| This thesis chose the accumulation of pyruvate andα-ketoglutaric acid in in Torulopsis glabrata, a multi-vitamin auxotrophic and pyruvate over-producing yeast, as a model system to study the effect of manipulation intracellular acetyl-CoA concentration on the distribution of carbon flux. Three different strategies for manipulating acetyl-CoA level in T. glabrata were investigated in detail. The main results were described as follows:1. The growth of T. glabrata increase with increasing of acetate sodium concentration on the fermentation medium when it as the sole carbon source. The highest value was achieved. The highest DCW (2.1 g/L) andα-ketoglutaric acid (13.5 mmol)were acquired when 6 g/L acetate sodium was presented. As a consequence, the intracellular acetyl-CoA level was 36.63±2.08 nmol/gDCW. However, the acetyl-CoA level and the concentration ofα-ketoglutaric acid did not increase with more 6 g/L acetate was supplement to the fermentation medium.The intracellular acetyl-CoA concentration was increased with increasing the concentration of VB1 and acetate in fermentation medium. As a consequence, the increased intracellular acetyl-CoA level promoted the accumulation ofα-KG by T. glabrata. The maximum concentration ofα-KG were achieved at 13.02 g/L and 13.35 g/L when 0.015 mg/L VB1 and 6 g/L acetate presented in fermentation medium, respectively.2. With the aim to increase theα-ketoglutaric acid production of a multi-vitamin auxotrophic yeast Torulopsis glabrata, by increasing the availability of acetyl-CoA. For this, we expressed ACS2 encoding acetyl-CoA synthase from Saccharomyces cerevisiae in the pyruvate producer Torulopsis glabrata WSH-IP303. Compared with that of the parent strain, the acetyl-CoA synthase activity of the mutant ACS2-1 increased about 920% and the mutant could utilize acetate as the sole carbon source for growth (2.6 g/L dry cell weight). When growing with glucose, the acetyl-CoA concentration,α-ketoglutaric acid, and the value of Cα-KG/Cpyr was higher 222%, 105% and 152% than the corresponding values of the parent strain WSH-IP303, respectively. The addition of 4 g/L acetate to the mutant ACS-1 and the parent strain WSH-IP303 culture broth led to a significant increase these values to 355%, 147% and 275%, respectively, compared with that of the parent strain WSH-IP303. And theα-ketoglutaric acid concentration achieved 17.8 g/L the strategy for increasing theα-ketoglutaric acid production by increasing the availability of acetyl-CoA may provide an alternative approach to enhance the novel metabolites production in yeast.3. A gene, PDC1, which encoding pyruvate decarboxylase from Saccharomyces cerevisiae expressed in the pyruvate producer Torulopsis glabrata WSH-IP303. Compared with that of the parent strain, the pyruvate decarboxylase activity of the mutant PDC1-1 increased about 14.2 fold(7.22±0.09 U/mg protein-1). When grown on 30 g/L pyruvate, the concentration ofα-ketoglutaric acid and the value of Cα-KG/Cpyr was:182%(38.8 g/L) and 284% (1.39) higher than the corresponding values of the controlled strain T. glabrata::pYES2, respectively. The mutant PDC-1 led to a significant increase of the intercellular acetyl-CoA levels and acetyl-CoA/CoA values to 238% and 376%, respectively, compared with that of the controlled strain T. glabrata::pYES2. And compare with T. glabrata CCTCC M202019 the pyruvate decarboxylase activity of the mutant PDC1-1 was 15.63 fold higher, andα-ketoglutaric acid concentration,and the value of Cα-KG/Cpyr was achieved 4.55(31.2 g/L) and 7(1.68) fold more than T. glabrata CCTCC M202019. |
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