应用高通量基因合成与筛选技术优化变形菌视紫红质表达及光驱动质子泵功能

Proteorhodopsin (PR) is an integral membrane light-powering proton pump that is found in bacteria distributed throughout global surface waters. Its excellent photochemical activity, cyclicity and thermal stability have made it considered as the most promising biomolecular copmonent for numerous comercial applications. However, its practical use has been obstructed by the low expression level in E.coli and the fact that the unique and complex spectral tuning mechanism is still not clear. The color-changing residues are infered to be distributed through the protein according to the results of random mutation by error-prone PCR which obviously cannot produce the exhaustive set of all color-affecting residues. To solve the problems, we plan to apply the high-throughput on-chip gene synthesis and screening technology to optimize the expression and the light-driven proton-pumping function of PR. The highly expressed variants with optimal proton-pumping activity are expected to be obtained by residue-by-residue mutation and screening based on the high-throughput gene synthesis and screening technology. This study may help understand the potentially significant contribution of PR to the solar energy input in the biosphere and accelerate the application of PR in bioenergy and optoelectronic devices. Besides, the application of high-throughput gene synthesis and screening in this study can also open up a new way for functional studies of proteins and protein engineering.

变形菌视紫红质（PR）广泛存在于全球各种水域的菌体中，是具有光驱动质子泵功能的膜蛋白，因其优异的光化学活性、光循环性和热稳定性而被认为是生物能源、生物光电材料领域最具应用潜力的蛋白。目前，对PR结构、功能和应用的研究受到两个主要瓶颈制约，即PR在大肠杆菌中表达水平偏低,及影响其功能的氨基酸位点分布广、影响因素异常复杂，常规或易错PCR突变手段难以全面探查。本项目拟采用前期开发出的高通量芯片基因合成与筛选技术优化PR在大肠杆菌中的表达，在此基础上通过"地毯式"大规模氨基酸突变全面发掘与PR光化学功能相关的重要氨基酸位点，以期通过进一步优化和表征最终获得具有最优光驱动质子泵功能的PR基因。本研究除帮助深入了解PR在全球水域碳循环和能量代谢中的作用、加快PR在光能转化利用领域的应用进程外，还将为蛋白质功能研究和优化改造开辟一条新的技术途径。

以广泛存在于全球各种水域菌体中的变形菌视紫红质（proteorhodopsin，PR）为研究目标，采用自主研发的长序列DNA中连续多点饱和突变库构建方法、多功能基因库的快速组装方法和大片段DNA及基因（库）组装技术，对PR从基因局部连续多点突变和全基因简并突变两个方向展开研究，通过“地毯式”大规模氨基酸突变全面筛选获得了功能性变异的突变体，并发掘与PR光化学功能相关的重要氨基酸位点。在此基础上，我们利用PR的突变体，开发了基于PR的新型系列光控相变光刻胶及一种适合于高通量生化反应的微阵列生物芯片。本项目的顺利实施，加快了PR在光能转化利用领域的应用进程，所研发的多项新技术在基因合成及蛋白质功能研究和优化改造领域具有良好的应用和产业化前景。