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Recombinant escherichia coli capable of producing O-acetyl-L-homoserine at high yield and application of recombinant escherichia coli

A technology of recombinant Escherichia coli, homoserine, applied in the field of metabolic engineering

Active Publication Date: 2020-12-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the metabolic pathway modification of OAH biosynthesis using glycerol as carbon source by modified strains has not been reported yet.

Method used

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  • Recombinant escherichia coli capable of producing O-acetyl-L-homoserine at high yield and application of recombinant escherichia coli
  • Recombinant escherichia coli capable of producing O-acetyl-L-homoserine at high yield and application of recombinant escherichia coli
  • Recombinant escherichia coli capable of producing O-acetyl-L-homoserine at high yield and application of recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of plasmid pTrc99A-metX expressing L-homoserine acetyltransferase

[0035] Using the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template, together with primers MetX-F and primers MetX-R, PCR amplification was carried out. PCR reaction conditions: pre-denaturation at 95°C for 5min, 95°C for 30s, 60°C for 30s, 72°C for 1min, a total of 30 cycles, and finally extension at 72°C for 10min. The PCR product was detected by 1.0% agarose gel electrophoresis, and the fragment was recovered and purified by cutting the gel.

[0036] The empty vector pTrc99A was used as a template, together with primers pTrc99A-F and primers pTrc99A-R for PCR amplification. PCR reaction conditions: pre-denaturation at 95°C for 5min, 95°C for 30s, 60°C for 30s, 72°C for 4min, a total of 30 cycles, and finally extension at 72°C for 10min. The PCR products were detected by 1.0% agarose gel electrophoresis, and the gel-cut gel was used to recover and purify t...

Embodiment 2

[0040] Embodiment 2: Construction of bacterial strain OAH-1

[0041]Using CRISPR-Cas9 gene editing technology, the metJ, metI, metB, thrB, metA, lysA and iclR genes in E. coli W3110 (purchased from The Coli Genetic Stock Center) were knocked out to obtain large intestine E. coli W3110ΔmetJΔmetIΔmetBΔthrBΔmetAΔlysAΔiclR (Peng Liu et al. 2020. Multiplex design of metabolic network for production of L-homoserine in Escherichia coli. Applied and Environmental Microbiology). Escherichia coli W3110ΔmetJΔmetIΔmetBΔthrBΔmetAΔlysAΔiclR was used as the starting strain, and the CRISPR-Cas9 gene editing technology was used (Yu Jiang et al.2015Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System.Applied Environmental Microbiology.81,2506-r4tA). The in situ promoters of the thrA and eamA genes were replaced by the trc promoter to obtain the recombinant strain OAH-1:

[0042] Construction of pTarget plasmid: Using the pTarget F plasmid (Addgene Plasmid#62226) as a tem...

Embodiment 3

[0046] Example 3: Construction and Shake Flask Fermentation of Strains OAH-2 / pTrc99A-metX, OAH-3 / pTrc99A-metX, OAH-4 / pTrc99A-metX Enhancing the Flux of Glycerol Metabolic Pathways to Produce O-Acetyl-L-Homoserine

[0047] Starting from the strain OAH-1 (W3110, ΔmetJΔmetIΔmetBΔthrBΔmetAΔlysAΔlacI::Trc-rhtAΔiclR Trc-metL Trc-thrA Trc-rhtA Trc-eamA), the gene editing technology mediated by CRISPR-Cas9 (Yu Jiang et al.2015Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System.Applied Environmental Microbiology.81:2506-2514), respectively knock out the glpR gene, overexpress the glpF gene and overexpress the glpD gene (the method is the same as in Example 2), and obtain bacterial strain OAH-2, OAH -3 and OAH-4. Transform the constructed plasmid pTrc99A-metX into the above-mentioned genetic engineering bacteria and the control strain OAH-1 to obtain recombinant strains OAH-1 / pTrc99A-metX, OAH-2 / pTrc99A-metX, OAH-3 / pTrc99A-metX and OAH -4 / pTrc99A-metX.

[0048] ...

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Abstract

The invention relates to recombinant escherichia coli capable of producing O-acetyl-L-homoserine at high yield and application of the recombinant escherichia coli in preparation of O-acetyl-L-homoserine by microbial fermentation. The method comprises the following steps that knocking out of a gene glpR responsible for encoding a repression protein in a glycerol metabolic pathway is carried out, sothat constitutive expression of a glycerol kinase gene glpK and a glycerol 3-phosphate dehydrogenase gene is realized, and the flux of the glycerol metabolic pathway of a strain is enhanced; secondly, the gene is found to be a key gene in a glycerol metabolic pathway of the strain by overexpression of glpD gene; and finally, an in-situ promoter of the glpD gene is replaced with a trc promoter toindicate that the level of O-acetyl-L homoserine produced by strain fermentation is closely related to the expression level of the glpD gene. Through the combination of the modification strategies, the escherichia coli strain capable of producing the O-acetyl-L-homoserine at high yield is obtained.

Description

[0001] (1) Technical field [0002] The invention belongs to the field of metabolic engineering, in particular to a high-yield O-acetyl-L-homoserine recombinant Escherichia coli and its application. [0003] (2) Background technology [0004] In the metabolic process of microbial cells, the derivatives of L-homoserine include O-succinyl-L-homoserine (OSH) and O-acetyl homoserine (OAH). OSH can not only be used as a precursor for the biosynthesis of L-methionine, but also a platform compound for the synthesis of various C4 compounds. In addition, OAH is also a precursor compound with potential industrial application value, which can be used to prepare homoserine and methionine. OAH can be hydrolyzed to produce homoserine, and homoserine can be used as a raw material for the synthesis of platform compounds such as L-homoserine lactone, γ-butyrolactone, 1,4-butanediol, and the pesticide glufosinate-ammonium. In addition, OAH can directly react with methyl mercaptan to generate m...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N15/67C12N15/54C12N15/53C12P13/06C12R1/19
CPCC12N15/52C12N9/1029C12N9/1205C12N9/0006C12N15/70C12N15/902C12N15/67C12P13/06C12Y203/01031C12Y207/0103C12Y101/05003
Inventor 柳志强刘鹏张博牛坤郑裕国
Owner ZHEJIANG UNIV OF TECH
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