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We contributed an innovative foundational component to the Registry platform: the artificial glucan-binding protein CMC, designated as BBa_K5332002. This component ingeniously integrates the mechanism of glucan-binding proteins with the scaffold protein CipC from Clostridium cellulovorans. Additionally, we incorporated the mCherry fluorescent protein sequence and the outer membrane protein A (OmpA) signal peptide, designing proteins with multiple copy numbers to optimize performance. To enhance the persistence of engineered strain FMK in the gut and its response in expressing anti-inflammatory factors, we designed a novel artificial glucan-binding protein CMC by simulating the natural mechanisms of glucan-binding proteins. CMC acts as a "bridge" between gut microbiota and the intestinal surface, effectively attracting and recruiting beneficial gut probiotics. This design not only promotes stable adherence of the engineered strain in the gut but also significantly enhances its efficacy in alleviating intestinal inflammation and modulating the gut microenvironment.

To ensure the effective and sustained retention of engineered bacteria in the gut, and their responsive expression of anti-inflammatory factors, we identified this as a key to enhancing therapeutic efficacy. After reviewing extensive literature, we found that the main component of intestinal mucus is the highly glycosylated glycoprotein MUC2, which contains various glycan structures such as Core1, Core2, and Core3. Additionally, glucans are important polysaccharides produced by bacteria and fungi. The beneficial properties of probiotics are often related to their production of extracellular polysaccharides (EPS), with many probiotics exposing glucans on their surfaces, such as the α-glucans of Lactobacillus. Some pathogenic bacteria, like Salmonella, have cell walls containing endotoxin lipopolysaccharides (LPS) with O antigens.

We realized that leveraging glucan-binding properties could lead to the design of an adhesion factor that acts as a "bridge" between gut microbiota and the intestinal surface, thereby stabilizing the attachment of engineered bacteria in the gut. Inspired by the binding of glucan-binding proteins to glucan substrates, we designed the CBMcipc domain. CBMcipc, derived from the scaffold protein CipC of *Clostridium cellulovorans*, includes a type III cellulose-binding domain (CBD), a hydrophilic domain, and two hydrophobic domains. The CBD domain endows CBMcipc with glucan-binding capability.

To evaluate how the copy number of CBMcipc might affect its glucan-binding ability, we developed a mathematical model to simulate changes in probiotic and pathogenic bacteria on intestinal cells after introducing different engineered strains: EcM, EcCM, EcCMC, and EcCMCC into a diseased gut. The results were expressed using a health status index, calculated as the number of intracellular probiotics minus the number of pathogens. Negative values are shown in blue, positive values in red, with deeper colors indicating higher quantities of probiotics or pathogens.

In the visualization, CM (Fig. 1a) had the poorest outcome, with pathogens predominantly occupying the gut. Although the differences between CMC (Fig. 1b) and CMCC (Fig. 1c) were not substantial, CMC achieved a healthy state—where probiotics dominate—more rapidly than CMCC. Therefore, we selected CMC as the adhesion factor for our experiment.

To identify the most effective glucan-binding protein, we introduced the mCherry fluorescent protein sequence and designed Clostridium cellulolyticum CipC (CBMcipc) proteins with varying copy numbers: M, CM, CMC, and CMCC. We cloned the corresponding genes (M, CM, CMC, CMCC) into the pET-28a plasmid and introduced them into the E. coli BL21 strain, constructing synthetic strains EcM, EcCM, EcCMC, and EcCMCC.

As shown in Fig. 2a, the proteins M, CM, CMC, and CMCC consist of mCherry alone, mCherry with one copy of CBMcipc, mCherry with two copies of CBMcipc, and mCherry with three copies of CBMcipc, respectively. The N-terminally linked outer membrane protein A (OmpA) signal peptide facilitates translocation across the inner membrane, localizing the proteins on the outer membrane surface, thus enabling surface display.

To validate these properties, we stained the cells with FITC-labeled glucan (FITC-glucan) and examined them using flow cytometry and confocal microscopy. After incubating the cells with FITC-glucan for 10 minutes, EcM cells exhibited almost no FITC fluorescence, while EcCM, EcCMC, and EcCMCC cells showed significant FITC fluorescence. Among the three strains containing CBMcipc, EcCMC cells displayed the highest FITC fluorescence (Fig. 2b and Fig. 2c), indicating that EcCMC has the greatest glucan-binding capability.

Confocal microscopy images and quantitative fluorescence analysis further revealed that EcM, EcCM, and EcCMCC cells exhibited a uniform distribution of mCherry fluorescence, while EcCMC cells showed a bilateral distribution of mCherry (Fig. 2d). This indicates that CMC demonstrates stronger surface display capability. Since the surface display of binding proteins is essential for extracellular glucan binding, this characteristic of EcCMC likely contributes to its enhanced glucan-binding ability.

Next, we conducted oral gavage experiments in mice using EcCM, EcCMC, and EcCMCC mixed with *Lactobacillus plantarum* (a probiotic). We evaluated the adhesion rate, defined as the ratio of beneficial bacteria in the gut 12 hours post-gavage to the amount administered. Results showed that EcCMC had the highest adhesion rate (Fig. 3). When the probiotic was replaced with *Salmonella enterica* serovar Typhimurium (a pathogen), the adhesion rates were much lower (Fig. 3), demonstrating the adhesion factor's preference for binding with probiotics.

In summary, we ingeniously leveraged the mechanism of glucan-binding proteins to design the artificial glucan-binding protein CMC. This bio-inspired approach ensures both effectiveness and biocompatibility, providing a novel solution for the stable adhesion of engineered bacteria. Through extensive literature review, we selected the type III cellulose-binding domain (CBD) from the scaffold protein CipC of *Clostridium cellulovorans* due to its superior glucan-binding capability. This choice not only enhanced binding efficiency but also maintained stability in the complex gut environment.

The successful design of the CMC protein was a result of thorough literature research and systematic experimental validation by our team. By designing CBMcipc proteins with different copy numbers (M, CM, CMC, CMCC), we precisely regulated binding strength and stability to optimize protein performance and identify optimal binding conditions. The incorporation of the mCherry fluorescent protein allowed for direct observation of binding results through fluorescence intensity, enhancing the clarity and accuracy of experiments. Using the outer membrane protein A (OmpA) signal peptide, we localized the protein to the cell outer membrane, ensuring full exposure and functionality of the binding domain, thus improving the efficiency of protein display.

Our design not only increased the adhesion of engineered bacteria but also facilitated the recruitment and stabilization of probiotics in the gut, achieving dual functionality. This binding domain can serve as a modular component in our plasmid, offering wide adaptability and flexibility. Through these innovative designs, our CMC protein demonstrated significant novelty and practicality both theoretically and practically, making it an excellent foundational component.

Details available at https://parts.igem.org/Part:BBa_K5332001