Metabolic Evolution Of Yeast For Improving Pyruvate Production

Pyruvate is the important intermediate by organic synthesis, which has widespread application in medicine, cosmetics, chemical production. In recent years, the increasing requirement of of pyruvate, especially used as diet products, resulted in the boom of manufacturing and research. Thermostable yeast can decrease troublesome of cooling and energy consumption in industrial production, and ensure that industrial fermentation can operate at high temperature. Therefore, obtaining the new strain which can grow at high-temperature by adaptive evolution is the main content of this study.A pyruvate-producing yeast, Torulopsis glabrata TFO005-3672which can normally grow and accumulate pyruvate at30℃was choosen as the starting strain. Through the method of adaptive evolution, a new evolved strain T. glabrata TIB-G90mutant was obtained which can grow well at40℃and accumulate pyruvate. Then the fermentation conditions for pyruvate production of this mutant were optimized. At last, the transcriptomic analysis between the parent strain and evolved strain was carried out by RNA-seq to preliminary elucidate the mechanism of high temperature tolerance of this evolved strain.The main results of this thesis are decribed as follows:1) Through the method of adaptive evolution, a new evoled strain, T. glabrata TIB-G90was obtained. The new strain can grow well at40℃, and its pyruvate production and glucose metabolic rate is better than parent strain. In the shake flask, the glucose metabolic rate of evolved strain is higher than that of parent strain about13.31%, and the pyruvate production rate is higher about1.88%at30℃. Similarly, the glucose metabolic rate of evolved strain is higher than that of parent strain about30.65%, and the pyruvate production rate is higher about7.38%at40℃.2) After the optimization of fermentation condition, we obtained that the optimized condition is that we cultivated the strain in250ml shake flask within10ml medium with the supplement of4vitamins, including nicotinic acid, thiamine, pyridoxal and biotin. In this condition, the evolved strain showed the highest pyruvate production. It can accumulate pyruvate about32.41g/L in24h at40℃, which is higher than the production of about18g/L before optimization, and the yield increased55%. In the scale-up fermentation in7.5fermentor, we found that the rate of glucose consumption for the evolved strain is2.06g·L-1·h-1at40℃. The yield of pyruvate accumulation is0.94g·L-1-h-1and the conversion is0.453g/g. It’s higher than that of parent strain about16.7%.3) Through the analysis of transcriptome sequencing between the evolved strain and parent strain. We found that the expression of tR(CCU)J, which encoded tRNA for transporting Arginine, was up-regulatedabout6.85fold in evolved strain. The tR(CCU)J is necessary for synthesis of heat shock protein Ssclp, which related to the cell growth at high temperature. We also found that the expression of YGR192C, which encoded glyceraldehydes-3-phosphate dehydrogenaseisozyme3, was up-regulated over1.7fold. This enzyme is involved in glycolysis and gluconeogenesis and necessary for facilitate glycolysis. Thus, the overexpression resulted that the consumption of glucose and the production of pyruvate evolved strain are improved at high temperature.