未培养厌氧甲烷氧化古菌来源细胞色素c的异源表达优化

Enhanced heterologous expression of the cytochrome c from uncultured anaerobic methanotrophic archaea

摘要:

细胞色素c是一类在生物体内广泛存在的血红素蛋白，由亚铁血红素和细胞色素c前体组成，在生物电子学、生物医药以及污染物降解等领域具有很大的潜在应用价值。然而，细胞色素c很难通过异源表达而大量获取。对于未培养厌氧甲烷氧化古菌来源的细胞色素c (CytC4)，目前尚无成功表达和功能研究。本研究首先通过在CytC4的N端分别引入组氨酸标签和凝血酶位点，同时过量表达可帮助细胞色素c分子成熟的CcmABCDEFGH，成功在大肠杆菌中异源表达了CytC4。接下来又以含有细胞色素c分子成熟系统的奥奈达希瓦氏菌为表达宿主，提高了CytC4的表达量。未培养厌氧甲烷氧化古菌CytC4的异源表达，为其生理功能的探索及生物工程的应用奠定了基础。

Abstract:

Cytochrome c is a type of heme proteins that are widely distributed in living organisms. It consists of heme and apocytochrome c, and has potential applications in bioelectronics, biomedicine and pollutant degradation. However, heterologous overexpression of cytochrome c is still challenging. To date, expression of the cytochrome c from uncultured anaerobic methanotrophic archaea has not been reported, and nothing is known about the function of this cytochrome c. A his tagged cytochrome c was successfully expressed in E. coli by introducing a thrombin at the N-terminus of CytC4 and co-expressing CcmABCDEFGH, which is responsible for the maturation of cytochrome c. Shewanella oneidensis, which naturally has enzymes for cytochrome c maturation, was then used as a host to further increase the expression of CytC4. Indeed, a significantly higher expression of CytC4 was achieved in S. oneidensis when compared with in E. coli. The successful heterologous overexpression of CytC4 will facilitate the exploitation of its physiological functions and biotechnological applications.