Genetic Engineering Of Thermotolerant Saccharomyces Cerevisiae On Bioethanol Fermentation Using Xylose Carbon

Lignocellulose is the most abundant biomass resource for biofuels and chemicals on the earth.Lignocellulose consists mainly of cellulose,hemicelluloses and lignin,and xylose is a major monosaccharide of hemicelluloses.As the traditional fermentation yeast strain,such as Saccharomyces cerevisiae,can not use xylose as carbon source for bioethanol fermentation,genetic engineering technology has been applied by introducing exogenous genes into yeast strain,but much remains unknown about the optimal gene recombination in transgenic yeast strainHere,this study focused to express three key genes(XYL1,XYL2,XKS1)involved in xylose metabolism in the thermotolerant Saccharomyces cerevisiae SF7 strain using a specifically integrated expression vector(pAUR101).Then,a classic genetic and biochemical analysis was performed in two transgenic strains(SF4,SF5),and their ethanol fermentation rates were measured by using either xylose as carbon source or soluble sugars released from dilute H2SO4 pretreatment and sequential enzymatic hydrolysis in two typical Miscanthus accessions.The main findings were described below:1.RT-PCR analysis:the XYL1 gene expression level in SF4 strain is much higher than that of control strain(SF-CK),whereas no alteration was observed in the SF5 strain.By comparison,XYL2 gene was highly expressed in both SF4,SF5 strains.In addition,the XKS1 gene expression was not much changed in both strains and control.2.SDS-PAGE analysis:XYL1-(G4S1)3-XYL2 fusion protein was determined,while no significantly different protein level was detected between single gene and multi gene tandem expression.3.SF4 and SF5 strains respectively show increased XYL2 enzyme activities by 17.3 and 10.8 folds,compare with the control,but XYL1 enzyme activity was only slightly increased.4.Recombinant strains could grow on the medium containing xylose as sole carbon source,in particular,SF4 strain grew up to 1.5 times higher than that of control.SF4 strain can utilize 95%xylose for fermentation,but the ethanol production only reach to 4.6%,which corresponds to 10%of the theoretical yield.Using both xylose and glucose as carbon sources,strain SF4 only utilizes 53.8%xylose for bioethanol production.5.In terms of total soluble sugars(hexoses and pentoses)released from dilute H2SO4 pretreatment and sequential enzymatic hydrolysis of Miscanthus biomass samples,SF4 strain could use more pentoses as carbon sources and the ethanol productions were respectively increased by 4.5 and 0.6 folds in two biomass samples with relatively high and low enzymatic saccahrification.