The Metabolic Engineering Of Trehalose And Related Mechanisms Of Stress Resistance In Saccharomyces Cerevisiae

Alcohol is one kind of most important and well-established reproducible biofuel. However, there are several issues and obstacles reminded in the technology of alcohol production, for example, high biomass-consuming, low efficiency of energy production, extremely large amount of wastewater produced, and arduous environmental management. So the novel technology with high efficiency and conservation has attracted more and more attentions. The achievement of fermentation under ultrahigh alcohol concentration opens a new and important avenue to increase the efficiency of alcohol production, and the basic requirement is the developing of Saccharomyces cerevisiae with highly potential ability of stress resistance during fermentation. The one of most important factors for the ability of stress resistance in Saccharomyces cerevisiae is the intracellular trehalose content. Researchers have found that Saccharomyces cerevisiae can increase the content of trehalose inside the cells to resist the potential damage in the extreme environment, but the study on the relationship between the accumulation of trehalose in Saccharomyces cerevisiae and their capability of alcoholic fermentation was rare.Herein, we reconstructed the correlation metabolism pathway of trehalose in yeasts to increase intracellular content of trehalose by cell and molecular biology technologies. The objective of this study is to establish one kind of Saccharomyces cerevisiae with excellent stress resistance and ability of high fermentation rate, which may have great potential in the applications of fuel industry. We also investigated the possible protection mechanism of trehalose and the evolution of HOG (High Osmolarity Glycerol) pathway in yeast to demonstrate the difference of stress resistance among yeasts. There are majorly four parts in this thesis:1. Construction of trehalose metabolic engineering strains. We separated 60 haploids from industrial strain D308. Then we analyzed and investigated the relationship between intracellular trhalose content and capability of alcoholic fermentation, and found there is the positive correlation of them. We reconstructed the metabolism process of trehalose by recombining the plasmid and produced two over-expressing trehalose-6-phosphate synthase gene (TPS1) strains: H11ptps1, H13ptpsl, two acid hydrolase gene (ATH1) knock-out strains:H11△athl, H13△athl, and two both over-expressing TPS1 and ATH1 knock-out strains:H11pTAA, H13pTAA. We further hybridized the above strains to produce three engineered diploid strains:D309ptpsl, D309Aathl, and D309pTAA.2. The comparison of fermentation performance between engineered strains and parental strains. The engineered diploid strain D309pTAA combined the excellent features of each haploid and increased the accumulation of trehalose inside cells. The maximal specific growth rate of D309pTAA was significantly higher than that of parental strain D308 under the same extreme stress environments such as high concentration of glucose, high concentration of alcohol, low pH value and high temperature. Moreover, the fermentation rate of D309pTAA was also higher than that of parental strain D308 under the same fermentation environments such as high concentration of glucose, high temperature, and low pH value. The intracellular trehalose content in engineered strain D309pTAA was higher than parental strain D308 over the duration of fermentation. Comparing with the parental yeast D308, the alcohol production rates from Oh to 46h of engineered strains D309ptpsl, D309Aathl, and D309pTAA were 2.253,2.243, and 2.264 (g h-1L-1), which increased 13.22%, 12.20%, and 13.76%, respectively; moreover, the alcohol yields of engineered strains D309ptpsl, D309Aathl, and D309pTAA were 117.171g/L,117.135g/L,117.555g/L, and increased 4.71%,4.67%, and 5.05%, respectively than parental strain D308 (110.905g/L) after 82 h corn mash (containing 254.906g/L glucose) fermentation.3. The reasons about the high stress resistance of engineered strains. The existence ratio of respiratory deficient yeast, cell membrane integrality, the accumulation of ROS (reactive oxygen species), and the activity of SOD (superoxide dismutase) were investigated. Under the alcohol stress condition, we found that the existence ratio of respiratory deficient yeast in engineered yeast was dramatically lower than parental yeast. The analysis of cell membrane integrality indicated the cell viability in engineered yeast was much higher than parental yeast under the same stress conditions such as high osmolity, high temperature, low pH value, and high concentration of ethanol, especially in the case of engineered strain D309pTAA. For example, the cell viability of D309pTAA still can reach to about 48.98% after the treatment of 50℃high temperature incubation for 1 h, which was 0.7 times higher than that of parental strain. Such different stress conditions indeed induce the accumulation of ROS inside cells. The maximal specific growth rate of D309pTAA was 7.95% higher than that of parental strain D308 under the existence of H2O2.The level of reactive oxygen species in engineered yeast was significantly lower, while the SOD activity in engineered yeast was higher than parental strain under the same conditions from 9.98% to 19.25%. The data shown here indicated there are two possible reasons about the protection mechanism of trehalose in yeast under stress conditions, one is the protection of cell membrane integrality in order to insure the high cell viability, and the other one is the maintenance of ROS metabolism relative enzyme (e.g., SOD) structure to increase the oxidation resistance, so that the overall stress resistance of engineered yeast was evidently higher than that of parental strain.4. The relationship between gene mutation of HOG pathway and osmotic tolerance of yeast. The sequence mutation of HOG signaling pathway within 23 Saccharomyces cerevisiae (including industrial yeast H13 and yellow wine yeast WH1) and among 10 different yeast species was investigated. For example, the number of mutant in upstream genes SLN1 and MSB2 was significantly higher other genes, especially the MSB2 gene in H13 and WH1 yeasts containing the segment of Indel (378bp). These variations may induce the different mechanisms to environmental osmotic stress resistance in different yeasts. Meanwhile, we further analysised the evolution of HOG pathway based on the pathway analysis and other statistical analysis methods. We found that the location of gene in the pathway, codon usage, protein length, protein interaction, and gene expression all play important roles in the evolution of HOG pathway. This could be the theoretical basis to further study on stress resistance mechanism of different yeasts.