利用代謝工程提升大腸桿菌酒精生產能力

Abstract

生質酒精在經濟與環保方面是極具潛力之再生能源，有鑒於纖維素生質原料具有全球儲存量高、來源多樣化及不易與糧食生產發生衝突等優點，將纖維素轉化為酒精已被視為現階段生質酒精產製過程中最具潛力的發展方向。Escherichia coli本身具有可廣泛利用木質纖維素水解後的產物xylose及glucose，來進行酒精發酵的優點，是具有潛力的酒精生產菌株。利用不同濃度酒精之培養基馴化並搭配突變劑NTG (N-methyl-N-nitrosoguanidine) 進行酒精耐受菌株篩選，篩得可耐5%酒精濃度之WSJ 5-1突變菌株，與KO11原始菌株相比，其發酵葡萄糖(glucose)酒精產量提升約10 %。 為了減少酒精發酵副產物有機酸的產生，發酵菌種篩選與改良十分重要，本論文研究目標為提升E. coli BW25113酒精生產能力，進行代謝路進改良，阻斷酒精發酵過程中副產物生成路徑，並利用ㄧ厭氧的基因啟動子，表現Zymomonas mobilis之pyruvate dehydrogenase (pdc) 及alchol dehydrogenaseⅡ ( adhB ) 兩酵素基因，配合發酵時厭氧誘導因子，期望能夠以基因重組方式加強並穩定大腸桿菌的高酒精產量表現。以含有7 %葡萄糖的 LB 培養液進行發酵槽酒精產量測試，實驗結果發現基因剔除菌株WSJ008發酵過程中有機酸產量與野生株BW25113 相比下降80%；酒精產量與野生株BW25113 相比則提升31 %，基因突變株之酒精轉換率達94.5 %。

Ethanol is an important and renewable fuel. Lignocellulose comprising glucose and xylose represents the most abundant biomass in the world. It is important to study the bioconvertion of lignocellulose into ethanol. Escherichia coli can metabolize both glucose and xylose to ethanol under anaerobic condition. In this study, the E. coli mutant with ethanol tolerance was successfully screened by NTG (N-methyl-N-nitrosoguanidine) and adaptive evolution using ethanol as a selection pressure. The ethanol tolerance of mutant WSJ 5-1 (5%) was higher than wild type KO11 (3.5%). Moreover, the ethanol production in WSJ 5-1 was 10% more than KO11 under glucose fermentation. During ethanol fermentation, a lot of by-products such as succinic acid, lactic acid, acetic acid, ethanol, and formic acid were formation. In this study, eight genes of E. coli encoding seven metabolic reactions were deleted, which can reduce the organic acids production and efficiently produce ethanol from glucose. The engineered E. coli (WSJ008) was obtained by P1 transduction to achieve the gene knockout. Also, pdc and adhB genes from Zymomonas mobilis encoding pyruvate decarboxylase and alcohol dehydrogenase II driven by the anaerobic promoter were used to improve ethanol production. The fermentation was carried out by 1 liter working volume with 7% glucose. The wild type BW25113 and gene knockout strain (WSJ008) were compared. The organic acid production from the WSJ008 strain was decreased 80%, whereas ethanol production was 31% more than the wild type. In addition, the carbon recovery rate of the WSJ008 strain was 91%, and the ethanol theoretical yields of WSJ008 was 94.5%.