| Xylose is the most important component of five-carbon sugar in lignocellulose, and second sugar sourceto glucose in nature. Effective use of Xylose is one of the key links of bioconversion of lignocellulose toproduce ethanol. Currently, microbial strains that have been found in nature are still unable to meet theneeds of commercial production. Natural yeast (Saccharomyces cerevisiae) can effectively use glucosefermentation to produce ethanol, but does not have the capacity to utilize Xylose. It is become the focus ofresearch and development currently to take advantage of new technologies and means to transform yeast,improve its ability of Xylose fermentation.The research by means of genetic engineering, will be introduce it to Saccharomyces cerevisiae after fivelab cloned Xylose metabolic genes tandemed splice to expression vector, to built a recombinant Escherichiafermentation Xylose and glucose to produce ethanol. First, the fusion PCR technology will stitching themthat come from the nearly smooth Candida (Candida parapsilosis) Xylose reductase gene (xyl1), tropicalCandida (Candida tropicalis) xylitol dehydrogenase gene (xyl2), pichia stipitis (Pichia stipitis), woodenone sugar kinase gene (xks1), Saccharomyces cerevisiae in the transaldolase gene (tal1) and transketolasegene (tkl1), to get segments connect xyl1-xyl2-tal1(X12A) and xks1-tkl1(SK). Reaction across the doublerestriction validation and T4connection, you will get mosaic fragment cloned into plasmid pAUR123Saccharomyces cerevisiae additional carrier, successfully construct plasmid pAUR123-X12A(xyl1-xyl2-tal1), pAUR123-SK (xks1-tkl1). Second, with recombinant plasmid pAUR123-X12A,pAUR123-SK for the template, design primer contains enzyme cut bit points, and clone out gene fragmentscontains enzyme cut bit points of X12A and SK. After double enzyme cut and the T4connection reaction,to wine yeast attached type mass grain pAUR123as vector, built them one has contain xyl1, xyl2and tal1gene of mass grain pAUR123-X12A and another contain xyl1, xyl2, tal1, xks1and tkl1plasmidpAUR123-XL (xyl1-xyl2-tal1-xks1-tkl1known as XL) successfully. Using lithium acetate transformation totransform the Saccharomyces cerevisiae INVSC1bacteria, resistances screening positive clones. Usingmolecular biological method to detect the positive clones and access engineering strain of recombinantplasmid containing S.c INVSC1-XL and INVSC1-X12A. Through the detection of interest protein, candetermine the construction of engineering bacteria at the same time to express Xylose reductase gene,wooden one sugar xylitol dehydrogenase gene kinase gene, transketolase and transaldolase gene. Thenthrough biochemical experiments show that each enzyme has a certain activity, provides germplasm sourcefor the study of Xylose metabolism.To further verify recombinant bacteria to use Xylose, use S.c INVSC1-XL and INVSC1-X12A with6%xylose as a single carbon source for fermentation. Found that recombinant bacteria can utilize Xylose,Xylose utilization rate were74.8%and79.5%respectively, ethanol yield of31.14%and39.88%respectively. In a different ratio of glucose/sugar fermentation under the condition of limited oxygenmixture, glucose/Xylose in recombinant bacteria the best mixing ratio of5:1, fermentation time of60h,then S.c INVSc1-X12A ethanol yield is74.85%at this time, sugar utilization of ethanol yield of96.61%,S.c INVSc1-XL of76.92%, sugar utilization of96.78%. This suggests that nearly smooth Candida Xylosereductase gene, tropical xylitol dehydrogenase gene of Candida, wooden one sugar kinase gene of thePichia stipitis, Saccharomyces cerevisiae endogenous transketolase and transaldolase gene gene expression protein Xylose-fermenting Saccharomyces cerevisiae alcohol can play a facilitating role. This study hassuccessfully acquired the yeast fermenting Xylose to produce ethanol in Saccharomyces, achieved thedesired purpose. |
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