Discovery, application and protein engineering of Baeyer–Villiger monooxygenases for organic synthesis

Abstract

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.

Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

“…However, while a variety of singlecomponent Type I BVMOs have been discovered, characterised, and in some cases targeted for successful protein engineering to the extent that they are now becoming applied in industrial processes (Balke et al 2012;Leisch et al 2011), relevant multi-component (Type II) BVMOs such as the isoenzymic DKCMOs from P. putida NCIMB 10007, have been relatively neglected so far even though their powerful catalytic qualities have been recognised for almost 20 years (Willetts 1997). An important drawback in the application of MCMOs has been the requirement for a FR-type protein that can serve as a robust effective shuttle for the requisite reduced FMN.…”

“…Specifically, enzymes were identified that catalyse the Baeyer-Villiger (BV) oxidation by incorporating one atom of molecular oxygen from air into a ketone substrate. This is advantageous compared to traditional chemical processes that are using environmentally harmful peracids (recent reviews: Balke et al 2012;Leisch et al 2011). The relevant enzymes, termed BaeyerVilliger monooxygenases (BVMOs), are members of a superfamily of flavoprotein monooxygenases mechanistically related by their ability to activate molecular oxygen by generation of a covalent adduct with flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) (Harayama et al 1992;Massey 1994;Walsh and Wencewicz 2013).…”

Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization

“…Viable recombinant E. coli (Escherichia coli) cells overproducing cyklohexanone monooxygenase (CHMO) from the group of BVMOs were used as a model biocatalyst in this study. Currently, there is increased interest in research into novel enzymes and the industrial applications of BVMOs, as they catalyse the enantioselective BV biooxidation of a wide range of cyclic ketones to the corresponding lactones as chiral precursors of natural and bioactive compounds as well as potential drugs [16][17][18][19]. The study also sought to determine the operational stability of PEC beads with immobilized recombinant E. coli cells with CHMO during repeated Baeyer-Villiger (BV) biooxidations as well as the influence of repeated biotransformations on the viability of cells.…”

Microbial Flavoprotein Monooxygenases as Mimics of Mammalian Flavin-Containing Monooxygenases for the Enantioselective Preparation of Drug Metabolites

“…Human FMOs and their mammalian orthologs are typically membrane associated and often thermolabile, which appear to be the major reasons for their problematic isolation from tissue (Cashman et al, 1995;Wu et al, 2004) and inefficient recombinant production. Although human FMOs can be studied using microsomal preparations and some human FMOs were expressed as functional enzymes in heterologous hosts (Motika et al, 2009;Balke et al, 2012;Geier et al, 2015;Shimizu et al, 2015), these enzyme preparations involve costly and cumbersome isolation procedures, and often suffer from low activity and stability (Cashman et al, 1992;Cashman and Zhang, 2006).…”