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標題: Clostridium pasteurianum F40 的產氫酶和鐵硫蛋白基因之異源表現
Heterologous expression of the hydrogenase 1 and ferredoxin genes of Clostridium pasteurianum F40
作者: 紀映光
Ji, Ying-Wuang
關鍵字: Clostridium pasteurianum;產氫酶鐵硫蛋白;hydrogenase 1;ferredoxin;異源表現
出版社: 生命科學系所
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氫氣是在石油面臨枯竭的當今,極具發展性且潔淨的能源之一。由於許多微生物能夠在利用各種碳水化合物進行生長代謝的過程中產生氫氣、乙醇及丁醇等可作為燃料的產物,藉由微生物產氫是各種產氫方式中,最不具地域性、成本較低廉且能永續發展的方式。梭狀芽孢菌屬(Clostridium)細菌是目前最常被用來深入研究微生物產氫的細菌之一,這類細菌能夠在厭氧的狀況下,利用醣解作用所產生的NADH,經由鐵硫蛋白(ferredoxin)的攜帶,將電子傳遞至產氫酶(hygrogenase),在產氫酶蛋白中,再經由4個鐵硫集團(Fe-S cluster)的傳遞,將電子送至產氫酶活性中心的H-cluster和氫氣離子形成氫氣。因此在梭狀芽孢菌屬細菌的產氫途徑中,產氫酶以及鐵硫蛋白扮演著不可或缺的重要角色。實驗室前人由白蟻腸道分離出來一株具有極佳半纖維素分解能力,但只稍具產氫能力的菌株Clostridium xylanolyticum Ter3,本研究希望藉由遺傳工程的方式提升其產氫能力,使之成為碳源利用產氫能力俱佳的菌種。實驗的策略是將實驗室另一株優良產氫菌株Clostridium pasteuriaum F40的產氫酶基因和鐵硫蛋白基因選殖到C. xylanolyticum Ter3。從C. pasteuriaum F40的染色體得到完整的兩個基因並經定序確認之後,將兩個基因一起用E.coli與Clostridium屬細菌的 shuttle vector pIMP1構築成為重組質體pIMP1F40d。為了避免將此重組質體送入C.xylanolyticum Ter3時發生新宿主菌的限制酶切割外源 DNA的問題,除了將重組質體送入E.coli DH5α之外,同時也將其送入E.coli ER2275及E.coli JM110,以便利用ER2275的甲基酶ψ3t1和JM110的甲基酶Dam及Dcm進行特定限制酶辨識位置的甲基化。在將pIMP1F40d送入E.coli DH5α之後,RT-PCR實驗分析證明選殖的產氫酶基因與鐵硫蛋白基因均能轉錄出mRNA,而分析E.coli DH5α、E.coli ER2275及E.coli JM110的轉殖株其氫氣能力皆較個別的wild type有4.7%~108%的增加。然而進一步嘗試藉由電穿孔轉形作用將重組質體送入C.xylanolyticum Ter3卻未能成功,可能是由於C.xylanolyticum Ter3有其特有的限制酶會辨認外源 DNA並加以切割所致。

Hydrogen is one of the most developmental clean energy under the oil shortage of today. Some microorganisms can break down cellulose to produce biofuels like hydrogen, ethanol and butanol. Using microorganisms to produce hydrogen is the least regional, lower-cost, and sustainable way among various hydrogen production methods. The genus Clostridium are one of the bacteria most often used for studying hydrogen production, they are strict anaerobes, when they carry out fermentation, NADH is produced in the glycolytic pathway and its electrons are transferred to hydrogenase by ferredoxin. In hydrogenase, Fe-S cluster transfers electrons to active site, H-cluster, to form hydrogen with proton. In this hydrogen production pathway, both hydrogenase and ferredoxin are indispensable. Clostridium xylanolyticum Ter3 is an early isolate of our lab, it has good hemicellulolytic ability but moderate hydrogen-producing ability. In this study an attempt was made to improve its hydrogen production by transforming C. xylanolyticum Ter3 with the hydrogenase gene and ferredoxin gene of C. pasteurianum F40, which is a very good hydrogen producer isolated by our lab. After both whole genes were obtained from C. pasteurianum F40 chromosome and sequenced, they were cloned into a shuttle vector, pIMP1,of E.coli and Clostridium. The resulting recombinant plasmid pIMP1F40D was used to transform E.coli DH5α first, in order to protect pIMP1F40D from degradation by specific clostridial restriction endonuclease, pIMP1F40D was also introduced into E. coli strains ER2275(pAN1) and JM110 to carry out in vivo methylation with then known methylases, ψ3t1 and Dam/Dcm, respectively. The results of RT-PCR indicated that both hydrogenase gene and ferredoxin gene could be transcribed in E.coli DH5α, but SDS-PAGE did not clearly show the translating products of both genes. When these transformed strains were grown anaerobically in LB medium containing 10 g/l glucose for 16 hours, they produced 25%~114% more hydrogen than their wild type strains. Unfortunately the transformation of C. xylanolyticum Ter3 with pIMP1F40D by electroporation was unsuccessful, it seems that C. xylanolyticum Ter3 possesses unknown restrictiction - modification system to be disclosed further.
其他識別: U0005-2908201112330300
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