Recombinant escherichia coli capable of producing L-methionine at high yield without action of exogenous amino acid and application of recombinant escherichia coli

A technology for recombining Escherichia coli and exogenous amino acids, applied in application, recombinant DNA technology, microorganism-based methods, etc., to achieve the effect of enhancing utilization capacity, improving energy and precursor substances, and reducing operational difficulty

Pending Publication Date: 2022-06-10
ZHEJIANG UNIV OF TECH
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, lysine is an essential amino acid required for the growth of microorganisms, and the knockout of the lysA gene will significantly affect the growth of microorganisms. Therefore, in the subsequent fermentation process, the method of supplementing lysine in the medium is usually used to solve this problem. This leads to increased fermentation costs, operational complexity and uncertainty

Method used

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  • Recombinant escherichia coli capable of producing L-methionine at high yield without action of exogenous amino acid and application of recombinant escherichia coli
  • Recombinant escherichia coli capable of producing L-methionine at high yield without action of exogenous amino acid and application of recombinant escherichia coli
  • Recombinant escherichia coli capable of producing L-methionine at high yield without action of exogenous amino acid and application of recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Fermentation method and content determination of L-methionine high-yielding strain

[0043] 1. Fermentation method:

[0044] Inoculate the constructed strain into 10 mL of LB medium containing 50 mg / L kanamycin (Kan), culture at 37°C for 8-12 hours, and then inoculate the culture solution to 20 mL containing 50 mg / L In the Kan's fermentation medium, the fermentation culture was carried out at 30° C. and 180 rpm for 48 hours to obtain a fermentation broth.

[0045] The composition of the fermentation medium 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, CaCO 3 10g / L, VB 12 0.2μg / L, 1mL / L trace element solution, the solvent is deionized water, the pH value is natural, and CaCO 3 and VB 12 Added at the time of inoculation; the composition of the trace element solution is: MgSO 4 ·7H 2 O500g / L, FeSO 4 ·7H 2 O 5g / L, MnSO 4 ·8H 2 O 5g / L, ZnSO 4 5g / L, the solvent is deionized water. ...

Embodiment 2

[0050] Example 2: Construction of effective bacterial strain E.coli W3110 M2-lysA-ATG / pAm and its shake flask fermentation E.coliW3110 M2 / pAm was used as the starting strain, using CRISPR-Cas9-mediated gene editing technology (Yu Jiang et al.2015Multigene Editing in the Eshcrichia coli Genome via the CRISPR-Cas9System.Applied Environmental Microbiology.81:2506-2514), in situ complementation of the lysA gene on the genome, the specific operations are as follows:

[0051] (1) Construction of pTarget-lysA plasmid: Use pTarget F plasmid (Addgene Plasmid#62226) as template, lysA-PAM-F / lysA-PAM-R in Table 2 as primers for PCR amplification, and the PCR product is incubated at 37°C with DpnⅠ Digested for 3 hours, then transformed into E.coli DH5α, inoculated on LB plates containing 50mg / L spectinomycin, cultured at 37°C for 12 hours, screened the colonies that could grow, and sequenced to verify that the correct pTarget-lysA plasmid was obtained for subsequent connection with Donor D...

Embodiment 3

[0061] Embodiment 3: Construction effective strain E.coli W3110 M2-PfliA-lysA / pAm and shake flask fermentation thereof

[0062] (1) Construction of pTarget-lysA-2 plasmid: use pTarget F plasmid (Addgene Plasmid#62226) as template, use lysA-PAM-F-2 / lysA-PAM-R-2 in Table 2 as primers for PCR amplification, PCR The product was incubated and digested by DpnI at 37°C for 3 hours, then transformed into E.coli DH5α, inoculated on LB plates containing 50 mg / L spectinomycin, cultured at 37°C for 12 hours, and the colonies capable of growth were screened, and the correct pTarget-lysA was obtained through sequencing verification -2 plasmids for subsequent ligation of DonorDNA.

[0063] (2) Construction of pTD-lysA-2 plasmid: using the E.coli W3110 genome as a template, L-fliA-up-F, L-fliA-up-R, L-fliA-down-F, L-fliA-down- R is the primer to obtain the upstream and downstream PCR products of the lysA gene regulated by the PfliA promoter. After the Clean up kit is recovered, the fusion PC...

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Abstract

The invention discloses recombinant Escherichia coli capable of producing L-methionine at high yield without the action of exogenous amino acid and application of the recombinant Escherichia coli. L-methionine high-yield strain E.coliW3110M2 / pAm knocked out in a lysine way is used as a starting strain, lysA genes are subjected to in-situ back-supplement on a genome, a promoter of the lysA genes is replaced with a PfliA promoter, and the recombinant Escherichia coli is obtained. And over-expressing a gltA gene and a malY gene on the plasmid of the pAm to obtain the gene. The strain disclosed by the invention can grow well without externally adding essential amino acid in the fermentation process, so that the fermentation cost is reduced, the uncertainty of the adding time of the essential amino acid in the fermentation process is solved, and the operation difficulty of fermentation regulation and control is reduced; the yield of L-methionine in a shake flask is increased from 2.44 g / L obtained by adding lysine to 2.96 g / L obtained by adding no exogenous amino acid.

Description

(1) Technical field [0001] The invention relates to a recombinant Escherichia coli capable of high-yielding L-methionine, a construction method thereof, and an application in preparing L-methionine by microbial fermentation. (2) Background technology [0002] L-methionine, also known as L-methionine, is the only sulfur-containing amino acid among the eight essential amino acids for the human body, and plays a key role in the metabolism of organisms. Sulfur-containing amino acids were first discovered by Fleitmann in the laboratory in 1847. Later, Osborne identified two sulfur-containing amino acids in high-purity proteins, and classified one of them as cysteine. In 1922, Mueller isolated another sulfur-containing amino acid from protein, and then in 1928 Barger and Coyne determined the chemical formula of this sulfur-containing amino acid and officially named it L-methionine. L-methionine is widely used in the fields of food, feed and medicine because of its unique structur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P13/12C12N15/113C12N15/53C12N15/60C12R1/19
CPCC12N15/70C12N1/20C12P13/12C07K14/245C12N9/0016C12N9/88C12Y404/01
Inventor 牛坤傅强柳志强周海岩张博汤晓玲徐建妙郑裕国
Owner ZHEJIANG UNIV OF TECH
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