Gloeobacter violaceus视紫红质在大肠杆菌中的异源表达与功能评价

Heterologous expression and function evaluation of Gloeobacter violaceus rhodopsin in Escherichia coli

摘要:

质子泵型视紫红质是一种在自然界广泛存在的简单光合系统，它可以结合视黄醛，在光照下将质子由胞内泵向胞外，形成质子梯度势，在一定程度上促进ATP的合成。在非光合工程菌中引入视紫红质将光能转化为化学能有助于促进微生物生长、生产以及提高细胞耐受性。文中在大肠杆菌Escherichia coli中异源表达了来自Gloeobacter violaceus PCC 7421的质子泵型视紫红质Gloeorhodopsin (GR)，并验证了它的功能活性。在大肠杆菌中表达时，GR可以正确行使光驱质子泵功能，最大吸收波长位于539 nm。GR分布在细胞膜表面，没有在胞内形成包涵体。在通过核糖体结合位点 (Ribosome binding site，RBS) 优化的手段提升GR的表达水平之后，观察到了胞内ATP水平的提高，证实在特定的条件下，GR可以为异源宿主带来额外的能量补充。

Abstract:

Proton-pumping rhodopsin (PPR) is a simple photosystem widely distributed in nature. By binding to retinal, PPR can transfer protons from the cytoplasmic to the extracellular side of the membrane under illumination, creating a proton motive force (PMF) to synthesize ATP. The conversion of light into chemical energy by introducing rhodopsin into nonphotosynthetic engineered strains could contribute to promoting growth, increasing production and improving cell tolerance of microbial hosts. Gloeorhodopsin (GR) is a PPR from Gloeobacter violaceus PCC 7421. We expressed GR heterologously in Escherichia coli and verified its functional activity. GR could properly function as a light-driven proton pump and its absorption maximum was at 539 nm. We observed that GR was mainly located on the cell membrane and no inclusion body could be found. After increasing expression level by ribosome binding site optimization, intracellular ATP increased, suggesting that GR could supply additional energy to heterologous hosts under given conditions.