本實驗利用分離自台灣各地區25株野生型Scheffersomyces stipitis菌株，分析木糖發酵酒精代謝過程相關基因多樣性，並在充分供氧、有限供氧及極限供氧等不同供氧條件下，分別進行各菌株木糖發酵之酒精產量分析，同時分析關鍵酵素木糖還原酶(xylose reductase)活性及轉錄組(transcriptome)分析，藉由基因表現差異性了解氧氣對於菌株發酵模式的影響，以做為解決木質纖維素生產酒精的瓶頸。
本實驗所使用S. stipitis菌株皆以分子生物特徵進行複核。各菌株間的大單元核醣體D1/D2區域序列差異少於3個核苷酸、核醣體內轉錄區間隔區差異少於5個核苷酸及真核延伸因子1 - α亞基序列差異少於42個核苷酸，因此鑑定皆為同種(co-species)。菌株間之遺傳相關性則根據木糖代謝相關基因(XYL1、XYL2、TKL1、TAL1、PGK1)序列，並利用親緣關係鄰聚法和最大似然法進行基因多樣性分析，結果共可區分為4大分群。所有菌株之酒精發酵試驗是以不同供氧條件發酵，分析酒精濃度後利用階層式分群法分析其酒精發酵能力與氧氣供給量關係性，共分出4大群，分群結果與基因多樣性結果相符。第一和第二群為偏好有限供氧發酵菌群，第三和第四群為偏好極限供氧發酵菌群，其中LA28B06不屬於任一群，其在充分供氧條件有20.25 g/l、有限供氧27.97 g/l、極限供氧28.30 g/l的酒精產量。各菌株之木糖還原酶之酵素動力學米氏常數(Michaelis-Menten constant)值，介於0.22 - 3.46間，顯示不同菌株內之酵素與NADPH或NADH兩種輔酶親和性差異極大，部分之輔酶親和性與遺傳親緣關係和發酵模式具有相關性，故仍有其他因子影響菌株之發酵模式。於發酵模式分群中篩選出4株菌代表各大族群分別為LA45B83、BCRC 21775、LA28B06和TC7S02，以qPCR分析基因表現及mRNA transcriptomes，結果顯示XKS1的基因會隨氧氣供應降低而表現量提升，PGK1則是隨氧氣供應降低而下降。LA45B83菌株屬有限供氧發酵模式代表菌株，在充分供氧下基因表現有上調的現象，而木糖發酵相關基因XYL1和XYL2在有限供氧條件下表現較佳。BCRC 21775菌株屬有限和極限供氧發酵皆佳之模式代表菌株，在充分供氧下基因表現有上調的現象，而木糖發酵相關基因XYL1和XYL2在有限供氧條件下表現較佳。LA28B06菌株三種供氧發酵皆佳之模式代表菌株，於充分供氧和有限供氧條件下基因有上調的現象，充分供氧條件更為明顯，木糖發酵相關基因XYL1、XYL2在充分供氧條件下表現較佳，且無蘋果酸去氫酶(malat dehydrogenase)的表現，除木糖發酵相關基因外，另有其他高表現基因，如TTP、ATPase相關基因。TC7S02菌株屬極限供氧發酵模式代表菌株，在各種供氧條件下與其他三株有明顯的基因表現差異性，木糖發酵相關基因XYL1和XYL2在極限供氧條件下表現較佳，且與其他菌株相比TAL1、PDC、G6PDH有高度的表現量。

