NSERC-DFG SUSTAIN: SysDevOx - Systematic development of new oxidative biocatalysts for the sustainable production of pharmaceutical compounds

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Although many drugs are still derived from natural products, drug manufacturing processes commonly require synthetic modifications, called semisynthesis, to convert metabolites found in nature into clinically relevant drugs. Whereas biological systems produce metabolites sustainably under mild conditions, semisynthesis often employs toxic chemicals, expensive catalysts and/or harsh non-sustainable conditions. Enzymes could provide a sustainable alternative for selective modifications of complex natural products. Oxidative enzymes such as cytochrome P450 monooxygenases are particularly relevant in this regard, as they can activate otherwise unreactive C-H bonds. Microbial cytochrome P450 monooxygenases have already been employed for chemoenzymatic syntheses. The biotechnological potential of cytochrome P450 monooxygenases from plants, however, has been drastically neglected so far. This stands in contrast to the fact that cytochrome P450 monooxygenases play key roles in plant metabolism and are therefore primed by evolution to oxidize plant metabolites. With this project, we want to close this gap and develop plant cytochrome P450 monooxygenases as oxidative biocatalysts for the sustainable functionalization of privileged alkaloid pharmacophores. Specifically, we will perform a targeted screening campaign to identify plant cytochrome P450 monooxygenases that oxidize plant alkaloids which are either already used as drugs or show high potential for drug development. Using a new sequence mining approach with sequence similarity networks and orthogroup inference analyses, we will rationally select a library of ca. 200 cytochrome P450 monooxygenases that will be screened against a panel of 30 commercially available plant alkaloids. The required throughput is achieved by transient co-expression of up to 25 cytochrome P450 monooxygenase genes simultaneously in the plant host Nicotiana benthamiana followed by dereplication. Positive hits will be verified in baker’s yeast. Large scale biotransformations in the 10-100 mg scale will demonstrate that the newly found biocatalysts are relevant for biotechnological applications and enable full structural characterization of oxidized products. This interdisciplinary collaborative project between a German (Franke, Leibniz University Hannover) and a Canadian group (Dang, University of British Columbia) is built on complementary expertise (Franke: sequence similarity networks, structure elucidation; Dang: alkaloid biosynthesis, oxidative enzymes, orthogroup inference analyses). Taken together, the novel biocatalysts developed in this project will facilitate the transition to sustainable production processes for medicinally relevant alkaloid derivatives.