Screening Of Ethanol-production Strains And Gene Cloning Of Key Enzymes For Xylose-metabolism

Bioethanol is taken into account as many countries' strategy as a sustainable, portable and renewable energy source.Cheap raw material and high-efficient conversion technology are required for commercial fuel-ethanol production;therefore the conversion of abundant and renewable lignocellulosics to bioethanol is a current hot spot,in particular,the production of ethanol from cellulose.The efficient conversion of xylose into ethanol is one of the key factors to such a process.Efficient xylose-fermenting to ethanol is the constraint of the technology industrialization. Research has been focused on the selection of microorganisms,the study of xylose metabolism,and the construction of efficient xylose-fermenting recombinant strains.In order to obtain efficient ethanol-producing microorganism using bagasse saccharification,two strains were isolated from mold culture and cattle feces.The strains were identified as Saccharomyces cerevisiae Q2-1 and Candida tropicalis H-1 respectively,via traditional classification and analysis of molecular biology.The bagasse saccharification fermentation conditions of S.cerevisiae Q2-1 for ethanol-producing were studied extensively.The optimum conditions for ethanol production were obtained as:nitrogen source 0.4%ammonium sulfate, fermentation temperature 30℃,pH5.5 and fermentation time 60h.The maximum zymotic ratio was obtained as 89.18%with an ethanol yield of 13.6% (W/W).Genomic DNA from C.tropicalis H-1 was extracted.Xylose reductase gene (xyl1) and xylitol dehydrogenase gene(xyl2) were cloned from genomic DNA of C. tropicalis H-1.No intron was observed after sequence analysis.Recombinant plasmid pPIC3.5K-xyll-xyl2 was constructed by inserting gene xyl1 and xyl2 into plasmid pPIC3.5K.The plasmid was then electroporated into strain GS115. Xylose reductase(XR) gene and xylitol dehydrogenase(XDH) gene were integrated to genomic DNA of GS115.The recombinant strains were obtained after screening. Xylose reductase preferentially uses NADPH as cofactor,whereas xylitol dehydrogenase exclusively uses NAD~+ as cofactor.XR and XDH were expressed with induction of methanol,and their enzyme activities were determinated.The relative activity of xylose reductase using NADPH as cofactor was 12.3U,while using NADH as cofactor was 1.1U.It determined that xylose reductase is an NADPH-preferring enzyme.The relative activity of xylitol dehydrogenase was determined as 2232.9U using NAD~+ as cofactor.