生物合成α-酮戊二酸的双酶共表达工程菌构建及发酵生产OA

[Objectives]α-ketoglutaric acid is an intermediate metabolic product of the tricarboxylic acid cycle,which is involved in the synthesis and energy metabolism of amino acids,vitamins and organic acids.α-ketoglutaric acid produced by chemical synthesis has some problems,such as toxic raw materials,which restricts the application of α-ketoglutaric acid in medicine and food.This article aimed to develop an efficient and environmentally friendly method for the synthesis of α-ketoglutaric acid.[Methods]In this study,an engineering strain of Escherichia coli co-expressing L-glutamate oxidase and catalase was constructed,and the whole cell transformation of L-glutamate sodium was used to produce α-ketoglutaric acid.The L-glutamic oxidase gene lgoX and catalase gene katE were cloned into expression vector pET-24a,and the constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3)with the α-ketoglutarate dehydrogenase gene sucA knocked out.The key parameters of whole cell transformation reaction of engineered bacteria were preliminarily studied.[Results]High-density fermentation and whole-cell transformation of engineered bacteria were carried out in a 5 L fermenter.After a 5-hour transformation reaction with 150 g·L-1 monohydrate glutamic acid sodium,110.5 g·L-1 α-ketoglutaric acid was obtained without the addition of exogenous catalase,with a molar conversion rate of 95.3%.[Conclusions]In this study,an engineered strain co-expressing L-glutamate oxidase and catalase was constructed for the production of α-ketoglutarate,laying a foundation for the development of an environmentally friendly and efficient industrial production process of α-ketoglutarate.