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A kind of genetically engineered bacteria for L-sarcosine production and its construction method and application

A technology of genetically engineered bacteria and sarcosine, applied in the field of genetic engineering, can solve problems such as undiscovered patent publications, achieve the effect of enhancing gene expression and improving metabolic flow

Active Publication Date: 2021-05-25
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Through detection, no patent publications related to the patent application of the present invention have been found

Method used

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  • A kind of genetically engineered bacteria for L-sarcosine production and its construction method and application
  • A kind of genetically engineered bacteria for L-sarcosine production and its construction method and application
  • A kind of genetically engineered bacteria for L-sarcosine production and its construction method and application

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Embodiment 1

[0074] Embodiment 1: genetically engineered bacterial strain Escherichia coli Escherichia coli Construction of SAR:

[0075] 1 Methods of gene editing

[0076] In the present invention, the CRISPR / Cas9-mediated gene editing method is used with reference to the literature (MetabolicEngineering, 2015, 31:13-21.), and the two plasmids used in this method are pGRB and pREDCas9 respectively. Among them, pREDCas9 carries gRNA plasmid elimination system, Red recombination system of lambda phage and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg / L), cultured at 32°C; pGRB plasmid, with pUC18 as the backbone, including promoter J23100 , gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg / L), cultured at 37°C.

[0077] 2 Specific process of strain construction

[0078] 2.1 will P T7 - wxya (contain wxya gene and T7 promoter) integrated in the pseudogene wxya site

[0079] by ...

Embodiment 2

[0108] Embodiment 2: bacterial strain E. coli Production of L-Sarcosine by SAR Shake Flask Fermentation

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Abstract

The invention discloses a genetically engineered bacterium for the production of L-sarcosine and its construction method and application. The genetically engineered bacterium takes Escherichia coli as a host and integrates a single copy of the imine reductase gene on its genome wxya ; single copy citrate synthase gene gltA ; Knockout of glyoxylate cycle inhibitor genes iCR ; Knockout of the malate synthase gene ace B ; single copy isocitrate lyase gene aceA ; single-copy membrane-bound transhydrogenase gene wxya ; Knockout of the 2‑ketoacid reductase gene wxya ; single copy phosphoenolpyruvate carboxylase gene ppc ; Knockout of pyruvate kinase gene pykF get. After undergoing systematic metabolic transformation, the engineered bacteria can synthesize L-sarcosine with glucose and methylamine as the main raw materials. After 30 hours of fermentation in a 5L fermenter, the output of L-sarcosine can reach 10g / L.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a genetically engineered bacterium used for L-sarcosine production, its construction method and application. Background technique [0002] L-sarcosine (N-methyl-L-glycine) is an N-methylated amino acid, and its sodium salt, sodium sarcosine, is often used as a precursor for the synthesis of some important products. L-sarcosine is an important amino acid product. Sarcosine has a considerable effect on the repair of brain and muscle damage. At the same time, L-sarcosine can provide quick-acting energy for the body, thereby reducing body fatigue. . Sodium sarcosinate and its downstream products basically have low toxicity and are easy to degrade in nature, which is more friendly to the environment. Therefore, L-sarcosine has wide application prospects in the fields of breeding, food, health care and medicine. [0003] The existing production of L-sarcosine is mainly f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N1/21C12N15/54C12N15/60C12N15/31C12P13/04C12R1/19
CPCC12N9/0006C12N9/0028C12N9/0036C12N9/1025C12N9/1205C12N9/88C07K14/245C12P13/04C12Y105/01001C12Y203/03003C12Y203/03009C12Y401/03001C12Y401/01031C12Y207/0104C12N2800/22
Inventor 范晓光田俊宇周宇航吴亚松陈夏非戈瑶谢希贤徐庆阳
Owner TIANJIN UNIV OF SCI & TECH
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