Baeyer-Villiger monooxygenases (BVMOs) are useful enzymes for organic synthesis as they enable the direct and highly regio-and stereoselective oxidation of ketones to esters or lactones simply with molecular oxygen. This contribution covers novel concepts such as searching in protein sequence databases using distinct motifs to discover new Baeyer-Villiger monooxygenases as well as highthroughput assays to facilitate protein engineering in order to improve BVMOs with respect to substrate range, enantioselectivity, thermostability and other properties. Recent examples for the application of BVMOs in synthetic organic synthesis illustrate the broad potential of these biocatalysts. Furthermore, methods to facilitate the more efficient use of BVMOs in organic synthesis by applying e.g. improved cofactor regeneration, substrate feed and in situ product removal or immobilization are covered in this perspective.

Joined forces: Alcohol dehydrogenase, enoate reductase, and Baeyer–Villiger monooxygenase are combined in a cascade reaction by coexpression in E. coli to have a recombinant whole‐cell biocatalyst. Such an artificial metabolic “mini”‐pathway provides access to functionalized chiral compounds in high yields and optical purities as exemplified for kinetic resolutions, desymmetrizations, and regiodivergent biotransformations.

PostprintThis is the accepted version of a paper published in Chemical Communications. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination.

The camphor-degrading Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas putida NCIMB 10007 have been of interest for over 40 years. So far the FMN- and NADH-dependent type II BVMO 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) and the FAD- and NADPH-dependent type I BVMO 2-oxo-∆3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) have not been entirely studied, since it was not possible to produce those enzymes in satisfactory amounts and purity. In this study, we were able to clone and recombinantly express both enzymes and subsequently use them as biocatalysts for various mono- and bicyclic ketones. Full conversion could be reached with both enzymes towards (±)-cis-bicyclo[3.2.0]hept-2-en-6-one and with 3,6-DKCMO towards (−)-camphor. Further OTEMO gave full conversion with norcamphor. OTEMO was found to have a pH optimum of 9 and a temperature optimum of 20 °C and converted (±)-cis-bicyclo[3.2.0]hept-2-en-6-one with a k cat/K M value of 49.3 mM-1 s-1.

Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit.