Klähn, S. | Search bioRxiv

bioRxiv2026

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

Hydrogen (H2) 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 H2 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 CO2 fixation toward H2 production. Combining these interventions increases H2 production by over two orders of magnitude relative to the previous benchmark system, demonstrating that sustained H2 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 H2 production and long-term metabolic stability.

bioengineering↗

bioRxiv2020

The novel PII-interacting protein PirA regulates flux into the cyanobacterial ornithine-ammonia cycle

Among prokaryotes, cyanobacteria have an exclusive position due to the fact that they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g. they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here we reveal another small, 51 amino acid protein, which is encoded by the ssr0692 gene, to regulate flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls the entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK). We suggest that Ssr0692 competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it PII-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen-control transcription factor NtcA. Consistently, deletion of PirA affects the cell to balance metabolite pools of the OAC in response to ammonium shocks. Moreover, its interaction with PII requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein-interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell. ImportanceCyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g. as major primary producers. Due to their photosynthetic lifestyle cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis in the cyanobacterial model strain Synechocystis sp. PCC 6803. Beside its role as proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. The obtained results will therefore not only enhance our understanding of flux control in these organisms, it will also help to provide a scientific fundament for targeted metabolic engineering and hence the design of photosynthesis-driven biotechnological applications.

microbiology↗