Overview
Dyeing processes have been an important part of human civilization since ancient times. However, modern dyeing methods widely rely on chemical dyes. These processes can place a burden on the environment and human health, while also struggling to meet the demands of modern green and sustainable development.
To address this challenge, we have developed a tyrosinase-based bio-dyeing technology leveraging innovations in synthetic biology. This technology utilizes E. coli to efficiently express tyrosinase, an enzyme that effectively catalyzes the oxidation of natural phenolic compounds to produce colored polymers, thereby achieving environmentally friendly dyeing. By introducing the gene encoding tyrosinase into E. coli, enabling it to produce large quantities of the enzyme protein, and combining it with tyrosine and its derivatives as substrates, this technology is applicable not only to silk but also to the coloring of cotton, synthetic fibers, and hair.
This method significantly reduces the environmental pollution associated with traditional dyeing processes while imparting more natural and unique color effects to fabrics and hair strands. This project not only promotes the innovative application of synthetic biology in the traditional dyeing industry but also demonstrates the important role of green manufacturing processes in connecting traditional culture with modern technology, offering new possibilities for achieving both environmental protection and economic benefits.
Cycle 1: TyrBm_ pET-21a(+)
We selected pET-21a(+) as the cloning vector, which contains a T7 promoter, ribosome binding site, and Lac operator. Using a homologous recombination method, the gene fragment TyrBm encoding tyrosinase was ligated with pET-21a(+) to construct the recombinant plasmid TyrBm_pET-21a(+). Figure 1 shows the plasmid map of TyrBm_pET-21a(+). The E. coli Rosetta strain was selected for tyrosinase expression.
First, the tyrosinase coding gene was optimized for E. coli codon usage and the sequence was synthesized by GenScript. The sequence was amplified by PCR, digested with BamHI and XhoI, and then ligated with the linearized pET-21a(+) vector. The ligation product was transformed into E. coli TOP10 competent cells. Verification by colony PCR and sequencing confirmed the successful construction of the TyrBm_pET-21a(+) recombinant plasmid, ready for subsequent experiments.
The verified correct recombinant plasmid was transformed into E. coli Rosetta competent cells, and successful transformation was confirmed by colony PCR. Expression was induced with 1 mM IPTG at 37°C for 15 hours. Cells were collected, ultrasonically disrupted, and protein expression was verified by SDS-PAGE (Fig.3).
To detect the activity of the recombinant tyrosinase TyrBm, the seed culture of the recombinant strain was spread on LB chromogenic screening medium containing the appropriate antibiotic and incubated at 37°C for observation. The blackening of the chromogenic plate indicated that the strain possessed tyrosinase activity under the culture conditions.
Cycle 2: TyrBm-L1-CipA_pET-21a(+) and CipA-L1-TyrBm_pET-21a(+)
To further enhance the dyeing effect of tyrosinase, we introduced the scaffold protein CipA, which can enhance enzyme activity and improve enzymatic properties. We connected CipA to the N-terminus and C-terminus of TyrBm via a flexible Linker (L1), constructing two recombinant plasmids: TyrBm-L1-CipA_pET-21a(+) and CipA-L1-TyrBm_pET-21a(+). Their plasmid maps are shown in Fig.5.
Using pET-21a(+) as the vector, the above two recombinant plasmids were constructed by homologous recombination. The gene sequences were codon-optimized and synthesized, amplified by PCR, ligated with the linearized vector, transformed into TOP10 competent cells, and successfully constructed as verified by colony PCR and sequencing. The construction process is shown in Fig.6.
The verified correct recombinant plasmids were transformed into the E. coli Rosetta(DE3) expression strain, respectively. Positive clones were picked, and expression was induced with 1 mM IPTG at 37°C for 15 hours before cell collection. Cells were ultrasonically disrupted and centrifuged. The supernatant and inclusion body samples were collected separately and analyzed by SDS-PAGE to detect target protein expression and solubility (Fig.7).
CipA can form a ring-like tetramer (Fig.8), with the N-terminus on the outer side of the tetramer and the C-terminus relatively inward. Structurally, when TyrBm is connected to the N-terminus of CipA, the three-dimensional space of the proteins is less likely to interfere with each other, minimizing the impact on their protein functions.
The synthetic protein connecting TyrBm and CipA can immobilize tyrosinase in the inclusion bodies. After tyrosinase catalyzes the substrate to form melanin, it can be recollected by centrifugation and reused (Fig.9). This can significantly reduce the cost of melanin production.
