The following illustration shows the architecture of our Bacterial Cellulose Modification System:

Module I. Bacterial Cellulose Production

Komagataeibacter is a microorganism widely utilized in large-scale bacterial cellulose production. We have optimized the production process, particularly by refining nitrogen source selection and cultivation conditions, thereby significantly improving both the yield and quality of the produced bacterial cellulose.

Module II. Scaffold Protein Design and Production

Through genetic engineering, we successfully constructed an Escherichia coli strain that expresses a scaffold protein, Curlis Fiber-Spytag, capable of binding to bacterial cellulose. In this system, the Curlis Fiber domain provides the bacterial cellulose-binding functionality, while Spytag enables the heterologous linkage via the SpyCatcher system.

Module III. Target Protein Design and Production

We designed a SpyCatcher-POI (protein of interest) fusion with a standardized interface for expression and purification. Using two distinct color proteins as demonstration models, we validated both the expression and purification processes. In future applications, we anticipate that this approach will allow the facile substitution of various POIs through the Golden Gate cloning method.

Composition

Finally, our experimental results confirmed the high-efficiency binding of bacterial cellulose, the scaffold protein Curlis Fiber-Spytag, and the target protein SpyCatcher-POI.

These findings demonstrate the effectiveness and feasibility of this modular system, providing a POC and novel strategy for the modification and application of bacterial cellulose.

Impact of Nitrogen Source and Cultivation Container Size on Bacterial Cellulose Production Efficiency

We utilized the Komagataeibacter xylinus strain CGMCC 17276, obtained from the China General Microbiological Culture Collection Center (CGMCC), submitted by East China Normal University, to explore the impact of alternative nitrogen sources on bacterial cellulose (BC) production. The genome-scale metabolic model (GEM) for this strain was constructed using the CarveMe pipeline, and simulations were performed in the CobraPy environment. Growth media formulations were based on standard Lysogeny Broth (LB) and Hestrin-Schramm (HS) media, with soytone serving as the original nitrogen source[Figure 1].

We aimed to reduce production costs by substituting soytone with alternative, non-food biomass nitrogen sources, specifically corn steep liquor and cottonseed meal. The nitrogen sources were tested individually and in equal mixtures, while maintaining the total nitrogen concentration equivalent to the nitrogen content in 5 g/L soytone. Using the GEM, we simulated the impact of these nitrogen sources on BC production and validated the results in a 250 mL HS media system, cultivated in 10 x 16 cm stainless steel plates. To calculate the conversion efficiency of bacterial cellulose (BC) production, we used the formula: Conversion Efficiency (%)=(Dry Weight of BC Produced / Total Sugar Input )×100

Figure2 demonstrated that the original soytone condition provided the highest conversion efficiency for BC production, with the mixture of corn steep liquor and cottonseed meal yielding higher conversion rates than either alternative used alone. This study provides insight into the potential of using cost-effective, alternative nitrogen sources for the production of bacterial cellulose while maintaining high efficiency.

We conducted an experiment to investigate how the dimensions of stainless steel plates affect the conversion rate of bacterial cellulose (BC) production. Using the same volume of Hestrin-Schramm (HS) media, we compared two stainless steel plates: one measuring 10 x 16 cm and the other 15 x 27 cm. Each plate was filled with 250 mL of HS media, which we prepared by dissolving 20 g of glucose, 5 g of peptone, 5 g of yeast extract, 2.7 g of disodium phosphate, and 1.15 g of citric acid per liter of distilled water, followed by autoclaving. After the media cooled, we inoculated both plates with the bacterial strain *Komagataeibacter xylinus*, ensuring equal inoculum concentration and uniform distribution across the surface of the media.

We incubated both plates statically at 30°C for 5 days, during which we monitored the formation of cellulose mats. At the end of the incubation period, we harvested the BC mats, washed them thoroughly with distilled water, and treated them with 2M NaOH at 80°C to remove residual cells. After neutralizing with water, we dried the cellulose mats in an oven at 80°C until constant weight was achieved.To calculate the conversion efficiency of bacterial cellulose (BC) production, we used the formula: Conversion Efficiency (%)=(Dry Weight of BC Produced / Total Sugar Input )×100

Figure 3 showed that the smaller plate (10 x 16 cm) resulted in a conversion rate of 11.5% on average, while the larger plate (15 x 27 cm) achieved a conversion rate of over 20%. We believe that the larger surface area of the 15 x 27 cm plate allowed for better gas exchange and nutrient distribution, leading to more efficient cellulose production. This experiment demonstrates that the dimensions of the cultivation container play a critical role in bacterial cellulose production efficiency, even when the media volume remains constant.

1.Congo Red Staining Experiment

Congo Red Staining Confirms Expression and Self-Assembly of Curlis-Spytag Protein.[Figure 4]

We used the Congo Red staining experiment to detect the expression and self-assembly of Curlis-Spytag protein. Strains expressing curlis fiber and strains carrying the empty pET28a+ vector were separately plated on Congo Red plates and control plates with 0mM, 0.5mM, and 1mM IPTG. We pipetted 30 μL of overnight culture onto plates with varying concentrations of Congo Red and control plates, then incubated them at 30°C for 48 hours to observe changes in colony color and self-assembly.

