Team:BJU China/Design
Design
Overall Objective:
The tyrian purple was originated in Byzantium, a dye extracted from Mediterranean Sea snails. It is one of the derivatives of indigo dyes. Discovered at a time when purple dyes were quite pre-existent, it quickly became popular and very expensive because of its rarity.
In order to achieve our goals of producing Tyrian purple and other halogenated indigos, we decide to use Escherichia coli to synthesis the dyes. We selected Trp 6-halogenase (Stth) from Streptomyces toxytricini to brominate tryptophan to 6-br-Trp in E.coli, and monooxygenase from Methylophaga aminisulfidivorans (MaFMO) to produce 6BrIG. There are two strains of E.coli utilized in our program—ΔtnaA Fre-L3-SttH and ΔtnaA TnaA+MaFMO. We plan to convert tryptophan to 6-Br-Trp in the pathway of bromination of Trp followed by conversion to 6-Br-indole (Trp→6-Br-Trp→6-Br-indole), utilizing Fre-L3-SttH and TnaA. Compared to the traditional pathway of synthesizing tyrian purple (Trp→indole→6-Br-indole), this route is proved to give a better yield of 6-Br-indole and advantageous for 6BrlG production.
In our specific pathway design, we split the process of synthesizing the tyrian purple into two steps. Adopting a two-cell reaction system to temporally separate the two reactions, we aim for higher output of 6-Br-Indole with minimum amount of indole formation.
Pathway:
In the first step, we will utilize ΔtnaA Fre-L3-SttH to convert tryptophan to 6-Br-Trp. To convert tryptophan to 6-Br-Trp first instead of to indole, we plan to mutate the TnaA gene in the E.coli. strain first. We plan to do this by using gene editing technology—CRISPR-Cas9. Since the enzyme SttH shows strict bromination regiospecificity for both Trp and indole, we select it for the first reaction. However, SttH alone shows low reactivity because it mostly exists in an insoluble form. Therefore, to improve the bromination action rate, we construct a fusion enzyme of SttH with flavin reductase (Fre) at the N terminus, which is highly expressed in a soluble form in E.coli and can also regenerate FADH2 efficiently, increasing the solubility. We plan to select the plasmid pACYC backbone and use the IPTG induction to express the inserted genes.
In the second step, we utilize the strain ΔtnaA Tna A+MaFMO to convert the 6-Br-Trp to 6-Br-IG. We select the plasmid pET28b backbone for this process, still using IPTG induction technology. In this step, after 6-Br-indole is produced from 6-Br-Trp by TnaA, 6BrIG is produced from 6-Br-indole by MaFMO.
Extension:
To expand this process to other halogenated indigos, we design to apply the fusion strategy to other halogenases as well. In our project, we choose the Trp 5-halogenase PyrH and Trp 7-halogenase RebH. As wild-type PyrH and RebH also exists mostly in insoluble form, we construct the fusion enzymes using Fre as well. Using the L3 as a linker, we obtain Fre-L3-PyrH and Fre-L3-RebH, respectively.
As Tyrian purple has the properties of low toxicity, chemical stability and its feature of good performance in inverters, it has great potential in becoming photosensitive semiconductors as an environmentally friendly material. Other indigos have similar traits and are therefore suitable candidates for photosensitive semiconductors as well.
Furthermore, we plan to use them to make dye-sensitized solar cells. Using dye solution, we plan to dip conductive glass with a thin coating of TiO2 in the diluted solution to make dye sensitized photoanode. With carbon counter electrodes and copper sheet, we plan to assemble the compenents into a cell and construct a series circuit with a buzzer. When the light radiates on the photoanode side of the cell, the buzzer will ring.