This study explored the characterization of 25 wild-strain Scheffersomyces stipitis isolated from Taiwan to analyze the genetic diversity of the xylose ferment ethanol process. S. stipitis were analyzed xylose ferment ethanol concentration in maximum oxygen supplied, limited oxygen supplied, and minimum oxygen three different oxygen supplied conditions respectively. The other, S. stipitis were analyzed the enzyme activity of xylose reductase and transcriptome analysis. This study used differences in gene expression to find out the effect of oxygen on the fermentation mode to solve the bottleneck of ethanol fermentation under lignocellulose.
The S. stipitis strains were all rechecked with molecular biological characteristics. Because of the large unit ribosomal D1/D2 domain sequence difference was less than 3 nucleotides, the internal trainscribed spacer difference was less than 5 nucleotides, and the eukaryotic elongation factor 1-αsequence difference was less than 42 nucleotides, as a result, the 25 strains were all identified as co-species. The genetic correlation between the 25 strains was based on the sequence of xylose metabolism genes (XYL1, XYL2, TKL1, TAL1, PGK1), and the genetic diversity analysis was carried out using the neighbor clustering methods and the maximum likelihood methods. The results could be divided into 4 clades. The ethanol fermentation test of all strains was based on different oxygen supply conditions. Through ethanol concentration, the strains were analyzed the relationship between the alcohol fermentation capacity and the oxygen supply by hierarchical clustering methods. The results could be divided into 4 clades, and the clustering results were consistent with the genetic diversity results. The first and second clades were prefer limited oxygen supply, and the third and fourth clades were prefer the limited oxygen supply. In addition to LA28B06, it didn’t belong to any clades. It had 20.25 g/l, 27.97 g/l, and 28.30 g/l in three different conditions. The Michaelis-Menten constant value of xylose reductase was between 0.22-3.46, indicating that the NADPH or NADH coenzymes affinity was an insignificant difference between strains. Affinities of coenzymes were related to fermentation mode, so there were still other factors that affect the fermentation mode of strains. Four strains LA45B83, BCRC 21775, LA28B06, and TC7S02 represent the major clade were selected by fermentation mode. The results of qPCR gene expression and mRNA transcriptomes showed the expression of XKS1 gene was increased and PGK1 was decreased while the oxygen supply decreases. The LA45B83 strain was a representative strain of the limited oxygen fermentation mode, and its genes were increased in maximum oxygen supply, while the xylose fermentation-related genes XYL1 and XYL2 performed better in limited oxygen conditions. The BCRC 21775 strain was a representative strain that had the best fermentation ability in limited and minimum oxygen supply. In maximum oxygen supply, its genes were increased, while xylose fermentation-related genes XYL1 and XYL2 performed better in limited oxygen supply. The LA28B06 strain was a representative strain that had the best fermentation ability in three different oxygen supply, and genes were increased in maximum oxygen supply. Xylose fermentation-related genes XYL1 and XYL2 performed better in maximum oxygen conditions, and there was no malat dehydrogenase performance. In addition to xylose fermentation-related genes, there were other genes, such as TTP and ATPase-related genes had high performance. The TC7S02 strain was a representative strain of the minimum oxygen fermentation mode. It’s had obvious genetic differences from the other three strains. The xylose fermentation-related genes XYL1 and XYL2 performed better in the minimum oxygen conditions, and compared with other strains TAL1, PDC, G6PDH had a high degree of expression.

中文摘要 I
英文摘要 III
誌謝辭 V
目錄 VI
表目錄 VIII
圖目錄 IX
壹、前言 1
一、替代能源發展歷史 1
二、生質酒精發展 1
三、酒精發酵菌種 3
四、Scheffersomyces stipitis分類與生理特徵 5
五、Scheffersomyces stipitis之發酵瓶頸 5
六、酒精發酵代謝途徑 7
七、木糖發酵的基因工程研究 8
八、研究動機 10
九、目的 11
貳、材料與方法 12
一、藥品 12
二、器材 12
三、儀器及設備 12
四、實驗菌株 13
五、研究方法 13
(一) 形態與生理鑑定 13
(二) 分子鑑定 15
(三) 基因親緣關係及相似度分析 18
(四) 木糖酒精發酵試驗 18
(五) 酵素活性分析 20
(六) 基因表現定量分析 22
(七) 轉錄組分析 23
參、結果與討論 25
一、菌種複核 25
二、序列比對與分子鑑定 25
二、木醣酒精發酵試驗 34
三、木糖代謝相關酵素序列比對 43
四、酵素動力學分析 56
六、轉錄組分析 65
肆、討論 85
伍、結論 88
陸、參考文獻 90

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