| With the rapidly development of economy, there is a lack of mineral resource of sorts day by day. Energy sources scarcity threatened the continuable development of society. The importance of alternative energy sources gave birth to initiatives. Fuel ethanol is regarded as one of the most promising environmentally friendly renewable recosource. Expansion of production of bioethanol utilizing lower-value Hgnocellulose offers a great potential for not only reducing the production cost but also protecting the environment as it produces far less air pollutants than gasline. D-Xylose is the most abundant monosaccharide in Hgnocellulose hydrolysates after glucose. Consequently, ethanolic fermentation of xylose is of major concern for the efficient utilization of lignocellulosic hydrolysates to produce fuel ethanol. It is of course the proper way to make ethanol producing industrial strains coferment glucoseand xylose by pathway engineering and improve the recombinant strains' utmostability of xylose utilization through optimizing the downstream fermentation by bioengineering technics.Saccharomyces cerevisiae has been traditionally used in producing ethanol. However, applying S. cerevisiae for fermentation of lignocellulosic hydrolysates has the drawback that it cannot naturally utilize xylose, only its isomer xylulose. Metabolic engineering can be used to extend the substrate range for growth and product formation of an organism. In this study, genes encoding enzymes for xylose metabolism have been introduced into an industrial strain of Saccharomyces cerevisiae. The xylose metabolic pathway was established in the industrial strain of Saccharomyces cerevisiae. The recombinant strains have the ability of utilization the xylose to produce ethanol. To enhance xylose consumption and ethanol productivity.the downstream formentation of industrial recombinant strains were improved through bioengineering technics. In this study, we improved the composition of fermentation medium, optimized the cofermentation of glucose and xylose by industrial recombinent strains, investigated the influence of dissolved oxygen and pH on ethanol production, ultimately got the formentation parameter that can be universally used in.cofermentation of glucose and xylose by industrial ï¿¡ cerevisiae .The main objectives of this research are as follows:The transformation system of yeast industrial strain has been established. Industrial strains can only use dominant selection marker genes such as G418, because industrial strains lack auxotrophic markers for genetic manipulations. Furthermore, there are some differences of transformation technology between industrial and laboratory strains. In this study, sensitivity of industrial strains, NAN-27> SPSC-1 > 6508 > 1308 to G418 were tested. The sensitivity of these strains to G418 was very different. Self-flocculating yeast SPSC-1 could grew in medium having 700ng/mL G418. 6508 couldn't grow when medium had G418 exceeding 200(ig/mL. The transformation method by Li Ac was modified and the transformation systems for the three industrial yeast strains were established.The strategy for direct integration of a target gene into the host chromosome is an effective method for the stable expression of foreign genes in the industrial strain of Saccharomyces cerevisia. We constructed integrating vector pYMIK and pYIK.Vector contain PGKl promoter and terminator and the G418 resistance gene (KanMX) as dominant selection marker. pYMIK contains a rDNA portion as a homologous recombination sequence to obtain multicopy integrants and pYIKcontains URA3. The XYL1 and XYL2 gene, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, and the XKS1 gene, which encodes xylulokinase (XK) from S. cerevisiae, were ligated into the low copy of integrating plasmid pYIK and the multiple copies of integrating plasmid pYMIK. The recombinant plasmids were named pYIK-xyl23 and pYMIK-xyl27, respectively. The plasmid pYMIK-xyl23 and pYMIK-xyl27 were transformed into the industrial strain of S. cerevisiae and laboratory strain. Transfonnants were selected on YPX plateswith G418 that have xylose as the sole carbon sourc. Further screening was performed by increasing the concentrations of G418 that provids a stronger selective pressure.and measuring the biomass following growth on YPX, resulting in recombinant strains containing the xylose metabolizing genes with a low copy number and mutiple copy numbers. The results of enzyme activities in YEPD without G418 show that genes encoding enzymes for xylose metabolism have been introduced into the industrial recombinant strainsXylose glucose co-fermentations of the & cerevisiae recombinant strains were performed under C^-limited conditions. The result of growth curves for those recombinant and parent strains indicates that the intrduced foreign genes are not effect the growth of recombinant strains. The amounts of consumed substrates and the products yield were determined by HPLC. The results show the recombinant strains have the ability of fermentation xylose to produce ethanol. The best recombinant strains of fermentation xylose to produce ethanol is NAN-127 which is used industrial strain of S. cerevisiae NAN-27 as the host strain and multiple copies of the xylose metabolizing genes were integrated into the host genome. Strain NAN-127 consumed 20.6 g/L xylose that consumed 18.6g/l more xylose than the parent strain, and produced 9.3 g/L ethanol that is 31% more than the parent strain.In this study, we ascertained the ratio (2:1) and concentration of glucose and xylose in fermentation mudium according to the composition of glucose and xylose in lignocellulose biomass and requirment of industrial production. The result of flask fermentation using improved facultative aerobe fermentation technics indicated that the recombinant industrial strains had much more defferences from the parent strains in ethanol production and xylose consumption. The best recombinant strains of fermentation xylose to produce ethanol is NAN-127 which is used industrial strain of S. cerevisiae NAN-27 as the host strain and multiple copies of the xylose metabolizing genes were integrated into the host genome. Strain NAN-127 consumed 53.6% of xylose in the substrate and the ethanol productivity was 0.44g/g suger consumed . The ability of xylose utilizating and ethanol productivy is much higer than that before improvement of fermentation technics. As a result we optimized the... |
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