cyAbrB蛋白在高产氢蓝藻—蓝杆藻氢代谢中的调控作用

Among the emerging renewable and green energy sources, biohydrogen stands out as an appealing alternative for fossil fuels due to its high calorific value and cleanness. Cyanothece is a group of unicellular, diazotrophic cyanobacterium with the capacity to generate higher level of hydrogen, compared with any previously described wild-type hydrogen-producing photosynthetic microbe. However the regulatory mechanism of the hydrogen assimilation is still unclear. The previous study shows that the A type cyAbrB protein, CalA, functions as a regulator binding to upstream of uptake hydrogenase gene(hup) and hydrogen maturation protein gene(hyp) in filamentous diazotrophic cyanobacteria. Hydrogenase activity and hydrogen production change dramatically in the mutant strain of CalA overexpression. CalB (B type of cyAbrB) was co-purified with CalA, which implies the two cyAbrBs may function as a pair. In this proposal, the solid binding of cyAbrB to the upstream of genes involved in hydrogen metabolism will be investigated. In order to study the function of the cyAbrB, a serial of mutants including deletion mutants and overexpression mutants will be constructed. The phenotype, enzymes activity and hydrogen production will be analyzed and compared among the mutants. In addition, research of the mutual relationship between the two types of cyAbrBs will be conducted. This proposal aim to reveal the regulatory function of cyAbrBs in the hydrogen metabolism process and to obtain higher hydrogen producing Cyanothece strains.

氢气由于其热值高且燃烧后无任何污染是最理想的化石燃料的替代品。单细胞固氮蓝藻蓝杆藻(Cyanothece) 的产氢效率远远高于目前已检测的野生型藻类。以蓝杆藻为对象研究单细胞固氮蓝藻的产氢调控机制对获得高产氢微藻有重要的意义。但迄今为止，对于这类蓝藻产氢的调控没有系统性的研究。此前，申请人在丝状固氮蓝藻的几种氢代谢酶基因上游到了A型cyAbrB蛋白CalA，CalA过表达突变体的氢酶活性和产氢都有很大的变化。蓝杆藻中，也发现了两种AbrB蛋白的同源蛋白。利用蓝杆藻吸收氢酶和双向氢酶上游序列和蛋白的亲和反应得到了CalA的结合。本课题以此为出发点，拟通过构建突变体来分析表型，检测酶活和测量产氢, 结合转录组数据，确定cyAbrB在氢代谢过程中的调控作用。另外也探求蓝杆藻中两种类型的cyAbrB的相互作用关系。最终旨在阐明cyAbrB在产氢过程中的调控作用并筛选获得高产氢的基因工程藻。

蓝杆藻(Cyanothece)是一种单细胞固氮蓝藻，其产氢效率远远高于目前已经检测的野生型藻类。本研究以蓝杆藻为研究对象，通过DNA Pull-down实验得到与产氢相关基因hup和hox启动子序列相结合的蛋白Cyano_1550，经比对与CalA蛋白属于同一家族。通过EMSA实验验证了Cyano_1550与吸收氢酶的启动子序列的特异性结合，并且结合位点在序列的前半部分。构建部分缺失突变株和过表达突变株与野生型细胞比较了的产氢效率，结果表明，缺失突变株的产氢效率低于野生型，而过表达突变株高于野生型。同时研究了缺氮条件下，12L/12D光暗交替培养条件下蓝杆藻中固氮酶基因、吸收氢酶基因和双向氢酶基因的表达呈现24小时的周期节律变化，并且在黑暗条件下，固氮酶和吸收氢酶的表达较高而光照条件下表达较低而双向氢酶的表达不受光暗条件的限制，始终稳定低水平表达。在丝状蓝藻Nostoc punctiforme中也分离得到了Npr5944蛋白结合在吸收氢酶的启动子序列， CalA型的Npr5944蛋白能与CalB型的蛋白共同纯化，说明两种蛋白也有可能形成复合体形式调控作用。Npr5944蛋白基因的过表达突变株的产氢效率同样较野生型细胞增强，说明CalA蛋白在蓝杆藻和念珠藻种可能是抑制了吸收氢酶的表达，从而促进了产氢效率。. 研究固氮蓝藻的产氢调控机制对获得高产氢微藻有重要的意义。通过构建突变体来分析表型，检测酶活和测量产氢,确定其在氢代谢过程中的调控作用。