Construction Of Strain Of Yogurt And Glucose Fermentation High - Yield Ethanol - Brewing Yeast

Fuel ethanol is one of the wide-accepted and most potential renewable clean energies. Using lignocelluloses as raw material to produce fuel ethanol will not only efficiently reduce the cost of production, but also play a significant role in waste management and environment protection. Glucose and xylose are the main saccharide elements of lignocelluloses digest.However, for the conventional alcohol fermentation industrial strains, such as Saccharomyces cerevisiae, they lack the ability to utilize xylose economically due to the absent of xylosmetabolism flow. Therefore, to implement the production of fuel ethanol using lignocelluloses as raw stuff by microorganism fermentation, the primary problem to solve is strain.Choosing Saccharomyces cerevisiae W5, the one has superior ferentation function, and Candida shehatae 20335, the one possesses xylosmetabolism pathyway, as the original strain to select engineering strains by means of protoplast fusion and genetic engineering breeding. This process will make the ability to utilize xylose generally available; what’s more, will favor the utilization of the admixture of xylose and glucose to produce alcohol in a relatively high efficience.By means of protoplast fusion, using W5 and 20335 as parent strain, we grope and determine the optimum condition for protoplast preparation and inactivation dose. We adopt the inactivated protoplast as screening marker, fusing the parent strain treated by different inactivation ways, ultimately screening four fusion stocks, in all of which the yield are higher than the parents. Among them, the yield of HDY2-14, 0.31g/g, is the highest,20.2% and 15.2% higher than W5 and 20335, respectively.By means of genetic engineering breeding, we respectively construct URA3-directed low copy integration-expression vector pZMYBXl and rDNA-directed high copy integration-expression vector pZMYX2. Between them, the former contains the Blasticidin resistance gene bsd as the selection marker for recombination strain and xylose reductases genes xyl1; the other one contains G418 resistance gene KanR, xylitol dehydrogenase xyl2 and xylulokinase xks1. By lithium acetate transformation method, cotransformating the linearization plasmid vector pZMYBXl (StuⅠ) and pZMYX2(HpaⅠ) into the integrated Saccharomyces cerevisiae cell. Ultimately we obtain three recon HDY-ZMYWBG1、HDY-ZMYWBG2、HDY-ZMYWBG3. The yield of HDY-ZMYWBG1 and HDY-ZMYWBG3 is 0.368g/g and 0.365g/g, respectively, both of which are higher than W5. The improved percentage is 11.5% and 9.7%.The success of the subject provides new example for the reforming of the xylosemetobolism pathway in Saccharomyces cerevisiae, supplies the alternative strain for the production of the biology alcohol using lignocelluloses as substrates.