Genome mining leads to the identification of a stable and promiscuous Baeyer-Villiger monooxygenase from a thermophilic microorganism

Baeyer-Villiger monooxygenases are NAD(P)H-dependent flavoproteins that catalyze oxygen insertion reactions which convert ketones to valuable 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 Baeyer-Villiger monooxygenases that are both stable and substrate promiscuous. Our genome mining efforts led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssn BVMO that exhibits i) the highest melting temperature recorded to date for a naturally sourced Baeyer-Villiger monooxygenase, ii) a remarkable kinetic stability across a wide range of conditions, and iii) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Kinetic characterization of this enzyme was undertaken to identify the optimal conditions for ssn BVMO catalysis, and a subsequent quantitative assay using propiophenone as a substrate afforded more than 95% conversion. To spur the implementation of this enzyme as an oxidative biocatalyst, several fusion proteins were constructed that linked ssn BVMO to a thermostable phosphite dehydrogenase. These self-sufficient enzymes can recycle NADPH and permit oxidations to be run with sub-stoichiometric quantities of this expensive cofactor. Extensive characterization of these fusion enzymes permitted identification of PTDH-L1- ssn BVMO as the most promising oxidative biocatalyst. Results described herein demonstrate that this new monooxygenase has significant potential as a useful industrial biocatalyst for Baeyer-Villiger oxidations.