Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97563
標題: 建構重組大腸菌株提升異源 ectoine 之生產
Construction of recombinant E. coli strains for enhancing heterologous ectoine production
作者: 陳震
Chen Chen
關鍵字: 耐鹽菌株;相容性物質;最適碳氮源;ectoine;Halomonas magadiensis;compatible solute
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摘要: 
耐鹽菌株 Halomonas magadiensis 可以生產ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecar boxylic acid),它是一種胺基酸類的相容性物質(compatible solute),可以有效的阻止滲透壓緊迫而造成菌體的破裂或是脫水,進而避免菌體的死亡。本研究首先探討H. magadiensis 菌株在ectoine生產的最適化條件,發現以無機氮源並將培養液之起始pH值先用NaOH調整至9後,於28℃ 培養48小時,ectoine的生產量可達1,000 mg/L左右。若以1% MSG為碳源,在搖瓶培養 36小時後,胞內 ectoine 生產量更可以達 2,000 mg/L。在培養基碳氮源比例上,發現以1% 麩胺酸鈉(monosodium glutamate, MSG)為碳源配合0.05% 的 (NH4)2SO4作為氮源,可使 ectoine 的最大生產量增至 2,500 mg/L。在研究中亦將H. magadiensis之ectoine生合成基因ectABC進行選殖,並構築在pET21bT-ectABC表現載體上,當導入E. coli Rosetta (DE3)pLysS,可於SDS-PAGE上確認EctABC 三種蛋白皆有表現,並於胞內及胞外偵測到ectoine 的生成。為進一步提升ectoine在E. coli菌株之生產,對ectoine生合成途徑上游之lysC及asd進行選殖,並分別構築於可被arabinose 誘導之pBBad22k表現載體,或帶有可持續表現啟動子之pMTA1載體上。當與pET21bT-ectABC載體在Rosetta (DE3)pLysS菌株進行共表現時,帶有pBBad22k-asd 載體的菌株,會比單獨表現之控制組多出了10% 的ectoine總生產量;而帶有pMTA1-asd 載體的菌株,雖然 Asd 蛋白的表現量較多,但在ectoine總生產量尚反而下降。帶有lysC 之兩種載體與 ectABC共表現時,LysC 蛋白表現不多且ectoine總生產量與單獨表現之菌株差異不大。為避免LysC 本身會受 lysine負回饋而影響到ectoine 的生產,研究中亦構築了兩種lysC之突變株LysCT344M及LysCS321T,並分別置於 pET21b 表現載體上,與在pET24 載體上之ectABC 於Rosetta (DE3)pLysS菌株進行共表現。結果發現帶有LysCT344M及LysCS321T的共表現株,比控制組增加了73% 及17% ectoine 的總表現量,若與帶有正常之lysC 之載體相比,ectoine 總表現量則分別提高3.9倍及 2.9倍。若將 pMTA1-asd 轉殖到 Rosetta(DE3)pLysS(pET21-lysCT344M)( pET24-ectABC)後,在誘導表現36小時後 ectoine 的總量可到達 5,292 ± 250 mg/L。總之,本研究提供了H. magadiensis 菌株在ectoine生產上的最適化條件,並且藉由代謝工程技術可以有效在大腸菌株中進行ectoine的異源生產。
URI: http://hdl.handle.net/11455/97563
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