重组脱硫弧菌NiFeSe氢化酶的有效翻译及其耐氧性能的分子机理研究

Hydrogenase enzymes, which catalyze the formation and dissociation of hydrogen that is thought to be an attractive carbon-neutral fuel alternative, are heteromeric metalloenzymes extremely sensitive to oxygen due to its labile catalytic site. This research project proposal aims to isolate hydrogenase recombinants with lower oxygen sensitivity by attempts including construction of E. coli overexpression kit for NiFeSe containing enzyme from Desulfovibrio vulgaris Hildenborough (DvH NiFeSe Hase), and setup of a screening platform for oxygen tolerant mutants via in vivo high-throughput measurement of enzymatic activity. In order to build the efficient E.coli host system for heterological expression of selenium containing DvH Hase, studies to be carried out include investigating how to co-express proteins/tRNA for the synergetic maturation process of the catalytic center, codon optimiaztion,complemental expression of homologs required for processing of catalytic center, and introduction of heterologous translation system for selenocysteine (Sec)encoded by opal stop code UGA. Meanwhile, a high-throughput screen method is going to be developed in term of detection of enzymatic activity of hydrogenase using microplate reader by recording the spectrum fluctuation of artificial dye upon redox potential change during the enzymatic assay.The project proposed here is an enabling step toward photoinduced hydrogen production by the development of valuable oxygen-resistant biocatalyst and synthetic hydrogenase-mimic electrocatalysts.

脱硫弧菌镍铁硒(NiFeSe)氢化酶是研究氢化酶耐氧及催化产氢机制的模式蛋白之一，对该酶耐氧性能结构生物学的研究对电子化学催化模拟物的研制有重要影响。本项目拟以大肠杆菌为重组表达宿主细胞，对涉及该酶催化中心成熟途径及硒代半胱氨酸(Sec)的胞内翻译系统进行基因组学、转录组学的全面分析，通过人工序列设计和基因拼接等分子生物学方法，构建在厌氧环境下高效表达重组氢化酶并进行有效的催化中心加工和Sec翻译的大肠杆菌表达平台，并利用大肠杆菌基因缺失突变株，对含硒NiFe氢化酶有效翻译的分子机制进行综合分析。同时借助色谱、光谱等现代分析化学手段，研究脉冲氧化对氢化酶动力学的影响，建立对氢化酶突变体文库进行高通量筛选的方法，以获得具有不同耐氧度的氢化酶突变体，通过对它们结构生物学的研究，为解开氢化酶氧耐受科学难题的基础研究提供技术支持，为生物制氢、氢电池研究相关领域提供理论依据。

本项研究针对脱硫弧菌镍铁硒氢化酶（DvH NiFeSe Hase）含镍铁硒的特点，综合应用合成生物学、生物信息学、分子生物学等多种研究手段，通过分析氢化酶表达及成熟所需的基因、启动子等分子元件，进行了人工操纵子设计，构建了可共存的多质粒的表达系统。同时，对大肠杆菌原始菌BW25113进行了自身氢化酶及含硒的三个甲酸脱氢酶的无痕敲除；在第一轮没有得到有氢化酶活性的重组大肠杆菌之后，针对氢化酶序列中的硒代半胱氨酸进行了点突变，将其变成半胱氨酸，突变后的重组氢化酶没有检测到活性；利用厌氧箱进行了氢化酶高通量筛选平台的研究，利用氢气进行氢化酶的氧化测定，并开发出了从大肠杆菌厌氧破碎到酶活批量测定的技术方案。后续，本项研究将利用已经建好的大肠杆菌表达系统和检测方案，开展针对高温可溶性氢化酶的外源重组构建和厌氧性能的优化研究。