利用大腸桿菌代謝工程進行甘油發酵酒精之研究

Abstract

製備生質柴油大多以植物性油質或動物性脂質為原料經過水解、皂化及轉酯化等程序，除了製造出生質柴油之外，一部分會合成副產物甘油，而過剩的甘油處理也將對環境造成問題，所以透過微生物發酵將甘油轉換成具有經濟價值的產物成為當前重要的研究議題。大腸桿菌進行甘油厭氧代謝需要額外提供電子接受者如tryptone、fumarate及nitrate等，本研究選擇使用nitrate作為電子接受者除了其還原電位較高之外，其成本也較兩者為低，再針對代謝路徑上表現量較低的gldA及dhaK兩基因分別利用厭氧啟動子進行大量表現(pGA-DK)以促進酒精之產量，結果顯示這兩個基因的表現可提高酒精產量為野生株的三倍之多，再以發酵槽進行甘油發酵之最佳化建構。藉由通入發酵槽不同空氣流速(0 ml/min、10 ml/min、20 ml/min、40 ml/min)製備出不同微氧的發酵環境，其中以空氣每分鐘供給10 ml的條件下酒精產量最高，添加酵母萃取物更可提高三倍酒精產量。甘油代謝路徑上之發酵酒精的合成需要NADH參與反應，因此進一步構築及表現可促進NADH合成之glpD基因，在空氣流速每分鐘10 ml及有添加酵母萃取物的條件下進行發酵代謝，實驗結果顯示可以促進酒精產量的上升，從原本的0.34 %增加到0.53 %的酒精濃度。由以上結果證明我們成功建構具有gldA、dhaK以及glpD三個表現基因之質體菌株BW25113 ( pGA-DK +pND)，並建立此菌株以nitrate為電子接受者之甘油發酵酒精的最佳化條件。

Biodiesel produced from animal fats and vegetable oils generates about 10% (w/w) glycerol. The excess glycerol becomes an environmental problem because it cannot be disposed as a waste. In this study, in order to enhance glycerol utilization, glycerol dehydrogenase (gldA), dihydroxyacetone kinase (dhaK) and glycerol-3-phosphate dehydrogenase (glpD) involved in glycerol metabolism were cloned on the overexpression plasmids. The results revealed that ethanol production from E. coli harboring gldA and dhaK was 3-fold higher than that of wild-type in anaerobic condition with sodium nitrate as electron acceptor. Moreover, E. coli can produce the highest ethanol under flow rate of 10 mL/min air and the presence of yeast extract. Coexpression of gldA , dhaK and glpD genes was conducted for ethanol fermentation. The result showed that ethanol production in optimal condition was 7-fold higher than that of wild type in the absence of yeast extract and anaerobic fermentation. Therefore , we have successfully constructed the plasmids harboring gldA , dhak and glpD genes in E. coli and demonstrated the optimal fermentative condition in glycerol metabolism with nitrate as electron acceptor.