Study On Construction Of Recombinant Strain Of Saccharomyces Cerevisiae And Ethanol Production By Xylose Fermentation

Lignocellulose hydrolysate is abundant with xylose,which is only second to glucose.Wild-type strain of Saccharomyces cerevisiae can produce ethanol from hexose fermentation efficiently, but they are unable to utilize xylose. Hence, making full use of Woodiness fiber materials, promoting biotransformation from xylose to ethanol,can be an effective way of lowering ethanol production cost. Recombinant strain of Saccharomyces cerevisiae introduced genes coding xylose reductase and xylitol dehydrogenase from yeast Pichia stipitis can ferment xylose. However, this kind of strain has a very limited ability to utilize xylose in ethanol production. A main reason is that,there is an intracellular redox imbalance between PsXR(with NADPH) and PsXDH(with NAD+) caused by different coenzyme specificity. As a consequence,xylitol was accumulated and xylose metabolism was influenced.To solve intracellular redox imbalance problem,coenzyme specificity of PsXR was modified by protein engineering methods,which would improve this situation effectively. In this study,we introduced genes of modified XR and XDH into wild-type strain of Saccharomyces cerevisiae and then studied on ethanol production by xylose Fermentation. Section I: Pri mary Study on Construction of Recombinant Strain of Saccharomyces cerevisiae and Xylose FermentationIn the Recombinant Saccharomyces cerevisiae which was introduced genes expressing Pichia stipitis xylose reductase and xylitol dehydrogenase,activity of xylose reductase depends on NADPH,while xylitol dehydrogenase depends on NAD+. Intracellular redox imbalance caused by the differences in the coenzyme between xylose reductase (XR) and xylitol dehydrogenase (XDH) has been thought to be one of the main factors of xylitol accumulation,lower xylose utilization and lower ethanol yield. Gene m1 and xyl2 respectively expressing NADH-preferring xylose reductase (NADH-XR) and xylitol dehydrogenase were introduced into Saccharomyces cerevisiae AH109 to obtain the recombinant strains AH-M-XDH,with the recombinant strains AH-XR-XDH expressing gene xyl1 and xyl2 as the control group. In this study,we used SC/-Leu/-Trp nutrient-deficient medium to screen recombinant strains. Positive transformants were cultured in shake-flask under O2-limited conditions in a yeast minimal medium with glucose plus xylose,and then HPLC was used to measure ethanol fermentation. Comparing to the strains AH-XR-XDH,xylose utilization of the strains AH-M-XDH increased significantly,ethanol yield increased by 16%,and xylitol excretion decreased by 41.4%. The results showed that key enzymes of xylose-metabolism constructed using gene engineering can be applied to ethanol production by xylose fermentation in Saccharomyces cerevisiae,increase xylose utilization and ethanol production in recombinant Saccharomyces cerevisiae by means of improving intracellular redox imbalance. SectionⅡ: Opti mization of Reco mbinant Strain of Saccharo myces cerevisiae and Ethanol Production by Xylose Fer mentationUnder regular fermentation conditions,xylose utilization and ethanol production of the Recombinant Saccharomyces cerevisiae was increased , while xylitol accumulation has also been improved though there is still a certain distance with theoretical value. Hence,we improved conditions of ethanol production by xylose fermentation in the recombinant Saccharomyces cerevisiae in terms of fermentation medium components and fermentation conditions. Our experimentation studied on the influence of total sugar in medium,different proportion of sugar, original pH,fermentation temperature,inoculum concentration and speed of wave bed on fermentation. The results showed that,besides original pH,all the other factors has a significant influence on fermentation optimization. Optimized conditions are as follows :total sugar in medium 5%,ratio of glucose and xylose 2:1,original pH=5.0,inoculum concentration 8%,fermentation temperature 30℃,speed of wave bed 120r/min. Recombinant Strain of Saccharomyces cerevisiae xylose fermentation with optimized conditions, xylose utilization increase by 7.19%,while ethanol production increase by 8.36%.