The Effect Of Pyruvate Carboxylase On Regulating The Synthesis Of Fumaric Acid In Saccharomyces Cerevisiae

Ethanol formation is one of the major products of carbon flow in yeast cells growing under aerobic conditions with excess sugar supply. Reducing or eliminating ethanol production is one of the metabolic engineering strategies for increasing production of organic acids in budding yeast. In this study, we show that deletion of both the pyruvate decarboxylase(PDC1) and the alcohol dehydrogenase(ADH1) genes decreases ethanol production and accumulates the pyruvate level under optimal fermentation conditions of yeast cells. Deletion of the FUM1 gene in the pdc1adh1 cells leads to a fumaric acid accumulation of 194.0±4.0 mg×L-1 at 96 h fermentation. Pyruvate carboxylase is one of the factors limiting fumaric acid production in yeast cells. We have made saturation mutagenesis for the Rhizopus oryzae Ro PYC P474 residue. The sole P474 N point mutation increases its enzymatic activity. Heterologous overexpression of this P474 N mutant enzyme leads to a further accumulation of fumaric acid to a titer of 357.0±1.4 mg×L-1. Main results were described as following:(1)Deletion both of PDC1 and ADH1 gene can weaken the activity of two key enzymes PDC and ADH in the alcohol synthesis pathway, but leading to cell growth weakly. By medium and culture conditions optimization, we got the optimal medium: glucose 40 g×L-1, YNB 3.4 g×L-1,(NH4)2SO4 5 g×L-1, medium volume was 40 m L/250 m L. Under these fermentation conditions, the biomass of the pdc1adh1 strain is almost equivalent to that of control strain. However, after 48 h fermentation, the maximum ethanol yield of the pdc1adh1 strain was 2.1 g×L-1, decreasing by 63.8% comparing to that of the wild-type strain, and the pyruvate yield was 2.0 g×L-1.The maximum ethanol production. Thus, knockout of PDC1 gene and ADH1 gene can weaken the activity of two key enzymes PDC and ADH, and then reduce the formation of ethanol effectively and promote the accumulation of pyruvate, which also provided an alternative strategy for the study to reduce the formation of by-product ethanol and promote the formation of C4-dicarboxylic acid.(2)The fum1 strain and pdc1adh1fum1 strain have been constructed. The ethanol yield reached a maximum after 36 h shaking flask fermentation. For wild-type strain CEN.PK2-1C and fum1 strain, the ethanol yield can accumulate to 5.8 g×L-1, but only 2.0 g×L-1 for pdc1adh1fum1 strain, reducing by 65.5%. After fermentation 96 h, fumaric acid yield of wild-type strain can not get accumulate, fumaric acid yield of fum1 strain can accumulate to 113.0 mg×L-1, meanwhile, fumaric acid yield of pdc1adh1fum1 engineered strain accumulated to 194.0 mg×L-1, increasing by 71.7% compared to that of fum1 strain.(3)The position of Ro PYC pyruvate carboxylase in Saccharomyces cerevisiae have been studied by constructing an expression vector p GFP33-TEF1p-Ro PYC. By microscopic observation of the DIC, GFP and DAPI horizons, the result showed that pyruvate carboxylase is located in the cytoplasm of S. cerevisiae. At the same time, the successfully expression of Ro PYC-GFP protein in S. cerevisiae got proved by SDS-PAGE and Western blot experiments.(4)The pyruvate carboxylase pathway was strengthen. Through saturation mutagenesis for the R. oryzae Ro PYC P474 residue, the P474 N mutation having increased pyruvate carboxylase activity was obtained. The Ro PYC activity of wild-type strain is 3.9 U×mg-1, butthat of P474 N mutant strain is 4.4 U×mg-1, increasing by 12.8%. After 96 h fermentation, the fumaric acid yield of pdc1adh1fum1-Ro PYC P474 N engineering strain was 285±5.7 mg×L-1, increasing by 13.3% compared to that of the control strain(252±17.0 mg×L-1). By adding 32, 64, 96, 128 mg×L-1 of biotins, which is the pyruvate carboxylase cofactor, to the culture medium, the yield of fumaric acid could significantly be improved. When 64 mg×L-1 of biotins was added which has been proved to be the best concentration, the fumaric acid yield can reach to 357.0±1.4 mg×L-1 after fermentation 96 h, increasing by 37.0% compared to that of the control group without biotin added(256.7±3.0 mg×L-1).