The Construction And Application Of Thermo-tolerant Molecular Regulation System Of Saccharomyces Cerevisiae

Energy consumption and environmental pollution could be greatly reduced if the microbial fermentations are performed at higher temperature. In this study,thermo-tolerant functional parts consisting of heat shock protein genes from thermophilic bacteria Thermoanaerobacter tengcongensis and Thermus thermophilus were mined, and the strong constitutive promoter(FBA1p) from Saccharomyces cerevisiae was chosen as regulatory part. Based on these parts, thermo-tolerant devices were designed and constructed through two different methods: single and dual functions assembly. Then these thermo-tolerant devices were transformed into S.cerevisiae in the form of plasmids or genome integration for improving its thermo-tolerance. The main results were as follows:The thermo-tolerance of unifunctional engineered strains was primarily characterized by the gradual increase of temperature from 35℃ to 45℃ or constant high temperature 42℃. The results showed that the engineered strains constructed with ubiquitin and small heat shock protein gene from T. tengcongensis had better thermo-tolerance. The OD660 and cell viability were improved by more than 30% and1.5-4.0 fold compared with the control, respectively. The engineered strains constructed using homologous recombination showed 21.6% increase in OD660 and2.1-3.0 fold increase in cell viability compared to the control.Physiological characteristics studies revealed that the thermo-tolerant engineered strains possessed better cell wall integrity, higher trehalose content and enhanced antioxidant activity, ethanol-resistance and metabolic energy.Promoters with different strength were introduced as regulatory part to reconstruct the unifunctional thermo-tolerant engineered strains. The transcription level studies showed that the growth ability of strains was relevent to the expression level of thermo-tolerant genes. The fermentation results revealed that the bifunctional thermo-tolerant engineered strains constructed by using DNA assembly showed better thermo-tolerance than that of the unifunctional thermo-tolerant engineered strain.when cultured at 42℃, the ethanol yield of bifunctional thermo-tolerant engineered strains was increased by 47.8% and 23.6% compared to the control and unifunctional engineered strains, respectively.Better thermo-tolerant devices were transformed into β-amyrin S. cerevisiae engineered strain and industrial ethanol yeast to verify their functions, respectively.The β-amyrin yield of thermo-tolerant β-amyrin S. cerevisiae engineered strains was improved by 33.4%, and the optimum growth temperature of S. cerevisiae was broadened to 35 ℃. The ethanol yield of all thermo-tolerant ethanol yeast engineered strains was improved above 10% compared to the control strain, especially the engineered strain T.te-IbpA, whose ethanol yield was increased 35.6%.This study provides a platform to substantially reduce energy consumption and production cost by improving microbial bio-synthesis efficiency at higher temperature and offers a valuable scientific reference to design and construct other resistant strains.