Indigo is an important natural dark blue dye that has been known about for more than 4000 years [1]. It has been widely used in the textile, cosmetic, pharmaceutical, plastic, food and paint industries worldwide [2]. Indigo is extensively employed for foods, cosmetics, and medical applications as an important additive owing to its distinct blue coloration and functional effects including antioxidant and radical scavenging capabilities [3]. The traditional production method of indigo is extraction and purification from plants, but there are obvious disadvantages including high cost, low production efficiency, and seasonal and regional limitations. Chemical synthesis methods could overcome the disadvantages of plant extraction methods, and their high yield could meet the needs of fast-growing markets [3]. Nevertheless, chemical synthesis requires harsh manufacturing conditions and inevitably generates toxic byproducts and causes environmental pollution [4]. Therefore, to produce indigo by a greener and cleaner method, continuous attention has been given to the microbial biosynthesis of indigo [2].

The biosynthesis of indigo has been reported in various microorganisms including Pseudomonas, Methylophage, Bacillus megaterium, and Sphingomonas macrogolitabida [5], [6], [7], [8], [9], [10]. The expression of the toluene-4-monooxygenase from P. mendocina KR1 led to indigo formation (29 μg/mg of protein) from indole (1 mM) in the recombinant E. coli strain [5], 90 μM indigo was detected in P. putida CA-3 incubated with indole for 2 h at 30 °C with shaking [6], the recombinant E. coli expressing the flavin-containing monooxygenases from Methylophaga sp. SK1 produced up to 160 mg/L indigo in the tryptophan medium after 12 h cultivation [7], the expression of the mutant cytochrome P450 BM-3 enzyme from B. megaterium produced several milligrams of indigo in the recombinant E. coli strain [8], the indigo-formation rates by the Acinetobacter sp. ST-550 cells grown on phenol and indole were 4.2 μM/mg/min and 0.2 μM/mg/min [9], and the wild type strain S. macrogolitabida TFA accumulated indigo from indole at a rate of 603 nM/min [10]. Although these wild strains can produce indigo, they cannot meet the needs of large-scale production due to low yields or unknown metabolic pathways. Therefore, the construction of genetically engineered strains based on the indigo biosynthesis pathway is an attractive approach with great potential for microbial indigo production. Indigo biosynthesis from indole via the aromatic hydrocarbon compound catabolism pathway in Pseudomonas strains has been further studied and proposed [11], [12], [13]. Generally, indole is first converted to indoxyl (3-hydroxyindole) and oxindole (2-indolinone) by dioxygenase; two indoxyl molecules form indigo by spontaneous dimerization reactions, while indoxyl and oxindole are dimerized into indirubin. If dioxygenase is replaced by monooxygenase, only indoxyl is generated from indole and pure indigo is obtained without the formation of the dark red byproduct indirubin (Fig. S1). It is evident that indigo biosynthesis by the catalysis of monooxygenase is a more economic and efficient method for natural indigo production. Previous studies showed that the styrene monooxygenase (SMO) StyAB was the key enzyme responsible for indigo biosynthesis from indole in Pseudomonas putida [14]. The SMO-encoding genes styA and styB are located in the styrene degradation gene cluster sty in P. putida and are under the transcriptional control of the two-component regulator styS/styR [15], [16]. The sensor kinase StyS detects styrene and activates the transcriptional activator StyR by phosphorelay, which initiates transcription of the styAB genes [17]. Expression of the sty operon genes under the control of the promoter Psty is regulated via the StyS/StyR system in response to the presence of a range of similar compounds including styrene, epoxistyrene, 2-phenylethanol, 2-phenylethylamine, and phenylacetaldehyde [18]. Therefore, the two-component StyS/StyR regulator system could be utilized to control the expression of SMO StyAB for indigo production from indole. In this work, the gene styAB and its regulator gene styS/styR were cloned from P. putida and coexpressed in E. coli for indigo production. The presence of the StyS/StyR system significantly enhanced the transcriptional levels of styAB and consequently remarkably promoted indigo biosynthesis from indole in the recombinant E. coli strain. This work provides a new strategy for the construction of cell factories for indigo production.