鼠李糖乳杆菌D-乳酸脱氢酶基因ldhD的敲除

Knockout of D-Lactate Dehydrogenase Gene ldhD in Lactobacillus rhamnosus

聚乳酸由可再生原料L-乳酸合成,是目前应用的最环保的生物塑料之一。鼠李糖乳杆菌JCM1553中的L-乳酸和D-乳酸,它们是由代谢途径中的L-乳酸脱氢酶和D-乳酸脱氢酶分别催化丙酮酸而生成。L-乳酸的光学纯度对于L-乳酸的应用至关重要。因此,为了获取光学纯的L-乳酸,需要敲除该鼠李糖乳杆菌编码D-乳酸脱氢酶的基因ldhD以阻断相关的D-乳酸代谢途径。本研究采用pK18mobsacB自杀质粒运用重叠延伸PCR和同源重组技术成功构建得到重组鼠李糖乳杆菌菌株JCM1553-△ldhD。构建的缺失突变体JCM1553-△ldhD菌株没有引入外源基因,完全符合食品、药品安全要求,发酵液中检测到的L-乳酸含量为99.92%,光学纯度达到99.84%,显著优于野生型菌株。

Polylactic acid（PLA） is synthesized with L-lactic acid,i.e.from renewable raw materials,and it is one of the most ecofriendly bioplastic available today.The L-lactic acid and D-lactic acid in Lactobacillus rhamnosus JCM1553 are derived from pyruvate by the catalysis of L-lactate dehydrogenase and D-lactate dehydrogenase in the metabolic pathway,respectively.The optically purity of L-lactic acid is important to its application.So in order to obtain optically pure L-lactic acid,the D-lactic acid metabolic pathway involved needs to be blocked by deleting the gene encoding D-Lactate dehydrogenase（ldhD） in Lactobacillus rhamnosus.In this work,a genetically modified strain JCM1553-△ldhD was successfully constructed by SQE-PCR and homologous recombination method with suicide plasmid pK18 mobsacB.The deleted strain JCM1553-△ldhD was free of exogenous gene,perfectly matched the safety requirements of food and drug.L-lactic acid with high product concentration（99.92%） and optical purity（99.84%） was detected in the fermentation solution of JCM1553-△ldhD,which was significantly higher than those in wild-type strain.This work provided a foundation for further study on L-lactic acid and its fermentation in Lactobacillus rhamnosus by reforming metabolic pathway.