During its second period, the PhotoSynH2 project transitioned from foundational tool-building to demonstrating integrated biological systems, marking significant progress across all areas of its research programme.
A major breakthrough was the demonstration of biological hydrogen production in engineered Synechocystis strains without the need for external chemical activation. The systems biology and strain engineering team at Uppsala University (WP3) achieved this by successfully co-expressing both an engineered [FeFe] hydrogenase and its specific maturation enzymes. This creates a self-sufficient biological system where the cell can assemble its own active catalyst in vivo, a critical step towards industrial feasibility.
This achievement was built upon the advanced genetic toolkit developed by the CSIC team (WP1). They designed and validated a powerful suite of synthetic biology tools, including novel CRISPR-based systems for gene activation and repression, as well as RNA-based regulators. These function like precise genetic switches, allowing the consortium to fine-tune metabolic pathways, downregulate competing processes, and control the expression of the project's engineered components.
The hydrogen-producing enzyme itself was the focus of the second research team at Uppsala University, specialising in biocatalysis (WP4). This team identified novel [FeFe] hydrogenases from nature and, through rational protein engineering, created new variants with significantly enhanced catalytic activity. Crucially, they engineered enzymes with improved tolerance to oxygen, a key bottleneck that has long hampered biological hydrogen production. This work has already resulted in a peer-reviewed publication in a high-impact journal.
To create a more resilient industrial chassis, the i3S team in Portugal (WP5) successfully engineered Synechocystis strains with enhanced tolerance to high-salinity conditions, demonstrating robust growth in media mimicking seawater. This innovation reduces the reliance on precious fresh water. Moreover, to create an intracellular environment appropriate for hydrogenases activity/H2 production, Synechocystis strains were further engineered to harbor oxygen scavenging devices. Taking a complementary approach to sustainability, the CNR team in Italy (WP6) demonstrated that the cyanobacteria could be cultivated using nutrient-rich wastewater from a municipal treatment plant. Their experiments showed not only successful growth but also sustained, long-term hydrogen production, showcasing a truly circular economy model.
Following a detailed techno-economic analysis, the project strategically pivoted to include the co-production of a high-value pigment. The CSIC team (WP2) is leading the work to integrate the genetic pathway for astaxanthin synthesis into the cyanobacterial host, aiming to create a highly profitable biorefinery.
Finally, the project's hardware has seen substantial development. The M2M team in Italy (WP6) designed an improved 1,300-litre multiplate photobioreactor (MPL). A 150-litre first improved MPL pilot version has been constructed and installed at the CNR site for outdoor testing under real-world solar conditions. In parallel, Algreen in the Netherlands (WP6) has identified a potential industrial site for future large-scale deployment and has mapped the complex regulatory pathway needed to operate with genetically engineered microorganisms at this scale in Europe.
