Effect Of NADH Metabolism On The Glycolysis In Torulopsis Glabrata

This thesis chose the metabolism of energy and carbon in Torulopsis glabrata, a multi-vitamin auxotrophic and pyruvate over-producing yeast, as a model system to study the principal physiological and biochemical characteristics. The effects of NADH metabolic pathway and its intracellular content on the glycolytic pathway, the rate of glucose consumption and the pyruvate production of T. glabrata were investigated in detail. The main results were described as follows:1. T. glabrata could use gluconate as sole carbon and energy source for growth, 3.5 g/L DCW was achieved when 30 g/L gluconate presented in the fermentation medium. When 10 g/L gluconate was supplemented to the culture broth at 16h, the growth, the rate of glucose consumption and the pyruvate production were increase 7.96%,13.04% and 32.81%, respectively. The enhancement of glucose consumption and pyruvate production rate were due to the fact that the concentration of NAD+ increase 29.73%, but the concentration of ATP and the ratio of NADH/NAD+ decrease 26.89% and 8.5%, respectively.2. A water-forming NADH oxidase which encoded by noxE, from Lactococcus lactis, was expressed in the T. glabrata and a mutant strain T. glabrata-PDnoxE, as a result specific NADH oxidase activity of 34.8 U/mg proteins, was achieved. During batch fermentation with 100 g/L glucose, the dry cell weight, the glucose consumption rate and the pyruvate production rate were higher 168%, 44.9% and 12% than the corresponding values of the parent strain. Only 2.5 g/L residual glucose was detected in the fermentation broth after 36 h culture. With 50 g/L glucose added, the pyruvate concentration increased to 67.2 g/L. As a result of NADH oxidase over-production, the intracellular NADH, NAD+ and ATP concentrations of the mutant and the parent strain were measured. The NADH and ATP content decreased by 18.1% and 15.8% respectively, while the NAD+ increased by 11.1%.3. A heterologous NADH alternative oxidase which encoded by AOX1 from Histoplasma capsulatum was expressed in the T. glabrata and a mutant strain T. glabrata-AOX1, as a result specific NADH alternative oxidase activity of 1.64 U/mg proteins, was achieved. During batch fermentation with 100 g/L glucose in fermentation medium, the dry cell weight decrease from 12.12 g/L to 9.55 g/L, whereas the rate of glucose consumption and pyruvate production were increase 31.17% and 75.12%, compared with that of the parent strain, respectively. The NADH and ATP content decreased by 23.1% and by 50.1%, respectively, but the NAD+ increased by 69.0% with the over-production of NADH alternative oxidase in T. glabrata.