強化Saccharomyces cerevisiae代謝木糖能力之菌株構築及其合成生質酒精之應用

Construction and application for the enhanced xylose-assimilating Saccharomyces cerevisiae strain on the production of bioethanol

楊曉涵(Siao-Han Yang)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技與工程研究所/碩士(2011年)

摘要

Saccharomyces cerevisiae 為目前工業上普遍用來生產乙醇之菌株，但未經改殖的Saccharomyces cerevisiae野生菌株，只能代謝以葡萄糖為主的六碳糖，無法代謝木糖，對於以木質纖維素為料源來生產乙醇方面，將會浪費額外的碳源。因此，本研究利用基因重組方式將具有木糖代謝能力之Pichia stipitis菌株中的XYL1、XYL2及Saccharomyces cerevisiae中的XKS1轉殖到Saccharomyces cerevisiae Hansen BY4742與Saccharomyces cerevisiae Hansen BY4742 Δzwf1中，並且比較刪除ZWF1之Saccharomyces cerevisiae與完整基因的Saccharomyces cerevisiae對於菌體生長及乙醇產量之影響。
研究顯示，藉由RT-PCR分析能明顯的看出Saccharomyces cerevisiae Hansen BY4742與Saccharomyces cerevisiae Hansen BY4742 Δzwf1中XYL1、XYL2、XKS1有mRNA的表現；而SDS-PAGE也顯示有蛋白質的產生。
發酵測試方面，刪除ZWF1基因確實能有效增加木糖代謝途徑的流暢度，初步結果顯示乙醇的產量為7.6 g/L，與對照組相比增加3.33 g/L的產量。而與其他菌株相比，Saccharomyces cerevisiae Hansen BY4742 Δzwf1-X3有較高的Yp/x，為0.52 gEthanol/gSugar-1。然而，馴養過後的Saccharomyces cerevisiae Hansen BY4742 Δzwf1-X3 & Saccharomyces cerevisiae Hansen BY4742-X3與之前相比在木糖消耗及乙醇產量上都有明顯提高，乙醇產量分別增加0.8及1.9 g/L，其中又以Saccharomyces cerevisiae Hansen BY4742-Δzwf1-X3有最高的產量，8.42 g/L。

關鍵字

Saccharomyces cerevisiae ； Pichia stipitis ； 乙醇

並列摘要

The yeast Saccharomyces cerevisiae is the most commonly used microorganism in traditional industrial fermentations. Although Saccharomyces cerevisiae is suitable for ethanol fermentation, it is unable to metabolize pentose (such as xylose); therefore, the resources used for ethanol fermentation are limited. For the lignocellulose material into fuel ethanol, there will be waste the extra carbon source. In this study, we were using metabolic engineering to improve ability of xylose utilization. Therefore, in this study, we have successfully cloned the pentose utilization genes (XYL1, XYL2 and XKS1) from Pichia stipitis and Saccharomyces cerevisiae into Saccharomyces cerevisiae Hansen BY4742 and Saccharomyces cerevisiae Hansen BY4742 Δzwf1 for biosynthesis of bioethanol. Data shown that the Saccharomyces cerevisiae Hansen BY4742-X3 and Saccharomyces cerevisiae Hansen BY4742 Δzwf1-X3 has mRNA expression for XYL1, XYL2, XKS1 by RT-PCR analysis; and also showed XYL1, XYL2, XKS1 protein production by SDS-PAGE. We also found xylose metabolic pathway can be more smooth for the over expression of XKS1 (Xylulokinase) gene. Moreover, the recombinant Saccharomyces cerevisiae Hansen BY4742 Δzwf1-X3 strain was able to synthesize significant concentration of bioethanol (7.6 g/L), compared with the control strain to increased 0.33g/L. The conversion rate of bioethanol also improved to 0.52 g Ethanol/g Sugar-1. Furthermore, the strain Saccharomyces cerevisiae Hansen BY4742 Δzwf1-X3 can obtain the highest bioethanol production (8.42 g/L) after the enrichment of high concentration of xylose.

並列關鍵字

Saccharomyces cerevisiae ； Pichia stipitis ； Ethanol

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