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.

中文翻译：

---

构建多酶级联反应以在大肠杆菌中从色氨酸选择性生产 6-溴靛玉红

在骨螺海螺中发现的 6-溴靛玉红 (6BrIR)是靛玉红衍生物抗癌药物的前体。然而，由于未表征的生物合成途径以及吲哚环上位点特异性溴化和氧化的困难，其合成仍然受到限制。在这里，我们提出了一种在大肠杆菌中高效的 6BrIR 生产策略通过使用四种酶，即融合黄素还原酶 Fre (Fre-L3-SttH)、色氨酸酶 (TnaA)、甲苯 4-单加氧酶 (PmT4MO) 和含黄素单加氧酶 (MaFMO) 的色氨酸 6-卤化酶。尽管大多数吲哚加氧酶优先氧化吲哚的电子活性 C3 位置，但 PmT4MO 的新特征在于以 45% 的产率对 6-溴吲哚进行 C2 氧化以产生 6-溴-2-羟吲哚。此外，通过控制 MaFMO 反应过程中半胱氨酸和氧气供应的还原能力，选择性地生成了 6BrIR，而没有靛蓝和靛玉红副产物。这些方法产生了 34.1 mg/L 的 6BrIR，使得仅从色氨酸、溴化钠和氧气等天然成分生产抗癌药物的关键前体成为可能。