Light-Driven Photobiocatalytic Oxyfunctionalization in a Continuous Reactor System without External Oxygen Supply

Oxygenases catalyze C-H oxyfunctionalization under mild reaction conditions and often display outstanding selectivity. However, their utilization is hampered by the difficulty of transporting oxygen across the gas-liquid interface, which is particularly problematic for continuous reactor systems and can only be alleviated by high pressure or the use of complex oxygen-permeable materials. Herein, oxygen is directly released into the medium by the phototrophic cyanobacterium <i>Synechocystis</i> sp. PCC 6803 expressing the genes of a Baeyer-Villiger Monooxygenase from <i>Burkholderia xenovorans</i> to drive the oxidation of cyclohexanone for the production of the polymer precursor, ε-caprolactone. The rates at which photosynthetic oxygen can solely drive the oxidation were determined by performing the reaction in a continuous coil reactor with a very limited external oxygen supply. In heterotrophic nonoxygen-producing <i><i>Escherichia coli</i></i> expressing the same gene, a 10-fold lower specific activity was observed when the oxidation was performed in the coil reactor compared with batch mode underlining the impact of oxygen-limitation on the volumetric productivity. In contrast, cyanobacterial whole cells showed activities of 16.7 and 13.5 U g_DCW ^-1 in nonoxygen-limited batch and oxygen-limited continuous flow, respectively. Net oxygen production of the whole-cell biocatalyst during the reaction led to a steady-state oxygen concentration allowing volumetric productivities as high as 3 mmol L^-1 h^-1 highlighting the advantages of photoautotrophic production systems for oxyfunctionalization under oxygen-limiting conditions. Moreover, the space-time yield of the reaction was improved 7-fold (2.8 vs 0.4 g L^-1 h^-1) by utilizing the continuous coil reactor compared to the batch mode. The combination of flow catalysis and photosynthetic oxygen production can overcome current limitations in photo(bio)oxidation and achieve significant improvements in terms of volumetric productivity enabling more sustainable chemical synthesis. This approach using whole-cells of cyanobacteria achieves a notably lower ratio of waste to product (E-factor) and higher atom economy compared with oxidation mediated by <i><i>Escherichia coli</i></i> .