Double Marker Screening For Xylose And Glucose Co-fermentation Of High-yield Ethanol Saccharomyces Cerevisiae Fusion

Using lignocelluloses as raw material to produce fuel ethanol has been coming a hot issue in the field of resource development.Glucose and xylose are the main saccharide elements of lignocelluloses hydrolyzate.Saccharomyces cerevisiae can just metabolize glucose due to the absence of complete xylose metabolic pathway.Consequently,it limits the efficient utilization of lignocellulosic materials for ethanol production.Engineering strains which have the ability to utilize xylose availably can be obtained by the means of protoplast fusion for transforming Saccharomyces cerevisiae.However,screening of fusants is especially important in the process of protoplast fusion.It is the crucial link in success of protoplast fusion that how to screen the fusants with excellent characters accurately,quickly and easily.Saccharomyces cerevisiae W5,the one has superior fermentation function;and Candida shehatae 20335,the one possesses xylose metabolic pathway were chosen as the original strains to make the xylose metabolic pathway unobstructed by the means of protoplast fusion.Resistance marker and reporter gene were used as the double markers to screen engineering strains of Saccharomyces cerevisiae which have the ability to utilize xylose availably and produce ethanol in a relatively high efficience when using xylose and glucose as hybrid carbon source.In this study,two yeast episomal plasmids which named pZLYl and pZLY2 have been constructed.The plasmid pZLYl whose size is 4949bp has G418 resistant marker and reporter gene gfp while the plasmid pZLY2 whose size is 7713bp has Blasticidin resistant marker and reorter gene gusA.By lithium acetate transformation method,the plasmid pZLYl and pZLY2 have been transformed into Candida shehatae 20335 and Saccharomyces cerevisiae W5 respectively in order to obtain transformants.Then the fusions have been screened using the four selective markers above by means of protoplast fusion with the transformants.Ultimately,ten fusants were screened,in all of which the yield were higher than the parents.Among them,the yield of ZLYRHZ7,0.427g/g,was the highest.37.3%and 21.0%higher than W5 and 20335,respectively.The success of the subject provides a new example for optimizing the method for screening of fusants and reforming of the xylose metabolic pathway in Saccharonmyces cerevisiae.It also supplies the alternative strain for the production of the biology ethanol using lignocelluloses as substrates.