Analysis Of Genetic Variations And Construction Of Knockout Mutants Of Saccharomyces Cerevisiae Rice Wine Strain

The rice wine (Huangjiu) is the Chinese national alcohol brewery. It is brewed by using the main raw materials, such as rice, corn, wheat, and a variety of microorganisms in the wheat koji. Rice wine, rich in amino acid, vitamins and trace elements, has the function of easily digestible, relaxing tendon activating blood, maintaining appetite and protecting heart. Saccharomyces cerevisiae, as the key microorganism, played a leading role in the fermentation. In the fermentation process of rice wine, yeast cells are subjected to stress of various environmental factors, such as temperature, osmotic pressure, pH, and ethanol concentration. When stress conditions exceed the physiological limits of the cell, it will inhibit yeast cell growth and ethanol fermentation efficiency. Therefore, it is essential for rice wine fermentation industry to select excellent rice wine yeast strains with improved stress-tolerant capbility.Previously, we have already separated one Saccharomyces cerevisiae haploid named D2, which have high tolerance to high stress environment. Through whole genome sequencing of D2and its comparative analysis with S288c, we found some new genes which exist in D2only, such as new gene g5170, and some missed genes in D2, such as gene AAD10.To investigate the potential effects of a novel gene g5170, the knockout strain D2-△g5170was constructed using Cre/loxP system. The g5170gene disruption cassette with the short flanking homologous regions to the g5170gene and Kanr as selectable marker was amplified and transformed into S. cerevisiae through LiAc/SS carrier DNA/PEG methods. The positive transformants were screened on G418plates and verified by PCR. The Kanr marker was removed by transforming plasmid pSH65into positive transformants and inducing expression of Cre recombinase by galactose. The procedure was repeated to obtain the knockout strain D2-△g5170, in which two g5170copies were completely deleted.Compared to wild type strain, the mutant D2-△g5170showed no significant alterations of tolerance to heat stress and osmotic stress, but exhibited significant lower tolerance to ethanol stress. The growth rate result also indicated that mutant D2-△g5170showed high tolerance to ethanol stress. The survival rate analysis of two strains under8%ethanol showed that the survival rate of the mutant strain reduced about1.16to1.71-fold compared with wild strain D2. Moreover, the results of simulated rice wine fermentation showed that the growth rate, ethanol production and other parameters of mutant was similar with that of wild strain. Taken together, these results suggested that novel gene g5170was associated with ethanol stress response in S. cerevisiae rice wine strain.