Construction And Studies Of Saccharomyces Cerevisiae Ethanol Responsive Reporter Vector

Ethanol produced by yeast fermentation is inhibitory to both yeast cell growth and metabolisms,research on the molecular mechanism of yeast ethanol tolerance can provide basis for breeding of yeast strains with improved ethanol tolerance.In this study,the Trehalose-6-phosphate synthase gene TPS1 promoter(P_TPS1spsc) was amplified from self-flocculating yeast SPSC01.Due to the 21 bp insertion comparing with the TPS1 promoter from the model yeast strain S288C,P_TPS1spsc harbors one more stress response element(Stress Responsive Element,STRE) and therefore is a novel promoter.Reporter vector containing the green fluorescent protein encoding gene EGFP directed by P_TPS1spsc was constructed based on the pYES2.0 plasmid,and transformed to yeast strain ATCC4126.The EGFP fluorescence intensity of the transformant harboring the reporter vector was assayed in the medium containing different concentrations of ethanol,glucose and under different temperatures by Flow Cytometry.The results showed that P_TPS1spsc responses specific to ethanol,and is insensitive to high-temperature and high glucose concentration,and thus can be used to detect the status of ethanol stress.Studies on the effect of zinc on P_TPS1spsc promoter activity showed that addition of zinc to the culture of ATCC4126 transformant containing the reporter vector did not affect the P_TPS1spsc promoter activity,which indicated that there is no direct interaction of zinc with P_TPS1spsc under the experimental conditions tested.The addition of zinc also did not affect the intracellular trehalose content of the transformant and the ethanol concentration,which suggest that effect of zinc on ethanol fermentation and ethanol tolerance may be influenced by the genetic background of the yeast strains.Studies on ARO80 encoding gene which is the transcription vector of the quorum-sensing circuit revealed that the promoter of ARO80 cannot induce the expression of EGFP in the reporter vector under the experimental conditions employed in this study,and the effect of ARO80 in yeast ethanol tolerance needs to be further explored.This work provides basic for further construction of ethanol responsive reporter vector library from the genome of SPSC01 and future studies on the molecular basis of improved ethanol tolerance of self-flocculating yeast.