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The overuse of antibiotics can lead to the rapid spread of antibiotic-resistant bacteria, becoming a significant challenge to global public health. Currently, there is a worldwide call for alternatives to antibiotics [1]. We are committed to developing a more efficient method for producing β-glucan, aimed at serving as an alternative to antibiotics in response to stringent global restrictions on antibiotic use, while ensuring production efficiency and sustainability in the livestock industry. β-glucan, as a natural immunomodulator and functional ingredient, has garnered increasing attention due to its good safety and biocompatibility. Many traditional glucan synthesis techniques currently suffer from low yields, high costs, and insufficient purity, which severely restrict the application potential of β-glucan in industrial and agricultural sectors [2]. To address these challenges, this project aims to enhance the capacity of Aureobasidium melanogenum BZ-11 for large-scale fermentation production of β-glucan through innovative protein engineering and metabolic engineering strategies. Aureobasidium spp. has been widely used in synthetic biology, with extensive research conducted over the years [3]. However, A. melanogenum BZ-11 has not previously been used as a chassis strain for fermentation production. Therefore, during the implementation of the project, we have undertaken a series of systematic experiments to validate its feasibility and effectiveness as a β-glucan production platform, as well as to verify the practicality of the hardware design used.
To verify the extracellular polysaccharide components of A. melanogenum BZ-11 and to clarify the direction for purifying β-glucan production, we analyzed the extracellular polysaccharides in the culture medium of this strain using High-Performance Liquid Chromatography (HPLC).
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Figure 1: HPLC analysis of the extracellular polysaccharide components of A. melanogenum BZ-11. |
The results indicate that A. melanogenum BZ-11 can effectively secrete abundant extracellular polysaccharides, with the main components being pullulan and β-glucan. This finding not only provides a clear direction for further purification but also facilitates the construction of a metabolic network for A. melanogenum BZ-11 in dry experiments, allowing for targeted gene knockouts, particularly focusing on the removal of pullulan to enhance the purity and yield of the target product. This result also provides a theoretical basis for our subsequent applications, indicating that A. melanogenum BZ-11 has the potential to become an efficient platform for β-glucan production.
Previous studies have shown that β-1,3-1,6-glucan has good immunostimulatory effects and exhibits broad application potential in various fields, especially in antibiotic alternatives. In contrast, β-glucan lacking 1,6 branches has low immunological activity and is less effective as an antibiotic substitute [3]. To verify the structure of β-glucan produced by A. melanogenum BZ-11, we employed Nuclear Magnetic Resonance (NMR) analysis.
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Figure 2: NMR results. |
The NMR analysis results reveal peaks at 86.84 ppm, 77.38 ppm, 73.34 ppm, 70.84 ppm, and 61.44 ppm, corresponding to the signals of carbons C3, C5, C2, C4, and C6, respectively. A signal at 103 ppm indicates the presence of terminal carbon, while a signal peak for terminal hydrogen appears at 4.5 ppm. These data collectively confirm that the extracellular β-glucan produced by A. melanogenum BZ-11 has the structure of β-1,3-1,6-glucan, ensuring its broad applicability in food, feed, and other industrial applications. This analytical result further demonstrates the potential of our chassis strain in terms of specificity and functionality for the target product, laying a foundation for future modifications and applications.
Vitreoscilla Hemoglobin (VHb) is a hemoglobin from Vitreoscilla, known for enhancing microbial survival under low oxygen conditions and promoting total protein synthesis, and it has been applied in industrial fermentation [4]. We found that the oxygen uptake capacity of A. melanogenum BZ-11 is relatively poor, which limits its β-glucan synthesis. Therefore, we designed a strategy to combine cell wall anchor proteins with VHb to enhance the oxygen uptake ability of the cells, thereby promoting β-glucan synthesis.
Cell wall proteins are a series of cell wall anchor proteins, and we identified their presence in A. melanogenum BZ-11 as well as several other species. To determine the most effective cell wall anchoring protein, we fused predicted N-terminal and C-terminal sequences with the target protein in preliminary experiments and used SignalP to simulate cleavage sites. Ultimately, we identified AM.CWP as the ideal anchoring protein for A. melanogenum BZ-11 based on the simulation results. To validate the effectiveness of the predicted cell wall anchoring protein, we fused several cell wall proteins with EGFP using restriction enzyme ligation, and connected them to the pNATX13-loxp plasmid for expression in Aureobasidium melanogenum BZ-11, aiming to confirm localization by observing fluorescent signals on the cell wall.
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Figure 3: Plasmid maps of CWP, Sed, and Tip cell wall proteins fused with EGFP. |
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Figure 4: A. melanogenum BZ-11 expressing the cell wall protein-EGFP expression vector. |
Fluorescence microscopy observations confirmed the fluorescence intensity of EGFP connected to different cell wall proteins under 488 nm excitation light. The results indicated that AM.CWP displayed better anchoring effects compared to AM.Sed and AM.Tip. This not only validates our previous predictions but also confirms AM.CWP as an ideal anchoring protein for connecting VHb, providing strong support for further enhancing β-glucan production efficiency. The success of these results offers guidance for the design of subsequent engineered strains, suggesting that we can achieve efficient production of the target product through this strategy.
To assess the feasibility of large-scale production, we conducted small-scale fermentation experiments to evaluate the impact of different cultivation conditions on β-glucan production, optimize the culture medium, and attempt to further increase the β-glucan yield of BZWΔags2-1/2. First, we optimized the type of nitrogen source and found that the extracellular β-glucan yield was significantly higher when using NaNO3 compared to the previously used nitrogen source NH2SO4. We then established a NaNO3 concentration gradient of 0.1 g/L, 0.2 g/L, 1 g/L, 2 g/L, and 3 g/L for optimization. Further optimization of the carbon source concentration was performed, selecting a carbon source concentration gradient of 90 g/L, 100 g/L, 110 g/L, 120 g/L, 130 g/L, and 140 g/L. We measured extracellular β-glucan, cell wall β-glucan, and cell biomass content under different conditions.
