产气肠杆菌(E. aerogenes)是近年研究较多的兼性产氢菌，其NADH产氢途径倍受关注。如何实现NADH高效供给是产气肠杆菌制氢研究的核心问题。课题组提出“NAD+/NADH Biosensor “智能”调控好氧/厌氧耦合供给NADH产氢”的攻关设想，围绕调控NADH供给与强化产氢两个方面开展工作:1)以T-Rex–cpYFP标记菌为出发菌株，基于cpYFP Biosensor侦测氧和NAD+/NADH的特性，让细胞“智能”调控氧和NADH供给，建立荧光可视和氢酶仍活跃的培养体系；确定耦合体系DO、NAD+/NADH和H2的函数关系，阐明耦合NADH供给产氢机理；2)组合敲除/组合表达二维设计，探究候选基因工程菌的代谢情况，NADH供给调控信息；整合组合敲除和组合表达优秀工程菌，提高氢气得率。本项目为提高NADH产氢途径的氢气得率，奠定理论基础；也为生物化工耦合培养技术提供理论借鉴。

The hydrogen production pathway of NADH of facultative anaerobic Enterobacteriaceae has higher known theoretical hydrogen yield and thus has attracted much attention. How to improve the actual hydrogen production by NADH-dependent pathway and break through the bottleneck of low hydrogen conversion rate is a research hot spot. In this research proposal, E. aerogenes (osp:: T-Rex–cpYFP) is employed to investigate the following aspects. First, key intelligent technologies by NAD+/NADH biosensor to regulate and control aerobic–anaerobic coupling process will be developed. A new process will be designed based on the aerobic–anaerobic coupling theory to intelligently regulate by NAD+/NADH biosensor in cells to supply NADH to produce hydrogen. A new theory and technology of biological hydrogen production will be derived from this study. Second, Genetic or cofactor engineering combined knock-out and overexpression gene targets through systematic and combinatorial approaches, respectively, in order to provide a basis to increase the low yield through increased flux for hydrogen production in E. aerogenes. A new ways of NADH supplied were rebuilt by knockout, overexpression and combination these targets. The combination of knockout and expression of excellent engineering strains can further improve hydrogen production, and as well as to enhance hydrogenase activity in cell. Moreover, some higher hydrogen-producing strains would be obtained and be used in the following assays. The bottleneck to hydrogen production would be resolved based on these theories. It also provides a theoretical reference for the aerobic–anaerobic coupling cultural technology using in industrial bio-fermentation.