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Construction and applications of corynebacterium glutamicum mutant strain for producing L-homoserine

A technology of Corynebacterium glutamicum and homoserine, applied in the field of fermentation engineering

Active Publication Date: 2020-07-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although Corynebacterium glutamicum is one of the main strains for producing amino acids, there is no report on the use of Corynebacterium glutamicum to produce L-homoserine. At present, E. coli is mainly used as the production strain, but E. coli is used as the host for production. L-homoserine will be accompanied by a large amount of acetic acid production (Li et al., Metabolic engineering of Escherichia coli W3110 for L-homoserine production. Process Biochemistry, publication date 2016)

Method used

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  • Construction and applications of corynebacterium glutamicum mutant strain for producing L-homoserine
  • Construction and applications of corynebacterium glutamicum mutant strain for producing L-homoserine

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Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: Construction of bacterial strain Cg01

[0041] 1. Construction of gene knockout plasmid

[0042] The plasmid pK18mobsacB was digested with restriction endonucleases HindIII and BamHI to obtain a linearized pK18mobsacB fragment;

[0043] According to Corynebacterium glutamicum genome (NCBI accession number is NC_003450.3), synthetic nucleotide sequence is as shown in the fragment mcbR-U of SEQ ID NO.1; Nucleotide sequence is as the fragment mcbR shown in SEQ ID NO.2 -D; the knockout plasmid pK-mcbR was obtained by assembling mcbR-U, mcbR-D and linearized pK18mobsacB by Gibson assembly method;

[0044] The fragment thrB-U with the nucleotide sequence shown in SEQ ID NO.3 and the fragment thrB-D with the nucleotide sequence shown in SEQ ID NO.4 were assembled with linearized pK18mobsacB by Gibson assembly to obtain a knockout plasmid pK-thrB;

[0045] The fragment metD-U with the nucleotide sequence shown in SEQ ID NO.5 and the fragment metD-D with the nuc...

Embodiment 2

[0050] Embodiment 2: the construction of bacterial strain Cg03

[0051] 1. Plasmid construction

[0052] The plasmid pK18mobsacB was digested with restriction endonucleases Hind III and BamH I to obtain a linearized pK18mobsacB fragment;

[0053] The fragment lysC(P)-U whose nucleotide sequence is shown in SEQ ID NO.7, the fragment lysC(P)-D whose nucleotide sequence is shown in SEQ ID NO.8, and the nucleotide sequence such as SEQ ID NO. The fragment Psod shown in ID NO.11 was assembled with linearized pK18mobsacB by Gibson assembly method to obtain the expression plasmid pK-lysC(P);

[0054] The fragment hom(P)-U whose nucleotide sequence is shown in SEQ ID NO.9, the fragment hom(P)-D and Psod whose nucleotide sequence is shown in SEQ ID NO.10 are assembled by Gibson, Assembly with linearized pK18mobsacB to obtain overexpression plasmid pK-hom(P);

[0055] Use primers lysC(m)-U-R and lysC(m)-D-F to perform site-directed mutagenesis on plasmid pK-lysC(P) by PCR, and replace...

Embodiment 3

[0062] Embodiment 3: the construction of bacterial strain Cg04

[0063] 1. Plasmid construction

[0064] The plasmid pK18mobsacB was digested with restriction endonucleases Hind III and BamH I to obtain a linearized pK18mobsacB fragment;

[0065] The fragment pyc(P)-U whose nucleotide sequence is shown in SEQ ID NO.12, the fragment pyc(P)-D whose nucleotide sequence is shown in SEQ ID NO.13, and the fragment Psod and Linearized pK18mobsacB was assembled to obtain the expression plasmid pK-pyc(P);

[0066] The fragment aspC(P)-U with the nucleotide sequence shown in SEQ ID NO.14, the fragment aspC(P)-D with the nucleotide sequence shown in SEQ ID NO.15, the fragment Psod, The fragment aspC with the nucleotide sequence shown in SEQ ID NO.16 was assembled with the linearized pK18mobsacB to obtain the expression plasmid pK-aspC(P);

[0067] Use the primers pyc(m)-F and pyc(m)-F to perform site-directed mutation on the plasmid pK-pyc(P), replace the proline at position 458 of py...

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Abstract

The invention discloses construction and applications of a corynebacterium glutamicum mutant strain for producing L-homoserine, and belongs to the technical field of fermentation engineering. Corynebacterium glutamicum ATCC 13032 is taken as a starting strain to knock out regulatory protein McbR, homoserine kinase, transport protein MetD, phosphoenolpyruvate carboxykinase; the expression of isocitrate dehydrogenase is down regulated; transport protein BrnFE, aspartic semialdehyde dehydrogenase and homoserine dehydrogenase are overexpressed; and the expression of aspartate kinase, pyruvate carboxylase and the aspartate kinase I derived from escherichia coli are enhanced. Shake flask culture can be performed on the mutant strain for 48 h, and the yield of L-homoserine can reach 8.8 g / L.

Description

technical field [0001] The invention relates to the construction and application of a mutant strain of Corynebacterium glutamicum producing L-homoserine, belonging to the technical field of fermentation engineering. Background technique [0002] As a four-carbon amino acid, L-homoserine is a potential platform compound for the synthesis of methionine, γ-butyrolactone and other compounds. Although L-homoserine is not an amino acid for protein synthesis, it can be a precursor of some important sulfur-containing compounds (L-methionine, S-adenosylmethionine), and L-methionine has been widely used In food, medicine, animal feed and other fields. Because the biosynthesis of L-methionine is strongly regulated, the industrial synthesis method is mainly a combination of enzymatic conversion and chemical synthesis. For the enzymatic conversion method, O-acetyl-L-homoserine can be used as a precursor to form L-methionine through enzymatic conversion with methyl mercaptan; for the ch...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/06C12R1/15
CPCC07K14/34C12N9/0006C12N9/0008C12N9/1205C12N9/1217C12N9/88C12N9/93C12N15/52C12N15/77C12P13/06C12Y101/01003C12Y102/01011C12Y207/01039C12Y207/02004C12Y401/01032C12Y604/01001
Inventor 周景文陈坚李宁曾伟主堵国成
Owner JIANGNAN UNIV
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