LAUSR

Abstract Metabolically diverse organisms can serve as valuable source for Cytochrome P450 monooxygenases (CYPs), extending the repertoire of biocatalysts for C–H bond oxyfunctionalization. Here, we report on the cloning, expression and characterization of two new P450 s from the gram-positive papaverine degrading Arthrobacter sp. The activity of one of these P450 s, CYP199A25, could be reconstituted enabling its detailed biochemical characterization. CYP199A25 has shown to be a regioselective benzoic acid hydroxylase which demethylates or hydroxylates only in para position using the non-physiological redox partners CamA and CamB from P. putida. In accordance with the previously published CYP199A members, the defined enzyme architecture determines the high regioselectivity. While the construction of a fusion protein gave low activity in vitro and in vivo, a whole-cell system comprising of the compatible pBAD18/33 plasmids produced 1.3 g L−1 of the final product 4-hydroxybenzoic acid corresponding to a total conversion of 94 %. CYP199A25 represents a highly selective and efficient biocatalyst complementing the interesting CYP199A subfamily for future applications.