Carbonyl reductase variant and its use in preparation of (R)-4-chloro-3-hydroxybutyrate

Patent metadata

Field	Value
Publication number	US-10961515-B1
Application number	US-202016904450-A
Country	US
Kind code	B1
Filing date	Jun 17, 2020
Priority date	Feb 26, 2020
Publication date	Mar 30, 2021
Grant date	Mar 30, 2021

Abstract

Disclosed herein are a carbonyl reductase variant and its use in the preparation of (R)-4-chloro-3-hydroxybutyrate. The carbonyl reductase variant is obtained by mutating phenylalanine-85 in an amino acid sequence as shown in SEQ ID NO:4 to methionine. An amino acid or amino acids at one or more positions other than position 85 in the amino acid sequence of the carbonyl reductase may be further replaced. The application also provides a recombinant expression vector carrying the gene encoding the carbonyl reductase variant, a genetically-engineered bacterium carrying the carbonyl reductase variant gene and glucose dehydrogenase gene, and an application of this bacterium in the asymmetric reduction of 4-chloroacetoacetate to prepare (R)-4-chloro-3-hydroxybutyrate.

First claim

What is claimed is:

1. A carbonyl reductase variant, wherein the carbonyl reductase variant is obtained by:
mutating phenylalanine-85 in the amino acid sequence as shown in SEQ ID NO:4 to methionine.

2. A carbonyl reductase variant, wherein the carbonyl reductase variant is obtained by:
mutating phenylalanine-85 in the amino acid sequence as shown in SEQ ID NO:4 to methionine; and
replacing an amino acid at one or more positions other than position 85 in the amino acid sequence as shown in SEQ ID NO: 4; wherein the amino acid at one or more positions is tyrosine-128, phenylalanine-132 or valine-162, or a combination thereof.

3. The carbonyl reductase variant of claim 2 , wherein the tyrosine-128 is replaced with alanine, methionine, glycine, leucine, valine or isoleucine; the phenylalanine-132 is replaced with alanine, methionine, glycine, leucine, valine or isoleucine; and the valine-162 is replaced with alanine, methionine, glycine, leucine or isoleucine.

4. An isolated nucleic acid encoding the carbonyl reductase variant of claim 1 .

5. A recombinant expression vector comprising the nucleic acid of claim 4 .

6. A genetically-engineered bacterium, comprising:
the recombinant expression vector of claim 5 ; and
a recombinant expression vector carrying glucose dehydrogenase gene.

7. A method for preparing (R)-4-chloro-3-hydroxybutyrate, comprising:
reducing substrate 4-chloroacetoacetate (I) at 20-50° C. in an initial reaction system at pH of 6-10 to produce the (R)-4-chloro-3-hydroxybutyrate (II), as shown in the following reaction scheme:

wherein:
R is a linear or branched-chain C 1 -C 8 alkyl, a C 3 -C 8 cycloalkyl, or a mono-substituted or poly-substituted aryl or aralkyl;
the initial reaction system comprises the substrate 4-chloroacetoacetate (I), an enzyme catalyst, glucose, an organic solvent immiscible with water and a buffer solution; and
the enzyme catalyst is a whole cell of the genetically-engineered bacterium of claim 6 , or a crude enzyme obtained by the lysis of the whole cell of the genetically-engineered bacterium.

8. The method of claim 7 , wherein, based on a total volume of the initial reaction system, the substrate 4-chloroacetoacetate (I) has a concentration of 0.10-0.30 g/mL; the organic solvent has a volume percentage of 5%-40%; the buffer solution has a volume percentage of 95%-60%; the enzyme catalyst calculated as wet bacterial cells is 20%-200% by weight of the substrate 4-chloroacetoacetate (I); and a molar ratio of the glucose to the substrate 4-chloroacetoacetate (I) is 1-4:1.

9. The method of claim 7 , wherein the organic solvent is toluene.

10. The method of claim 7 , wherein the buffer solution is phosphate buffered solution.

11. The method of claim 7 , wherein an alkaline solution is added to keep the pH at 6-10 during the reaction.

Assignees

• Univ Fudan

Inventors

• Chen Fener
• Huang Zedu
• Wang Zexu
• Liu Minjie

Classifications

• C12P7/62

  Carboxylic acid esters · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title