Metabolic engineering of Escherichia coli MG1655 for indigo production using naphthalene dioxygenase from Comamonas sp. MQ

Indigo, one of the oldest dyes still widely used in textiles, food, and pharmaceuticals, requires sustainable microbial production due to the high environmental burden and energy intensity of its chemical synthesis. In this study, indigo biosynthesis was enabled in E. coli MG1655 through heterologous expression and optimization of tryptophanases (TnaAs) and naphthalene dioxygenases (NDOs). To improve strain robustness for production, genes involved in the oxidative stress response were integrated into the chromosome, and gldA was overexpressed to ensure cofactor NADH availability. Furthermore, co-expression of molecular chaperone genes was employed to enhance the soluble expression of NDO. Following these multi-step optimizations, the final engineered strain achieved an indigo titer of 2.38 g/L in shake-flask fermentation. Finally, we demonstrated that fed-batch cultivation in a 5 L bioreactor yielded 6.90 g/L of indigo.