Study On Directely Transform Saccharomyces Cerevisiae For Accumulating Pyruvic Acid

Pyruvic acid is the intermediate in the EMP cycle of most biological cells. It is not only very important in the biological energy metabolism, but also precursors of a variety of useful material. Therefore, it has been used widely in the chemical, pharmaceutical, agricultural and other industries and scientific reseach. At present, the yeast Torulopsis glabrata is the major strain used in the production of pyruvic acid, and the mostly-used strain for pyruvic acid production is the auxotrophic mutant of thiamine, biotin, pyridoxine, nicotinic acid. However, T.glabrata is a conditioned pathogen and may cause potential healthy problems. In contrast, Saccharomyccs cerevisiae normally used in the production of alcohol is acid torent and generally recongnized as safe. So in this paper, the mutant which can produce pyruvic acid is developed from S.cerevisiae.Firstly, the pdc1knockout fragment was transformed into S. cercvisiae Y2by using electroporation, transformants were screened on YEPD plates containing G418resistant and were validated by PCR and southern blot to get the pdcl gene disrupted strain S. cerevisiae Y2-1.Then, the pdc5gene knockout fragment was transformed into the Y2-1by lithium acetate, transformants were screened on YNBG plates and were validated by PCR and southern blo to get the S. cerevisiae Y2-15with both pdc1and pdc5gene disrupted.At last, the seed medium, fermentation medium and conditions of S. cerevisiae Y2-15were optimized. The best composition of seed medium is(g/L):glucose30, peptone10. yeast extract5. KH2PO41. MgSO4-7H2O0.5. sodium acetate2. corn steep liquor0.5%. pH5.6. The best composition of fermentation medium is(g/L):glucose80. peptone20. yeast extract10, sodium acetate2.5. corn steep liquor1%. KH2PO41. MgSO4·7H2O0.5. metal ions liquor5mL, pH5.6. The best fementation condition is:seed age26h.12%inoculum. medium volume70mL/250mL flask. S. cerevisiae Y2-15accumulated24.85g/L pvruvie acid after optimization, which increased by168%compared to the production before optimization.