A carbonic anhydrase-based nanogel for cyanobacterial growth enhancement

Sustainable bioproduction requires improved carbon fixation in phototrophic microorganisms. Cyanobacteria such as <i>Arthrospira platensis</i> and <i>Synechocystis</i> sp. PCC 6803 convert CO₂ into biomass using sunlight, yet their growth under ambient conditions is often limited by low CO₂ solubility. Here, we report the development of self-assembling all-enzyme nanogels composed of carbonic anhydrase (CA) to enhance CO₂ bioavailability in cyanobacterial cultures. These carrier-free nanogels were formed through covalent self-assembly of CA variants fused to SpyTag or SpyCatcher domains and characterized using electrophoresis, dynamic light scattering, atomic force, environmental scanning electron, and confocal laser scanning microscopy. A custom pH-based CO₂ hydration assay showed that the nanogels retained high catalytic activity and stability for several weeks, and systematic analyses of buffer capacity revealed CA-dependent changes in the extracellular environment. Although CA supplementation had only minor effects on <i>A. platensis</i>, <i>Synechocystis</i> cultures exhibited a clear increase in biomass and chlorophyll content. Overall, CA nanogels represent a robust, carrier-free platform for enhancing CO₂ utilization and hold promise for applications in carbon capture, biomass production, and multi-enzyme cascade systems.