| Lactic acid is an important organic acid. It can be used in food, medicine, tanning, textile, cigarette industry, chemical industry, environmental protection, agriculture and many other applications. In addition, L-lactate can produce a new-type polyester. This polylactic acid (PLA), a renewable, biodegradable plastic, were paid more attention for its specific applications. In this study, we reported the cloning of the Pediococcus acidilactici L-Lactate Dehydrogenase gene and expressing in Escherichia coli. And then disrupted the adhE gene for the production of L-lactic acid.P. acidilactici could produce L-lactate dehydrogenase (L-LDH), and its genome was used as template to amplified the gene encoding L-lactate dehydrogenase. The PCR fragment was double digested with restriction endonucleases, and the target fragment was recovered. Then it was inserted into pSE-380 vector and pET-22b(+) vector, respectively. The recombinant expression plasmids pSE-ldhL and pET-ldhL were obtained, and then they were transformed into E.coli .Two positive recombinants, E.coli JM109 containing pSE-ldhL vector and E.coli BL21 harboring pET-ldhL vector,were obtained. After the two recombinants were induced to express LDH with IPTG, SDS-PAGE showed that they all expressed the gene of ldhL. The levels of LDH activity expressed by E. coli JM109/ pSE-ldhL and E. coli BL21/pET-ldhL were up to 114.8U/ml (4.1 times as that of P. acidilactici) and 97.95U/ml (3.4 times as that of P.acidilactici),respectively.The biochemical properties of L-LDH showed that it had an optimum temperature of 27~30°Cand pH of 5.0-5.3.In this study, replacement plasmid for homologous recombination was constructed by inserting a DNA fragment which contain tetracycline-resistant gene flanked with a homologous region of adhE into pEGM-3zf plasmid. After amplifying the fragment, the PCR products were elector-transferred into E.coli strain BL21. We want to obtain an E.coli strain whose adhE gene were inactive by tetracycline-resistant gene.
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