# US 8,541,652 B2 · Mutants of PHA synthase from Pseudomonas sp. 6-19 and method for preparing lactate homopolymer or copolymer using the same

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- Number: US 8,541,652 B2
- Document ID: US8541652B2
- Status: Granted
- Kind: B2
- Application number: 12/312,676
- Filed: 2007-11-21
- Published: 2010-02-25
- Granted: 2013-09-24
- Earliest claimed: 2006-11-23
- Assignees: LG Chem, Ltd.
- Applicants: Taek-Ho Yang; Hye-Ok Kang; Si-Jae Park; Sang-Hyun Lee; Eun-Jeong Lee; Tae-wan Kim; Sang-Yup Lee
- Inventors: Taek-Ho Yang; Hye-Ok Kang; Si-Jae Park; Sang-Hyun Lee; Eun-Jeong Lee; Tae-wan Kim; Sang-Yup Lee
- Web page: https://patentgrep.com/patent/US8541652B2

## CPC

- C12N 9/1025: {Acyltransferases (2.3)}
- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- C12P 7/625: Polyesters of hydroxy carboxylic acids
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}

## Abstract

The present invention relates to polyhydroxyalkanoate synthase (PHA synthase) mutant originated from Pseudomonas sp. 6-19 (KCTC 11027BP) which can prepare lactate polymer and/or copolymer by using lactyl-CoA as a substrate. The present invention relates to a method for preparing lactate polymer and/or copolymer with the synthase mutant. The polyhydroxyalkanoate synthase mutants of the present invention originated from Pseudomonas sp. 6-19 can efficiently prepare lactate polymer and/or copolymer by using as a substrate lactyl-CoA which is difficult to be used as a substrate by conventional polyhydroxyalkanoate synthase.

## Claims

### Claim 1

A recombinant vector comprising:
a gene encoding propionyl-CoA transferase (pct), and
a gene encoding polyhydroxyalkanoate synthase mutant using lactyl-CoA as a substrate to produce lactate polymer or lactate copolymer,
wherein the polyhydroxyalkanoate synthase mutant has an amino acid sequence of SEQ. ID No: 10, wherein at least the glutamine at position 481 of the amino acid sequence of SEQ. ID No: 10 is mutated.

### Claim 2 (depends on 1)

The recombinant vector of claim 1, wherein at least one amino acid selected from the group consisting of glutamate at position 130; serine at position 325; and serine at position 477 in SEQ. ID No: 10 is further mutated.

### Claim 3 (depends on 2)

The recombinant vector of claim 2, wherein the amino acid sequence in SEQ. ID No: 10 has any mutation of:
a) S325T and Q481M;
b) E130D and Q481K;
c) S325T and Q481K;
d) E130D and Q481M;
e) E130D and Q481R;
f) E130D, S325T and Q481M;
g) E130D, S325T and Q481K;
h) E130D, S477R and Q481K;
i) E130D, S477R and Q481M;
j) E130D, S477R and Q481R;
k) E130D, S477H and Q481K;
l) E130D, S477H and Q481M;
m) E130D, S477H and Q481R;
n) E130D, S477F and Q481K;
o) E130D, S477F and Q481M;
p) E130D, S477F and Q481R;
q) E130D, S477Y and Q481K;
r) E130D, S477Y and Q481M;
s) E130D, S477Y and Q481R;
t) E130D, S325T, S477R and Q481M;
u) E130D, S325T, S477R and Q481K;
v) E130D, S325T, S477F and Q481M;
w) E130D, S325T, S477G and Q481M; or
x) E130D, S325T, S477F and Q481K.

### Claim 4 (depends on 1)

A cell or plant transformed with the recombinant vector of claim 1.

### Claim 5 (depends on 1)

A cell or plant obtained from transformation with the recombinant vector of claim 1, wherein the original cell or plant does not have a gene encoding propionyl-CoA transferase.

### Claim 6 (depends on 5)

A method for preparing lactate polymer or 3-hydroxyalkanoate-lactate copolymer, wherein the method comprises culturing the cell or plant of claim 5.

### Claim 7 (depends on 6)

The method of claim 6, wherein the culturing is performed in a medium comprising 3-hydroxyalkanoate and the copolymer is a copolymer comprising 3-hydroxyalkanoate monomer unit and lactate monomer unit.

### Claim 8 (depends on 7)

The method of claim 7, wherein the 3-hydroxyalkanoate is at least one selected from the group consisting of 3-hydroxybutyrate, 3-hydroxyvalerate, (D)-3-hydroxycarboxylic acid of the medium chain length (C6-14), 3-hydroxypropionic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, 3-hydroxy-4-pentenoic acid, 3-hydroxy-4-trans-hexenoic acid, 3-hydroxy-4-cis-hexenoic acid, 3-hydroxy-5-hexenoic acid, 3-hydroxy-6-trans-octenoic acid, 3-hydroxy-6-cis-octenoic acid, 3-hydroxy-7-octenoic acid, 3-hydroxy-8-nonenoic acid, 3-hydroxy-9-decenoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-hydroxy-6-cis-dodecenoic acid, 3-hydroxy-5-cis-tetradecenoic acid, 3-hydroxy-7-cis-tetradecenoic acid, 3-hydroxy-5,8-cis-cis-tetradecenoic acid, 3-hydroxy-4-methylvaleric acid, 3-hydroxy-4-methylhexanoic acid, 3-hydroxy-5-methylhexanoic acid, 3-hydroxy-6-methylheptanoic acid, 3-hydroxy-4-methyloctanoic acid, 3-hydroxy-5-methyloctanoic acid, 3-hydroxy-6-methyloctanoic acid, 3-hydroxy-7-methyloctanoic acid, 3-hydroxy-6-methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid, 3-hydroxy-8-methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy-7-methyl-6-octenoic acid, malic acid, 3-hydroxysuccinic acid-methyl ester, 3-hydroxyadipinic acid-methyl ester, 3-hydroxysuberic acid-methyl ester, 3-hydroxyazelaic acid-methyl ester, 3-hydroxysebacic acid-methyl ester, 3-hydroxysuberic acid-ethyl ester, 3-hydroxysebacic acid-ethyl ester, 3-hydroxypimelic acid-propyl ester, 3-hydroxysebacic acid-benzyl ester, 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxybutyric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid, para-cyanophenoxy-3-hydroxybutyric acid, para-cyanophenoxy-3-hydroxyvaleric acid, para-cyanophenoxy-3-hydroxyhexanoic acid, para-nitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5-phenylvaleric acid, 3-hydroxy-5-cyclohexylbutyric acid, 3,12-dihydroxydodecanoic acid, 3,8-dihydroxy-5-cis-tetradecenoic acid, 3-hydroxy-4,5-epoxydecanoic acid, 3-hydroxy-6,7-epoxydodecanoic acid, 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3-hydroxyheptanoic acid, 9-cyano-3-hydroxynonanoic acid, 3-hydroxy-7-fluoroheptanoic acid, 3-hydroxy-9-fluorononanoic acid, 3-hydroxy-6-chlorohexanoic acid, 3-hydroxy-8-chlorooctanoic acid, 3-hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid, 3-hydroxy-11-bromoundecanoic acid, 3-hydroxy-2-butenoic acid, 3-hydroxy-2-methylbutyric acid, 3-hydroxy-2-methylvaleric acid and 3-hydroxy-2,6-dimethyl-5-heptenoic acid.