目的 钝齿棒杆菌厌氧发酵时，其主要代谢产物为琥珀酸和乳酸。敲除乳酸脱氢酶基因（LdhA）可以减少乳酸的生成，提高钝齿棒杆菌产琥珀酸代谢途径的通量。方法 为获得乳酸脱氢酶基因缺失的钝齿棒杆菌突变株，先通过PCR扩增获得LdhA基因的上、下游同源重组臂及卡那霉素抗性基因（Kn），然后通过融合PCR技术将LdhA基因上、下游同源重组臂与Kn抗性基因连接构建打靶片断与打靶质粒。结果 先后成功构建了打靶片段?LdhA::Kn、打靶质质粒pCVD442-?LdhA::Kn与供体菌株E.coli β2155/pCVD442-?LdhA::Kn，并成功筛选到具有卡那霉素抗性的钝齿棒杆菌单克隆菌株。结论 PCR鉴定与乳酸脱氢酶活力分析结果表明，钝齿棒杆菌基因组中LdhA基因被Kn抗性基因替换，LdhA基因敲除成功，为后续利用钝齿棒杆菌高产琥珀酸奠定了基础。

[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.