Gene Knockout Of D-lactic Acid Produced Strain

D-lactate is an important precursor of many chiral compounds, which have been applied in many fields, such as chemical engineering, agriculture, food industry, pharmacy, e nvironmental pr otection. At p resent, D -lactate is m ainly p roduced b y lactobacillus with t he s hortcoming of hi gh cost and low yield. Corynebacterium glutamate shows obvi ous a dvantages of s imple f eedstock a nd hi gh production i n lactate production by anaerobic fermentation, compared with Lactobacillus. Hence, C. glutamicum was co nsidered a s an ideal hos t f or l actate p roduction. I n t his w ork, D-lactate lactate biosynthesis pathway was reconstructed in C. glutamicum Res167 for th e f irst tim e. T he L-lactate de hydrogenase gene (ldh) w as knocked out successfully. Further, D-lactate d ehydrogenase (ldhA) overexpression v ector w as contrusted. The researches above lay a solid foundation for the high-efficiency and low-cost production of D-lactate.In order to obtain D-lactic acid of high purity, the L-lactic acid metabolic pathway needs to be blocked in Corynebacterium gl utamicum. Firstly, th e ldh gene w as knockout in C. glutamicum Res167. The upstream homologous arm of ldh (Ldh1) and downstream homologous arm of ldh (ldh2) were cloned from C. glutamicum Res167 genome and linked by overlap PCR resulted in ldh1-ldh2. Then it was inserted in pK18mobsacB to form the knockout vector. After transformed into C. glutamicum Res167, C. glutamicum Res167Δldh recombinants were screened by positive selection of Kmr, then by reversed selection of sucrose medium. The cultures in plats showed that C. glutamicum Res167Δldh recombinants could not grow in basal medium with the sole carbon source of L-lactate, indicating that the L-lactate metabolism of the microorganism was interrupted owing to the deletion of L-lactate dehydrogenase.The pXJM19 is a shuttle expressed plasmid between Corynebacterium glutamate and Escherichia coli. In order to confirm its genetic stability, 100 generations were subcultured. The results showed that stability rates reached to 98% and 94% with and without the antibiotics pressure respectively, which indicated good genetic stability of pXJM19 i n C. gl utamicum Res167. Then, t he e xpression pl asmid pXJM19-ldhA containing heterogeneous ldhA from Lactobacillus bulgaricus was constructed.