建立 Escherichia coli NAG 基因表達系統之研究

Abstract

Zymomonas mobilis 為一革蘭氏陰性菌，具有良好的酒精生產能力而在生質酒精發展中受到重視。Z. mobilis 和一般常用來生產酒精之酵母菌相比，有較高的酒精產量和較高的酒精耐受性等優勢，然而 Z. mobilis 可代謝之基質範圍狹窄，僅能使用葡萄糖、蔗糖和果糖，因此在應用上受到限制。幾丁質是由 N -乙醯葡萄糖胺 (GlcNAc) 聚合而成的分子，其含量僅次於纖維素，是自然界中含量第二高的多醣類，故具有作為生質酒精基質的潛力。 Z. mobilis 中缺乏代謝 GlcNAc 所需之基因，故無法順利使用 GlcNAc 作為其生長所需之碳源。我們試圖使 Z. mobilis 得以利用 GlcNAc 作為其生長之碳源，因此在 Z. mobilis 中建構並表現 Escherichia coli 的三個基因 (nagA, nagB, nagE)，此三個基因分別可轉譯出 GlcNAc6P deacetylase 、 GlcN6P deaminase 和 GlcNAc 的運輸蛋白。在基因的建構設計上，我們參考乳糖操作組的模式，在相連接的每段基因中額外設計了一組 RBS 序列。本研究中，我們複製了這三個基因並且成功的送入 pET-28a 載體中。起初在基因的表現上遭遇了一些問題，但在經過多次調整後，我們成功在 E. coli 中表現了這三個蛋白。現在我們嘗試將這三個基因次轉殖送入可在 Z. mobilis 中表現異源蛋白的載體 pKT230 。最後，我們期望所建構的 Z. mobilis 得以利用 GlcNAc 做為其生長所需之碳源而生產酒精。

Zymomonas mobilis is a gram-negative bacterium which is notable for its bioethanol-producing capabilities. Compared to yeasts, Z. mobilis has better ethanol tolerance and higher ethanol yield. One limitation of Z. mobilis is that it can only use glucose, fructose, and sucrose as substrates for ethanol fermentation. Chitin is a polysaccharide consisting of N-acetylglucosamine (GlcNAc) monomers, and it is the second most abundant polysaccharide after cellulose on earth. Chitin can be hydrolyzed into GlcNAc by microbial chitinases, and the aim of this study is to engineer Z. mobilis to utilize GlcNAc for bioethanol production. Our strategy is to clone and express three E. coli genes:NagE, NagA,and NagB in Z. mobilis. NagE encodes a GlcNAc transporter, NagA encodes a GlcNAc-6-phosphatedeacetylase, and NagB encodes aglucosamine-6-phosphate deaminase. We have successfully cloned these three genes and expressed them in E. coli. By placing a ribosome binding site (RBS) in front of each gene, we have engineered a bicistronic construct that can co-express NagA and NagB. We have also successfully cloned and expressed NagE, which is a membrane protein and can only be detected in the enriched membrane fraction. The cloned NagA, NagB in E. coli, and NagE genes are being subcloned into the broad-host-range plasmid pKT230 and will be expressed in Z. mobilis. The use of GlcNAc for ethanol fermentation in the engineered Z. mobilis will be further investigated.