Study On Breeding Of High-ethanol-yield Strains Of Saccharomyces Cerevisiae By Inactivated Biparental Protoplast Fusion

A fusant displaying higher ethanol production ability,as compared to that of its parents, was obtained by PEG-induced fusion between UV-inactivated haploid protoplasts of Saccharomyces cerevisiae GGSF16 and heat-inactivated haploid protoplasts of S. cerevisiae GJ2008.In shaking flask fermentation trials, the ethanol fermentation performances of S.cerevisiae GGSF16 and GJ2008 were studied. The results showed that the ethanol produced by S.cerevisiae GGSF16 and GJ2008 from cassava mash (21.5% of initial sugar concentration) for 60h and from sugar juice (12.6% of initial sugar concentration) for 24h were 7.20%(v/v) and 13.2%(v/v), respectively. Moreover, different tolerences of S.cerevisiae GGSF16 and GJ2008 were tested by using acid, sugar and ethanol tolerance tests. The least level of acid tolerence for the growth of S.cerevisiae GGSF16 and GJ2008 was pH3.0 and S.cerevisiae GJ2008 showed stronger acid resistance when grown at pH3.0. The growth inhibition of S.cerevisiae GGSF16 and GJ2008 was observed at the sugar concentration exceeding 20%(w/v) and S.cerevisiae GGSF16 showed stronger sugar resistance at 20%(w/v) sugar concentration. The? growth? of? S.cerevisiae GGSF16 and GJ2008 was suppressed at the ethanol concentration exceeding 10%(v/v) and S.cerevisiae?GGSF16 showed stronger ethanol resistance at 10%(v/v) ethanol concentration.The conditions for sporulation and ascospore isolation of S.cerevisiae GGFS16 and GJ2008 were studied. The sporulation rates of GGFS16 and GJ2008 were 91.46% and 87.20% when the strains were incubated on McCLary medium at 22℃for 7d. The ascospore isolation rates of S.cerevisiae GGSF16 (66.15%) and GJ2008 (67.35%) were obtained with the treatment of 0.3g/L snailase for 8h. By analysis of the DNA contents, weight and volume of haploid and diploid cells of S.cerevisiae GGSF16 and GJ2008, the results showed that the cells from ascospore separation trials( diploids by heat-inactivation at 60℃for 10min) were haploid. Comparing to the ethanol fermentation performences of diploid and haploid cells of S.cerevisiae GGFS16 and GJ2008, no significant differences were observed. The haploid cells of S.cerevisiae GGFS16 and GJ2008, served the most similar ethanol fermentation performance to its corresponding diploids, were used for inactivated biparental protoplast fusion.The conditions for the generation and regeneration of haploid protoplast S.cerevisiae GGSF16 and GJ2008 were obtaind when the strains(8h) were pretreated by 0.2%β-mercaptoethanol and 0.1%EDTA-Na2 at 37℃for 20mins, then treated with 0.15g/L snailase at 37℃for 30min and taken 170g/L sucrose as osmotic stabilizer. Under the conditions, the generation rates of haploid protoplast S. cerevisiae GGFS16 and GJ2008 were 98.14% and 97.16%, respectively and the regeneration rates were 17.29% and 15.78%, respectively. The protoplast fusion rate could achieve 2.7×10^-6 when the protoplasts from haploid S. cerevisiae GGFS16 (treatment by UV-inactivation for 70s) and haploid S. cerevisiae GJ2008 (treatment by heat-inactivation at 60℃for 840s) were fused by polyethylene glycol treatment.Finally, a fusant with high-ethanol-yield was selected by the trials of screening, rescreening and stability tests. The selected single fusant RH3 has higher ethanol production compared to that of its parents.