# US 10,865,429 B2 · Microorganism having PHA synthase-coding genes and method for producing PHA using same

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- Number: US 10,865,429 B2
- Document ID: US10865429B2
- Status: Granted
- Kind: B2
- Application number: 15/763,323
- Filed: 2016-09-15
- Published: 2018-10-25
- Granted: 2020-12-15
- Earliest claimed: 2015-09-28
- Assignees: Kaneka Corporation
- Applicants: Kaneka Corporation
- Inventors: Shingo Kobayashi; Hitoshi Sashiwa; Tetsuya Fujiki
- Web page: https://patentgrep.com/patent/US10865429B2

## CPC

- C12P 7/625: Polyesters of hydroxy carboxylic acids
- C08G 63/06: derived from hydroxycarboxylic acids
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12Y 203/01: transferring groups other than amino-acyl groups (2.3.1)
- 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)}
- C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- 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

An object of the invention is to improve the crystallization speed of a PHA copolymer which is to be slowly crystallized, and improve the melt workability and productivity. A microorganism is used which has genes encoding two or more different PHA synthases derived from the genus Aeromonas. The genes encoding the PHA synthases derived from the genus Aeromonas preferably include genes encoding at least two PHA synthases which are capable of synthesizing a copolymer PHA including, as monomer unit species, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and which are different in substrate specificity toward 3-hydroxyhexanoic acid from each other. When this microorganism is cultured, a PHA mixture can be produced which includes three or more PHA species different in melting point from each other.

## Claims

### Claim 1

A microorganism, comprising genes encoding at least two PHA synthases which are different in substrate specificity toward 3-hydroxyhexanoic acid from each other and are obtained from the genus Aeromonas,
wherein the microorganism is a transformant of a host, and wherein the host is a microorganism belonging to the genus Cupriavidus, and
wherein one of the at least two PHA synthases which has a higher substrate specificity toward 3-hydroxyhexanoic acid has an amino acid sequence in which 149th asparagine in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, and/or 171th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is substituted with glycine.

### Claim 2 (depends on 1)

The microorganism according to claim 1, wherein the at least two PHA synthases are capable of synthesizing a copolymer PHA comprising, as its monomer unit species, 3-hydroxybutvric acid and 3-hydroxyhexanoic acid.

### Claim 3 (depends on 1)

The microorganism according to claim 1, wherein one of the genes encoding the at least two PHA synthases encodes a PHA synthase which has a lower substrate specificity toward 3-hydroxyhexanoyl-CoA and has the amino acid sequence of SEQ ID NO: 1.

### Claim 4 (depends on 1)

The microorganism according to claim 1, wherein one of the genes encoding the at least two PHA synthases encodes a PHA synthase which has a lower substrate specificity toward 3-hydroxyhexanoic acid and has an amino acid sequence in which 505th alanine in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than alanine.

### Claim 5 (depends on 1)

The microorganism according to claim 1, further comprising a gene encoding a PHA synthase which is derived from the genus Cupriavidus.

### Claim 6 (depends on 1)

The microorganism according to claim 1, wherein the microorganism belonging to the genus Cupriavidus is Cupriavidus necator.

### Claim 7 (depends on 1)

The microorganism according to claim 1, further comprising a gene encoding R-body-specific enoyl-CoA hydratase.

### Claim 8 (depends on 1)

The microorganism according to claim 1, wherein one of the at least two PHA synthases that has a lower substrate specificity toward 3-hydroxyhexanoic acid is a PHA synthase comprising the amino acid sequence of SEQ ID NO: 1.

### Claim 9 (depends on 1)

A method for producing a PHA mixture, comprising culturing the microorganism of claim 1.

### Claim 10 (depends on 9)

The method for producing a PHA mixture according to claim 9, wherein at least one of the at least two PHAs in the PHA mixture comprises, as its monomer unit species, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

### Claim 11 (depends on 9)

The method for producing a PHA mixture according to claim 9, wherein from the at least two PHAs in the PHA mixture, a PHA having a highest melting point is a PHA having an endothermic peak at 160 to 185° C. in a DSC yielded by annealing the mixture at 160° C.

### Claim 12 (depends on 9)

The method for producing a PHA mixture according to claim 9, wherein from the at least two PHAs in the PHA mixture, a PHA having a lowest melting point is a PHA having an endothermic peak at 90 to 135° C. in a DSC of the PHA mixture.