Protoplast Fusion To Construct A Fuel Ethanol Production Strain Capable Of Co-fermenting Pentose And Hexose Released From Lignocellulose

Haploid strains were obtained from Saccharomyces cerevisiae strain 1308 by optimum sporulation conditions,and fermentation activity of them was analyzed.The results suggest that haploid D-12 was the best.So we took D-12 as one of parent strains.The optimum conditions of formatting and regenerating of parent strain D-12 were: the cells which had been cultured for 9h,were pretreated withβ-ME(0.1%)for 30min, then broke cell walls with snailase(4ml/mL)and cellulose(4mg/mL)for 1.5h,the protoplast formation rate and regeneration rate were 93.6%and 15.9%;The optimum conditions of formatting and regenerating of parent strain P-01 were:the cells which had been cultured for 21h,were pretreated withβ-ME(0.1%)for 30min,then broke cell walls with snailase(4ml/mL)and cellulose(4mg/mL)for 1.5h,the protoplast formation rate and regeneration rate were 97.7%and 9.9%.The protoplast of parent strain D-12 was inactivated with heating,the optimum conditions were:treating for 40min in electric-heated thermostatic water bath(70℃).The protoplast of parent strain P-01 was inactivated with UV,the optimum conditions were:treating for 60s below the UV lamp(15W).The inactivated protoplast fusion were induced by PEG.We obtained a fusant F10 which can co-ferment pentose and hexose into ethanol after initial screening by TTC plates and secondary screening by fermenting and identification by isozyme electrophoresis.The ethanol concentration of F10 in fermentation broth reached 1.3ml/100ml(v/v),and 36.8%higher than parent strain P-01(0.95ml/100ml).At last,we analyzed the fermentation activity of F10 in corn straw hydrolyzates. The ethanol concentration of F10 reached 1.1ml/100ml,and far higher than the parent strains P-01(0.6ml/100ml)and D-12(0.3ml/100ml).