Metabolic engineering of Escherichia coli BL21(DE3) for efficient production of indigo using Methylophaga aminisulfidivorans flavin-containing monooxygenase

Abstract

Biological production of indigo can be achieved by enzymatic hydroxylation of indole, the degradation product of tryptophan. Many oxygenases have been identified to be capable of producing indigo from indole, among which flavin-containing monooxygenase (FMO) is the most extensively studied. In this study, Escherichia coli BL21 (DE3) was metabolically engineered to efficiently produce indigo using FMO. Five reported indigo-producing FMOs and five homologous human FMOs were selected to evaluate the in vivo indigo production abilities, in which Methylophaga aminisulfidivorans FMO showed the highest indigo titer. Various metabolic engineering strategies were performed to stepwise enhance the indigo titer, including strengthening tryptophan transportation, blocking tryptophan synthesis-related competitive pathways, regulating key metabolic genes for tryptophan synthesis. When supplemented with tryptophan, the final engineered strain produced 532 and 1492 mg/L of indigo by shake-flask and fed-batch cultivation, respectively.