Expanding the set of rhodococcal Baeyer-Villiger monooxygenases by high-throughput cloning, expression and substrate screening

Abstract

To expand the available set of Baeyer-Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.

Fig. 1

Radial branching diagram of the comparison of active site residues of the selected BVMO protein sequences. The included sequences are: AlmA1, Acinetobacter sp. DSM 17874 (ABQ18224.1); EtaA, Mycobacterium tuberculosis H37Rv (NP_218371.1); HAPMO, Pseudomonas fluorescens ACB (Q93TJ5.1); MoxY, Aspergillus flavus (AAS90037.1); PtlE, Streptomyces avermitilis MA-4680 (NP_824170.1); CDMO, Rhodococcus ruber SCI (AAL14233.1); CPDMO, Pseudomonas sp. strain HI-70 (BAE93346.1); CHMO, Acinetobacter calcoaceticus NCIMB 9871 (BAA86293.1); PAMO, T. fusca (YP_289549.1); STMO, Rhodococcus rhodochrous (BAA24454.1); ACMO, Gordania sp. strain TY-5 (BAF43791.1); MEKMO, Pseudomonas veronii MEK700 (ABI15711.1); CPMO, Comamonas sp. strain NCIMB 9872 (Q8GAW0)

Fig. 2

Absorbance spectra of a cleared cell extract containing overexpressed BVMO. The spectra of the oxidised flavin cofactor in BVMO14 (continuous line) and after reduction by NADPH (broken line) are shown. The difference in absorbance at 440 nm that can be used to determine the BVMO concentration is indicated