Investigation On The Ethanol Fermentation From Crop Straw By The Recombination Technology Of Saccharomyces Cerevisiae

Our country is a big country in crop cultivation. And crop straws and other crop wastes were produced up to 700 million tons every year. These crop straw have not been utilised effectivly, even were burned and cause serious environmental pollution. T-hese abundant cheap crops straw can be used to make non-grain fuel ethanol instead of food and the sweet sorghum is one of the most potential engergy crops. Sweet sorghum(Sorghum bicolor(L) Moench) has good prospect as biomass engery, because it has rich germplasm resources,planting range, strong ability to resist adversity high ste-m yield and sugar content of stem juice, and it is one of the highest biomass crops.The sugar content of the sweet sorghum stem juice is very high and its juice and re-sidue can be used to produce ethanol. And Xylose is the second most abundant carbohydrate in hydrolysates of croplignocellulosic biomass. Efficient fermentation of xylose is one of the key factors to a-ttain economical feasiblity for ethanol production from lignocellulosic biomass. By using engineering Saccharomyces cerevisiae strains, which can metabolize xylose efficiently, the cost of cellulosic ethanol of crop straw couled be decreased, and at the sametime the planting cost of farmers also couled be reduced. And by using the constucted metabolism of xylose strains, crop straw such as s-weet sorghum straw material can used to produce fuel ethanol, therefore relieve the e-nergy crisis and environmental protectionIn this study, with the sweet sorghum straw as raw materials, we applied geneticengineering approach for construction of efficient xylose-fermenting S. cerevisiae, and optimized the condition of ethanol fermentation of the mixed stem juice and bagasse by using the engineering strain. Thereby improved the sweet sorghum straw ethanol pr-odution and provided the mode for comprehensive utilization of crop straw.1. The background of S. Cerevisiae T1308 was analysized. Its size and sequence of 5.8 S rDNA-ITS is consistent with sequence databases of S. cerevisiae, 100% homologous sequence, so the strain is conformed as S. cerevisiae. It was conformed that T1308 is heterozygous diploid(aα) strain by the analysis of spore stainined by methylene blue and analysis of the sequences of the MAT locus. And constructed the ura-cil auxotrophy of T1308 by two steps of knockout of URA3 and got the recombinant strain T1308-U.2. The xylose isomerase expression vectors were constructed with japonica and fu-ngi xylose isomerase gene and expressed in S. Cerevisiae. According the xylose isom-erase enzyme activity analysis of the recombinant strains, the fungus xylose isomerase gene is determined that it is more suitable for expression in S. Cerevisiae and its suitable promoter. Xylose utilasition of the recombinant strains were 1.78-1.99 times higher than reference strain and its highest ethanol yield reached 0.31g/g consumed sugar.3. Vectors of promoter TPI with two different selective markers were constucted,respectively. TPI promoter were respectively inserted in the front of xylulokinase gene and key genes involved in PPP of reference strain. Then the vector of XylA of Piromyces sp. E2 and Oryza sativa subsp were transformed.Then the recombinants which xylose metablic flow were enhanced were constructed. The transcription levels of the gene of XKS1, RPE1, RKI1, TAL1 and TKL1 of PXI-T1308-U were increased 4.08、4.17、17.36、1.62 and 3.98-fold, respectively, compared to control strain T1308-U. The xylose utilization ratio was 3.28 times higher than control strain, and the ethanol yield was 0.33g/g consumed sugar.4. With the Sweet sorghum straw as raw material, the stem juice and bagasse we-re fermented ethanol. And the condition of the simutaneous saccharification fermentati-on process was optimized. The ethanol concentration of the fermentation of recombinant strain was 31.79g/L, which was 5.45g/L higher than the fermentation of the sweet sorghum stem juice.This is the first study on the construction of recombinant industrial S. Cerevisiae by using XylA of Oryza sativa subsp japonica, and inserted the promoter in the front of XKS1 and the gene of PPP directly. We successfully constructed efficient xylose-fer-enting S. Cerevisiae. We also optimized the simutaneous saccharification fermentation process of the stem bagasse and juice for ethanol production in this study. All this is provided the reference and application foreground of the fuel ethanol production from the straw biomass.