Evolving a P450BM3 Peroxygenase for the Production of Indigoid Dyes from Indoles

Abstract Herein, we describe an environmentally benign enzymatic approach for the preparation of indigoid from indole derivatives. A series of beneficial P450BM3 mutants were obtained using a stepwise approach involving site‐directed, random, and combinatory hot‐site mutations. Using H 2 O 2 as the terminal oxidant and N ‐( ω ‐imidazolyl)‐hexanoyl‐ L ‐phenylalanine as a co‐catalyst, the quadruple‐mutant F87G/T268V/F77I/E140D efficiently catalyzed the 3‐hydroxylation of indole and subsequent autooxidation to indigo with higher efficiency than the flavin‐containing monooxygenase, PTDH‐mFMO, which was the best oxidizing enzyme for indigo synthesis reported to date. Following this procedure, 12 indigoid compounds were prepared using indole derivatives with various substituents as starting materials with moderate to high isolated yields (31.3–79.5 %). The catalytic efficiencies ( k cat / K m ) of the beneficial mutants for typical indole substrates ranged from 182–2849 mM −1 ⋅ min −1 , almost 2‐ to 712‐fold higher than those of the reported P450 enzymes. This study provides a new enzymatic approach for the biosynthesis of indigoid dyes from indole derivatives.