Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids.

Abstract

The hydroxylation of fatty acidsis an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for thew-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutationnear the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced byPichia pastorisat 0.4 g/L in a fed-batch bioreactor and used in thepreparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83%eethrough the (S)-enantiomer.The hydroxylation of fatty acidsis an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for thew-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutationnear the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced byPichia pastorisat 0.4 g/L in a fed-batch bioreactor and used in thepreparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83%eethrough the (S)-enantiomer.