We aimed to construct fusion proteins of TasA and binding proteins, which would be expressed on the bacterial surface to mediate adhesion to materials. We selected three peptides or proteins that might adhere to polystyrene: Mfp5 derived from mussel foot protein, and two computer-simulated polystyrene-binding peptides. These were fused with TasA to enhance the affinity of Bacillus subtilis for polystyrene fillers. Among them, Mfp5 exhibited broad adhesion to materials underwater with low specificity, while the PS-tags, generated via computer simulation, showed certain specificity for polystyrene.

Mfp5 is the major protein component in the distal region of mussel byssal plaques, and its core function is to mediate the firm binding of mussels to various underwater surfaces, serving as a key molecule for the biological property of "strong underwater adhesion" in mussels. Studies have shown that Mfp5 chains can adhere to various surfaces through interactions such as bidentate hydrogen bonds and metal complexation. They can also interact with adjacent Mfp5 chains via "double DOPA hydrogen bonds, aryloxy radical cross-linking, and physical chain entanglement" to form a stable network, thus exhibiting broad adhesive activity. Therefore, we aimed to design the TasA-Mfp5 fusion protein to enhance the adhesive capacity of the engineered bacteria(Kim et al., 2018).

They are essentially short peptides with PS-targeting binding ability. Through specific interactions such as hydrophobic interactions and van der Waals forces with the PS surface, they achieve precise localization on PS, while simultaneously possessing biocompatibility and biodegradability(Alshehri et al., 2025).

PS tags optimally designed by Alshehri et al. based on Evidence Deep Learning (EDL) and Biased Random-Key Genetic Algorithm (BrKGA) exhibit stronger adhesive performance. The table below lists three polystyrene-binding peptides with relatively high adsorption free energy designed by this method(Alshehri et al., 2025). We selected the two with the highest △G values as our binding moieties, aiming to design TasA-PS tag fusion proteins to enhance the adhesive capacity of the engineered bacteria

Since the two fusion proteins differ in their adhesive force to polystyrene materials, we intended to compare their adhesive abilities to polystyrene materials and select the fusion protein more suitable for polystyrene materials.

Peptides sequence	△G (kcal mol−1)
WWMRHMFAWRIF	-35.0
FWWRTIVWRHIR	-28.3
YFIWWWRMFFFR	-27.2

Table1. Best peptides found using EDL for Peptides sequence

During the design process of TasAnchor, we identified significant limitations in the directly constructed macromolecular fusion proteins: such proteins might exert adverse effects on the secretion function and self-assembly ability of TasA---two capabilities that are precisely the key factors determining the adhesive performance of TasAnchor, ultimately leading to impaired adhesive capacity(Reddington et al., 2015).

To address this issue, we proposed to engineer the TasA protein using the SpyTag-SpyCatcher system. The core reason for selecting this system lay in the small molecular weight of its components, which could minimize interference with the inherent secretion efficiency and self-assembly properties of TasA, thereby ensuring the stability of the basic adhesive function of TasAnchor.

Furthermore, by replacing the adhesion protein moiety in the SpyCatcher-Binding Protein complex, this engineering strategy could achieve high-strength adhesion adaptation to different filter media materials. This allowed TasAnchor to break through the limitations of material compatibility, endow it with stronger application universality, and lay a foundation for its popularization and application in multiple scenarios. We designed the SpyTag-SpyCatcher system using the Mfp5 protein---with a relatively large molecular weight---as the binding protein.

The SpyTag-SpyCatcher system is a highly efficient covalent bioconjugation tool, whose core function lies in forming stable isopeptide bonds through specific interactions between a short peptide (SpyTag) and a protein (SpyCatcher). It has been widely applied in the fields of biotechnology, biomaterials, and synthetic biology. The working mechanism of SpyTag and SpyCatcher critically relies on the synergistic effect of three conserved amino acids: Glu77 mediates proton transfer, and the amino group of Lys31 nucleophilically attacks the relevant group of Asp117, ultimately forming a stable isopeptide bond. We aimed to construct a SpyTag-SpyCatcher system using Mfp5 as the binding protein to enhance the adhesion of TasAnchor to filter media.

Cadmium is a colourless, odourless heavy metal whose potent toxicity and persistent pollution pose severe threats to water resource safety and ecological stability (Sun et al., 2021). Research indicates that the core of whole-cell Cd²⁺ biosensors comprises transcription factors that specifically bind Cd²⁺ and the red fluorescent protein gene mCherry (Kim et al., 2018). The cadmium-responsive transcription factor cadR gene belongs to the MerR family, specifically recognising and binding Cd²⁺ to activate transcription of downstream genes via the pCadR promoter (Lee et al., 2001). Consequently, we modularly assembled the cadR gene with the mCherry gene to construct a tool for the specific detection of Cd²⁺ in wastewater. We measured the fluorescence protein expression intensity of the engineered strain under different Cd²⁺ concentrations to validate the pathway's ability to achieve specific detection of Cd²⁺ in solution.

Biosorption is a widely employed technique for treating heavy metal ions in wastewater, owing to its advantages of low cost, high efficiency, absence of secondary pollution, broad applicability, and excellent selectivity (Wang et al. 2024). Research has demonstrated that the metallothionein SmtA from cyanobacteria exhibits specific and highly efficient adsorption of Cd²⁺ (Cavet et al. 2003). Capitalising on this property, we designed a fusion protein combining the SmtA protein with the Bacillus subtilis biofilm-forming protein TasA. This fusion protein was expressed on the surface of tasA-knockout Bacillus subtilis, enabling the strain to restore biofilm formation while simultaneously adsorbing and capturing Cd²⁺, ultimately reducing Cd²⁺ concentrations in wastewater. To validate the efficacy, we shall test the engineered bacteria's biofilm formation capacity and the residual Cd²⁺ concentration in treated wastewater under varying Cd²⁺ concentrations. These two parameters will confirm the strain's Cd²⁺ tolerance and adsorption performance.

The adsorption of cadmium ions by SmtA protein is pH sensitive. During the adsorption process, the cationic Cd²⁺ is negatively charged and attracted to SmtA. Cd²⁺ can be captured by the functional groups of SmtA protein and then fixed on the surface of SmtA. During the desorption process in acidic solution, H⁺ replaces heavy metal ions in functional groups by competing with binding sites. Finally, due to the charge change under low pH conditions, Cd²⁺ separated from the surface of SmtA.

In the functional validation module, we combined the Cd²⁺ sensing module and the Cd²⁺ adsorption module with the TasA protein. The Cd²⁺ sensing module was fused with TasA to form a fusion protein, which was expressed on the surface of Bacillus subtilis to achieve specific detection of Cd²⁺ in water. The Cd²⁺ adsorption module was also fused with TasA to form a fusion protein, which was expressed on the surface of Bacillus subtilis to achieve specific adsorption of Cd²⁺ in water. We tested the fluorescence protein expression intensity of the engineered strain under different Cd²⁺ concentrations to validate the pathway's ability to achieve specific detection of Cd²⁺ in solution. We also tested the engineered bacteria's biofilm formation capacity and the residual Cd²⁺ concentration in treated wastewater under varying Cd²⁺ concentrations to confirm the strain's Cd²⁺ tolerance and adsorption performance.