Structure-Based Redesign of a Self-Sufficient Flavin-Containing Monooxygenase towards Indigo Production

• Published in: International journal of molecular sciences 2019-12, Vol.20 (24), p.6148
• Main Authors: Lončar, Nikola, van Beek, Hugo L, Fraaije, Marco W
• Format: Article
• Language: English
• Subjects: Biocatalysts; Cloning; Computer applications; Crystal structure; Dimethylaniline monooxygenase (N-oxide-forming); Engineering; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Flavin; Indigo Carmine - metabolism; Indoles; Indoles - metabolism; Libraries; Metabolism; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Mutagenesis; Mutation; Oxidation-Reduction; Redesign; Structural members; Thermal stability
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms20246148

Indigo is currently produced by a century-old petrochemical-based process, therefore it is highly attractive to develop a more environmentally benign and efficient biotechnological process to produce this timeless dye. Flavin-containing monooxygenases (FMOs) are able to oxidize a wide variety of substrates. In this paper we show that the bacterial mFMO can be adapted to improve its ability to convert indole into indigo. The improvement was achieved by a combination of computational and structure-inspired enzyme redesign. We showed that the thermostability and the for indole could be improved 1.5-fold by screening a relatively small number of enzyme mutants. This project not only resulted in an improved biocatalyst but also provided an improved understanding of the structural elements that determine the activity of mFMO and provides hints for further improvement of the monooxygenase as biocatalyst.