Institute of Biochemistry and Technical Biochemistry, Department of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
Alexander Seifert
Institute of Biochemistry and Technical Biochemistry, Department of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
Tim Häbe
Institute of Biochemistry and Technical Biochemistry, Department of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
Bettina M. Nestl
Institute of Biochemistry and Technical Biochemistry, Department of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
Bernhard Hauer
Institute of Biochemistry and Technical Biochemistry, Department of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
A novel approach for the synthesis of vanillin employing a three-step two-enzymatic cascade sequence is reported. Cytochrome P450 monooxygenases are known to catalyse the selective hydroxylation of aromatic compounds, which is one of the most challenging chemical reactions. A set of rationally designed variants of CYP102A1 (P450 BM3) from Bacillus megaterium at the amino acid positions 47, 51, 87, 328 and 437 was screened for conversion of the substrate 3-methylanisole to vanillyl alcohol via the intermediate product 4-methylguaiacol. Furthermore, a vanillyl alcohol oxidase (VAO) variant (F454Y) was selected as an alternative enzyme for the transformation of one of the intermediate compounds via vanillyl alcohol to vanillin. As a proof of concept, the bi-enzymatic three-step cascade conversion of 3-methylanisole to vanillin was successfully evaluated both in vitro and in vivo.