Inactivation Markers Can Be Used To Screen Yeast Fusants That Can Use Xylose To Produce High Ethanol

Ethanol fuel is a well-acknowledged and most promising renewable clean energy so far.Production of ethanol fuel by lignocellulosic biomass as raw material can reduce costs for raw materials and play an important role in environmental protection and disposal and reuse of the waste.Lignocellulosic raw materials can be hydrolyzed to produce a mass of monosaccharides,principally xylose and glucose.However,in terms of conventional industrial strains for alcoholic fermentation,i.e.,Saccharomyces cerevisiae,xylose cannot be effectively used because they themselves have incomplete metabolic flux of xylose.Hence,to effectively use lignocelluloses,it is imperative to dope out a solution to strains by producing ethanol fuel via microbial fermentation.In this study,protoplast fusion was performed with Saccharomyces cerevisiae TG and Pichia stipitis as original strains,and genomes of three strains,which have complete metabolic flux of xylose and can efficiently use xylose to produce fusants of ethanol,were screened and fused by fermentation and molecular methods.Based on confirmed optimum conditions of protoplast preparation and regeneration for all original strains,with Pichia stipitis and Saccharomyces cerevisiae TG as parental strains while inactivated protoplasts as selection markers,twenty fusants were fused and finally selected by fusion of parental strain protoplasts inactivated in different ways.Of them,TP-11 had the highest ethanol yield(0.2438g/g)when fermented with xylose as single carbon source,with an increase by 20.2%compared with Pichia stipitis,one of the two parental strains(with an ethanol yield of 0.1710 g/g);while TG,the other parental strain,could hardly produce ethanol by xylose fermentation.Genomes of fusants were verified by PCR,proving that the xylose reductase gene xyll and xylitol dehydrogenase gene xyl2 had stably been integrated into the chromosomes of fusants obtained.;analysis results of enzyme activities of xylose reductase(XR),xylitol dehydrogenase(XDH)and xylulokinase elated to the xylose metabolism in fusants also indicated that:within 48-hour measurement,enzyme activities of XR,XDH and xylulokinase of TP 11 were 6.5791U/mg(with increases by 409%compared with TG and by 27.8%with Pichia stipitis),16.1712U/mg(with increases by 104%compared with the parental TG strain and by 10.4%with the parental Pichia stipitis strain)and 115.1298U/mg(with increases by 17.3%compared with the parental Pichia stipitis strain),respectively,indicative of effective expression of these three genes.Success of this research provides a new successful example for the establishment of xylose metabolic pathways of Saccharomyces cerevisiae and optimization of screening methods for protoplast fusants,and lay a solid foundation for studies on xylose metabolic engineering of Saccharomyces cerevisiae and ethanol production by biotransformation of lignocellulose.