| Summary: | 碩士 === 大葉大學 === 分子生物科技學系碩士班 === 98 === ABSTRACT
With the development of modern industry, fossil fuel consumption is dramatically increasing. New energy research and development are urgent than ever. Biomass energy has some advantages, they are recycles and they are less CO2 is emitted. Both bioethanol fuel and gasoline mixed with ethanol are used worldwide. Currently, there are two so called ethanol crops which are corn and sugar cane. The consumption of corn and sugar in ethanol production results in the raising of the crops price. Therefore, wood fiber alcohol has become a major development. The wood fiber contains about 24 % to 45 % of cellulose (β-polymer of glucose), 22 % ~ 31 % of hemicellulose (contains 80 ~ 85% xylose) and others. The wine yeast, used to ferment the ethanol, use glucose as carbon source easily. However, it cannot directly use the xylose as carbon source. This will leave xylose as waste and contaminate our environment. If any organism can ferment both the cellulose and hemicelluloses, it will help lower the cost, produce more ethanol and leave less waste. In these criterions, P. stipitis is good candidate yeast for this wood sugar fermentation. But P. stipitis alcohol tolerance is poor and the accumulation of ethanol during fermentation causes the death of P. stipitis. This may the reason for poor ethanol production by P. stipitis fermentation. To solve this dilemma, we try to find out the ethanol stress response transcription factors, and transfected into the P. stipitis, expectedly ethanol tolerance will be increased in P. stipitis. In wine yeast, several stress transcription factors have been reported, they are Msn 2, Msn 4 and Skn 7. By semi-quantitative RT-PCR analysis, Msn 2 and Msn 4 have increased 40 % while Skn7 was not detected when 3% ethanol were included in the media. Since the Msn 2P can form either homodimer or heterodimer with Msn4, Msn 2 was cloned into pGAPZ vector, which contains a constitutive promoter. The pGAPZ-Msn2 plasmid was transformed into P. stipitis. The experimental results showed that P. stipitis transformants had higher ethanol tolerance compared to wild-type and pGAPZ transformant.
Keywords: bioethanol, xylose, P. stipitis
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