产甘油假丝酵母抗逆转录因子的过表达对酿酒酵母耐酸胁迫性的影响

Effect of overexpressing transcription factors of Candida glycerinogenes on acid tolerance of Saccharomyces cerevisiae

以具有优良环境耐受性的产甘油假丝酵母(Candida glycerinogenes)为研究对象,考察其抗逆转录因子对酿酒酵母(Saccharomyces cerevisiae)酸胁迫耐受性的影响.分别克隆获得C.glycerinogenes和S.cerevisiae的转录因子基因haa1和asg1,在S.cerevisiae W303-1A中分别过表达这4个基因,继而进行摇瓶试验考察重组菌株的酸耐受性.结果显示,过表达不同转录因子均能提高细胞酸耐受性,其中90 mmol/L乙酸时重组菌S.cerevisiae/Cghaa1和S.cerevisiae/Cgasg1的生物量与S.cerevisiae/Schaa1和S.cerevisiae/Scasg1相比分别提高了44.3%和18.9%.q RT-PCR发现,与Schaa1和Scasg1相比,过表达Cghaa1和Cgasg1能够显著上调下游酸耐受相关基因的表达水平.酸胁迫下乙醇发酵结果显示,相比对照组,重组菌S.cerevisiae/Cgasg1的乙醇产量提高11.1%.上述结果表明转录因子HAA1和ASG1均能提高酿酒酵母酸耐受性和酸胁迫下乙醇产量,其中Cghaa1和Cgasg1效果更为明显,结果可为提高酿酒酵母酸耐受性提供新的基因资源和思路,为进一步挖掘C.glycerinogenes抗逆基因提供借鉴.

The aim of this study was to examine the effect of antiretroviral transcription factors of Candida glycerinogenes on the tolerance of Saccharomyces cerevisiae to acid stress. The haa1 and asg1 genes of C. glycerinogenes and S. cerevisiae strains were obtained by cloning, and these four genes were subsequently overexpressed in S. cerevisiae W303-1A. The acid tolerance of the recombinant strains was examined using shake flask experiments. The results showed that overexpression of different transcription factors increased the acid tolerance. In comparison with S. cerevisiae/Schaa1 and S. cerevisiae/Scasg1, the biomass of S. cerevisiae/Cghaa1 and S. cerevisiae/Cgasg1 in 90 mmol/L acetic acid was 44.3% and 18.9% higher, respectively. Strains overexpressing Cgasg1 and Cghaa1 exhibited enhanced tolerance to a high concentration of acetic acid. qRT-PCR analysis revealed that overexpression of Cghaa1 and Cgasg1 significantly up-regulated the expression of downstream acid-tolerance-related genes. Under conditions of acid stress, the ethanol yield of S. cerevisiae/Cgasg1 was 11.1% higher than that of the control group. These results indicate that the transcription factors HAA1 and ASG1 can enhance the tolerance of S. cerevisiae to acid stress. This study provides a valuable insight into techniques for improving the acid tolerance of S. cerevisiae and for future research into C. glycerinogenes resistance genes.