代谢工程改造酿酒酵母生产L乳酸-发酵工程专业论文.docx

Abstract n Abstract Abstract As an important platform chemical，出e demands of L-Iactic acid are continously increasing with the depletion of oil resources. Among the L-Iactic acid production methods， microbial manufacture L-Iactic acid wi出 the renewable biomass is a potential alternative. However ，the high nutritional requirements and high susceptibilityωenvironmental stresses of lactic acid bacteria，and the formation of mycelium and byproducts of fungal limited the rapid development of L-lactic acid fermentation industry. Sacchromyces Cerevisiae，制 single-cell organisms，can utilize simple substrates for rapid growth and synthese the desired metabolites，which make S. cerevisi翩 a host strain for organic acids production. In this thesis，a serial of metabolic engineering strategies were appliedωconstruct an engineered S. cerevisiae for L-Iactic acid production. The main results are described as follows: 刀le gene LDH encoding lactate dehydrogenase (LDH) from the bovine was overexpressed and the free expression vector pY13TEFI-LDH was constructed. Then， pY13TE l-LDH was transformed into the S. cerevisiae and an engineered mutant strain S. cerevisiae CEN.PK2-1C-LDH was successfully constructed. The L-lactic acid concerntraion reached 3.6 g/L after incubation in YNB medium for 36 h. In order to further increase L-lactic acid titer，a 仕a伊lent for integrated expression of LDHw，制constructed 也rough fusion PCR.Then the fragment was transformed into 由e S. cerevisiae by the LiAc transformation ，and an engineering 附ain S. cerevisiae CEN.PK2-1C[LDH] wi由 LDH overexpression and PDCl defective wωsuccessfully constructed. This mutant could accumulate 14.7 g/L L-Iactic acid，however，no L-lactic acid was detected in the fermentation broth of the parent stra?n CENPK2-1C. Compared to 伽 p町ent strain CEN.PK2-1C，由e ethanol concentration of the engineered strain CEN.PK2-1C [LDH] decreased 仕om 27.3 g/L to 16.2 g/L，which decreased by 40.7%，and the yield of ethanol on gl