利用基因重組技術建構生產細胞保護劑ectoine之Escherichia coli菌株

Construction of recombinant Escherichia coli strain by genetic technique for ectoine production

劉惠雅(Hui-Ya Liu)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技與工程研究所/碩士(2009年)

https://doi.org/10.6838/YZU.2009.00247

摘要

Ectoine為許多嗜鹽菌或耐鹽菌在高鹽環境下，為維持及平衡細胞內外滲透壓所合成的一種compatible solute。研究顯示ectoine為兼具抗UV輻射、抗皮膚老化、抗皺、防曬、保濕等多功能之天然細胞保護劑，於產業應用特性上，屬於多功能之全方位生技化妝品。基於ectoine生產菌株主要為嗜鹽或耐鹽菌，必須以高鹽份含量之培養基進行培養以生產ectoine，長期下來將對醱酵槽及下游回收純化等設備造成極大侵蝕傷害；再者，為尋求能將菌體細胞內合成之ectoine大量釋放至胞外，便於分離回收之生產製程，因此本研究自高鹽環境所獲得之ectoine優質化生產菌Marinococcus sp.中選殖其ectoine合成基因ectABC，藉以建構高ectoine生產力之重組E. coli。本研究首先利用南方雜合以及inverse PCR (IPCR)的方法，自Marinococcus sp.中選殖出ectABC；接著設計專一性引子，以PCR的方式將ectABC進行放大，再經由限制酶作用放到pBAD24上，得一ectABC表現質體pECT1，之後將其轉型至E. coli進行表現。經SDS-PAGE以及LC-MS/MS分析實驗證實，已確認EctB及EctC成功被表現，EctA則經由酵素活性測試偵測其存在。最後以HPLC分析重組E. coli (pECT1) 胞內外ectoine的濃度，證實無論是胞內還是胞外培養液，皆可測得ectoine存在；就含量而言，胞外培養液ectoine濃度遠高於胞內，並且隨著誘導時間增加，ectoine釋放濃度也隨之增多。因此，本研究已成功利用基因轉殖的方式，構築一株能在低鹽環境下合成ectoine之重組E. coli (pECT1)。未來將進一步探討此菌株量產ectoine之相關製程及策略。

關鍵字

Ectoine ； compatible solute ； Marinococcus sp. ； IPCR

並列摘要

To master the osmotic stress of saline environments, halophilic bacterium accumulate highly water-soluble organic osmolytes, so-called compatible solutes. Ectoine, the compatible solute that was first discovered in Ectothiorhodospira halochloris, is one of the most commonly found osmolytes in nature. Ectoine is a natural cell protective agent that it can against UV radiation, protect skin from wrinkle, photoaging, and water loss. Therefore ectoine is widely used in cosmetic biotechnology. A large amount of salts must be added to the medium for the production of ectoine in the current process. Also, these salts will cause serious corrosion for fermentor and downstream processing equipments. Moreover, in order to let ectoine release into the medium and be purified easily, we construct the recombinant E. coli for ectoine production by genetic technique. First, we cloned the ectoine synthesis genes ectABC from Marinococcus sp. by Southern hybridization and inverse PCR (IPCR). Then, we amplified ectABC cluster by PCR, and the ectABC cassette was ligated into cleaved expression vector pBAD24. Finally, the resulting vector, pECT1, was transformed in E. coli DH5α. By SDS-PAGE and LC-MS/MS analysis, the expression of EctB and EctC in E. coli (pECT1) was successful; and the activity of the EctA was confirmed by an acylation assay. The concentration of ectoine in E. coli (pECT1) was analyzed by HPLC. We shown that whether intracellular or extracellular extracts could be measured the existence of ectoine, and the concentration of extracellular ectoine was much higher than intracellular. In this study, we have successfully constructed a recombinant E. coli (pECT1) for producing ectoine in lower salinity environment by genetic technique. In future, we will further explore the strategies to increase the ectoine production by this recombinant E. coli (pECT1).

並列關鍵字

Ectoine ； compatible solute ； Marinococcus sp. ； IPCR

參考文獻

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