iDEC 2022 | SUSTech

Background

Tyrian purple, mainly composed by 6,6'-dibromoindigo (6BrIG), is a dye extracted from the murex shellfish which was first produced by the Phoenician city of Tyre in the Bronze Age. It then spread in popularity and was adopted by the Romans as a symbol of imperial authority and status. The glands of thousands of putrefied crushed shellfish left to bake in the sun, with the overwhelming smell from the process. The resulting liquid was used to dye cloth fibres in manipulated variations of colours ranging from pink to violet. To obtain 1.4g tyrian purple, 12,000 Mediterranean snails would be killed. Its difficulty of manufacture, striking purple to red colour range, and resistance to fading made clothing dyed using Tyrian purple highly desirable and expensive.

For chemical synthesis, there are still no productive methods known for industrialization. This challenge often comes from the difficulty of introducing the two bromine atoms in 6BrIG.

First, most methods use bromine gas or hydrogen bromide for bromination, but they suffer from lack of regiospecificity, low product yield and environmentally toxic manufacturing processes. Second, when synthesis is started from brominated precursors, the high cost of these precursors becomes an obstacle for large-scale synthesis.

Therefore, the bromination process is still too inefficient and uneconomical to be feasible on an industrial scale for either biological or chemical synthesis of 6BrIG.

As a green and convenient production is preferred, syntheitc biology could be a better choice. Lee et al reported the first production strategy of Tyrian purple in E.coli using an accessible substrate tryptophan (Trp). Lee used three enzymes Trp 6-halogenase (Trp 6-Hal), tryptophanase (TnaA) and monooxygenase (MO) to finish this multiple-stepproduction.

In addition, a consecutive two-cell system is designed as a solution to the basic problem of byproduct indigo in tyrian purple. TnaA can react with both Trp and the first step product Br-Trp. So the first step using Fre-SttH and second step using TnaA have to be in two different strains. To prevent the effect of endogenous TnaA expression, a ΔtnaA strain of E. coli was used and TnaA was overexpressed using a plasmid when needed throughout this study. Thus, a ΔTnaA E. coli strain without endogenous TnaA expression and another ΔTnaA E. coli strain with extra TnaA expression are constructed to form a two-cell system for production of tyrian purple.

Optimization of dyeing method

We introduce a reversible glucose protection group to make the production of tyrian purple dye more stable and convenient for industrial scale-up.

The reported scheme of tyrian purple production in E.coli with the substrate of Trp is shown. We designed our optimization based on this scheme.

FIGURE 1

Reaction schemes of Trp 6-halogenase (Trp 6-Hal), tryptophanase (TnaA) and monooxygenase (MO)

The application of UGT and BGL in tyrian purple synthesis is inspired by 2013 iGEM team UC Berkeley with employing the glucose protecting group for a sustainable indigo dyeing strategy. Glucose acts as a protecting group for indoxyl inside the production host and in the fermenter until it is removed by co-application with β-glucosidase to cotton.

FIGURE 2

Reaction scheme of UGT and BGL

The current industrial process involves chemically synthesizing indigo and adding a reducing agent (typically sodium dithionite) to the indigo vat for reduction to dye competent, soluble leucoindigo. In the proposed microbial process designed by 2013 iGEM team UC Berkeley, E. coli biosynthesizes indoxyl and glucosylates it at the C3 hydroxyl group before its spontaneous air oxidation to indigo. The glucoside, indican, is stable in air and can be stored. The glucosyl group is removed only at the point of dyeing, allowing the regenerated indoxyl to oxidize to indigo crystals in cotton fibers. No reducing agent is required when dyeing with indican. More importantly, indican is water soluble but indigo is not. Thus, dyeing fabrics with soluble precursor indican is more even and has good dyeing quality, compared to direct dyeing with final product indigo.

FIGURE 3

A glucosyl protecting group enables control over the timing and location of indigo dyeing

As natural dye indigo and tyrian purple is hard to solute in water and cause difficulty in cloth dying. We applied the strategy that introduce a glucose moiety as a reversible protecting group to the reactive indigo and tyrian purple precursor indoxyl by glucosyltransferase PtUGT to form soluble indican as fermentation product. And β-glucosidase BGL is applied to remove the protecting group when cloth dying. Also, for the steps using TnaA and MO (using one of special kinds named flavin-containing monooxygenase, MaFMO or FMO), we linked them with a flexible linker to form a chimeric enzyme for higher field and efficiency.

