Recombinant Whole-cell Mediated Baeyer-Villiger Oxidations

Biocatalysis is a cost-efficient environment-friendly process for the preparation of fine chemicals and represents a "green chemistry" approach for the production of biologically active compounds. The introduction of enzymes as catalysts into synthetic organic chemistry enabled many new highly enantioselective transformations. The development of new biocatalytic conversions is dependent on finding the right biocatalyst, and therefore biocatalyst identification, characterization, and development is a key activity in research and development in this field. The employment of microorganisms provides access to enzymes that are difficult to use in an isolated form. Such organisms are self-propagating sources of the desired biocatalyst and all cofactors necessary for the reaction. Therefore, whole-cell systems are simpler to use even for chemists without special expertise in microbiology. In native whole-cells the efficiency of biotransformations is limited due to the large number of potentially competing enzymes present. To overcome these problems, recombinant expression systems offer the advantage to substantially increase the ratio of the required enzyme compared to all other biocatalytic entities present in a living cell. Major goal of this projects is to provide easy-to-handle biocatalysts for enantioselective Baeyer-Villiger oxidations, that possess a predictable pattern of reactivity and allow conversion of a large variety of compounds. The quest of substrate profiling of novel Baeyer-Villigerases requires the development of a rapid screening methodology with respect to chemo- and enantioselectivity to enable evaluation of the systems for subsequent biocatalytic applications. In order to allow high throughput analysis diverse libraries of potential substrates will be developed. Biotransformations using recombinant whole-cells will be analyzed by mass spectroscopy and a high degree of automation is attempted. Sophisticated labeling techniques will be applied to enable rapid evaluation of the enantioselectivity of the overexpression systems. Method development for high-throughput screening will be on a general basis to facilitate transfer onto other enzymatic systems. The chiral products obtained by the investigated systems will be utilized for the synthesis of biologically active compounds with pharamceutical relevance. A major goal is the development of environmentally friendly alternative routes for the synthesis of drugs used in the treatment of cancer and viral diseases.

Biocatalysis offers a highly environmentally compatible option for green chemistry oxidation processes, as the catalytic entities (enzymes, microorganisms) are completely biodegradable and molecular oxygen is utilized as oxidant. Due to the limited stability of oxygenases and their cofactor requirements, recombinant whole-cell mediated biotransformations offer a simple option to perform such reactions. This project was proposed to overcome some of the limitations of the enzymatic Baeyer-Villiger biooxidation at the beginning of this decade. Several novel Baeyer-Villiger monooxygenases (BVMOs) were becoming available due to progress in genome deciphering. Up to this point, the majority of results in the area of Baeyer-Villiger biocatalysis was generated upon studying very few enzymes and the potential of BVMOs for applications in synthetic chemistry was unclear. With the discovery of a diversity of new enzymes, identification of the biocatalytic performance of these BVMOs was becoming a critical issue. Consequently, three major issues were addressed by the project: (i) development of a parallel screening format for substrate assessment of novel BVMOs; (ii) demonstration of the versatility of novel BVMOs in natural and bioactive product synthesis; (iii) scale-up of chiral lactone synthesis to gram-scale using benchtop fermentation equipment using E.coli based recombinant overexpression systems. With the results generated during this project, the promising prospects of biocatalytic oxidation processes - in particular for the Baeyer-Villiger reaction - have been clearly outlined. During this research study, several novel BVMOs have been characterized for their stereoselectivity and substrate acceptance. A particular contribution to the field was the identification of two groups of cycloketone accepting BVMOs generating antipodal product lactones. Hence, access to both optical forms of a required product is provided, a key aspect for subsequent utilization of this methodology in pharmaceutical chemistry. Within this program, a compound library was developed in combination with a parallel GC-based screening protocol, which allows the assessment of the catalytic repertoire of novel BVMOs within days at the expense of mg-quantities of compounds. This technology was used to develop substrate profiles for both wild-type and mutant BVMOs, which were acquired from cooperation partners. In order to demonstrate the applicability of whole-cell mediated Baeyer-Villiger reactions, up-scaling was performed using fermenter equipment, in order to provide gram quantities for subsequent utilization in organic synthesis. Satisfying results were achieved using a solid-state-reservoir technology acquired from a collaboration partner. Summarizing, the microbial Baeyer-Villiger biotransformation was successfully developed from analytical parallel- throughput screening to preparative laboratory scale.