Indigo, a natural dye derived from plants such as Polygonum tinctorium Ait., Isatis tinctoria Ait., and Baphicacan thuscusia Brem. Stasiak et al. (2014) has a rich history spanning over 6000 years. It is widely utilized in the textile industry for dyeing jeans and other fabrics. The chemical synthesis of indigo was perfected by BASF researchers in 1925 when they improved the synthesis of N-phenylglycine from aniline (Schmidt, 1997). However, the annual production of approximately 80,000 tons of indigo through chemical synthesis involves the use of toxic compounds like aniline, formaldehyde, and hydrogen cyanide (Linke et al., 2023; Paul et al., 2021). The wastewater, leakage of toxic compounds, and residual presence of these compounds in indigo products contribute to environmental pollution and pose health risks to workers. In response to these concerns, the concept of indigo biosynthesis through microbial fermentation has emerged as a sustainable and eco-friendly alternative.

The study of naphthalene dioxygenases (NDO) from Pseudomonas putida PpG7 marked the inception of indigo biosynthesis exploration (Doukyu et al., 2002; Groeneveld et al., 2016; Pathak and Madamwar, 2010). In 1983, Ensley pioneered constructing a synthetic indigo-producing E. coli strain, yielding 25 mg/L indigo by employing native E. coli tryptophanase (TRP/TnaA) to convert L-tryptophan to indole, catalyzed by NDO (Ensley et al., 1983). Subsequent efforts involved exploring NDO expression with various plasmids and inducers, culminating in the milestone construction of the pBS959 plasmid in 1989, enabling indigo synthesis within recombinant E. coli (Boronin et al., 1989). However, initial indigo expression levels in recombinant E. coli were lower compared to P. putida. In 1993, the indigo production was enhanced by incorporating a lac promoter and operator into the plasmid, resulting in a yield of 135 mg/L (Murdock et al., 1993). This approach achieved levels comparable to P. putida by inactivating the trpB gene to produce indigo from glucose. Despite progress, feedback inhibition during 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) formation posed challenges until Berry et al. engineered a feedback-resistant DAHP synthase in 2002, yielding a 60% increase in indigo production (Berry et al., 2002). Research expanded to explore NDO from different species, with notable instances such as Pseudomonas sp. HOB1 and Comamonas sp. MQ demonstrating significant indigo production. However, practical applications of multicomponent enzymes face constraints due to specific fermentation conditions, whereas simpler flavin-containing monooxygenases (FMOs) prove more operationally practical. Additionally, toluene dioxygenases have been extensively studied for indigo biosynthesis, with Stephens et al. integrating toluene dioxygenase from P. putida NCIB 11767 into E. coli HB101 using pIG, resulting in approximately 60 mg/L indigo synthesis with the supplementation of 5 mM tryptophan (Stephens et al., 1989). Styrene monooxygenase (SMO), belonging to the flavoprotein monooxygenases (FPMOs) class E, can convert indole to indigo (Cheng et al., 2016). The maximum yield of indigo produced by SMO was up to 787.25 mg/L after 24 h of fermentation with 2.0 g/L tryptophan as the substrate (Pan et al., 2023). Another enzyme in the FPMOs class E is indole monooxygenases (IMOs). The synthesis catalyzed by IMOs and SMOs has shown minimal by-products, indicating the excellent potential of SMOs and IMOs for pharma and drug discovery (Heine et al., 2019). Other enzymes, including Cytochrome P450 monooxygenases (CYP) (Fiorentini et al., 2018; Kim et al., 2018), and Multicomponent phenol hydroxylases (Doukyu et al., 2003; Qu et al., 2012), have shown the ability to synthesize bio-indigo. However, the yield of indigo synthesized by these enzymes has generally not exceeded 300 mg/L, except for CYP102A (Kim et al., 2017), due to the requirement of stable conditions for each component.

Flavin-containing Monooxygenases (FMOs) have emerged as promising enzymes to overcome the limitations of low yield in indigo biosynthesis (Fig. 1). In 2006, flavoprotein monooxygenases (FPMOs) were classified into six classes based on sequence and structural data(van Berkel et al., 2006). Flavin-containing Monooxygenase (FMO, EC 1.14.13.8), belonging to class B, is encoded by a single gene and contains a tightly bound FAD cofactor and depends on the coenzyme NADPH/NADP+ during catalysis (Fig. 4). FMO consists of two dinucleotide-binding domains (Rossmann fold) that bind FAD and NADPH, respectively (Fig. 2).Presently, the yield of bio-indigo synthesized by mFMO can reach 1.7 g/L in Escherichia coli NEB10β (Fabara and Fraaije, 2020).While several countries, including China (Daosheng biology), America (Tinctorium and Huue), and France (Pili Bio), have companies striving to commercialize indigo biosynthesis, there remain several challenges.

In addition to its use in the textile industry, FMO-catalyzed indigo biosynthesis finds particularly applications in biotechnology. It serves as a widely employed monitor for detecting enzyme assembly, colocalization, and co-compartmentalization, providing quantifiable color in the range of 600–620 nm(Alexander et al., 2023; Gambill et al., 2023; Giessen and Silver, 2016; Lv et al., 2020; Myhrvold et al., 2016).It can also act as a precursor reaction or be co-expressed with other industrial products. In Tyrian purple biosynthesis, indigo biosynthesis serves as a precursor reaction. The combined production of isobutanol and indigo presents a promising approach for waste reduction(Cho et al., 2023a). In PHB production, indigo is co-expressed with PHB to alter the color and mechanical properties of PHB (Cho et al., 2023b).

This paper provides a comprehensive review of indigo biosynthesis mediated by FMOs, addressing the challenges, and exploring potential strategies for industrialization. Other enzymes, which have characteristics in indigo biosynthesis, are also discussed. The industrialization of indigo biosynthesis using FMOs holds promise for achieving sustainable and eco-friendly production of this valuable dye.