Method for producing 1,2-amino alcohol compound by whole cell transformation

Patent metadata

Field	Value
Publication number	US-11060076-B2
Application number	US-201916682486-A
Country	US
Kind code	B2
Filing date	Nov 13, 2019
Priority date	Nov 13, 2018
Publication date	Jul 13, 2021
Grant date	Jul 13, 2021

Abstract

The present invention discloses a method for producing a 1,2-amino alcohol compound by utilizing whole-cell transformation, and belongs to the technical field of gene engineering and microorganism engineering. According to the present invention, engineered Escherichia coli co-expresses epoxide hydrolase, alcohol dehydrogenase, ω-transaminase and glutamate dehydrogenase, is capable of realizing whole-cell catalysis of an epoxide in one step to synthesize a 1,2-amino alcohol compound, and meanwhile, can realize regeneration of coenzyme NADP + and an amino doner L-Glu; alcohol dehydrogenase expressed by the engineered Escherichia coli is RBS optimized alcohol dehydrogenase, and such RBS optimization can control the expression quantity of alcohol dehydrogenase, so that the catalysis rate of alcohol dehydrogenase and transaminase can achieve an optimum ratio, to eliminate influence caused by a rate-limiting step in a catalyzing course.

First claim

What is claimed is:

1. An engineered Escherichia coli , wherein the engineered Escherichia coli comprises recombinant plasmid A and recombinant plasmid B; the recombinant plasmid A comprising a target gene A and an expression vector; the recombinant plasmid B comprising a target gene B, a target gene C, and an expression vector; the target gene A being a gene encoding epoxide hydrolase (SpEH) comprising SEQ ID NO: 1; the target gene B being a gene encoding alcohol dehydrogenase (MnADH) comprising SEQ ID NO: 2; the target gene C being a gene encoding w-transaminase (PAKω-TA), wherein the amino acid sequence expressed by target gene C comprises SEQ ID NO: 3;
and wherein the recombinant plasmid A also comprises a target gene D; the target gene D being a gene encoding glutamate dehydrogenase (GluDH) comprising SEQ ID NO: 5.

2. The engineered Escherichia coli according to claim 1 , wherein the alcohol dehydrogenase (MnADH) is optimized by RBS; RBS optimization of alcohol dehydrogenase (MnADH) meaning that an RBS sequence used for regulating alcohol dehydrogenase (MnADH) and located at the upstream of alcohol dehydrogenase (MnADH) on recombinant plasmid B is substituted; the substituted RBS sequence comprising SEQ ID NO: 4.

3. A method for producing a 1,2-amino alcohol compound, wherein the method uses the engineered Escherichia coli described in claim 1 .

4. A method for producing a 1,2-amino alcohol compound, the method comprising:
providing a catalysis system comprising a substrate selected from epoxyethylbenzene, epoxypropane, epoxybutane, epichlorohydrin or epoxypentane and the engineered Escherichia coli according to claim 1 ,
adding coenzyme NADP+, amino donor L-Glu, and ammonium chloride, and
reacting for 10-15 hours.

Assignees

• Univ Jiangnan

Inventors

• Rao Zhiming
• Liu Song
• Gao Renjie
• Zhang Xian
• Yang Taowei
• Xu Meijuan

Classifications

• C12P13/001

  Amines; Imines · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title

• C12N9/14 Primary

  Hydrolases (3) · CPC title

• C12Y206/01018

  Beta-alanine-pyruvate transaminase (2.6.1.18) · CPC title

• C12Y104/01002

  Glutamate dehydrogenase (1.4.1.2) · CPC title