关键词: 钝齿棒杆菌, 乳酸脱氢酶, 基因敲除, 融合PCR技术

Abstract: [Objective] Succinic acid and lactic acid are the main products when Corynebacterium crenatum were cultured under anaerobic conditions. The knockout of lactate dehydrogenase gene(LdhA), a key enzyme for lactic acid production, can decrease the production of lactic acid and increase the flux of the metabolic pathway for succinic acid production. [Methods] In order to obtain the C. crenatum ?LdhA mutant strain, the upstream and downstream homologous recombination genes of LdhA and kanamycin resistance gene (Kn) were obtained by PCR. Afterwards, the targeting homologue recombination DNA and plasmid were constructed by fusion PCR. [Results] The targeting homologue recombination DNA of ?LdhA::Kn, targeting plasmid of pCVD442-?LdhA::Kn and donor bacteria of E.coli β2155/pCVD442-?LdhA::Kn were constructed, and the mutant strain of C. crenatum which had resistance to kanamycin was screened. [Conclusion] The results of PCR and lactate dehydrogenase activity analysis indicated that the LdhA in genome of C. crenatum was knockout and replaced by Kn. This study provides a foundation for producing succinic acid efficiently using C. crenatum.

Key words: Corynebacterium crenatum, lactate dehydrogenase, gene knockout, fusion PCR technology