Constructing multi‐enzymatic cascade reactions for selective production of 6‐bromoindirubin from tryptophan in Escherichia coli

6‐Bromoindirubin (6BrIR), found in Murex sea snails, is a precursor of indirubin‐derivatives anticancer drugs. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and difficulties in site‐specific bromination and oxidation at the indole ring. Here, we present an efficient 6BrIR production strategy in Escherichia coli by using four enzymes, that is, tryptophan 6‐halogenase fused with flavin reductase Fre (Fre‐L3‐SttH), tryptophanase (TnaA), toluene 4‐monooxygenase (PmT4MO), and flavin‐containing monooxygenase (MaFMO). Although most indole oxygenases preferentially oxygenate the electronically active C3 position of indole, PmT4MO was newly characterized to perform C2 oxygenation of 6‐bromoindole with 45% yield to produce 6‐bromo‐2‐oxindole. In addition, 6BrIR was selectively generated without indigo and indirubin byproducts by controlling the reducing power of cysteine and oxygen supply during the MaFMO reaction. These approaches led to 34.1 mg/L 6BrIR productions, making it possible to produce the critical precursor of the anticancer drugs only from natural ingredients such as tryptophan, NaBr, and oxygen. Regulating the biosynthesis of indigo and indirubin has been continuously attempted. However, there is still no definitive way to control the production of each molecule due to the difficulties of regiospecific oxygenation and bromination at the indole ring. Here, we present an efficient 6‐bromoindirubin production strategy in Escherichia coli using an enzymatic system, that is, tryptophan 6‐halogenase SttH, toluene 4‐monooxygenase PmT4MO, and flavin‐containing monooxygenase MaFMO. Through the process, the critical precursor of indigoid drugs can be regiospecifically produced from tryptophan.