Xylulokinase Gene Cloning And Expression In Saccharomyces Cerevisiae Strain

Saccharymyces cerevisiae can ferment D-xylulose,however,because of the block of xylulose metabolic stream and infection of redox imbalance,recombinant Saccharymyces cerevisiae containing Pichia stipitis xylose reductase and xylitol dehydrogenase show xylitol accumulation phenomena which leads to the reduction of ethanol productivity.In this research, we clone Pichia stipitis xylose reductase gene,xylitol dehydrogenase gene and xylulokinase gene,express them in Saccharymyces cerevisiae to dredge xylose metabolic stream and rise ethanol productivity.Using Oligo and Primer PREMIER to design Pichia stipitis xylulokinase gene xy13 (gi8100399,2942bp)PCR primer.Upper primer is 5'-CCAACCAGCAGCGTGTG-3',lower primer is 5'-TCTATCGTGATATTCGCACAT-3'.Gene xy13 coding xylulokinase was cloned from the genome DNA of Pichia stipitis with the length of 2704bp,namely X3.The similarity value between X3 and the gene sequence of xy13 in GeneBank was 98%.Analysis of this gene showed that there were promoters and terminators but no intron in xy13 which is essential to the expression in Saccharomyces cerevisiae.The gene X3 was coded and a 623AAR polypeptide (69324Daltons)was produced.The DNA similarity between X3 and Saccharymyces cerevisiae xylulokinase gene xksl is 35.46%,protein similarity is 34.2%,and they have highly conserved regions.From yeast episome plasmid YEp24,successfully construct YEp24-X3.With plasmid YEp24-X1X2 and YEP24-X3,expression vector YEp24-X1X2X3 contained genes X1,X2 and X3,was recombined.Recombinant of Saccharomyces cerevisiae,namely R5(with YEp24-X1X2X3)was constructed by transferring mutant Saccharomyces cerevisaie 1949 with LiAc reagent.Saccharomyces cerevisaie R5 could grow well in xylose medium,molecule test indicate that xylose reductase gene,xylitol dehydrogenase gene and xylulokinase gene were contained.Apparently different has shown in xylose fermentation by Saccharomyces cerevisaie R5 on YCM medium.Saccharomyces cerevisaie R5 could grow well in xylose medium under aerobic and anaerobic conditions,under aerobic condition R5 can consume 99.5%xylose in the medium and ethanol concentration is 0.46g/l.Under anaerobic condition,ethanol yield was 19%of theoretical yield.This shows that expression of Pichia stipitis xylose reductase gene, xylitol dehydrogenase gene and xylulokinase gene is helpful to xylose ferment ethanol. Recombinant xylose-fermenting yeast strains were constructed,which expands and substantiates the strains' source and the theory for xylosc-metabolic engineering of microorganisms and the bioconversion of lignocellulosics to ethanol.