Construction Of Engineering Strain Saccharomyces Cerevisiae Metabolizing Xylose

Biomass is an important kind of renewable energy resource, which has broad market prospects as raw material to produce fuel ethanol. Many countries have integrated fuel ethanol into the development planning of energy strategy since it exhibits as a convenient, clean and renewable energy. The prerequisites for industrial production of fuel ethanol lie in cheap & readily available raw materials and efficient production process, therefore generation of ethanol via biological conversion of renewable lignocellulosic has become a research focus. Xylose is just less than glucose in the content of lignocellulose hydrolyzation, so how to obtain xylose by highly efficient conversion is one of the key technologies for bio-preparation of ethanol from lignocellulosic. Currently, it is a popular research topic on strains which can ferment xylose to ethanol efficiently.This paper mainly studied on two aspects. Firstly, genomic DNA was extracted from Candida shehatae, which was employed as template to amplify xylose reductase and xylitol dehydrogenase genes. Then they were cloned into the plasmid pYES2 to construct the recombinant plasmid pYES2-XYL1-XYL2. Recombinant Saccharomyces cerevisiae was obtained by transforming pYES2-XYL1-XYL2 to S. cerevisiae INVScl. Secondly, the corresponding enzyme activity was determined. The fermentation and preliminary research of fermentation conditions for the construction of Saccharomyces cerevisiae engineered strain were investigated. The main results are as follows:(1) Using laboratory-mutated and well-domesticated Candida shehatae as the initial strain, genomic DNA was extracted by phenol-chloroform method. As a template, the main xylose metabolic enzymes such as xylose reductase gene XYL1 and xylitol dehydrogenase gene XYL2 were amplified by PCR. The sequencing results blasted with the GenBank demonstrated that the nucleotide homology was 99% and 98%, respectively, and all the protein homology were 100%.(2) The recombinant plasmids pYES2-XYL1 and pYES2-XYL2 were constructed by the method that gene XYL1 and gene XYL2 were connected to plasmid pYES2, respectively. PGAL-XYL1, XYL1 haboring galactose promoter fragment, was amplified using pYES2-XYLl as template. The recombinant plasmid pYES2-XYL1-XYL2 was constructed by connecting the fragment PGAL-XYL1 to the recombinant plasmid pYES2-XYL2. The engineering strain S.cerevisiae metabolizing xylose was successfully constructed by transforming the recombinant plasmid into S. cerevisiae INVScl.(3) The xylose reductase and xylitol dehydrogenase activity of engineered strain S.cerevisiae were assayed by experiment to be 0.83U/mg and 1.04U/mg, respectively. In contrast, the xylose reductase and xylitol dehydrogenase activity of un-recombinant S.cerevisiae were both zero. It showed that the xylose reductase and xylitol dehydrogenase had been expressed in recombinant S.cerevisiae. The fermentation conditions were investigated, including the initial xylose concentration, initial pH, temperature, rotation speed and so on. The optimal fermentation condition for generation of ethanol is:initial xylose concentration 100g/L, initial pH 5.5, temperature 33℃, rotation speed 50 rpm after 150 rpm for 5h. Under this condition, the yield for ethanol and xylitol were 33.45g/L and 14.85 g/L, respectively.The engineering strain S.cerevisiae which could metabolize xylose was constructed successfully in this research. The strain could be resistant to high concentration of sugar and ethanol. In addition, the study using a single dual-gene vector method provides a feasible approach for cloning of a single multiple-gene vector.