| Summary: | 碩士 === 國立中正大學 === 化學工程研究所 === 107 === In industry, heat tolerance Saccharomyces cerevisiae (S. cerevisiae) can save a lot of cooling costs. Heat shock proteins (HSPs) are induced in order to prevent the increase of misfolded proteins in cells when S. cerevisiae is exposed to high temperature stress. HSP104 can disintegrate denatured protein. Although HSP104 can be induced by heat, it cannot be expressed for a long time during fermentation. In order to change the HSP104 activity during fermentation, we replaced the promoter of HSP104 with the TEF1 promoter. The DNA fragment containing the TEF1 promoter flanked with two 40 bp homologous sequences was obtained using the polymerase chain reaction with plasmid pTEF1-pta as a template. The linear DNA fragments was then transformed into S. cerevisiae Kyokai NO.7 using the lithium acetate method. The promoter of the original HSP104 was replaced with the TEF1 promoter by means of chromosomal homology substitution. The clone with the promoter replacement was selected by the colony PCR and further confirmed by sequencing. In high temperature and high concentration ethanol fermentation, it was found that the native strain exhibited better temperature and ethanol tolerance than the recombinant strain. In the Real-time PCR experiments, the expression level of the HSP104 mRNA in recombinant strain was higher than wild type at 14 hours of fermentation. However, the wild-type strain still showed a higher temperature tolerance than the recombinant strain at the prolonged fermentation experiment after 14 hours.
|
|---|