We observed that on the Congo Red plates containing 0.5mM and 1mM IPTG, the strain expressing curlis fiber exhibited a pink color (colonies on the sides), while the strain with the empty pET28a+ vector (middle colony) remained white. Based on these results, we preliminarily conclude that the curlis fiber-expressing strain is capable of expressing CsgA, secreting it extracellularly, and assembling into curlis fibers, which appear pink in the presence of Congo Red. The same strain on control plates containing 0.5mM and 1mM IPTG appeared entirely white, indicating that the results observed on the Congo Red plates are specific.

2.Expression and Purification in Liquid

Successful Expression, Purification, and Detection of Curlis-Spytag Fibers via Western Blot

We took samples from the glycerol stock of Curlis-Spytag and streaked them onto LB agar plates containing kanamycin to isolate single colonies. Simultaneously, we streaked pET28a+ empty vector colonies as controls onto LB agar plates containing kanamycin. From the agar plates, we selected three Curlis-Spytag single colonies and inoculated them into LB liquid medium containing kanamycin, followed by overnight incubation at 37°C, 220 rpm. On the second day, we diluted the overnight cultures 1:100 into fresh LB medium and induced the cultures with IPTG at different concentrations when the OD600 reached 0.3. After induction, the cultures were incubated at 30°C, 220 rpm for another 24 hours. After 24 hours, we observed significant fibrous precipitates in liquid cultures, indicating that the Curlis fibers. were successfully expressed and self-assembled into fibrous structures[Figure 5].

After washing with ddH2O, 1X PBS, and 5X PBS, we discovered that the fibrous precipitates in the culture could not dissolve uniformly. To dissolve the Curlis fibers and detect the expression of Curlis through electrophoresis, we decided to use 8M urea and eventually succeeded in dissolving the fibrous precipitates. We then used the GST purification kit from Beyotime to purify the protein solution after dissolution in 8M urea, removing nonspecific proteins and obtaining high-purity Curlis target proteins. The purified Curlis protein samples were then analyzed using Western Blot (WB). The designed Curlis-Spytag fusion protein carried a GST tag, and we were able to detect the target protein bands using anti-GST tag antibodies.[Figure 6]

The results confirmed that the Curlis-Spytag fibers could be efficiently dissolved in 8M urea into a soluble state, and the fibrous precipitates obtained from two independent colonies could be detected in subsequent immunoblot experiments.

Successful Expression and Purification of Fusion Proteins

We streaked bacteria from glycerol stocks containing the GFP-SpyCatcher and amilCP-SpyCatcher plasmids, selected single colonies, and inoculated them into 50 ml of LB ste liquid medium for overnight incubation at 37°C. The bacterial cultures were collected by centrifugation at 10,000 rpm in 50 ml centrifuge tubes, and the pellets were resuspended and washed with 50 ml of 1X PBS buffer. Next, we used Thermo B-PER reagent to lyse the bacteria through sonication, and then performed protein purification using the His-Tag purification kit from Beyotime. The experimental results demonstrated that the plasmids we designed successfully expressed the GFP-SpyCatcher and amilCP-SpyCatcher fusion proteins in Escherichia coli. SDS-PAGE and Western blotting results showed that the fusion proteins GFP-SpyCatcher and amilCP-SpyCatcher had molecular weights of 40.38 kDa and 39.38 kDa, respectively. However, the molecular weight bands of the individual proteins GFP (26.8 kDa), amilCP (25.8 kDa), and SpyCatcher (12.58 kDa) were not observed on the gel, indicating that no monomeric proteins were expressed in our experiment. This confirmed the successful expression and purification of fusion proteins. Furthermore, the presence of the SpyCatcher peptide did not affect the properties of the color proteins.

We designed a control experiment to verify the binding of bacterial cellulose, Curlis-Spytag, and color protein-Spycatcher.

A piece of BC gel is cut into small blocks of similar size. Six stainless steel bowls (304 grade) are prepared, each containing 50 ml of ddH2O, 50 ml of POI-Spycatcher solution, and 50 ml of POI-Spycatcher-Curlis-Spytag binding solution. The similarly sized gel blocks are then placed into the solutions and incubated overnight at room temperature with shaking at 80 rpm. The following day, the cellulose is removed from the stainless steel bowls and placed into 50 ml of ddH2O. This is incubated at room temperature with shaking at 80 rpm for 6 hours. Subsequently, the gel is removed from each group, drained, and photographs are taken.

Figure 9 and Figure 10 represent the experiments where amilCP and sfGFP were bound to bacterial cellulose via scaffold proteins. A comparative analysis revealed that the experimental groups with mixed scaffold protein solutions exhibited a noticeably deeper color than those without the scaffold proteins. This indicates that our scaffold protein design enhances the binding efficiency between the Protein of Interest (POI) and bacterial cellulose.