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Figure 5: Optimization of the nitrogen source in the culture medium. |
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Figure 6: Optimization of the carbon source in the culture medium. |
From the experimental data, we found that the highest β-glucan concentration, reaching 26.97 g/L, occurred at a nitrogen source concentration of 0.2 g/L ammonium nitrate; the highest β-glucan concentration of 29.64 g/L was achieved at a glucose concentration of 130 g/L. We discovered that the yield under optimal conditions surpassed many previous methods for producing β-glucan [5][6]. These experiments lay a solid foundation for subsequent industrial production and demonstrate the significant potential of A. melanogenum BZ-11 as an industrial fermentation platform.
Feed is one of the main expenses in intensive aquaculture, and optimizing food intake can promote growth and body composition, improve feed conversion efficiency, and reduce nutrient loss, which is crucial for fish farmers [7]. Based on feedback from our iHP, the amount of feed for fish in closed water bodies requires long-term experiential knowledge; if too little feed is given, fish cannot obtain sufficient nutrition. Additionally, the rapid sedimentation of feed means that fish do not consume all the food they encounter, and most aquatic animals rarely forage at the bottom. Overfeeding can lead to feed accumulation on the bottom, resulting in pollution. To assess the specific pollution level caused by feed on water quality, we conducted validation experiments. We added 15 g of fish feed (3 mm pellets, consisting of fish meat, extruded soybean meal, seaweed powder, brewer's yeast powder, flour, fish oil, soybean lecithin, ferrous sulfate, sodium selenite, manganese sulfate, zinc sulfate, vitamins, microorganisms, and natural minerals) to a 50 L water body (60 cm x 30 cm x 35 cm) and observed the water at around 29°C at 0 h, 8 h, and 16 h intervals.
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Figure 7: The impact of a certain amount of sinking feed on water quality in 50 L of water. |
We found that after 8 hours, the water showed some degree of pollution, changing from clear to yellow-brown. By 16 hours, the pollution became severe, with the water turning from clear to murky yellow-green and emitting an unpleasant odor. This indicates that excessive feed significantly pollutes water quality, adversely affecting aquaculture development. This deepened our understanding of feed pollution levels and highlighted the seriousness of sinking feed on water quality. Consequently, we began designing appropriate hardware to seek solutions in future research, ensuring the sustainability and ecological friendliness of aquaculture.
We measured the settling rate of fish feed and found that in our simulated aquatic environment, the settling speed was approximately 0.075 m/s. This means that in a 3-meter-deep pond, the feed can sink to the bottom in just 40 seconds. This realization highlighted the need for a device capable of holding the feed to effectively prevent it from sinking. Consequently, we began designing the hardware, creating a feeder consisting of three parts: a float, a connecting rod, and a feed carrier. We used SolidWorks to establish the model, designing the float as a spherical body with a diameter of 3 cm and a thickness of 2 mm; the connecting rod was 8 cm long with a diameter of 3 mm, and the feed carrier was a curved disc with a thickness of 3 mm and a diameter of 10 cm. We produced a physical prototype of the hardware using 3D printing for testing.
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Figure 8: The impact of a certain amount of sinking feed on water quality in 50 L of water. |
After conducting feed placement tests, we found that the feeder could stably float on the water surface while holding the feed. This confirmed the feasibility of our hardware design. However, we also observed that it was challenging to precisely direct the feed onto the carrier in open water, resulting in a significant amount of feed still sinking to the bottom during feeding.
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[2] Zhu, F., Du, B., & Xu, B. (2016). A critical review on production and industrial applications of beta-glucans. Food Hydrocolloids, 52, 275–288. https://doi.org/10.1016/j.foodhyd.2015.07.003
[3] Wang, P., Jia, S.-L., Liu, G.-L., Chi, Z., & Chi, Z.-M. (2022). Aureobasidium spp. and their applications in biotechnology. Process Biochemistry, 116, 72–83. https://doi.org/10.1016/j.procbio.2022.03.006
[4] Suzuki, T., Kusano, K., Kondo, N., Nishikawa, K., Kuge, T., & Ohno, N. (2021). Biological Activity of High-Purity β-1,3-1,6-Glucan Derived from the Black Yeast Aureobasidium pullulans A Literature Review. Nutrients, 13(1), 242. https://doi.org/10.3390/nu13010242
[5] Zhang, L., Li, Y., Wang, Z., Xia, Y., Chen, W., & Tang, K. (2007). Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. Biotechnology Advances, 25(2), 123–136. https://doi.org/10.1016/j.biotechadv.2006.11.001
[6] Maheshwari, G., Sowrirajan, S., & Joseph, B. (2017). Extraction and Isolation of β-Glucan from Grain Sources-A Review. Journal of Food Science, 82(7), 1535–1545. https://doi.org/10.1111/1750-3841.13765
[7] Varelas, V., Liouni, M., Calokerinos, A. C., & Nerantzis, E. T. (2015). An evaluation study of different methods for the production ofβ-D-glucan from yeast biomass. Drug Testing and Analysis, 8(1), 46–55. https://doi.org/10.1002/dta.1833
[8] Assan, D., Huang, Y., Mustapha, U. F., Addah, M. N., Li, G., & Chen, H. (2021). Fish Feed Intake, Feeding Behavior, and the Physiological Response of Apelin to Fasting and Refeeding. Frontiers in endocrinology, 12, 798903. https://doi.org/10.3389/fendo.2021.798903