Cycle 3: TyrBm-L2-CipA_pET-21a(+) and TyrBm-L3-CipA_pET-21a(+)
Based on the results of Cycle 2, the introduction of the CipA protein significantly enhanced the enzyme activity of TyrBm, especially when CipA was located at the C-terminus of TyrBm. To further optimize the conformation and catalytic efficiency of the fusion protein, we redesigned the linkers. Building upon the previously used 45 bp (Gly4-Ser)3 Linker (referred to as L1), we introduced two new linkers: L2 (Gly-Gly) and L3 ((Gly4-Ser)2), aiming to improve protein folding and functional expression. The newly constructed recombinant plasmids were named TyrBm-L2-CipA_pET-21a(+) and TyrBm-L3-CipA_pET-21a(+). Their molecular designs were completed using SnapGene software, and the plasmid maps are shown in Fig.10.
Using the same strategy as the previous cycles, with pET-21a(+) as the expression vector, the two new plasmids were constructed by homologous recombination. The gene sequences for TyrBm-L2-CipA and TyrBm-L3-CipA were codon-optimized and synthesized by GenScript. The synthesized fragments were amplified by Phusion PCR and ligated into the linearized pET-21a(+) vector using a homologous recombination kit. The ligation products were transformed into E. coli TOP10 competent cells. After plating, single colonies were picked for colony PCR verification and sequencing to ensure correct plasmid construction. The specific construction process and verification results are shown in Fig.11.
The verified correct recombinant plasmids were transformed into theE. coliRosetta(DE3) expression strain, respectively. Positive clones were picked, and expression was induced with 1 mM IPTG at 37°C for 15 hours before cell collection. Cells were ultrasonically disrupted and centrifuged. The supernatant and inclusion body samples were collected separately and analyzed by SDS-PAGE to detect target protein expression and solubility (Fig.12).
To systematically evaluate the effect of different linkers on the enzyme activity of the fusion proteins, we measured the enzyme activity of each protein sample using L-tyrosine as the substrate under optimized reaction conditions (containing 0.2 mmol/L CuSO44, pH 7.0 buffer system). The change in absorbance at 475 nm (A475) was dynamically monitored using a microplate reader, the initial reaction rate was calculated, and one unit of enzyme activity (U) was defined as the amount of enzyme producing 1 μmol/L of dopachrome per minute (ΔA475=0.001/min).
Figure 13 shows the enzyme activity curves and calculated enzyme activity units for the five proteins – TyrBm, CipA-L1-TyrBm, TyrBm-L1-CipA, TyrBm-L2-CipA, and TyrBm-L3-CipA – in both the supernatant and inclusion body fractions. The experimental results indicate that among all constructs, the TyrBm-L1-CipA fusion protein (i.e., using the (Gly4-Ser)3 Linker with CipA at the C-terminus) exhibited the highest tyrosinase activity in both the soluble supernatant and inclusion bodies, and was therefore selected as the best candidate protein for subsequent dyeing experiments.
The superior enzymatic activity of the TyrBm-L1-CipA construct, utilizing the (Gly4-Ser)3 linker (L1), is likely attributable to an optimal balance of flexibility and spatial organization. The extended length and inherent flexibility of L1 provide sufficient scope for both the TyrBm and CipA domains to fold correctly without steric hindrance, while still allowing for productive interactions that enhance stability and catalytic efficiency. The shorter linkers, L2 and L3, may restrict this conformational freedom, impairing proper folding or the scaffold function of the CipA tetramer. For future enhancement, a rational design approach could create a library of linkers with fine-tuned rigidity and length, or alternatively, fermentation conditions could be optimized to further boost soluble expression and specific activity.
Dyeing
Comparing the detected enzyme activities of TyrBm, CipA-L1-TyrBm, TyrBm-L1-CipA, TyrBm-L2-CipA, and TyrBm-L3-CipA revealed that the enzyme activity of TyrBm-L1-CipA was the highest, both in inclusion bodies and supernatant. Therefore, we selected TyrBm-L1-CipA as the target protein for subsequent dyeing experiments. The dyeing experimental procedure is described in the Experiment section. To enhance color diversity, we selected four substrates: L-tyrosine, and the L-tyrosine derivatives Fmoc-L-Tyr, Boc-L-Tyr, and Boc-L-Tyr-OMe. The dyeing results are shown in Fig.14. Except for leather, all other fibers showed good dyeing effects.
The suboptimal dyeing performance on leather, compared to other substrates, can be primarily explained by its distinct physicochemical properties. Leather, being a tannified collagen fiber, possesses a dense surface structure and is often negatively charged under dyeing conditions. This creates an electrostatic repulsion with the similarly charged melanin polymers, preventing effective adsorption and penetration. In contrast, fibers like cotton, polyester, and hair offer more accessible surfaces for pigment deposition through hydrogen bonding or hydrophobic interactions. To address this, potential strategies could include pre-treating the leather with cationic agents to modulate its surface charge, or optimizing the reaction to promote covalent anchoring of reactive quinone intermediates to leather proteins. These approaches may potentially improve dye uptake and fastness, and would require further investigation.
