Biocatalytic Oxyfunctionalisation using Unspecific Peroxygenases (UPOs)

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The oxygenation of organic compounds presents an ongoing challenge to synthetic chemistry as conventional reagents present difficulties with toxicity, harsh reaction conditions and a lack of selectivity. Enzymatic oxygenations, by contrast, present many advantages with respect to both sustainability and selectivity. Recently, the discovery of unspecific peroxygenases (UPOs) has identified new possibilities for scalable biocatalytic oxygenations. UPOs are secreted enzymes from fungi that contribute to the degradation of lignin. UPOs present major advantages over the better-studied cytochromes P450 as they catalyze selective oxygenations at the expense of only hydrogen peroxide, without the addition of cofactors or auxiliary proteins. They also exhibit higher turnovers, superior stability and can be produced in large amounts amenable to lyophilization, resulting in an easy-to-use biocatalysts for selective oxygenation reactions. In a previous PhD project at York, we applied a robust system for the expression of AaeUPO from Agrocybe aegerita in the yeast Pichia pastoris and used the enzyme for the oxygenation of a preliminary series of compound classes. We now aim to explore the potential of UPOs with further studies on natural homologs and engineered enzymes, with a focus on the generation of agrochemical metabolites and the transformation of biomass-derived building blocks (BDBBs). The following major objectives will be pursued: 1) To clone genes encoding native UPOs and express in Pichia pastoris, to expand the library of enzymes we can use to oxygenate organic molecules.2) To characterise the activities of the new UPOs through substrate synthesis and biotransformation studies using substrates including agrochemicals and biomass-derived building blocks.3) To engineer reaction conditions for UPO-catalysed biotransformations using Design-of-Experiment (DoE) approaches4) To rational engineer UPOs to improve their catalytic properties, based on structure determination using X-ray crystallography.While UPOs have been investigated for the oxygenation of model substrates, their application to selective synthesis of agrochemical metabolites or BDBBs has not been explored. Neither has the optimization of process conditions particular to these targets, nor the engineering of the enzymes to address performance optimization. Selective oxyfunctionalisations are major reactions in industrial chemistry requiring urgent green and sustainable alternatives and many industrial collaborators highlight the need for biocatalytic alternatives. An increased understanding of UPOs, their mechanisms and process requirements is timely, as it will help to enable their industrial application.