Multilevel engineering of cyanobacterial energy metabolism advances photosynthetic hydrogen production while revealing its constraints

Hydrogen (H ₂ ) is a promising sustainable energy carrier, and its direct production from photosynthetic water splitting is appealing. Yet long-term photosynthetic hydrogen production by cyanobacteria remains inefficient despite decades of engineering. Here, we systematically dissect the hierarchical and state-dependent constraint architecture governing sustained H ₂ evolution in Synechocystis sp. PCC 6803. We show that hydrogenase overexpression relieves the primary enzymatic limitation, exposing ATP/NADPH balancing and competing electron sinks as successive metabolic constraints. Inspired by cyanophage strategies, we engineered synthetic CP12-based regulatory proteins that redirect photosynthetic electrons from CO ₂ fixation toward H ₂ production. Combining these interventions increases H ₂ production by over two orders of magnitude relative to the previous benchmark system, demonstrating that sustained H ₂ production requires coordinated management of metabolism and regulation rather than elimination of a single bottleneck. However, overcoming these constraints also promotes metabolic adaptations and genetic instability, illustrating the trade-off between maximal H ₂ production and long-term metabolic stability.