微生物細胞表面顯示系統及 合成多樣化金屬奈米粒子的開發與應用

Abstract

微生物細胞表面顯示(Microbial cell-surface display)，即利用暴露在細 胞表面的蛋白質作為載體蛋白(carrier protein) ， 將乘客蛋白或胜肽 (passenger protein/peptide)顯示在細胞表面的技術。因此，載體蛋白能夠有效並成功地將乘客蛋白顯示於細胞表面是極為重要的。本實驗藉由來自 Escherichia coli 的outer membrane iron transporter protein (FhuA) 作為載體蛋白，發展一個可以使用於不同菌種及顯示不同的異源性乘客蛋白/胜肽的細胞表面顯示平台，同時比較來自E. coli，截短的outer membrane protein(OmpA)，以及來自Neisseria gonorrhoeae 的 immunoglobulin A protease(IgA protease)作為載體蛋白，將乘客胜肽顯示於一革蘭氏陰性菌，Ralstonia eutrpha 之表面。透過實驗分析，三種載體蛋白無論於E. coli或R. eutropha中，皆成功易位於細胞外膜，並將不同的乘客胜肽顯示於細胞表面，而乘客胜肽之功能依舊，表示此使用E. coli，FhuA 作為載體蛋白之策略適合顯示異源性胜肽在細胞表面的生物工程應用。 在自然界中，許多微生物本身就具有在細胞內或細胞外合成金屬奈米粒子的特性。由於生物性的合成方法具有無毒並對環境友好性的特質。因此，利用微生物作為合成金屬奈米粒子反應器已廣泛的被研究，除了使用本身具有合成金屬奈米粒子特性的微生物外，也有許多利用重組菌株的方式以合成更多樣化的金屬奈米粒子。Rhizobium etli，是一種固氮菌，其所含的melA 基因序列被證實為tyrosinase 的基因序列，且被使用於重組E.coli 並藉由外加的L-DOPA 產生黑色素。本實驗藉由帶有melA 基因片段的重組E. coli，表現tyrosinase 並催化L-DOPA，產生出黑色素並合成多種的金屬奈米粒子。Microbial cell-surface display allows the passenger protein/peptide to be displayed on the surface of microbial cells by fusing them with the carrier protein. Therefore, it’s important that carrier proteins display passenger proteins/peptides efficiently and successfully on the cell surface. In this study, a system for the display of heterologous passenger proteins/peptides on the surface of different strain was developed using the Escherichia coli outer membrane iron transporter protein (FhuA) as a carrier protein. Simultaneously, a truncated outer membrane protein A (OmpA) from E. coli and a immunoglobulin A protease (IgA protease) from Neisseria gonorrhoeaen were compared in a Gram-negative bacteria, Ralstonia eutropha. Through the experimental analysis, the three carrier proteins and different passenger peptides are located and displayed on the cell surface in both E. coli and R.eutropha. These results suggest that the strategy employing the E. coli FhuA as a carrier protein is suitable for the display of heterologous peptides on the cell surface for biotechnical applications. In nature, microorganisms are capable of reducing the metal ions to form metal nanoparticles intracellularly or extracellularly. Biosynthesis of metal nanoparticles has been studied extensively because of their unique properties such as non-toxicity and environmental friendly. The protein encoded melA by the nitrogen-fixing bacterium, Rhizobium etli, is a tyrosinase. It has been shown that recombinant E. coli produced melanin with melA gene from R. etli. In the present study, we demonstrate that the melanin biosynthesis pathway in R. etli can be exploited for the in vivo synthesis of metal nanoparticles with the inexpensive L-DOPA using recombinant E. coli.