone deletion or knockout of the genes encoding lactate dehydrogenase, phosphotransacetylase, and alcohol dehydrogenase.

### Claim 9 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the recombinant microorganism reduces or inhibits conversion of pyruvate into lactate, conversion of acetyl-CoA into acetyl phosphate (acetyl P), or conversion of acetyl-CoA into ethanol.

### Claim 10 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the recombinant microorganism is a microorganism deposited in the Korean Collection for Type Cultures as Deposit No. KCTC11947BP, KCTC11948BP, KCTC11949BP, or KCTC11950BP.

### Claim 11 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the gene encoding an enzyme that reduces malonyl-CoA into malonic semialdehyde is a gene encoding a malonyl-CoA reductase (mcr), and the gene encoding an enzyme that reduces malonic semialdehyde into 3-HP is a gene encoding a malonate semialdehyde reductase (msr).

### Claim 12 (depends on 1)

The recombinant microorganism for producing 3-HP of claim 1, wherein the recombinant microorganism further comprise a gene encoding an NADPH regeneration enzyme.

### Claim 13 (depends on 12)

The recombinant microorganism for producing 3-HP of claim 12, wherein the gene encoding the NADPH regeneration enzyme is a gene encoding a transhydrogenase or a gene encoding a glyceraldehyde-3-phosphate dehydrogenase.

### Claim 14 (depends on 13)

The recombinant microorganism for producing 3-HP of claim 13, wherein the gene encoding the transhydrogenase is pyridine nucleotide transhydrogenase (pntAB) or soluble pyridine nucleotide transhydrogenase (udhA), and the gene encoding the glyceraldehyde-3-phosphate dehydrogenase is non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (gapN).

### Claim 15 (depends on 1)

A method of producing 3-HP in a recombinant microorganism according to claim 1, the method comprising:
converting pyruvate into acetyl-CoA;
converting acetyl-CoA into malonyl-CoA;
converting malonyl-CoA into malonic semialdehyde; and
converting malonic semialdehyde into 3-HP,
wherein, during the conversion of pyruvate into acetyl-CoA, an activity of at least one enzyme selected from the group consisting of lactate dehydrogenase, phosphotransacetylase, and alcohol dehydrogenase is inhibited in the microorganism.

### Claim 16 (depends on 15)

The method of producing 3-HP of claim 15, wherein the inhibition of the enzyme activity is performed by deletion or knockout of at least one gene selected from the group consisting of adhE, pta, and adhE in the microorganism.

### Claim 17 (depends on 15)

The method of producing 3-HP of claim 15, wherein the method increases malonyl-CoA pools in a microbial cell.

### Claim 18 (depends on 15)

The method of producing 3-HP of claim 15, wherein the method is performed by incubating the recombinant microorganism in a medium containing a carbon substrate.

### Claim 19 (depends on 18)

The method of producing 3-HP of claim 18, wherein the carbon substrate is at least one selected from the group consisting of glucose, sucrose, cellulose, and glycerol.