Screening And Molecular Biological Modification Of Yeast Strains For Ethanol Production From Xylose

As the fossil fuel resources decreasing and the energy crisis strengthening in the world, ethanol fermentation using fibre resources has become a hotspot recently. The key point of this research field is microbial strain selection capable of producing ethanol from various carbohydrates efficiently. In this thesis, the wild Candida tropicalis was chosen as the original strain. Metabolic engineering breeding strategy was applied to modify the metabolic flux in order to increase the ethanol yield.One yeast strain, which was isolated from 256 natural samples, was found to be able to utilize D-xylose effectively. On the basis of assimilation physiological tests and Molecular biology teast, all of the detection led to the identification of yeast strain as a strain of Candida tropicalis. GenBank accession number of ITS nucleotide sequence was EU121523. The strain was submitted to the Culture and Information Centre of Industrial Microorganisms of China Universities and the corresponding serial number was CICIM Y0092.The Candida tropicalis obtained was taken as the further research strain. It was found that the main product of xylose metabolism is xylitol and that of the glucose metabolism is ethanol. By means of metabolism analysis and determination of the key enzyme activity in xylose metabolism, we ascertained the method of metabolic engineering as following, the Candida tropicalis was taken as the host strain, and over expression of Pichia Stipitis Xylitol Dehydrogenase (XDH) Gene XYL2 was expected. It was attempted to modify the metabolic flux guiding into the direction of ethanol producing in order to increase the ethanol yield.The pYX212-XYL2-hygro was based on shuttling expression vector pXY212-XYL2.With hygromycin B resistance gene acted as a dominant selectable marker, pXY212-XYL2-Hygro (Recombinant plasmid which was used integration was digested by Not I) was transformed into Candida tropicalis by electroporation. Through PCR identification, the integration was proved. One band were got, which was 1.7 kb XYL2 of Pichia Stipitis. Specific XDH activities of free expression transfornants were 0.5 U/mg protein, which was 3 times more than that of the parent strain. 15% increasing was also acquired for the specific XDH activities of intergrative expression transfornants.It was detected by the xylose fermentation test that, the free expression transformants yield of xylitol was 3 times less than that of the parent strain, and the yield of ethanol increased by 5 times; the xylitol yield of the integrative expression transfornants also decreased, but no significant difference has been detected regarding the ethanol yield, owe to the expression level. It could also indicate from the fermentation test that, a higher XDH expression level contributes to decreasing xylitol accumulation and better ethanol yield. The feasibility of ethanol production from xylose fermentation by C. tropicalis was verified, which possesses great significance of utilizing the abundant cellulose resources.