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

Abstract

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 kcat 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.

Nitrogen‐Neutral Amino Acids Refinery: Deamination of Amino Acids for Bio‐Alcohol and Ammonia Production

“…Subsequent oxidation of indoxyl leads to the generation of indoxyl radicals, and two molecules of indoxyl radicals are converted to a well‐known blue‐colored indigo dye‐a symmetric dimer‐through a self‐dimerization reaction 53–57. According to recent studies, several monooxygenases such as flavin monooxygenase (FMO), cytochrome P450 monooxygenase (CYP), toluene dioxygenase (TDO), and naphthalene dioxygenase (NDO) were involved in indole C3‐hydroxylation and bio‐indigo formation from L‐tryptophan 58–64.…”

Constructing multi‐enzymatic cascade reactions for selective production of 6‐bromoindirubin from tryptophan in Escherichia coli

“…In the case of indole oxidation, several oxygenases have been reported for the production of indigoids, since IG and IR are produced from the dimerization of C3‐C3′‐oxygenated indoles and C3‐C2′‐oxygenated indoles, respectively (Choi, 2020; Fabara & Fraaije, 2020). However, most oxygenases including MaFMO (Loncar et al, 2019), CYP102G4 (J. Kim et al, 2018), and naphthalene dioxygenase (Cheng et al, 2016) preferentially oxygenate the C3 position of the indole ring, as electron‐deficient C3 position is relatively more susceptible to oxygenation than the C2 position (J. Kim et al, 2019; Leone et al, 2021) (Supporting Information: Figure S3). Among the oxygenases, toluene 4‐monooxygenase from Pseudomonas mendocina KR1 (PmT4MO) can noticeably generate 2‐oxindole from indole, and its F196L mutant enables to direct 74% of the oxygenated indole toward 2‐oxindole formation (McClay et al, 2005).…”

“…However, most oxygenases including MaFMO (Loncar et al, 2019), CYP102G4 (J. Kim et al, 2018), and naphthalene dioxygenase (Cheng et al, 2016) preferentially oxygenate the C3 position of the indole ring, as electron-deficient C3 position is relatively more susceptible to oxygenation than the C2 position (J.…”

Discovery of New Phenylacetone Monooxygenase Variants for the Development of Substituted Indigoids through Biocatalysis

“…Due to the importance of indigoids for textile and pharmaceutical industries, and looking for environmentally friendly manufacturing strategies, several research groups are working on their production using biocatalytic processes, such as the use of recombinant bacteria, especially in Escherichia coli , overexpressing enzymes to catalyze the synthesis of these molecules [ 14 , 19 , 20 , 21 , 22 , 23 ].…”