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Genetically engineered bacteria with high production of l-methionine and its construction and application

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 limited methionine production capacity and methionine dependence on imports, and achieve enhanced internal transportation. , reduce metabolic stress, enhance the effect of expression

Active Publication Date: 2022-07-08
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Nowadays, my country has a great demand for methionine, but the domestic production capacity of methionine is limited, and most of the required methionine depends on imports.

Method used

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  • Genetically engineered bacteria with high production of l-methionine and its construction and application
  • Genetically engineered bacteria with high production of l-methionine and its construction and application
  • Genetically engineered bacteria with high production of l-methionine and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Fermentation method of L-methionine high-yielding strain and determination of content

[0046] The fermentation method is as follows:

[0047] The constructed strain was inoculated into 10 mL of LB medium containing 50 mg / L 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, and the lysine stock solution was 20 μl / mL, and the fermentation culture was carried out under the conditions of 28 ° C and 180 rpm for 48 h.

[0048] Described MS fermentation medium composition is 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 the 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 Zn...

Embodiment 2

[0053] Example 2: Construction of effective strain E.coli W3110 M2 / pAm metF and its shake flask fermentation

[0054] (1) Construction of pAm metF plasmid: using the previously constructed pAm plasmid (see the plasmid map Figure 7 ) as template, using pAm-line-F / pAm-line-R as primers to obtain PCR linear amplification product pAm-line plasmid, the PCR product was digested by DpnI at 37°C for 3h and the DNA fragment was recovered with Clean up kit; The genome of E.coli W3110 was used as the template, and pmetF-F / pmetF-R was used as the primer to obtain the PCR amplification product metF, and the DNA fragment was recovered by the Clean up kit; according to ClonExpress (One step clone kit, Vazyme Biotech, Nanjing, China) The instructions link the linearized pAm-line plasmid and the fragment metF together, and transform the ligated product into DH5α competent cells by chemical transformation; finally select clones and verify by sequencing to obtain the pAm metF plasmid.

[0055]...

Embodiment 3

[0059] Example 3: Construction of effective strain E.coli W3110 M2(Trc-fliy) / pAm metF and its shake flask fermentation

[0060] Using Escherichia coli W3110 M2 / pAm metF as the starting strain, CRISPR-Cas9-mediated gene editing technology was used (Yu Jiang et al.2015Multigene Editing in the Escherichia coliGenome via the CRISPR-Cas9 System.Applied Environmental Microbiology.81:2506-2514 ), using the trc promoter derived from pTrc99A (nucleotide sequence shown in SEQ ID No. 1) to replace the original promoter of fliY in the genome to enhance the expression strength of fliY.

[0061] (1) Construction of pTarget-fliY plasmid: using pTarget F plasmid (Addgene Plasmid #62226) as a template, using pT-fliY-F / pT-fliY-R as primers for PCR amplification, the PCR product was digested by Dpn I at 37°C After 3h, it was transformed into E.coli DH5α, screened by Spectinase, and verified by sequencing to obtain the correct pTarget-fliY plasmid, which was used for subsequent ligation of DonorD...

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Abstract

The present invention relates to a genetically engineered bacterium with high L-methionine production and a construction method and application thereof. The invention transforms the L-methionine synthesis network of Escherichia coli, and enhances the utilization of methylenetetrahydrofolate by Escherichia coli by strengthening metF and GCV in the one-carbon module of the L-methionine synthesis pathway of Escherichia coli Ability; by replacing the original promoters of fliY and malY with the Trc promoter derived from pTrc99A, the cysteine / cystine transport pathway and the cysteine ​​utilization pathway are enhanced, and the cysteine ​​production in E. coli is relieved. The resulting metabolic inhibition; by knocking out the glyA of E. coli itself and introducing the glyA derived from Arthrobacter sp.FB24 to overexpress on the plasmid, the restriction of serine hydroxymethyltransferase in the large intestine itself is relieved, and finally high-yielding bacteria containing the plasmid are obtained. , the L-methionine yield increased from 2.8g / L to 3.83g / L.

Description

(1) Technical field [0001] The invention relates to a genetically engineered bacterium with high production of L-methionine, a construction method thereof, and its application in the preparation of L-methionine by microbial fermentation. (2) Background technology [0002] 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, which is equivalent to threonine and lysine, is an essential amino acid and ketogenic amino acid for mammals. It was first isolated from casein by Mueller in 1922. Methionine is abundant in nuts, meat and some plants, but animals and humans cannot do it themselves. Lack of methionine can cause human toxemia, muscle paralysis, schizophrenia and dysplasia. L-methionine is currently widely used in medicine, food, feed and other fields. [0003] At present, the production methods of methionine are mainly divided i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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