Enabling Unspecific Peroxygenases (UPOs) for Applications in Industrial Biocatalysis

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Oxygenation reactions catalysed by microorganisms are valued in industry for the selective functionalisation of complex pharmaceuticals such as steroids. In these cases, the hydroxylations are catalysed by cytochromes P450 (P450), usually associated with the cell-membranes of the fungi that are employed for the reactions. The potential of P450 for industrial hydroxylations has thus resulted in a large volume of research on the enabling of these enzymes for many other selective reactions on a range of industrially relevant substrates. However, many of the envisaged applications require the heterologous expression of P450 in a convenient laboratory host, such as E. coli, but the complexities associated with this expression strategy have frustrated efforts to provide usable catalysts. The reasons include poor solubility (especially of eukaryotic P450) and stability, very low catalyst turnover, but especially the requirements of P450 for nicotinamide cofactors and redox transfer proteins for activity. These difficulties have now stimulated the search both for alternative hosts for P450 expression, but also for more stable and active enzymes that catalyse equivalent processes. The field of biocatalytic oxygenations was recently energised by the discovery of 'unspecific peroxygenases' (UPOs), which are secreted by filamentous fungi as part of their catalytic arsenal for the degradation of recalcitrant plant matter. UPOs have attracted a great deal of interest as, although they are 'unspecific' - meaning that a wide range of substrates is transformed - they are also highly selective in terms of both regio and enantioselectivity. However, the most compelling advantages of UPOs over P450s are their stability - as secreted proteins they have evolved to be stable under challenging conditions of pH, temperature, and also organic solvents; activity - turnovers of 200 s-1 can be achieved, and their dependence only on hydrogen peroxide for full activity, removing the need for either expensive nicotinamide cofactors or redox transfer proteins. As UPO work is in its infancy, most groups have dependent on growing the wild-type strain of fungus Agrocybe aegerita for its production, with the attendant complexities of fungal fermentation and purification of the enzyme from the complex mix of secreted proteins. In recent work, we have expressed AaUPO in a heterologous system - the yeast Pichia pastoris. This gives the advantage of superior control of fermentative production of the enzyme, and also huge improvements in yield and simplicity of purification. We are currently characterising the recombinant UPO and assessing its suitability for scaleable asymmetric oxygenations of benzylic carbons, alkenes, and sulfides. Working in collaboration with GSK, in this studentship project, we will look to exploit the superior activity of recombinant UPOs in the transformation of a range of synthetically relevant organic substrates.