建立釀酒酵母菌以phosphoketolase 代謝途徑生產酒精之質體建構

Plasmid Construction for Producing Ethanol Using the Phosphoketolase Pathway in Saccharomyces cerevisae

許舜閔(Shun-Min Hsu)

指導教授 ： 黃光策

國立中正大學/工學院/化學工程學系/碩士(2015年)

摘要

全球石油日漸缺少、在避免環境汙染下，尋找替代能源已是重要課題。生質能、太陽能、風能、地熱能…等，不僅是可用的再生資源，也能降低二氧化碳的排放，避免全球暖化。其中生質酒精的生產菌種主要為釀酒酵母(Saccharomyces cerevisiae) ，在自然狀態下代謝六碳葡萄糖形成乙醇需經丙酮酸的去羧基反應，此代謝途徑因產生兩個二氧化碳分子造成碳損失，使乙醇理論產率約0.51g 乙醇 / g葡萄糖。因菌種自然代謝途徑的碳損失，降低了其與其他替代能源產業的競爭性。 因此本研究利用聚合酶連鎖反應(polymerase chain reaction, PCR)從Bifidobacterium longum和Escherichia coli K12 wild type擴增xfp、pta、mhpf基因片段，並以Gibson接合至pTEF1/Zeo，形成pTEF1-xfp、pTEF1-pta、pTEF1-mhpf質體。質體建構完成後，利用聚合酶連鎖反應擴增含promoter和terminator 的xfp、pta、mhpf片段，同時和2μori接合至pTEF1/Zeo，引入到酵母菌中，使葡萄糖以非氧化途徑生成乙醇，進一步提高乙醇產率。額外加入的glycerol則可提供所需之NADH。

關鍵字:釀酒酵母菌、磷酸酮醇酶

關鍵字

磷酸酮醇酶 ； 釀酒酵母菌

並列摘要

The extreme climate change is correlated to the increased greenhouse gas emission. Finding new alternative energies such as biomass energy, solar energy, wind energy, and biothermal energy instead of using fossil fuel become an important issue these days. The ethanol production from glucose in Saccharomyces cerevisiae involves decarboxylation reaction of pyruvate in nature. This metabolic pathway produces two molecules of carbon dixoide per molecules of glucose, resulting in carbon loss. Theoretical yield of ethanol is about 0.51 g ethanol /g glucose. The carbon loss in producing bioethanol reduces its potential as compared with other regenerative energies. In this study, we amplified xfp gene from Bifidobacterium longum and pta and mhpf genes from Escherichia coli K12 by polymerase chain reaction (PCR) for future introducing a phosphoketolase pathway into Saccharomyces cerevisiae. The PCR-amplified genes were cloned into pTEF1/Zeo vector using the Gibson mixture reagent and were designated as pTEF1-xfp ,pTEF1-pta, and pTEF1-mhpf, respectively. xfp, pta, and mhpf will be amplified to include a promoter and a terminator and insert into pTEF1/Zeo with 2μori. Once this metabolic pathway is introduced into Saccharomyces cerevisiae which can produce ethanol using a non-oxidative pathway, resulting in a higher ethanol yield. The additional need for NADH for producing ethanol could be supplemented using a portion of glycerol as substrate.
Keywords: Saccharomyces cerevisiae, phosphoketolase

並列關鍵字

phosphoketolase ； Saccharomyces cerevisiae

參考文獻

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