Saccharomyces Cerevisiae Recombinant Strain Of The Metabolism Of Xylose To Ethanol Is Initially Built

With the constant rise of international energy price,it is of great practical importance of using cheap lignocellulosic materials to produce fuel ethanol.Xylose is monosaccharide whose content is higher than other sugars except glucose in most cellulosic hydrolysates.As the traditional ethanol fermentation strain Saccharomyces cerevisiae is not able to utilize xylose,the difficulty of producing ethanol from cellulosic material arises.People have been trying to modify the existing microorganisms for many years by genetic engineering and cell fusion technology to make them enable to metabolize xylose and gain the characteristics for working in cellulosic hydrolysate.This article introduced the progress in this field home and abroad.In order to make Saccharomyces cerevisiae utilize and metabolize xylose to produce ethanol, it is indispensable to express at least two enzymes of xylose metabolism,XYL1 encoding xylose reductase and XYL2 encoding xylitol dehydrogenase,in the the cytosol of Saccharomyces cerevisiae.This article first cloned the genes XYL1 from Candida shehatae and XYL2 from Candida tropicalis,respectively.Due to the relative expression level of above two enzymes has a great influence in the accumulation of xylitol and yield of ethanol,this research constructed an yeast expression plasmid pYES2-xyl1 containing XYL1 under the control of an inducible promoter and another yeast expression plasmid pDR195-xy12 containing XYL2 under the control of a constitutive strong promoter.Afterwards,the two constructed plasmids were used to transform S.cerevisiae host cell.Enzyme assay demonstrated genes XYL1 and XYL2 were both functionally expressed in the transformants.Protein electrophoresis further proved that the two enzymes were over-expressed in the cytosol of host cell,respectively.By sequencing and analyzing the unknown DNA fragment on the vectors,the design of constructing the recombinant yeast chromosome integration plasmid was confirmed.The gene XYL1 and its expression element was cloned from previously established yeast expression plasmid pACT2-xyl1 and then fused with yeast chromosome integration vector YIp5-KanR, resulting in recombinant plasmid YIp5-KanR-x1.This article has accomplished preliminary construction work of recombinant S.cerevisiae metabolizing xylose to ethanol.It is expected to further construct the recombinant chromosome integration plasmid YIp5-KanR-x12,and then integrate the above genes into the genome of the Baker's yeast possessing good fermentation abilities by homologous recombination to acquire a recombinant S.cerevisiae that could steadily metabolizing xylose to ethanol.