Tyrian purple (6,6′-Dibromoindigo) is an ancient precious dye, which possesses remarkable properties as a biocompatible semiconductor material. Recently, biosynthesis has emerged as an alternative for the sustainable production of Tyrian purple from a natural substrate. However, the selectivity issue in enzymatic tryptophan (Trp) and bromotryptophan (6-Br-Trp) degradation was an obstacle for obtaining high-purity Tyrian purple in a single cell biosynthesis. In this study, we present a simplified one-pot process for the production of Tyrian purple from Trp in Escherichia coli (E. coli) using Trp 6-halogenase from Streptomyces toxytricini (SttH), tryptophanase from E. coli (TnaA) and a two-component indole oxygenase from Providencia Rettgeri GS-2 (GS-C and GS-D). To enhance the in vivo solubility and activity of SttH and flavin reductase (Fre) fusion enzyme (Fre-L3-SttH), a chaperone system of GroEL/GroES (pGro7) was introduced in addition to the implementation of a set of optimization strategies, including fine-tuning the expression vector, medium, concentration of bromide salt and inducer. To overcome the selectivity issue and achieve a higher conversion yield of Tyrian purple with minimal indigo formation, we applied the λpL/pR-cI857 thermoinducible system to temporally control the bifunctional fusion enzyme of TnaA and monooxygenase GS-C (TnaA-L3-GS-C). Through optimization of the fermentation process, we were able to achieve a Tyrian purple titer of 44.5 mg L⁻¹ with minimal indigo byproduct from 500 μM Trp. To the best of our knowledge, this is the first report of the selective production of Tyrian purple in E. coli via a one-pot process.

泰尔紫（6,6'-二溴靛蓝）是一种古老的珍贵染料，作为生物相容性半导体材料具有卓越的性能。最近，生物合成已成为从天然基质中可持续生产泰尔紫的替代方案。然而，酶促色氨酸（Trp）和溴色氨酸（6-Br-Trp）降解的选择性问题是在单细胞生物合成中获得高纯度泰尔紫的障碍。在这项研究中，我们提出了一种简化的一锅法，使用来自毒三链霉菌( SttH ) 的色氨酸 6-卤化酶、来自大肠杆菌(TnaA) 的色氨酸酶和一种大肠杆菌 ( E. coli ) 中的色氨酸生产泰瑞安紫。来自Providencia Rettgeri GS-2的双组分吲哚加氧酶（GS-C 和 GS-D）。为了增强SttH和黄素还原酶（Fre）融合酶（Fre-L3-SttH）的体内溶解度和活性，除了实施一套优化策略外，还引入了GroEL/GroES（pGro7）伴侣系统，包括表达载体、培养基、溴化盐浓度和诱导剂的微调。为了克服选择性问题并在靛蓝形成最少的情况下实现更高的泰尔紫转化率，我们应用 λpL/pR-cI857 热诱导系统来临时控制 TnaA 和单加氧酶 GS-C 的双功能融合酶 (TnaA-L3-GS- C）。通过优化发酵过程，我们能够实现 44.5 mg L ^-1的泰尔紫滴度，并且来自 500 μM Trp 的靛蓝副产物最少。据我们所知，这是通过一锅法在大肠杆菌中选择性生产泰尔紫的第一份报告。