School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea.
Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Republic of Korea.
Biotechnol Bioeng. 2022 Oct;119(10):2938-2949. doi: 10.1002/bit.28188. Epub 2022 Aug 4.
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.
6-溴靛红（6BrIR）存在于石磺海蜗牛中，是靛玉红衍生类抗癌药物的前体。然而，由于其生物合成途径尚未阐明，并且吲哚环的特异性溴化和氧化存在困难，其合成仍然受到限制。在这里，我们通过使用四种酶，即与黄素还原酶 Fre（Fre-L3-SttH）融合的色氨酸 6-卤代酶、色氨酸酶（TnaA）、甲苯 4-单加氧酶（PmT4MO）和黄素单加氧酶（MaFMO），在大肠杆菌中提出了一种有效的 6BrIR 生产策略。尽管大多数吲哚加氧酶优先氧代吲哚的电子活性 C3 位，但新表征的 PmT4MO 能够以 45%的产率对 6-溴吲哚进行 C2 氧代反应，生成 6-溴-2-氧吲哚。此外，通过在 MaFMO 反应过程中控制半胱氨酸的还原能力和氧气供应，可以选择性地生成 6BrIR，而不会产生靛蓝和靛玉红副产物。这些方法导致 6BrIR 的产量达到 34.1mg/L，使得仅从天然成分（如色氨酸、NaBr 和氧气）就可以生产抗癌药物的关键前体。
