Sustainable biocatalytic generation of bioactive tropolone natural products

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PROPOSAL SUMMARY For years, nature has been the source for health remedies in traditional medicine, using plants and fungi for their curative benefits for over 2000 years. We can now attribute the benefits of these traditional treatments to natural products generated in their biosynthetic pathways. Moreover, natural products have been a consistent source of inspiration and resource in the development of alternative therapeutics. However, studies show that there is a gap in knowledge on these alternatives which is rooted in a lack of evidence-based information on their efficacy, hindering their application in mainstream medicine. Therefore, it is imperative to develop strategies that could allow to reach the valuable molecules that give these sources their medicinal power. A subset of natural products contains tropolone rings in their structures and have shown to have great therapeutic potential for treating cancer, malaria, bacterial and fungal infections as well as cardiovascular, renal, and inflammatory diseases. The tropolone moiety commonly acts as a pharmacophore, making it a valuable target to synthetically access and evaluate. However, the complex nature of the aromatic seven-membered tropolone ring limits the sustainability of their large-scale production, this reflected in common synthetic methods being hindered by low yields, diverse functional group tolerance, and the need for hazardous and costly reagents. Conversely, nature has evolved biocatalysts that enable direct routes to diversely functionalized tropolones such as the fungal α-ketoglutarate dependent non-heme iron dioxygenase XenC, bypassing the general setbacks of traditional synthetic methods. Nonetheless, the applicability of this enzymatic method remains hindered by the concentration of substrate that XenC can tolerate, the required two-step reaction sequence, and the limited substrate scope that prevents the practical applications of this approach to access a plethora of tropolone natural products. In the efforts of improving the scalability, sustainability, and broader applicability of this biocatalytic method towards accessing bioactive tropolones, I aim to engineer XenC into an enzyme that tolerates higher substrate loadings, works in a sustainable one-pot cascaded at pH 8, and has a broader non-native substrate scope. The successfully evolved enzymes and the developed biocatalytic platform will enable novel chemoenzymatic syntheses of tropolone sesquiterpenes, fungal marine tropolones, and tropolone alkaloids, providing a scalable and sustainable strategy that allows to reach diverse tropolones and analogs to facilitate their in-depth pharmacological studies. In turn, this will impulse the development of novel medicine alternatives with the purpose of treating human-health concerns including infections, inflammation, malaria, and cancer.