FIGURE 4

Optimized reaction scheme with the reversible protection of glucose

Optimized Tyrian purple synthesis in one-cell system

We use directed evolution to develop the process of synthesis from two-cell system to one-cell system, with an expected optimization controlling system of endogenous TnaA enzyme in E.coli.

In this study, we optimized two-cell system to one-cell system with a spatio- temporal well-controlled TnaA synthesis. We applied a synthetic phylogeny of programmable Trp repressor with directed evolution to E.coli, which simplifiedthe process of reacting with bromotryptophan not tryptophan. Iterative rounds of positive and negative compartmentalized partnered replication (CPR) led to the exponential amplification of variants that responded with high affinity and specificity to halogenated tryptophan analogs and novel operator sites. We empowercells to perceive the relative ratio of bromotryptophan to tryptophan to control TnaA synthesis. Withthe optimization of directed evolution, we achieved the goal of producing Tyrian purple dye in an environmentally friendly process with a higher level of purity and yield.

The tryptophan repressor (TrpR) is a negative feedback repressor that is responsible for regulating the expression of genes involved in the production of aromatic amino acids, in particular, L-tryptophan. TrpR is a homodimeric protein with an uncommon structure of highly interlocked interface regions between the two subunits. This interface forms a highly rigid central core that acts as a brace to support the l-tryptophan binding pocket and the helix-turn-helix (HTH) motif that selectively binds to the operator sequence. The allosteric mechanism of activation has been extensively studied in the TrpR system. In essence, bound L-tryptophan stabilizes the HTH motif and subsequently leads to operator binding and inhibition of transcription. Both subunits of the dimer must bind L-tryptophan in a noncooperative fashion to initiate binding to two operator half-sites and induce repression.

FIGURE 5

The TrpR regulatory program controls the negative-feedback inhibition during L-tryptophan biosynthesis. The TrpR protein homodimerizes by formation of a highly interlocking interface region. Recognition of its effector, L-tryptophan, stabilizes an HTH motif, which drives sequence-specific recognition of two operator half-sites with dyad symmetry (axis indicated by black diamond).

Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype–genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection.

FIGURE 6

Circuit Dynamics of Positive and Negative CPR Selection. (A) The positive CPR selection circuit is initially in a state of repression due to the production of the Lambda phage repressorCI (λR) driven bythe tryptophan promoter (PTrp). Following IPTG induction of the Trp repressor and addition of the desired L-tryptophan analog, TrpR variants may repress PTrp-resulting in the depletion of λR(facilitated by a LVA degradation tag) and the subsequent expression of Taq polymerase from the λPR promoter. In this fashion, TrpR variants that most effectively repress their cognate promoter sequence will produce the greatest amount of Taq polymerase and will correspondinglybe more efficiently amplified in the following emulsion PCR. (B) The negative CPR selection circuit counter-selects TrpR variants that are activein the absence of the desired L-tryptophan analog.

With directed evolution, we optimized the reaction scheme into one-cell production system with an optimization of dyeing method at the same time.

FIGURE 7

Optimized reaction scheme of one-cell system adding directed evolution

References

[1] Lee, J., Kim, J., Song, J. E., Song, W. S., Kim, E. J., Kim, Y. G., Jeong, H. J., Kim, H. R., Choi, K. Y., & Kim, B. G. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nature chemical biology, 2021, 17(1), 104-112. DOI: https://doi.org/10.1038/s41589-020-00684-4
[2] Hsu, T. M., Welner, D. H., Russ, Z. N., Cervantes, B., Prathuri, R. L., Adams, P. D., & Dueber, J. E. Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nature chemical biology, 2018, 14(3), 256–261. DOI: https://doi.org/10.1038/nchembio.2552
[3] Ellefson, J. W., Ledbetter, M. P., & Ellington, A. D. Directed evolution of a synthetic phylogeny of programmable Trp repressors. Nature chemical biology, 2018, 14(4), 361–367. DOI: https://doi.org/10.1038/s41589-018-0006-7
[4] Abil, Z., Ellefson, J. W., Gollihar, J. D., Watkins, E., & Ellington, A. D. Compartmentalized partnered replication for the directed evolution of genetic parts and circuits. Nature protocols, 2017, 12(12), 2493–2512. DOI: https://doi.org/10.1038/nprot.2017.119

Design