Cloning Of Xylose Reductase Gene From Pichia Stipitis And Expression In Saccharomyces Cerevisiae

Xylose is one of the major components in cellulosic biomass,and the fermentation of xylose is essential for the bioconversion of biomass to ethanol.The commonly used Saccharomyces cerevisiae has many advantages as an ethanol producer, such as fast D-glucose consumption. However, a major drawback is that S. cerevisiae cannot utilize the pentose sugar xylose, only utilizing its isomer xylulose.we can attempt to clone and transform the key gene encoding enzymes for the conversion of xylose to xylulose in S. cerevisiae to gain the gene engineering S.cerevisiae that can stably and effectively utilizes various components in hydrolyzed lignocellulose to ferment form ethanol. Therefore, this research mainly aim is to clone xylose reductase gene which is the first key gene in fermenting xylose to ethanol from Pichia stipitis, and transformed it into the S.cerevisiae for laying the foundation to subsequently construct the ferment xylose genetic engineering microorganism.In this paper, Pichia stipitis CBS6054 provided the xylose reductase gene. The primers were designed according to the sequence of xyll reported on the NCBI.The gene encoding xylose reductase was amplified by using high-fidelity DNA Polymerase and sequenced without bases mutations. The target gene was cloned into a shuttle vector p424GPD and was placed under the strong constitutive promoter GPD.The recombinant vector p424GPD-xyll was transformed into E.coli TOP 10,extracting the Total protein and analysis by SDS-PAGE.The xylose reductase gene was expressed in E.coli by means of enzyme assay.The construction of this vector provides an important basis for the further establishment of recombinant Saccharomyces cerevisiae.The expression vector was digested to linear and transformed it into S.cerevisiae haploids, screened positive clonings through nutritional defect experiment and PCR detection,and determined xylose reductase activity. Xylose reductase activity was around 0.1-0.3U/mg in recombinant haploids S.cerevisiae 1916 and 1917.The highest xylose reductase activity recombinant haploids S.cerevisiae 1916-8 and 1917-1 were combined, screened diploid S.cerevisiae through dyeing ascospore experiment, and determined xylose reductase activity. Xylose reductase activity was around 0.1U/mg in diploid S. cerevisiae,compared with the expressing quantity of xylose reductase in haploid S.cerevisiae,the diploid S.cerevisiae had the lower activity,showed not to attain an intended result.Because expression vectors easily lost in S.cerevisiae proliferation process,so we constructed integration vector through digestion and ligation transcription components and xylose reductase gene coming froming the expression vector into integration vector,integrated xylose reductase gene into S.cerevisiae genome chromosomes through the integration vector,became a part of S.cerevisiae genome chromosomes,enhanced its genetic stability,and made gene efficient and stable to express,and layed the foundation to construct efficiency and stability genetic engineering S.cerevisiae which can ferment xylose to ethanol.