Construction Of Optical Pure D-lactic Acid Producing Strain NZ9000

Lactococcus lactis is a group of Gram-positive bacteria which ferment carbohydrate to produce lactic acid.As a food safety microorganism,Lactococcus lactis is widely used in the fields of fermented dairy products,production of industrial compounds,food preservatives and biomedicine.Under natural metabolic conditions,Lactococcus lactis can convert about 90% of the substrate glucose into lactic acid,which is an ideal strain for lactic acid production by microbial fermentation.Optically pure D-lactic acid(D-LA)and L-lactic acid(L-LA)are important monomers for the synthesis of biopolymer polylactic acid(PLA).The heat resistance and mechanical properties of PLA can be significantly improved by mixing D-polylactic acid(PDLA)and L-polylactic acid(PLLA)in a certain proportion.In this study,the L-lactate dehydrogenase(L-LDH)gene of NZ9000 was knocked out,and the D-LA synthesis pathway was reconstructed in the L-LDH gene deficient strain.In this study,Lactococcus lactis NZ9000 was selected as the host strain.The homologous double-exchange recombination method was used to knock out the L-LDH genes of the NZ9000 genome,and the final p H and lactic acid production of the deleted strains were explored.After NZ9000 fermented for 12 h,the p H ≈ 4.45,and the L-LDH gene deletion strain had a p H ≈ 5.45 after 12 h of fermentation.NZ9000 can produce about 17 g/L of L-LA,while the deletion strain produces almost no L-LA.In order to reconstruct the D-LA synthesis pathway in NZ9000,the potential of D-LDH gene(ldh A)derived from Escherichia coli,Lactobacillus bulgaricus and Corynebacterium glutamicum to produce D-LA in NZ9000 was explored.In order to improve the production of D-LA,the effects of glucose transport,glycolysis pathway,pyruvate to lactic acid transformation and acid tolerance on the yield of D-LA were investigated.Enhancing glucose transport had no effect on D-LA production.Integrating key genes pfk and pyk of glycolysis pathway into genome increased D-LA yield from 15.24 g/L to 15.96 g/L.On this basis,further enhancing the conversion of pyruvate to lactic acid through two strategies of tandem strong promoters and tandem enzymes can increase the production of D-LA by 25.9%,about 19.87 g/L.Further overexpression of acid tolerance gene msmk could increase the yield of D-LA by 6.8%.On the basis of the above endogenous strategy,the yield of D-LA was increased by 147% by adding glucose and Na OH into the medium,and a strain 2Pldh A-IN9040 was obtained which could produce 37.68 g/L D-LA after 48 hours fermentation.