Construction Of Lactobacillus Delbrueckii Sp. Bulgaricus Engineering For High Optical Purity D-lactic Acid And Application In Biorefinery

D-lactic acid is an important synthetic intermediate and is widely used in food,chemical,environmental protection,medicine and other fields.The annual demand for D-lactic acid is increasing with the rise of polylactic acid industry.However,there are many problems in the industrialized production of D-lactic acid.Among them,the chemical synthesis method has low optical purity,and is easy to produce racemic body(D / L-lactic acid).It has high pollution in the production process and does not adapt to the requirements of sustainable development.The production of D-lactic acid by microcial fermentation has the characteristics of no pollution and high optical purity of D-lactic acid,and has a better advantage over chemical synthesis.However,there are currently two major problems: 1)High cost: In the microbial fermentation process,the nutritional requirements of the bacterial substrate for the fermentation substrate are generally high,which leads to a rise in the production cost of D-lactic acid;2)The lower production efficiency: there are many natural strains producing D-lactic acid,such as Lactobacillus genus,Escherichia coli,Bacillus subtilis,etc.,but the yields of wild-type strains are relatively low,far from meeting the requirements of industrial production.In order to solve the two major problems of D-lactic acid produced by microbial fermentation,this study improved the metabolic pathway of Lactobacillus.delbrueckii sp.bulgaricus and knocked out the metabolic pathway of acetic acid and L-lactic acid to construct a strain for producing high-yields and high-purity D-lactic acid.With the purpose of reducing the cost of producing D-lactic acid by microbial fermentation,in this study,a rich lignocellulosic resource,corn stover,was used as fermentation substrate.Firstly,the pretreatment process of corn stover was studied,and the corn stover was pretreated by using new catalyst-solid acid.The optimal technology of pretreating corn stover in solid acid was studied.Finally,the pretreated corn stover was applied to simultaneous saccharification and fermentation for D-lactic acid produced by engineering bacterium.The main findings are as follows:1.The optimum growth conditions of Lactobacillus.delbrueckii sp.bulgaricus CICC 21101: temperature 37 ?,pH 6.0,rotation speed 120 rpm;optimum acid production conditions: temperature 42 ?,inoculum volume 5%,initial glucose concentration 5%.Under optimal fermentation conditions,the yield of D-lactic acid was 35 g/L and the optical purity was 87.5%e.e.2.Improving the metabolic pathway of the wild-type Lactobacillus.delbrueckii sp.bulgaricus CICC21101.Lactocate-specific knockout plasmid pGHost4 was used to knock out key enzyme L-lactate dehydrogenation gene(ldb0094,ldb0120)and acetate kinase of Lactobacillus.delbrueckii sp.bulgaricus CICC 21101 L-lactic acid metabolic pathway and acetate metabolic pathway.(1)Knockout of Acetate kinase gene: The Lactobacillus.delbrueckii sp.bulgaricus competent cells were electrotransformed by using the enzyme-catalyzed plasmid pGhost-?ack constructed in our laboratory,and positive clones were screened by using the knockout primer pGhost4-?ack–L-F/pGhost4-?ack-R-R verified that the acetate kinase was successfully knocked out.The D-lactic acid production and optical purity of the engineered bacteria(DS-A)fermentation broth was determined by HPLC,and the D-lactic acid production reached 35 g/L,optical purity 90% e.e.(2)Knockout of L-lactate dehydrogenase gene(ldb0094,ldb0120): upstream and downstream fragments of the L-lactate dehydrogenase gene(ldb0094,ldb0120)was selected and spliced by overlapping PCR to connect pGhost4 knockouts.Through construction of L-lactate dehydrogenase knockout plasmid pGhost-?ldh1/pGhost-?ldh2,electroporation of Lactobacillus.delbrueckii sp.bulgaricus competent cells,screening positive clones,and verifying their genes and fermentation products,PCR validation results showed that L-lactate dehydrogenase gene and acetate kinase gene were successfully knocked out.The results of fermentation production were determination by HPLC,which showed that after knocking out L-lactate dehydrogenase gene(ldb0094),engineering bacteria(DS-L1)L-lactic acid production and optical purity in the fermentation broth increased significantly,yield reached 29.8g/L,optical purity was 93.5%e.e.,D-lactic acid yield and optical purity decreased significantly,the yield was 2.6 g/L;knocking out L-lactate dehydrogenase gene(ldb0120),the D-lactic acid production and optical purity of the engineered strain(DS-L2)increased significantly.The final D-lactic acid yield was as high as 41 g/L and the optical purity was 99.5% e.e.3.The optimum technology of pretreatment of corn stover was studied by using crushed corn stover as raw material and solid acid as pretreatment agent.The optimal process of pretreatment of corn stover with solid acid was obtained by single factor and orthogonal experiment: temperature 120 ?,solid acid concentration 1.5%,time 80 min,cellulase addition amount 30 FPU / g cellulose.The final conversion rate was 71.06%.Scanning electron microscopy(SEM),X-ray diffraction(XRD)and Fourier transform infrared spectroscopy(FTIR)were used to determine the structural changes.SEM results showed that the surface of corn stover before and after solid acid pretreatment was smooth.The structure of the order becomes messy and coarse,and a large number of holes are produced;X-ray results showed that the crystallinity of corn stover is reduced after pretreatment,recrystallization occurs in the crystal area;FTIR results showed that the hemicellulose and lignin absorption peaks weakened and the corresponding cellulose absorption peaks increased.SEM,XRD,and FTIR results demonstrated that solid acid pretreatment of corn stover has a good result.4.Application of engineering bacterium DS-L2 in corn stover biorefinery: The use of pretreated corn stover as an alternative glucose supplement and sole fermentation substrate were studied in the Lactobacillus.delbrueckii sp.bulgaricus CICC21101 and the strain of engineering bacterium DS-L2 to produce D-lactic acid.The results showed that the highest yields of D-lactic acid produced by the wild-type and engineering strain DS-L2 were obtained when the amount of corn stover added was 10% in MRS+stover fermentation medium,the yield of D-lactic acid was 51 g/L,61 g/L.When corn stover was the only fermented carbon source,the yield of D-lactic acid was 18 g/L,the optical purity was 87.5% e.e,and the D-lactic acid yield of the engineering strain DS-L2 was 21 g/L,the optical purity was 99.8% e.e.