Using homologous recombination to knockout genes for hexose metabolism and construct the xylose-fermenting Pichia stipitis for ethanol production

• Main Authors: Chia-chun Chuang, 莊佳鈞
• Other Authors: Ween-chein Lee
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/51465191173676638133

碩士 === 國立中正大學 === 化學工程所 === 98 === P. stipitis is able to uptake xylose for ethanol production. When a hexose (glucose) appears in the fermentation medium, the affinity for xylose and the ability to produce ethanol by the yeast decreases. In this study, plasmids containing the genes of antibiotic resistance and sequences for gene deficiency of hexokinase and glucokinase were constructed and transformed to the yeast by eletroporation for homologous recombination. The hexokinase-deficient P. stipitis was screened using the antibiotic zeocin. Results from the fermentation of hexokinase-deficient P. stipitis in glucose and xylose indicted that the glucose uptake was delayed in the recombinant strain. However, the uptake of xylose by the recombinant strain was similar to that of wild-type yeast. Actually, the plasmid including the gene of kanamycin resistance and sequence for the deficiency of glucokinase gene was not properly constructed. Homologous sequences for the recombination of glucokinase were missing during the construction of plasmid due to probably a recombination event in Escherichia coli DH5α.