|a英文題名:Construction of Recombinant Saccharomyces cerevisiae with Ethanol and Aldehydes Tolerance by Overexpression of Aldehyde Reductase
|a醣類物質經高溫或酸水解後，常會伴隨著呋喃醛(furans)生成，呋喃醛主要為糠醛(2-furaldehyde，furfural)及5-羥甲基糠醛 (5-hydroxymethyl-furfural，HMF)；呋喃醛若存於發酵基質中，則被視為微生物生長之抑制物，因其會使釀酒菌株之遲滯期延長以致降低發酵效率。本研究將選殖Saccharomyces cerevisiae之醛類還原酶基因，並於Pichia pastoris X-33及S. cerevisiae細胞中大量表現此酵素，期望轉殖菌株因對抑制物有較佳之耐受性而提高其應用性。利用PCR (polymerase chain reaction)方法由S. cerevisiae BCRC (21685)選殖出醛類還原酶DNA，將之重組於pGAP質體後，轉型於P. pastoris及S. cerevisiae細胞並評估轉殖菌株對furfural 、 HMF及酒精之耐受性。PCR所得之醛類還原酶DNA含1044 bp，基因轉殖株經PCR、限制酶圖譜、基因定序及西方點墨分析確定目標基因成功殖入P. pastoris及S. cerevisiae細胞中，其蛋白質分子量約為38 kDa。相較於野生株，S. cerevisiae轉殖株對於HMF、furfural及酒精具有較高之耐受性；但， P. pastoris轉殖株之耐受性反而更差。本研究所得之醛類還原酶S. cerevisiae轉殖株因對發酵環境之壓力具有高耐受性，更適合作為酒精生產之發酵菌酛或培養於含高濃度HMF及furfural發酵基質中以提升產品之酒精濃度。|uThe breakdown of sugars either by thermal processes or acid hydrolysis often result in the generation of furans. Furfural and 5-hydroxymethylfurfural (HMF) are two common furans. The present of furans in fermentation matrix can inhibit microbial growth by extending the lag growth phase and thus reduce the fermentation efficiency. This study cloned aldehyde reductase gene from Saccharomyces cerevisiae, and then express this enzyme in Pichia pastoris X-33 and S. cerevisiae. Expression of this enzyme was to enhance the tolerance of the microbes against furans. Using polymerase chain reaction (PCR) method to clone the aldehyde reductase gene from S. cerevisiae (BCRC21685) and ligated into the pGAP plasmid. This plasmid was then transformed into and expressed in P. pastoris and S. cerevisiae. The tolerance of the recombinant cultures toward furfural, HMF and ethanol was evaluated. The PCR product of aldehyde reductase had 1044 bp. After PCR, mapping, gene sequencing and Western blot analysis, it was conformed that the target gene was successfully cloned into P. pastoris and S. cerevisiae. The protein size was about 38 kDa. This recombinant culture of S. cerevisiae had higher HMF, furfural and alcohol tolerance than the wild type. However, the recombinant culture of P. pastoris showed lower HMF, furfural and alcohol tolerance than the wild type. It was concluded that the recombinant culture of S. cerevisiae was suitable to serve as an alcoholic fermentation starter for biomass production and wine making because the recombinant culture can grow better in high HMF or furfural fermentation medium.
|aConstruction of Recombinant Saccharomyces cerevisiae with Ethanol and Aldehydes Tolerance by Overexpression of Aldehyde Reductase