Sustained isobutene production by Synechocystis sp. PCC 6803 entrapped in polyvinyl alcohol hydrogel beads

Cyanobacteria convert CO₂ into valuable compounds using solar energy, making them ideal for sustainable isobutene production, a key precursor for fuels and chemicals. This study aimed to enhance isobutene production in engineered Synechocystis sp. PCC 6803 strains: Syn-RnKICD, which produes isobutene from α-ketoisocaproate via Rattus norvegicus α-ketoisocaproate dioxygenase (RnKICD), and Syn-F336V, a mutant RnKICD variant with a phenylalanine to valine substitution at position 336 showing improved isobutene production. We investigated the effects of varying culture conditions, including light intensity, inorganic carbon, and nitrogen on isobutene production. Nitrogen limitation emerged as a critical factor, improving yields to 112 µg L^-1 OD₇₅₀^-1 by reducing growth and redirecting carbon toward isobutene synthesis. However, prolonged nitrogen limitation ultimately reduced productivity. To address this limitation, we employed a polyvinyl alcohol-sodium alginate (PVA-SA) hydrogel, crosslinked with B(OH)₄^- and Ca²⁺ to entrap cells. This approach restricted growth while maintaining cell viability and isobutene productivity. Optimizing crosslinking parameters such as time, pH, and the hydrogel-to-cell mass ratio improved bead stability under bicarbonate and nitrate supply. This strategy extended cell viability and isobutene productivity in Syn-RnKICD and Syn-F336V by nearly a month, increasing yields by 60 % and 80 %, respectively, compared to suspension cells, achieving a maximum yield of 94 mg/g DW at 744 h and reaching a highest production rate of 1 mg/g DW/h at 264 h. This study underscores the importance of optimizing environmental conditions for isobutene production in Synechocystis and highlights the effectiveness of PVA-SA cell entrapment as a biocatalyst platform for sustained chemical production.