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Genetically engineered bacterium for producing L-methionine at high yield as well as construction and application of genetically engineered bacterium

A technology of genetically engineered bacteria and methionine, applied to genetically engineered bacteria with high production of L-methionine and its construction and application fields, can solve the problems of methionine dependence on imports, limited methionine production capacity, etc.

Active Publication Date: 2021-05-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Nowadays, my country has a great demand for methionine, but the domestic production

Method used

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  • Genetically engineered bacterium for producing L-methionine at high yield as well as construction and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing L-methionine at high yield as well as construction and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing L-methionine at high yield as well as construction and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Fermentation method and content determination of L-methionine high-yielding strain

[0051] The fermentation method is as follows:

[0052] The constructed strain was inoculated into 10 mL of LB medium containing 50 mg / L of Kan, and after 8-12 h, the genetically engineered bacterial strain was transferred to 20 mL of MS fermentation medium containing Kan, and CaCO was added during inoculation. 3 0.3g / L, VB 12 The mother liquor was 20 μl / mL, the lysine mother liquor was 20 μl / mL, and the fermentation was carried out at 28° C. and 180 rpm until 48 hours.

[0053] The MS fermentation medium is composed as follows: glucose 20g / L, (NH 4 ) 2 SO 4 16g / L, KH 2 PO 4 1g / L, Na 2 S 2 o 3 2g / L, yeast extract 2g / L, 1ml / L trace element solution, the solvent is deionized water, the pH value is natural; the composition of trace element solution is: 500g / L MgSO 4 ·7H 2 0,5g / L FeSO 4 ·7H 2 O, 5g / L MnSO 4 ·8H 2 0,5g / L ZnSO 4 , the solvent is deionized water. ...

Embodiment 2

[0058] Embodiment 2: Construction of effective bacterial strain E.coli W3110 M2 / pAm metF and its shake flask fermentation

[0059] (1) Construct the pAm metF plasmid: use the previously constructed pAm plasmid (see the plasmid map Figure 7 ) as a template, using pAm-line-F / pAm-line-R as primers to obtain the PCR linear amplification product pAm-line plasmid, the PCR product was digested by DpnI at 37°C for 3 hours, and then the DNA fragment was recovered with the Clean up kit; Using the genome of E.coli W3110 as a template and using pmetF-F / pmetF-R as primers to obtain the PCR amplification product metF, the DNA fragment was recovered by the Clean up kit; according to ClonExpress (One step clone kit, Vazyme Biotech, Nanjing, China) The instructions connect the linearized pAm-line plasmid and fragment metF together, and transform the ligated product into DH5α competent through chemical transformation; finally select the clones, and obtain the pAm metF plasmid through sequencin...

Embodiment 3

[0064] Embodiment 3: Construction effective bacterial strain E.coli W3110 M2(Trc-fliy) / pAm metF and shake flask fermentation thereof

[0065] Using Escherichia coli W3110 M2 / pAm metF as the starting strain, use CRISPR-Cas9-mediated gene editing technology (Yu Jiang et al.2015Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System.Applied Environmental Microbiology.81:2506-2514 ), replace the original promoter of fliY in the genome with the trc promoter derived from pTrc99A (the nucleotide sequence is shown in SEQ ID No.1), so as to enhance the expression intensity of fliY.

[0066] (1) Construction of pTarget-fliY plasmid: Use pTarget F plasmid (Addgene Plasmid#62226) as a template, use pT-fliY-F / pT-fliY-R as primers for PCR amplification, and the PCR product is digested by Dpn I at 37°C 3h, and then transformed into E.coli DH5α, screened on the spectacle enzyme plate, and sequenced to verify that the correct pTarget-fliY plasmid was obtained, which was used...

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Abstract

The invention relates to a genetically engineered bacterium for producing L-methionine at high yield as well as a construction method and application of the genetically engineered bacterium. According to the invention, an L-methionine synthesis network of escherichia coli is modified, and the utilization capacity of escherichia coli on methylenetetrahydrofolic acid is enhanced by enhancing metF and GCV in a carbon module of an escherichia coli-L-methionine synthesis pathway; by replacing original promoters of fliY and malY with a Trc promoter derived from pTrc99A, a cysteine/cystine internal transport pathway and a cysteine utilization pathway are enhanced, and metabolic inhibition of escherichia coli caused by cysteine generation is relieved; and by knocking out glyA of escherichia coli and introducing glyA derived from Arthrobacter sp.FB24 for overexpression on plasmids, limitation of serine hydroxymethyltransferase of large intestines is relieved, finally, high-yield bacterium containing plasmids is obtained, and the yield of L-methionine is increased to 3.83 g/L from 2.8 g/L.

Description

[0001] (1) Technical field [0002] The invention relates to a genetically engineered bacterium capable of high-yielding L-methionine, a construction method thereof, and an application thereof in preparation of L-methionine by microbial fermentation. [0003] (2) Background technology [0004] Methionine, also known as methionine, English name Methionine, full name 2-amino-4-methylmercaptobutyric acid. A sulfur-containing non-polar α-amino acid, which belongs to aspartic acid family III like threonine and lysine, and is an essential amino acid and ketogenic amino acid for mammals. It was first isolated from casein by Mueller in 1922. Methionine exists in large quantities in nuts, meat and some plants, but animals and humans cannot make it themselves. Lack of methionine can cause human toxemia, muscle paralysis, schizophrenia and stunting. L-methionine is currently widely used in various fields such as medicine, food, and feed. [0005] At present, the production methods of m...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/90C12N15/113C12P13/12C12R1/19
CPCC12N9/0028C12N9/1014C12N15/902C07K14/245C12P13/12C12Y201/02001
Inventor 柳志强蒋浩然张博沈臻阳杨辉郑裕国
Owner ZHEJIANG UNIV OF TECH
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