Enhancement of Indigo Production by a Metabolically Engineered <i>Escherichia coli</i> MG1655 Using Membrane Engineering and Two-Stage Cultivation

Indigo, a natural blue pigment extensively used in the food and textile industries, faces sustainability challenges due to toxic chemicals in its industrial synthesis. In this study, an efficient <i>E. coli</i> MG1655 system was developed for indigo biosynthesis. The strain was engineered by genomic integration and plasmid-based expression of <i>Methylophaga aminisulfidivorans</i> flavin-containing monooxygenase (MaFMO) and endogenous tryptophanase (TnaA). To optimize metabolic flux toward indigo production, key competitive pathway genes were deleted, and membrane engineering genes were introduced to alleviate toxicity and improve product secretion. A two-stage fermentation strategy with controlled feeding of the tryptophan substrate and surfactants further optimized indigo production. The integrated approach achieved a maximum indigo titer of 3.9 g/L in 5 L of fed-batch fermentation. This study highlights the successful integration of genetic engineering and fermentation strategies to enhance microbial indigo production, offering a green alternative to chemical synthesis with potential applications in food-grade colorant production and the textile industries.