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Genetically engineered bacterium for L-sarcosine production as well as construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and sarcosine, applied in the field of genetic engineering, can solve problems such as undiscovered patent publications, achieve good industrial application prospects, and enhance the effect of gene expression

Active Publication Date: 2021-03-19
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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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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  • Genetically engineered bacterium for L-sarcosine production as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for L-sarcosine production as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for L-sarcosine production as well as construction method and application of genetically engineered bacterium

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Experimental program
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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 L-sarcosine production as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium is obtained by taking escherichia coli as a host, integrating and single copying imine reductase gene dpkA, single copying citrate synthase gene gltA, knocking out glyoxylic acid circulation inhibition gene iclR, knocking out malate synthase gene aceB, single copying isocitrate lyase gene aceA, single copying membrane combination transhydrogenase gene pntAB, knocking out 2-keto acid reductase gene ycdW, single copying phosphoenolpyruvate carboxylase gene ppc, and knocking out pyruvate kinase gene pykF on a genome of the escherichia coli. After system metabolism transformation, theengineering bacteria can synthesize the L-sarcosine by taking glucose and methylamine as main raw materials, and the yield of the L-sarcosine can reach 10 g / L after fermentation is carried out in a 5L fermentation tank for 30 hours.

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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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 UNIVERSITY OF SCIENCE AND TECHNOLOGY
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