Construction Of Recombinant Saccharomyces Cerevisiae For Fermenting Glucose And Xylose To Ethanol

Lignocellulose is the most abundant renewable organic resource on the earth.Xylose fermentation is the foundation and the key step for the successful biological conversion of lignocellulosic biomass to ethanol,but there is no natural microorganisms strain suitable of the business production.It was critical to enhance the capability of yeasts xylose-fermentation by gene engineering.The recombinant expression vector pACT2-xyl1 with the xylose reductase gene xyl1 from Candida shehatae has been established in our laboratory.The vector pACT2-xyl1 was then transformed into Saccharomyces cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-x1,and positive transformants were isolated on selective plate without Leu.The specific xylose reductase activity of two transformants S.cerevisiae YS58-x1a and YS58-x1b were 0.26U/mg and 0.32U/mg total protein respectively,which were 162.5-fold and 200-fold higher than that of gene donor YS58.This result indicated that the xyl1 gene from C.shehatae has been actively expressed in laboratory S.cerevisiae YS58.S.cerevisiae YS58-x1b was used to co-fermented glucose and xylose to produce xylitol.YS58-x1 consumed 6.688g/L xylose which was 3.87-fold more than that of the parent strain YS58,and produced 6.22 g/L xylitol which was 10.5-fold more than that of YS58.The result indicates that YS58-x1 can fenment xylose to xylitol with glucose as the assistant carbon source effectively.The xyl2 gene from S.cerevisiae TMB3000 encoding the xylitol dehydrogenase was amplified by PCR,and inserted into the expression vector pDR195,resulting in the recombinant vector pDR195-xyl2.Then the plasmid pDR195-xyl2 was transformed into S.cerevisiae YS58 to yield the recombinant yeast YS58-x2,and positive transformants were isolated on selective plate lacking of Ura.The specific xylitol dehydrogenase activity of two transformants S.cerevisiae YS58-x2a and YS58-x2b were 0.0656U/mg and 0.07U/mg total protein respectively,which were 16.4-fold and 17.5-fold higher than that of gene donor YS58.This result indicate that the xyl2 gene from S.cerevisiae TMB3000 has been overexpressed successfully in laboratory S.cerevisiae YS58.Under screening pressure by lacking Leu and Ura in the plate simultaneously,recombinant vectors pACT2-xyl1 and pDR195-xyl2 were both transformed to S.cerevisiae YS58 to produce recombinants YS58-x1-x2.The stability of two incompatible plasmids exsisting in the same host strain was also studied,it was found that 85%unborn recombinants were still alive in the plate without Leu and Ura after 8days,which meant that the recombinants with two incompatible plasmids were really steady with the selecting pressure.The XR and XDH activity in YS58-x1-x2 were equal to that of their expressing respectively.Furthermore,YS58-x1-x2 could consume 7.983g/L xylose which was 4.8-fold higher than that of YS58,and ethanol production was up to 14.486 g /L which increased by 23.5%than that of the host.This implied that two incompatible plasmids could exsist in the same S.cerevisiae with screening stress,and could even express two different kinds of proteins.The recombinant yeast created in this experiment could be the foundation of the further improvement of recombinant S.cerevisiae.