Engineered Saccharomyces Cerevisiae To Produce L-lactic Acid

As an important platform chemical, the demands of L-lactic acid are continously increasing with the depletion of oil resources. Among the L-lactic acid production methods, microbial manufacture L-lactic acid with the renewable biomass is a potential alternative. However, the high nutritional requirements and high susceptibility to environmental stresses of lactic acid bacteria, and the formation of mycelium and by-products of fungal limited the rapid development of L-lactic acid fermentation industry. Sacchromyces Cerevisiae, as single-cell organisms, can utilize simple substrates for rapid growth and synthese the desired metabolites, which make S. cerevisiae as a host strain for organic acids production. In this thesis, a serial of metabolic engineering strategies were applied to construct an engineered S. cerevisiae for L-lactic acid production. The main results are described as follows:(1) The gene LDH encoding lactate dehydrogenase (LDH) from the bovine was over-expressed and the free expression vector pY13TEF1-LDH was constructed. Then, pY13TEF1-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.(2) In order to further increase L-lactic acid titer, a fragment for integrated expression of LDH was constructed through fusion PCR. Then the fragment was transformed into the S. cerevisiae by the LiAc transformation, and an engineering strain S. cerevisiae CEN.PK2-1C[LDH] with LDH over-expression and PDC1 defective was successfully constructed. This mutant could accumulate 14.7 g/L L-lactic acid, however, no L-lactic acid was detected in the fermentation broth of the parent strain CENPK2-1C. Compared to the parent strain CEN.PK2-1C, the ethanol concentration of the engineered strain CEN.PK2-1C [LDH] decreased from 27.3 g/L to 16.2 g/L, which decreased by 40.7%, and the yield of ethanol on glucose of the parent strain and the engineered strain were 0.319 g/g and 0.20 g/g, respectively.(3) With the aim of decreasing ethanol titer, the Km value of key enzymes for NADH in L-lactic acid and ethanol synthesis pathway were carefully compared. We successfully over-expressed a NADH oxidase (nox) from Streptococcus pneumoniae into the cytoplasm for the construction of S. cerevisiae CEN.PK2-1C[LDH]-nox. The over-expression of nox led to the decrease of intracellular NADH content from 0.48μmol/g DCW to 0.24μmol/g DCW, and increase of NAD+ pool from 5.94μmol/g DCW to 8.10μmol/g DCW. Therefore, the NADH/NAD+ ratio decreased to 0.03. Results obtained from fermentation experiments by CEN.PK2-1C[LDH] and CEN.PK2-1C[LDH]-nox showed that the L-lactic acid concentration (19.4 g/L) of mutant CEN.PK2-1C[LDH]-nox was improved by 32.0% and the ethanol concentration (8.8 g/L) was decreased by 45.7% compared with the mutant CEN.PK2-1C [LDH].