Genome Mining Leads to the Identification of a Stable and Promiscuous Baeyer‐Villiger Monooxygenase from a Thermophilic Microorganism

Baeyer–Villiger monooxygenases (BVMOs) are NAD(P)H‐dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer‐Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 °C), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95 % conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub‐stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH−L1‐ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer‐Villiger oxidations. Using sequence similarity networks, a stable and substrate‐promiscuous enzyme from Chloroflexota bacterium (strain G233), named ssnBVMO, is identified. ssnBVMO exhibits high melting temperature, kinetic stability, broad substrate scope, and high conversion rates, making it a promising candidate for industrial biocatalyst engineering. Additionally, fusion proteins incorporating ssnBVMO and phosphite dehydrogenase enable NADPH recycling.