Co-Expression Of Trehalose And Heat Shock Proteins To Increase The Ethanol Tolerance Of Saccharomyces Cerevisiae

During the use of traditional fossil energy some greenhouse gases and pollutants which cause serious environmental pollution and destruction were exhausted. Seeking renewable clean energy is imminent which is not only related to the develpoment of global economy, but also related to the heritage of human civilization. As a representative of clean energy, clean fuel ethanol has become a hot spot among both research scholars and governments concern because of its merits such as enough engery and convenience for use. Currently fermentation is the main method of fuel ethanol production. However, some intractable problems arise during the fermentation process such as ethanol stress, contamination and other issues which directly affect the yield and economic benefits. Ethanol accumulated during the fermentation can inhibit the growth and metabolism of Saccharomyces cerevisiae. Although S. cerevisiae can tolerate a certain degree of ethanol, ethanol accumulated with the fermentation time going on leads to the serious degree of inhibition. High concentration of ethanol can also induce some stress responses in S. cerevisiae cells, such as the increased expression of heat shock proteins (HSPs), high accumulation of intracellular trehalose and so on. The accumulation of trehalose, HSPs, ergosterol and other protectants will improve the ethanol tolerance of S. cerevisiae.This paper aims to complete the co-expression of trehalose and heat shock proteins to increase the ethanol tolerance of S. cerevisiae. Growth curves and cell diameter of S. cerevisiae cultured under different ethanol concentrations (0%,5%,10%,15%v/v) were measured and analyzed. Growth and metabolism of A. cerevisiae cultured with exogenous ethanol were subject to different degrees of inhibition, and the degree of inhibition increased with high ethanol concentration. The contents of trehalose and ergosterol in S. cerevisiae cells cultured under different concentrations of ethanol were also determined and compared. The results showed that the contents of trehalose increases with ethanol concentration and reached maximum at 15%(v/v) ethanol concentration. The content of trehalose also increased with increasing ethanol concentration, but reached maximum 10%(v/v) ethanol concentration. In addition, trehalose and ergosterol were positively correlated, indicating that they played an important role in the ethanol tolerance of S. cerevisiae.The related genes of trehalose and heat shock proteins in S. cerevisiae cultured under different ethanol concentrations (0%,5%,10%,15% v/v) were determined by real-time quantitative PCR. The results showed that the expressions of trehalose synthase genes (tpsl, tps2, tps3, tsll) and HSPs synthase genes (hsp78, hsp82, hsp104, ssa1)increased with high ethanol concentration. The expressions of trehalose decomposition genes (ath1,nth2) decreased with high ethanol concentration, but nthl was significantly increased.On this basis, hsp104 and tps2 amplified by PCR method were selected to achieve their co-expressed in yeast cells in order to enhance the ethanol tolerance of S. cerevisiae. Plasmid pYSE2 and these two genes amplified by PCR method were double digested and connected. The recombinant plasmid was transformed into E. coli BMTOP10 and screened positive clones. Sequencing results showed there were no mutations and the sequence was completely correct. Both tps2 and hsp104 completed genic recombination with plasmid pYES2, and continue to build a co-expression recombination plasmid of tps2 and hsp104 which was used in the transformation of S. cerevisiae.