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, producing isobutene from -ketoisocaproate (KIC) via Rattus norvegicus - ketoisocaproate dioxygenase (RnKICD), and Syn-F336V, expressing a mutant variant of RnKICD with improved KIC specificity. 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 by reducing growth. This likely occurred due to decreased competition from branched-chain amino acid (BCAA) biosynthesis, redirecting carbon toward isobutene synthesis. However, prolonged nitrogen limitation ultimately reduced productivity due to impaired metabolic functions. To address this limitation, we employed a polyvinyl alcohol-sodium alginate (PVA-SA) hydrogel, crosslinked with B(OH)- and Ca{superscript 2}, 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. 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. HighlightsO_LIOptimized light, carbon, and nitrogen sources enhanced isobutene production. C_LIO_LIRate of isobutene production increased 10-fold compared to our previous report. C_LIO_LINitrogen limitation improved isobutene yield by redirecting carbon from growth. C_LIO_LIPVA-entrapped cells sustained isobutene productivity for nearly 31 days. C_LI