![]() These approaches led to 315.0 mg L −1 6, 6′-dibromoindigo production from 2.5 mM tryptophan. introduced a consecutive two-cell reaction system to overproduce regiospecifically brominated precursors of 6, 6′-dibromoindigo by spatiotemporal separation of bromination and bromotryptophan degradation ( Lee et al., 2021). To overcome this TnaA competes with SttH for tryptophan issue, Lee et al. coli would produce indigo and indirubin (isomer of indigo), Tyrian purple, and 6, 6′-dibromoindirubin (TP isomer, Tyrian purple isomer), simultaneously ( Figure 1), that is, TnaA competes with SttH for tryptophan, both TnaA and MaFMO are lack of selectivity that lead to the production of many byproducts, which are the limitations of Tyrian purple biosynthesis. As TnaA is effective enough to convert tryptophan or its halogenated derivative into the corresponding indoles, and MaFMO catalyzes the hydroxylation of indoles (indole or 6-bromoindole) to 2-hydroxyindoles and 3-hydroxyindoles ( Kim et al., 2019), the biosynthesis of Tyrian purple using three enzymes in E. It is often not feasible to obtain pure Tyrian purple by biosynthesis. coli using tryptophan 6-halogenase from Streptomyces toxytricini (SttH), tryptophanase from E. presented an alternative 6, 6′-dibromoindigo production strategy in E. Biocatalysis has emerged as an alternative for sustainable synthesis of Tyrian purple from a natural substrate through microbial fermentation, but the selectivity issue in enzymatic tryptophan and bromotryptophan degradation becomes an obstacle for large-scale biosynthesis. ![]() In addition, due to the difficulty in chemical or biological synthesis, there is still no method for its industrial production ( Wolk and Frimer, 2010). It is very difficult to obtain the dye in large quantities from natural sources as it requires euthanizing 12,000 snails per 1.4 g of dye ( Mcgovern and Michel, 1990). Tyrian purple has a range of striking purple to red, color-fast and resistance to fading, and also has a promising application in dye-sensitized solar cells, functional polymers, and conductive materials ( Głowacki et al., 2012 Głowacki et al., 2013 Guo et al., 2015 Kim et al., 2018 Schnepel et al., 2021). Tyrian purple, known as royal purple, and mainly composed of 6, 6′-dibromoindigo, is an ancient dye extracted from the murex shellfish ( Ngangbam et al., 2015 Lee et al., 2021). This is the first study to show the existence of an indole biodegradation pathway in Providencia rettgeri, and the indole biodegradation gene cluster can contribute to the selective production of Tyrian purple. Interestingly, the monooxygenase GS-C co-expressed with its corresponding reductase GS-D in the cluster has better activity for the biosynthesis of Tyrian purple compared with the previously reported monooxygenase from Methylophaga aminisulfidivorans (MaFMO) or Streptomyces cattleya cytochrome P450 enzyme (CYP102G4). To further explore the underlying mechanism of the selectivity, we explored the intermediates in this indole biodegradation pathway using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS), which indicated that the indole biodegradation pathway in Providencia rettgeri is the catechol pathway. The heterologous expression of the indole degradation gene cluster in Escherichia coli BL21 (DE3) and in vitro enzymatic reaction demonstrated that the indole biodegradation gene cluster may contribute to selectively biosynthesizing Tyrian purple. An indole degradation gene cluster for indole metabolism was identified from this GS-2 strain. This GS-2 strain was then identified as Providencia rettgeri based on bacterial genome sequencing analysis. In this study, we found Tyrian purple can be selectively produced by a bacterial strain GS-2 when fed with 6-bromotryptophan in the presence of tryptophan. Tyrian purple, mainly composed of 6, 6′-dibromoindigo, is a precious dye extracted from sea snails. 4Center for Synthetic Biochemistry, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.3CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.2Shenzhen Key Laboratory for the Intelligent Microbial Manufacturing of Medicines, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.1School of Life Sciences, Inner Mongolia University, Hohhot, China.
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