Team LINKS_China completed the validation of a two-cell system for the production of tyrian purple dyes in E. coli in 2021, and our team performed further experiments on this part this year, demonstrating the feasibility of using a one-cell system for the synthesis of tyrian purple dye in E. coli. As presented in [1], the synthesis pathway of tyrian purple can be seen in Figure 1. In the first E.coli, tryptophan (Trp) is firstly halogenated to bromotryptophan (6-Br-Trp) by Fre-SttH and then introduced into the second E.coli to synthesize tyrian purple. However, we found two-cell reaction system had two issues.

(1)The two-cell system is inefficient in practical production
(2)Tyrian purple insolubility in water affects dyeing quality

To solve these two problems, we referred to [2] and selected the repressor with Trp sensitive repressor (TrpR) by directed evolution to complete the basic components of synthetic biology, making single-cell production possible and greatly saving the costs ofproduction.

And as shown in Figure 2, inspired by optimization of indigo production made by Team Berkeley[3], the problem of water insolubility of tyrian purple was solved by utilizing a glucose group protection to prevent too early production of tyrian purple (6BrIG) from auto-oxidation reaction. Then, we added the step of transferring 6-Br-indoxyl into water soluble Br-Indican for binding to fabric fibers more evenly than dyeing with tyrian purple directly.

With the help of modeling, we simulated the selection step of directed evolution and verified the feasibility of one-cell production of tyrian purple through wetlab experiments.