| Pyruvate is the important intermediate metabolite in the cell of organism, which is related to metabolism of sugar and lipids. It takes the key part of metabolic pathways in the cells by connecting the Hexoe Diphosphaty Pathway with Tricarboxylic Acid Cycle. Lots of metabolic pathways could be linked to a network through the pyruvate. In daily life, pyruvate could be useful in many fields, for example, as an important chemical intermediate, it could be used to synthesize amino acids,vitamin or some other compounds. Besides that, it sometimes is applied to produce germicide or weedicide as part of raw materials. Especially in recent years, it is found that calcium pyruvate has good effect on losing weight, so research on producing pyruvate are becoming a focus. Methods for producing pyruvate include chemical synthesis, bio-catalysis and fermentation with microorganism. But there is always some problems on producing pyruvate through the above two ways, such as expensive costs, inactive raceme or even unsuitability for mass production, so it becomes some necessary to fermentate to synthesize pyruvate in microbe.As a wide useful eukaryote, Saccharomyces cerevisiae has good performance on producing ethanol, especially when yeast cells are cultured in anaerobic condition or Crabtree effect takes place, almost all of the pyruvate from glycolysis flow to pathway of ethanol synthesis. Considering that, it will be a new method for producing pyruvate by redirecting flux to ethanol to pyruvic acid. Pyruvate is converted to ethanol by the catalyzeing of PDC and ADH, during the process, PDC plays the first role in the conversion. To redirect flux to ethanol to accumulating pyruvate, series of PDC genes (PDC1/5/6) should be knocked out. However, pdc△S. cerevisiae strains have two growth defects:(ⅰ) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C2 compound, these strains cannot grow in batch cultures on synthetic medium with glucose. That greatly limits the utility of producing pyruvate by pdc△S. cerevisiae strains.Microbial populations and single-celled microorganisms respond in a flexible manner to environmental changes. For the growth defects of pdc△S. cerevisiae strains, Evolutionary engineering could be helpful for the optimizing phenotype of cells. An optimized yeast strain BY5419-A0 was selected by the directed evolutionary process, in which C2 compound was controlled to decrease gradually at first, then high glucose-tolerant strain was selected by raising the glucose concentration by gradient. At optimized fermentation condition, a pyruvate concentration of 66.4 g liter-1 was obtained, with an overall yield of 0.55g of pyruvate·g of glucose-1.However, the strain still growing slow, it lead to long fermentation period.In yeast cells, drop difference of co-enzyme factors concentration could redirect the fluxes of metabolism. When cell was accumulating pyruvate, redundant NADH need to be reoxidized to cycle, but sometimes it could be hard. In cofactor engineering, heterologous proteins could be expressed in S.cerevisiae to adjust the metabolic redox balance. NoxE encoding a water-forming NADH oxidase from Lactococcus lactis and udhA encoding transhydrogenase from E.coli were expressed in S.cerevisiae BY5419-A0. Both genes, by being constrcted in plasmids with different copy number after different promters, were controlled to regulate the expression in yeast. After synchronous fermentation of the Ao strain with the genes, four pyruvate-raising strain were selected-strains 1AN-A0,2TN-A0,1GU-A0,1G2U-A0 could accumulate 70.47g/L,70.86g/L,72.86 g/L,74.83 g/L pyruvate sepetately. |
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