Plasmid, alpha-ketoglutaric acid engineering strain and construction method and application thereof

By constructing the plasmid pETKG and strengthening the expression of related genes, the problem of hydrogen peroxide production in enzymatic transformation method is solved, and the efficient and stable production of α-ketoglutarate is achieved, which is suitable for industrial production.

CN120384091APending Publication Date: 2025-07-29TIANJIN UNIV OF SCI & TECH
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Patent Information

Application Number
CN202510873022.7
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-06-27
Publication Date
2025-07-29

AI Technical Summary

Technical Problem

The existing enzymatic conversion method has the problem of large-scale production of by-product hydrogen peroxide in the production of α-ketoglutaric acid, which inhibits the activity of L-glutamate oxidase, resulting in the inactivation of enzymes and the reduction of conversion efficiency. In addition, engineered bacteria are prone to oxidative stress accumulation at high concentrations of substrates, limiting their application under high load and long-term reaction conditions.

Method used

The plasmid pETKG was constructed, carrying the L-glutamate oxidase LGOX gene, and by strengthening the expression of katE, katG, sodB, grxA and catR genes, the antioxidant ability of the engineered strains was enhanced. At the same time, the expression of LGOX was enhanced by using the T7 promoter, and combined with the pETKG plasmid to express LGOX, forming an efficient whole-cell catalytic system.

Benefits of technology

It has achieved efficient production of α-ketoglutaric acid, with a yield of 64.85 g/L, a conversion rate of 83.15%, improved catalytic efficiency, stable strain performance, and suitable for industrial applications.

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Abstract

The invention provides a plasmid, an alpha-ketoglutaric acid engineering strain and a construction method and application of the alpha-ketoglutaric acid engineering strain, the strain is obtained by adopting a directional transformation method, transcriptional levels of katE, katG, sodB and grxA genes are up-regulated, a catR gene and a katA gene are heterologously expressed, a high-copy plasmid pETKG is carried, and the plasmid simultaneously expresses L-glutamate oxidase LGOX genes from Streptomyces sp and Chain A; the strain self-synthesized catalase eliminates H2O2 generated in the process of producing alpha-ketoglutaric acid through enzyme catalysis, engineering bacteria of L-glutamate oxidase LGOX expressed by pETKG plasmids are used for performing whole-cell catalysis on L-sodium glutamate to synthesize alpha-ketoglutaric acid, and the engineering strain is a high-level engineering strain capable of industrially and stably producing alpha-ketoglutaric acid.
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