Background
The Color of Power—Tyrian purple
The silk shroud of Charlemagne made with gold and Tyrian purple.
Tyrian Purple, aka Royal Purple or Imperial Purple, is a precious dye from the secretion of rock shell. With its vibrant color and gold comparable price, Tyrian Purple became the favorite color of the upper class of the Roman Empire.
In the traditional extraction method, 12,000 snails will be sacrificed for pre 1.4 grams of Tyrian purple, which will pay a huge environmental cost. Nowadays, Tyrian Purple is still an expensive dye due to the difficulty of introducing bromine atoms, and there is no mature chemosynthesis method at present.
6,6-dibromoindigo not only has a strong cultural capacity, but can be applied to dye-sensitized solar cells and conductive materials owing to its orderly stacked and repeated conjugatedπbonds.
Chemosynthesis
6,6-dibromoindigo (6BrIG)
In 1909, the German chemist Paul finally determined the main structure of Tyrain Pruple is 6,6’-dibromoindigo (6BrIG).
6BrIG is a bromide product of indigo, and different colors can be displayed by replacing the halogen atom species and its position on the benzene ring.
Methods
The method of 6BrIG’s production are as follows:
The Chemical method of 6BrIG’s production
Problem
In chemical process, bromide or hydrogen bromide are commonly used to brominate. It not only lacks regional specificity, but will produce a large number of toxic waste gas, causing serious environmental pollution and greatly raising the production cost.
So as we can see, this dye has never been produced industrially in the modern age. Can we produce this historical dye in an environmentally friendly, economically and easily way?
Inspiration
Biosynthesis pathways
By consulting the relevant materials, we decided to use tryptophan as the initial substrate to obtain 6BrIG by a series of biocatalytic reactions:
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·Tryptophan 6-halogenase(SttH) catalyzes the bromation of tryptophan:
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Tryptophan 6-halogenase is a biocatalyst with highly regional specificity capable of catalyzing the substrate NaBr and Trp to 6-Br-Trp. We found that SttH has a low solubility by reviewing information and SDS-PAGE,so we added the highly soluble protein Fre to its N terminus, and the fusion protein Fre-Linker-SttH was constructed to improve the solubility of SttH.
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·Tryptophanase (TnaA) breaks down 6-Br-Trp to 6-Br-indole:
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TnaA is an endogenous enzyme of E. coli, with capable of breaking down Trp or 6-Br-Trp to indole or 6-Br-indole.
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·Flavin-containing monooxygenase (MaFMO)catalyzes the autooxidation reaction
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Flavin-containing monooxygenase (MaFMO) catalyzes the autooxidation reaction and produce the 6-Br-indole dimer.
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Problem
When we add exogenous tryptophan to the medium and we hope SttH to catalyze it into 6-Br-Trp, but endogenous TnaA expression is higher than exogenous SttH, so there will be a competition between enzymes, thus producing indindex (indole), resulting in final product 6BrIG containing a large number of by-product indigo.
Our Solution
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· Knocked out the endogenous TnaA of E. coli BL21 (ED3) with CRISPR:
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The CRISPR plasmids were constructed to obtain the E. coli BL21 strain-TnaA(-).
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· Constructed a two-cell system:
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The TnaA(-) Fre-Linker-SttH and TnaA(-) TnaA+MaFMO expression vectors were constructed, and upon the maximum 6-Br-Trp yield of the TnaA(-) Fre-Linker-SttH system, the supernatant was mixed with the TnaA(-) TnaA+MaFMO resuspension to minimize the byproduct content and increase the 6,6-dibromoindigo yield.
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References
[1] Lee J, Kim J, Song JE, Song WS, Kim EJ, Kim YG, Jeong HJ, Kim HR, Choi KY, Kim BG. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat Chem Biol. (2021)
[2] Glowacki, E. D. et al. Indigo and tyrian purple—from ancient natural dyes to modern organic semiconductors. Isr. J. Chem. 52, 540–551 (2012).
[3] Glowacki, E. D., Voss, G. & Saricifci, N. S. 25th anniversary article: progress in chemistry and applications of functional indigos for organic electronics. Adv. Mater. 25, 6783–6800 (2013).
[4] Kim, H. et al. Ambipolar organic phototransistors based on 6,6'-dibromoindigo. RSC Adv. 8, 14747–14752 (2018).
[5] Wolk, J. L. & Frimer, A. A. A simple, safe and efcient synthesis of Tyrian purple (6,6’-dibromoindigo). Molecules 15, 5561–5580 (2010).
[6] Gaboriaud-Kolar, N., Nam, S. & Skaltsounis, A.-L. in Progress in the Chemistry of Organic Natural Products Vol. 99 (eds. Kinghorn, A. D., Falk, H. & Kobayashi, J.) 69–145 (Springer, 2014).
[7] Andorfer, M. C. & Lewis, J. C. Understanding and improving the activity of favin-dependent halogenases via random and targeted mutagenesis. Annu. Rev. Biochem. 87, 159–185 (2018).
[8] Costa, S., Almeida, A., Castro, A. & Domingues, L. Fusion tags for protein solubility, purifcation and immunogenicity in Escherichia coli: the novel Fh8 system. Front Microbiol 5, 63 (2014).