Analysis Of The Physiological Functions Of NADH In Pyruvate Production By Torulopsis Glabrata

This dissertation chose the pyruvate production by a multi-vitamin (i. e., thiamine, biotin, pyrodoxin and nicotinic acid) auxotroph Torulopsis glabrata strain, CCTCC M202019, as a model system to investigate the effect of NADH regulation on T. glabrata's metabolic characteristic, gene and protein (enzyme) expression profiles, and the adaptation to adverse environment. The main results were as follows:1) The glycolytic flux in T. glabrata was using as a model, to investigate the mechanisms in the role of extracellular oxidoreduction potential (ORP) in the intracellular NADH metabolism and the consequent effects on the glycolytic flux in T. glabrata. Potassium ferricyanide was added as electron acceptor to T. glabrata culture broth at 20% dissolved oxygen (DO) concentration, which redirected NADH oxidation from oxidative phosphorylation to membrane-bound ferric reductase. This novel pathway successfully enhanced NAD+ regeneration and decreased ATP production. It resulted in higher activities of hexokinase, phosphofructokinase, and pyruvate kinase, as well as high expression levels of the genes encoding these enzymes, and increases in the specific glucose consumption rate, the specific pyruvate production rate and pyruvate yield of T. glabrata was by 46%, 82% and 23%, respectively. Our results showed that membrane-bound ferric reductase offer an alternative and efficient NADH oxidation pathway at lower DO concentration, which increases the glycolytic flux of T. glabrata.2) A nox (encoding a cytoplasmic H2O-forming NADH oxidase), an AOX1 (encoding a mitochondrial alternative oxidase) and a PtxD (encoding a cytoplasmic phosphite dehydrogenase) were successfully expressed heterologously in T. glabrata, resulting in a decrease in the NADH contents, respectively, of 55% (in T. glabrata NOX) and 45% (in T. glabrata AOX) and -11% (in T. glabrata PtxD). The decreases in NADH concentrations led to increases in the pyruvate concentration, the pyruvate yield and pyruvate productivity of 13%, 15% and 20% (in T. glabrata NOX) and 19%, 21%, and 29% (in T. glabrata AOX), and -20%,-17% and -20% (in T. glabrata PtxD), respectively, compared with the corresponding values of the control.3) Decreased the cytoplasmic and mitochondrial NADH concentrations resulted in (1) high expression levels of the CAGL0A01782g gene, however, CAGL0A01782g knockout did not lead to decreased glycolytic flux in T. glabrata CON, T. glabrata NOX and T. glabrata AOX. It suggested glucose transporter encoded by CAGL0A01782g was not the key factor to control the glycolytic flux in T. glabrata, (2) high activities of the key glycolytic enzymes, hexokinase, phosphofructokinase, and pyruvate kinase, as well as high expression levels of the genes encoding these enzymes,(3) no significant effects on the expression levels of the genes encoding pentose phosphate pathway enzymes. In addition, modifying cytoplasmic NADH levels, not mitochondrial, more significantly affected the expression levels of the genes encoding glycerol synthesis pathway enzymes. Regulated cytoplasmic and mitochondrial NADH concentrations, had minor effects on the expression levels of the genes encoding pentose phosphate pathway enzymes. Increasing cytoplasmic NADH concentration (1) had no effect on the expression levels of the genes encoding glucose transporters, (2) down regulated the activities of the key glycolytic enzymes, as well as the expression levels of the genes encoding these enzymes, (3)up regulated significantly the expression levels of the genes encoding the glycerol synthesis pathway enzymes, as well as improved the specific activities of glycerol-3-phosphate dehydrogenase.4) There were no significant effects on the expression levels of the genes encoding TCA cycle pathway enzymes, as well as the enzymes contents and their specific activities, and TCA cycle intermediate metabolite concentrations. When more thiamin was in the culture broth, the NADH regulation could change the flux of TCA cycle pathway. It suggested that the suboptimal concentration of thiamine suppressed the activity of pyruvate dehydrogenase was responsible for the noneffective regulation. For alcohol synthetic pathway, the decreases in cytoplasm or mitochondrial NADH levels resulted in the down regulation of the expression levels of the genes encoding ethanol dehyrogenase, as well as the enzyme activities. However, accelerating mitochondrial NADH oxidation weaken the ethanol synthesis pathway more significantly. But, increase of the NADH level in cytoplasm activated the ethanol synthesis pathway.5) This study was aimed at understanding the physiological parameters that allow an industrial yeast strain, to rapidly regain its catabolic capacity during recovery from nutrient starvation. In the yeast, T. glabrata, intracellular ATP levels were modulated by overexpression of heterologous genes encoding either (i) the water-forming NADH oxidase (nox) or (ii) phosphite dehydrogenase (PtxD). Cultures of these two recombinant strains and the parental strain were grown aerobically in a chemostat and subjected to carbon or nitrogen starvation followed by recovery, and the content of various intracellular metabolites was monitored. The catabolic capacity was assessed in terms of yield of pyruvate per glucose utilized. Based on the comparative analyses of the recombinant strains and parental strain, intracellular ATP was positively correlated with recovery of catabolic capacity. Supplementation with the auxiliary energy substrate, citrate, in the starved cultures allowed a recovery of the catabolic capacity. Thus, generation of a high intracellular ATP concentration is a recommended strategy for a faster recovery of catabolic capacity during industrial fermentations.