Designing Chimera Baeyer-Villiger Biooxidation Catalysts

Oxygenases carry out the chemo-, regio-, and stereoselective introduction of molecular oxygen into organic molecules. Based on the nature of the oxidant (O2 ) and the degradability of enzymatic catalysts, this approach represents a green and highly sustainable methodology for environmentally benign oxidation reactions. The stereoselective Baeyer-Villiger oxidation of ketones to the corresponding chiral lactones is one of the prominent domains for biocatalysis, as enzymes are by far superior catalytic entities compared to de-novo designed organometal catalysts with respect to selectivity. However, the necessity to provide and recycle required cofactors and the limited stability of Baeyer-Villiger monooxygenases (BVMOs) has prevented wide-spread application among the community of synthetic chemists, so far. Recently, we could demonstrate the feasibility of whole-cell mediated Baeyer-Villiger biooxidations utilizing recombinant organisms designed to overexpress the required biocatalyst. Such microbial strains are easy-to-use catalytic systems and allow the gram scale synthesis of chiral lactones as key precursors for natural products and bioactive compounds on laboratory scale. This project aims at overcoming the last major obstacle en route to readily applicable BVMOs in every day synthetic applications by designing novel thermostable chimera enzymes. Such -BVMOs will be designed by gene shuffling of a thermostable enzyme from a moderately thermophilic organism (PAMO) with BVMOs of bacterial origin. While PAMO displays a very limited substrate profile hence, making it an unlikely candidate for general applications in catalysis there is a considerable number of other BVMOs with well described and broad ketone tolerance allowing in addition the preparation of enantiocomplementary lactone products. By combining PAMO and bacterial BVMOs as parent enzymes into new -BVMOs, high- hroughput screening techniques with fluorescence assays are expected to provide candidates with a recombination of beneficial properties. In a second stage, such novel biocatalysts will be optimized using random and knowledge-based approaches in molecular biology in an iterative process. At the end of these efforts, a set of -BVMOs will become available, which displays broad substrate specificities and high stereoselectivites combined with an improved thermal stability. This will facilitate the chiral Baeyer-Villiger biooxidation to become an easily applicable tool in stereoselective synthesis. In addition, novel indications on the key structural areas of high impact for biocatalyst efficiency are expected for BVMOs, in general, providing a better understanding of this fascinating enzyme family.

Biocatalysis offers a highly environmentally compatible option for green chemistry oxidation processes based on the bioavailability and biodegradability of the catalytic entities (enzymes, microorganisms). Biooxygenation reactions are particularly useful as molecular oxygen is employed as primary oxidant which is abundantly available and economically attractive. However, low operational stability of oxygenases is a major limitation together with the need to adapt biocatalytic performance to novel substrates. Hence, this project aimed at solving some of the problems associated to a particular sub-class of this enzyme group: Baeyer-Villiger monooxygenases (BVMOs), which catalyze the name-giving transformation usually in remarkable chemo-, regio- and stereoselectivity. Progress towards a more versatile application of BVMOs in organic synthesis was achieved in several areas: (i) Significant improvement of the storage and operational stability of BVMOs exemplified on a particular model enzyme. This part required intensive re-engineering of the protein scaffold and concomitant development of suitable screening methods. (ii) Profiling of novel wild-type enzymes and mutant libraries to identify important new transformations with respect to substrate acceptance and regioselectivity (in particular focussing on previously neglected structure types). (iii) Application of BVMOs in novel biotransformations enabling efficient access to novel chiral building blocks and products. One major operational route within the project was the identification of critical structural parameters of the protein scaffold to improve thermal stability of BVMOs. Several approaches were investigated and ultimately a knowledge-based strategy based on the comparison of consensus sequences in combination with structural information available from reported x-ray diffractions turned out to be highly successful. This project part required the design and validation of suitable screening protocols for efficient enzyme engineering. Within a second line of research, the applicability of wild-type and engineered BVMOs was investigated. Comprehensive substrate profiles of two novel wild-type BVMOs were determined and comparative screenings of stereoselectivity of two mutant libraries (based on cyclohexanone and cyclopentanone monooxygenase) were conducted. In addition, two new applications in synthesis were developed to enable access to fragrance compounds and ß-amino acids as important intermediates for pharmaceutical applications.