Construction Of Recombinant Plasmid For Yeast Ethanolic Xylose-Fermentation

Fuel ethanol is regarded as one of the most promising environment friendly fuel in the future.Lignocellulose is the most abundant renewable organic resource on the earth.Conversion of cellulosic biomass into ethanol would not only reduce the cost of production but also helpful to decrease the pollutant produced by consumpation of gasline.Ethanol fermentation from xylose is the foundation and the key step for cellulosic ethanol.However,most natural microorganisms lack the ability to ferment xylose into ethanol.So,it's critical to enhance the capability of yeasts ethanolic xylose fermentation by gene engineering.PCR Primers were designed using oligo and Primer premier 5 procedures. Xylose reductase gene(xyl1) was cloned from Candida shehate.Xylitol dehydrogenase gene(xyl2) was cloned from C.tropicalis H-1.Xylulokiase gene(xks1) was cloned from Saccharomyces cerevisiae BJ3505.Sequence analysis results showed that no intron existed in three genes.The alcohol dehydrogenase promoter ADHp and alcohol dehydrogenase terminator ADHt were cloned from plasmid pGADT7.In order to increase ethanol production and reduce xylitol production,xylose reductase gene and xylulokiase gene were expressed under the control of the ADH.Xylitol dehydrogenase gene was expressed under the control of the PGK.To obtain the ADHp-xyl1-ADHt and ADHp-xks1 -ADHt fragment,the xyl1 and xks1 was ligated with the ADHp and ADHt.Plasmid pMA91-xyl2 was constructed by ligating the xyl2 with plasmid pMA91.The orientation of the xyl2 gene was verified by restriction enzyme digestions.The xyl2 gene under the control of PGK promoter was excised from pMA91-xyl2 with Hindâ…¢,yielding the PGKp-xyl2 -PGKt fragment.To obtain the plasmid Ylp5-3,the ADHp-xyl1-ADHt,ADHp-xks1-ADHt and PGKp-xyl2-PGKt fragment were ligated at the EcoRâ… ,Hindâ…¢and BamHâ… sites of Ylp5.The orientation of the ligated fragments was verified by restriction enzyme digestions.Thus,the integrating plasmid Ylp5-3 contains xyl1,xyl2 and xks1 genes which require for ethanolic xylose fermentation was constructed,and the further work is undergoing.