改造产氢酶提高光合深红红螺菌产氢能力的研究

Hydrogen gas is regarded as a potential energy carrier of the future, since it is renewable and clean, with no carbon dioxide produced and water as the only product of its combustion. Biological H2 production gains more attention in recent years because it is the only way of H2 production independent of using traditional energy, and it can be produced from negative-value waste materials. Purple non-sulfur (PNS) bacteria have been extensively studied for biological H2 production, since they have relative high substrate to product conversion yield and can produce highly pure H2 without the contamination of oxygen, which a strict inhibitor of most H2-producing enzymes. ..Rhodospirillum rubrum is a PNS bacterium capable of producing H2 under specific conditions. Nitrogenase is the main H2-producing enzyme in R. rubrum. As H2 is produced as a by-product in the nitrogen fixation procedure, its production is limited by N2 competition. Genome sequences analysis of R. rubrum reveals a 30 kb gene cluster encoding two hydrogenases (other than nitrogenase) and their maturation proteins, including formate-hydrogen lyase, iron-only hydrogenase, NADH dehydrogenase, 4Fe-4S ferredoxin, hydrogenase expression/formation protein HypE, HypF, HypD etc, suggesting that R. rubrum may have potential to use those hydrogenases although hydrogenase activity was not detected under regular growth conditions. The objective of this project is to discover the mechanism that regulate the hydrogenases and nitrogenase in order to increase the H2 production of R. rubrum. The specific aims are:..(1) Define the most efficient nitrogenase for hydrogen production. Using site-directed mutagenesis strategy to modify the substrate-reduction sites on Mo-Fe protein of nitrogenase to construct the mutants with altered nitrogenase, which will no longer reduce N2 but still be able to reduce proton to produce hydrogen, thus increase the hydrogen production in the presence of nitrogen...(2) Explore the expression and regulation mechanism of two other hydrogenases in R. rubrum. we will use molecular biology and biochemistry approaches, try to find out how these hydrogenases genes are regulated and the conditions for expressing these hydrogenases activities to increase hydrogen production. ..Successfully expressing iron-only hydrogenase and formate-hydrogen lyase would highly increase the hydrogen production of R. rubrum. Combination of the altered nitrogenase and active hydrogenase will increase the potential of R. rubrum mutants for the biological production of hydrogen.

光合细菌制氢优势在于细菌将太阳能直接转化为氢能，最大程度解除对传统能源的依赖，具有高产氢开发潜力。固氮酶是光合细菌主要产氢酶，具有产氢纯度高、无产氢酶抑制物（氧气）产生等优势，但固氮酶产氢受到氮气竞争抑制。甲酸裂解氢酶和铁氢酶是非光合细菌的产氢酶。我们对光合细菌深红红螺菌的基因组分析发现，该菌具有表达甲酸裂解氢酶和铁氢酶所需的全部基因，但常规培养条件未检测到相应的酶活。本项目拟以深红红螺菌为研究对象，通过以下两方面的研究来提高该菌的产氢能力：第一，定点突变固氮酶的底物还原位点，使其失去还原氮气能力，只识别质子为底物进行产氢，解除氮气对固氮酶产氢的抑制；第二，探索该菌潜在的甲酸裂解氢酶和铁氢酶及其调控机制和表达条件，激活相应基因的表达，获得表达这两种氢酶活性的菌株，从而获得解除氮气竞争、可持续高产氢的深红红螺菌菌株，为生物产氢研究提供的理论和应用基础。

光合细菌制氢优势在于细菌将太阳能直接转化为氢能，最大程度解除对传统能源的依赖，具有高产氢开发潜力。固氮酶是光合细菌主要产氢酶，具有产氢纯度高、无产氢酶抑制物（氧气）产生等优势。固氮酶产氢受到氮气竞争影响，而氮气是空气的主要组分。为解除氮气抑制，降低光合细菌对产氢条件的要求，提高产氢，本项目通过改造固氮酶，证实了定点突变深红红螺菌固氮酶的底物还原位点，可以使固氮酶失去还原氮气能力，只识别质子为底物进行产氢。此外，固氮酶对氧敏感是又一利用固氮酶固氮或产氢的限制因素，本研究发现了固氮菌可以通过改变群体存在形式，通过形成生物膜来响应高碳低氮、高氧的环境，首次发现一些固氮菌可以形成胞囊来屏蔽氧，以保护固氮酶发挥活性。为固氮酶的研究和开发提供新的思路。