Research On Thermotolerance Of Saccharomyces Cerevisiae Based On Cellular Protein Homeostasis

In the process of the industrial fermentation, large quantities of heat constantly generated because of the metabolism and the mechanical mixing, causing the fermentation temperature continuously increased. However, heat will affect the growth and production of microorganisms, hence cooling control is needed, resulted consuming a large amount of energy. By enhancing the thermotolerance of microorganisms, fermentation is able to proceed at high temperature. In this way, not only the energy consumption will reduce, but also the rate of fermentation will be improved. Therefore, the genes related to protein quality control of Saccharomyces cerevisiae as functional parts and the strong promoter FBA1 p, were constructed into the genetic-devices with single and multiple function by using synthetic biology, respectively. The main results were as follows:2 genes from autophagy system, 12 genes belonged to 26 S ubiquitin-proteasome system and 2 genes related to chaperones were mined.16 PQC genetic-devices were assembled and the corresponding engineered strains were constructed as well. FBA1p-RSP5, FBA1p-ATG8, FBA1p-UBC4 and FBA1p-HSP104 were screened through gradually increased temperature incubation, as the heat-resistant PQC genetic-devices that could endow Saccharomyces cerevisiae thermotolerance. Compared with the control, the OD660 of the engineered strains of S.c-RSP5, S.c-ATG8, S.c-UBC4 and S.c-HSP104 were 32.5%, 43.6%, 38.0%, 49.7% higher when cultured at 40℃; physiological characteristics implied that their thermotolerant strains possessed higher heat shock survival rate, trehalose-6-phosphate synthase gene and chitin synthase gene expression.4 single functional PQC genetic-devices were rational designed, 3 multiple functional genetic PQC devices(FBA1p-HSP104-SLM5t-CDC60p-ATG8-MSR1 t,FBA1p-HSP104-SLM5t-CDC60p-UBC4-MSR1 t, SSA1p-UBC4-SLM5t- CDC60p- ATG8- MSR1t- FBA1pRSP5) were constructed. The result of thermotolerance experiment showed, the multiple functional PQC genetic-devices could endow better thermotolerance, cell wall integrity and higher cell activity than the single functional PQC genetic-devices. This suggested that genes from different mechanisms of protein quality control system could enhanced the thermotolerance of Saccharomyces cerevisiae synergisticly. Thermotolerant engineered strains were verified through 35℃ ethanol fermentation. The result showed that all the multiple functional Thermotolerant strains grew better than single functional Thermotolerant strains. What’s more, the OD660 of the engineered strains S.c-HA, S.c-HU, S.c-RAU were 17.3%, 42.9% and 56.9% higher than wildtype, which are close to WT cultured at general conditions; the ethanol production increased 37.0%, 40.7%, 37.0% than WT/V, which were no difference than that of WT cultured at general conditions. It showed that the heat-resistant PQC genetic-devices could enhanced the thermotolerance of Saccharomyces cerevisiae.