| This dissertation investigated the effect of hyperosmotic stress on physiological function, metabolic characteristic, gene and protein expression profiles and pyruvate production with a pyruvate producer, a multi-vitamin (i. e., thiamine, biotin, pyrodoxin and nicotinic acid) auxotroph Torulopsis glabrata strain, CCTCC M202019, as a model system. Based on the understanding of the mechanisms during the hyperosmotic stress, three strategies for process optimization of pyruvate production by T. glabrata were developed,using gene chip, real-time PCR, 2D electrophoresis, iTRAQ (isobaric tag for relative and absolute quantitation), key enzyme activity measurements and key metabolite analysis. The main results were as follows:1) Gene expression profiles of Torulopsis glabrata at different osmolarity (860 mOsmol/kg, 1765 mOsmol/kg, 2603 mOsmol/kg and 3324 mOsmol/kg) were investigated by microarray. Significantly upregulated genes were mainly involved in regulation of cell wall, cell membrane and pheromone-regulated membrane proteins. The genes in central metabolic pathway, such as glycolysis, Krebs cycle and oxidative phosphorylation pathway, were mainly upregulated, especially the genes associated with ATP or NADH formation. Futhermore, the genes associated with potential key compatible solutes, such as proline, arginine and glutamate synthesis pathway were upregulated, and urea carboxylase which involved in arginine catabolism was drasticly downregulated to only about 1/100.2) The proteome of the T. glabrata was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and isobaric tag for relative and absolute quantitation (iTRAQ). Significantly upregulated proteins were mainly involved in ribosomal subunit proteins, cell division control proteins, DNA topoisomerase, and transport proteins. The protein involved in central metabolic pathways and energy metabolism was also investigated, and overall moderate correlations between transcriptome and proteome were found. Futhermore, the expression levels of superoxide dismutase and verprolin increased under hyperosmotic conditions, of which the former may due to the accumulation of reactive oxygen species (ROS), and the latter may be related to the accumulation of proline.3) The proline could enhance the growth of the yeast T. glabrata under hyperosmotic stress. Osmolarity progressively increased from 860 mOsmol/kg to 2603 mOsmol/kg with the accumulation of sodium pyruvate in the culture broth, leading to a significant decrease in cell growth and pyruvate accumulation. When 1.0 g/L of proline as a compatible solute was added to the culture medium, it was imported and enhanced cell growth by 59.0% at 2603 mOsmol/kg. By addition of proline during pyruvate production, the concentration, productivity and yield of pyruvate increased 22.1%, 38.4%, and 14.3%, respectively. These results suggested that T. glabrata can import proline as an osmoprotectant against high osmotic stress, thus enhance pyruvate productivity. The enhancement of cell growth and viability under hyperosmotic stress by the addition of proline provided an alternative approach to enhance the organic acids production by yeast with both process optimization and metabolic engineering strategies.4) The effect of intracellular arginine accumulation either through imported from the extracellular environment or by intracellular synthesis, on the growth of the yeast, T. glabrata under hyperosmotic stress, was investigated. Compared with medium without arginine supplementation, in the presence of 0.5 g/L arginine, could improved cell growth by 173.7% (5.2 g/L) or 121.4% (0.31 g/L) in media with osmolarities of 2,603 mOsmol/kg or 3,324 mOsmol/kg, respectively. Under hyperosmotic conditions, transcription of genes encoding enzymes for arginine degradation decreased while that of genes encoding enzymes for arginine biosynthesis increased thus caused the accumulation of arginine. Overproduction of two key enzymes of arginine synthesis pathway, glutamate N-acetyltransferase and N-acetylglutamate kinase resulted in an increase in cellular arginine content and promoted cell growth under hyperosmotic stress. These results indicated that arginine functions as a compatible solute to improve the osmotic stress resistance of T. glabrata.5) A heterologous water-forming NADH oxidase was introduced into T. glabrata, and the effect on cell growth under hyperosmotic conditions was investigated. Expression of the noxE gene from Lactococcus lactis NZ9000 in T. glabrata resulted in a marked decrease in the NADH/NAD+ ratio, and higher activities of key enzymes in water-regenerating pathways, leading to an increase in intracellular water content. NaCl-induced reactive oxygen species production was also decreased by introduction of the NADH oxidase, resulting in a significant increase in the growth of T. glabrata under hyperosmotic stress conditions (3824 mOsmol·kg-1). The results indicated that the osmotolerance of cells can be enhanced by manipulating water-production pathways.
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