Construction Of An Engineering Strain Producing Ethanol From Straw

Metabolism of xylose is one of the important factors affecting the comprehensive cost of straw ethanol.Industrial Saccharomyces cerevisiae can not metabolize xylose due to the lack of enzymes that specifically convert xylose to xylulose.If the transformation of xylose into xylulose enzyme system into industrial Saccharomyces cerevisiae,it will promote and speed up the straw ethanol production technology practical process.In this paper,xylose isomerase was expressed by heterogenous genes to transform the utilization of xylose in saccharomyces cerevisiae.And the xylose isomerase pathway strains metabolize the other oxidase under almost all test conditions compared to xylose metabolism using xylose The reduction pathway strains had better yields.After D-xylose was catalyzed by xylose isomerase to produce D-xylulose,xylulose kinase needs to be overexpressed to perfect the downstream enzyme system of xylose metabolism.Although the genome of Saccharomyces cerevisiae contains the gene XKS1 encoding xylulokinase,its activity is too low to meet the need of xylose metabolic pathway engineered bacteria to ferment xylose to produce ethanol.Overexpression of xylulose kinase causes more metabolic flux of xylose to ethanol.Xylitol is one of the major by-products of yeast xylose metabolism ethanol production.The endogenous aldose reductase encoded by the GRE3 gene is an enzyme that reduces xylose to xylitol,while xylitol also inhibits xylose Enzyme activity.The constitutively translated elongation-elongation factor-1(TEF-1)promoter was first obtained from the Pichia pastoris genome and ligated into the xylulokinase(XK)sequencing plasmid obtained from the industrial S.cerevisiae group by restriction enzyme ligation.The xylulokinase(XK)with the translation elongation factor-1(TEF-1)promoter was ligated to the shuttle plasmid p YES2-XI with uracil-deficient screening factor through the seamless ligation of Clontech's In-Fusion Plasmid,so as to successfully construct the shuttle plasmid p YES2-XI-TEF1-XK.Although it had not yet been successfully transformed into T1308 industrial Saccharomyces cerevisiae and expressed,it is a preliminary work for the later transformation into T1308 industrial Saccharomyces cerevisiae so as to observe whether xylose metabolism has significantly increased.In order to reduce the toxic effects of byproducts,we constructed the 1308-GRE3?1 knockout 1 GRE3 gene and the 1308-GRE3?2 knockout 2 GRE3 gene,respectively,using industrial yeast T1308 as a starting strain.The experiment confirmed that engineered strain 1308-GRE3?1 can improve the ethanol fermentation efficiency of xylose,the by-product xylitol reduced by 75.1% compared with the starting strain.Moreover,1308-GRE3?1 also has the advantage of not affecting the original glucose utilization rate of the starting bacteria,and at the same time the inhibitor tolerance is high.