Carbon Metabolic Pathway Modification Of Lactobacillus Delbrueckii Subsp.bulgaricus For Producing D-lactic Acid

D-lactic acid is a synthetic precursor of many important chemical products and is widely used in the pesticide,pharmaceutical and material industries.With the rapid development of the biodegradable material polylactic acid,the demand for D-lactic acid is increasing.At present,the main production methods of D-lactic acid are chemical synthesis and microbial fermentation.The microbial fermentation method is widely concerned because of its low pollution,low cost,and high optical purity.Lactobacillus bulgaricus has the characteristics of high safety,strong acid tolerance,and strong acid-generating ability,and has become one of the widely used strains in industry.However,while L-Bulgaria produces D-lactic acid,it also produces acetic acid,L-lactic acid and other by-products,which reduces the optical purity and yield of D-lactic acid.Lignocellulose is a renewable biomass resource with abundant reserves.Lignocellulose biological refining can greatly reduce costs,and at the same time it can also solve the environmental pollution caused by the waste of crop straw lignocellulose.However,Lactobacillus bulgaricus itself has no xylose metabolism pathway and cannot use xylose.In order to solve the above-mentioned bottleneck problem,this article takes Lactobacillus delbrueckii subsp.Bulgaria CGMCC1.6970 as the starting strain,analyzes its carbon metabolism pathway,and uses homologous recombination technology to knock out the metabolism of L-lactic acid and acetic acid Related key genes,reduce the production of by-product acetic acid and improve the optical purity and yield of D-lactic acid.Using PMG36e as an expression vector,the key enzyme gene for xylose utilization(xylose isomerase gene xylulose kinase gene)was introduced into the above strains,and the above two genes were integrated into the L.bulgaricus metabolic pathway to give the engineering strain the use of five-carbon sugar The ability to expand the variety of carbon sources and reduce fermentation costs.The main findings are as follows:1.Through homologous recombination gene knockout technology,construct the knockout vector with the temperature-sensitive vector p G~+host4:successfully construct the L-lactic acid dehydrogenase gene ldb0094 knockout vector p G~+host4-?ldb0094;successfully construct and replace ldb0120 with D-lactate dehydrogenase gene(ldh A)knockout vector p G~+host4-?ldb0120::ldh A;the acetate kinase gene knockout vector p G~+host4-?Ack was successfully constructed.2.Optimized the method of electrotransformation of Lactobacillus bulgaricus:collect the cells grown in MRS medium with 0.1%glycine to OD600nm=0.4-0.6,warm bath at 45?for 20 min,prepare competent cells;add 1-3?g to be transformed Plasmid,electroporation conditions 1000 V,800?,25?F.Incubate in 0.4 mol/L sucrose,1 mmol/L magnesium chloride skim milk medium for 4 h.3.The knockout vector p G~+host4-?ldb0120::ldh A was transferred into Lactobacillus bulgaricus,and the L-lactate dehydrogenase gene ldb0094 was successfully replaced with the D-lactate dehydrogenase gene(ldh A)to obtain engineering bacteria.4.Amplify the xylose utilization key enzyme genes xyl A(xylose isomerase)and xyl B(xylulose kinase)from E.coli,successfully construct the xylose utilization key enzyme expression vector p MG36e-xyl AB.In this study,the acetate kinase knockout vector,L-lactate dehydrogenase gene(ldb0094 and ldb0120)knockout vectors were successfully constructed,and xylose used key enzyme expression vectors.The above results have laid the foundation for the carbon metabolism pathway of Lactobacillus bulgaricus and the construction of engineering bacteria,and also provide the basis for the utilization of xylose for the biological refining of straw,which has certain theoretical and practical significance.