Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids
Patricia Gomez De Santos (Evoenzyme S.L., Madrid)
Alejandro González-Benjumea (CSIC - Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS))
Angela Fernandez-Garcia (Institute of Catalysis, CSIC, Madrid)
Carmen Aranda (CSIC - Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS))
Y. Wu (TU Delft - BT/Biocatalysis)
A. But (TU Delft - BT/Biocatalysis)
Patricia Molina-Espeja (Institute of Catalysis, CSIC, Madrid)
W. Zhang (TU Delft - BT/Biocatalysis)
F Hollmann (TU Delft - BT/Biocatalysis)
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Abstract
The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal 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 by Pichia pastoris at 0.4 g L−1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.
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