Regulation Of Glycolytic Capability And Pyruvate Production In Torulopsis Glabrata

This dissertation chose the metabolism of energy and carbon in Torulopsis glabrata, a multi-vitamin auxotrophic and pyruvate over-producting yeast, as a model system to study the principal physiological and biochemical characteristics. The effects of energy metabolism on the glycolytic pathway, and the mechanism of product inhibitory in T. glabrata were investigated in detail. The main results were described as follows:1. This study aimed at increasing the glycolytic flux of a multi-vitamin auxotrophic yeast Torulopsis glabrata by defecting oxidative phosphorylation. We examined two different strategies to defect oxidative phosphorylation. First strategy is used to disrupt the activity of ETC by adding 10 mg/L specific inhibitor of complex I (rotenone) and bc1 complex (antimycin A) to the culture broth of T. glabrata CCTCC M202019, which resulted in a significantly decreased intracellular ATP level (43% and 27.7%) and a significantly increased rates of glucose consumption rate (qs) and pyruvate production (qp).For the second strategy, is used to decrease the specific activity of F0F1-ATP synthase. 0.05 mmol/L oligomycin (the specific inhibitor of F0F1-ATPase) was added to the culture broth of T. glabrata CCTCC M202019. In addition, a neomycin-resistant mutant N07, with 65% of F₀F₁-ATPase activity decrease, was screened. As a consequence, the intracellular ATP level of the mutant N07 decreased by 24%, which resulted in the glucose consumption rate and pyruvate productivity of the mutant N07 increased by 34% and 42.9%, respectively. Consistently, the activities of key glycolytic enzymes of the mutant N07, including phosphofructokinase, pyruvate kinase and glyceraldyde-3-phosphate dehydrogenase, increased by 63.7%, 28.8% and 14.4%, respectively.In summary, with the two strategies, the specific activity of PFK (R²=0.9971), the average specific glucose consumption rate (R²=0.9967) and the average specific pyruvate production rate (R²=0.965) were closely correlated with intracellular ATP level, and all of them were increased under a lower intracellular ATP level. Our studies showed that impaired oxidative phosphorylation in T. glabrata lead to a decreased intracellular ATP production thereby the glycolytic flux could be largely enhanced.