Molecular Evolution Of Xylose Isomerase From Thermus Thermophilus And The XylA Gene From Sorangium Cellulosum: Cloning And Expression

The production and application of fuel ethanol which is the most promising new renewable energy is paid much attention by many governments for the importance of economic development and strategic significance. There are abundance of lignocellulosic materials in nature, however only 3-4% of lignocellulosic materials can be utilized. D-xylose is the most abundant monosaccharide in lignocellulose hydrolysates after glucose.Utilizing xylose to produce ethanol adequately is one of the bottlenecks to ethanolic fementation of lignocellulosic materials.Saccharomyces cerevisiae has been traditionally used in ethanol producing, which has acquired qualities such as high ethanol productivity, tolerance to process hardiness, tolerance to fermentation by-products and is, therefore, preferred for ethanol production from crops. Although a few xylose-fermenting yeasts were found, the preferred organism for industrial ethanol production is the yeast S. cerevisiae which can not utilize D-xylose, only its isomer D-xylulose.Metabolic engineering can be used to extend the substrate range for growth and product formation of an organism. It is a useful way to establish the xylose metabolic pathway by expressing correlative gene in the S. cerevisiae. Xylose isomerase (XI) does not require any cofactors that can transform xylose to xylulose directly,which is regarded as the first way to establish the xylose metabolic pathway in the S. cerevisiae. Previous attempts to express xylose-isomerase gene from a variety of bacteria and Archaea in S. cerevisia have failed. Anotable exception involved xylose isomerases from thermophiles. How ever, exhibite only very low activities at temperatures that are permissive yeast to grow. We do molecular evolution to xylA from T. thermophiles and clone other xylose-isomerase gene which can be actively expression in S. cerevisia.This paper main work is the expectation that establish effective XI xylose metabolism pathway in S. cerevisiae.On the one hand, XI from Thermus thermophilus was mutanted by molecular evolution , on the other hand, the S.cellulosum xylA gene was cloned and active expressed in Saccharomyces cerevisiae.We choose two amino acid Pro137 and Asp247 for mutation to Gly basd on the...