Cyanobacterial carbonic anhydrases drive carbon recycling and prodrug biosynthesis in Escherichia coli.

Sustainable biomanufacturing in Escherichia coli is advancing through the integration of carbon dioxide (CO2)-fixing modules from algal systems. However, the assimilation efficiency remains limited by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) performance and intracellular CO2 availability. To address these constraints, we introduced carbonic anhydrases from Cyanobacteria aponinum (capoCAs) and repressed the native CA (can) gene using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) to enhance design specificity. Afterward, capoCAs were overexpressed in E. coli BL21(DE3) and its derivative strains of can deficient (dCA), Calvin-Benson-Bassham (CBB) (RuBisCO-Prk expressing), and dCA+CBB (coupled with dCA and CBB). For functional demonstration, the engineered strains were used to produce a valuable prodrug, 5-aminolevulinic acid (5-ALA). As a result, capoCAs modestly improved 5-ALA titers, with further increments upon CBB coexpression. The optimum dCA+CBB* strain achieved 7.04 g/l (1.46-fold improvement), alongside 9.59% carbon enrichment and a 39.4% reduction in CO2 release. The 13C-isotope confirmed CO2 incorporation during 5-ALA biosynthesis. This work provides tunable carbon-management modules for developing low-footprint microbial cell factories.