Corynebacterium glutmicum synthase gene cloning and L183Q site directed mutagenesis method

Abstract

The present invention discloses a high-new bio-technology, and particularly relates to a Corynebacterium glutamicum DAHP synthase gene cloning and L183Q site directed mutagenesis method, wherein an enzyme engineering technology and a molecular biology technology are adopted, and combined with a metabolic engineering theory, a fermentation engineering technology and other high-new bio-technologies, a substrate structure analogue resistance mutant and phenylalanine-tyrosine double auxotrophic Corynebacterium glutamicum are adopted as starting strains, researches on Corynebacterium glutamicum self-feedback regulation release, metabolic flow enhancement, improvement of catalysis activity of key enzymes in a tryptophan biosynthesis path, and substrate biosynthesis increase are performed respectively according to the metabolic engineering theory, and a tryptophan biological metabolism path of the Corynebacterium glutmicum is reasonably designed and modified to obtain the gene engineering bacterial, wherein the gene engineering bacterial has the following characteristics that: acid production rate and conversion rate meet contract requirements, and corn is adopted as a raw material to produce feed grade L-tryptophan.

Description

technical field [0001] The invention relates to a high-tech biological technology, in particular to a method for gene cloning of Corynebacterium glutamicum DAHP synthetase and L183Q site-directed mutation. Background technique [0002] DAHP synthase is the first key enzyme that metabolizes the flow of aromatic amino acids, and the encoding gene is AroI, which catalyzes the synthesis of 3-deoxy-D-arabino- 7 phosphate heptulose (DAHP), and then generate chorismate, which is finally converted into tryptophan under the joint participation of tryptophan operon and other enzymes, and secreted out of the cell. With the rapid development of molecular biology technology, on the basis of the combination of protein directed evolution technology and gene cloning expression method, it is possible to replace alleles of DAHP synthase mutant genes with improved catalytic activity into the genome or overexpress in engineering bacteria , so that the metabolic flow is conducive to the directi...

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Problems solved by technology

[0003] In recent years, people have made more in-depth research on the DAHP synthase of Escherichia coli, and the crystal structure of the enzyme has been included in the NCBI structure database. David L et al. made site-directed mutations on His180, Craig M et al. on Cys61 and Cys328, However, the catalytic activity has not been improved. Domestic Fudan University Hu Changyun and others determined that the catalytic activity of AroGL175Q, AroGL175A, AroGL175D, and Phe209Ser mutant enzymes increased by 1.33 times, 1.57 times, 1.65 times, and 1.8 times, respectively. There are few reports on the DAHP synthetase of Corynebacterium glutamicum, only Liao H-F et al reported that Ser187 plays an important role in the allosteric effect of the enzyme

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