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Our project was inspired by a student's shopping experience at the end of 2023. When he reached the checkout, he was shocked to see his total far exceeded his expectations. Panic-stricken, he asked the cashier if there was a mistake, since he was trying to save money for the iGEM competition. The cashier calmly assured him that the machine never makes errors and kindly pointed out the two boxes of antibiotic-free eggs he had picked up. It dawned on him that the eggs he had randomly selected were much more expensive than the ordinary eggs he usually bought. He finally managed to return the two cartons of eggs. And when these eggs were returned, they asked the salesperson why the price of the non-antibiotic eggs was so high. The salesman said that recently people have begun to pay attention to the antibiotic residues and use of animal products, and more and more people tend to choose non-antibiotic products, so supermarkets stock non-antibiotic products to cater to the needs of these people. These antibiotic-free products are often produced without the use of any antibiotics, and thus cost more to protect the health of farmed animals. The selling price will naturally be higher than the average product.
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Figure 1: Antibiotic-free eggs |
He shared this story with us after a group meeting. Subsequent further searches revealed that not only China, but the whole world is facing the same problem. As a result, we began to experiment with synthetic biology approaches to create practical alternatives to antibiotics, with a view to lowering the cost of production of antimicrobial-free animal products.
The discovery of antibiotics commenced with the introduction of penicillin in the early twentieth century, and their subsequent pervasive utilization in healthcare and agriculture has transformed modern medicine and animal husbandry.
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Figure 2: Antibiotics |
While the misuse of antibiotics in the healthcare industry is widely publicized, the reliance on antibiotics in agriculture, especially in livestock, is equally significant. In addition to therapeutic antibiotics, antibiotics are also added to feed. In the context of poultry production, the administration of antibiotics is a common practice with the objective of preventing the occurrence of diseases in livestock or limiting their spread. Additionally, various studies have demonstrated the efficacy of certain antibiotics in promoting weight gain and improving feed efficiency in livestock[1].
At the same time, irrational antibiotic use in animal husbandry is widespread. In 2020, the use of antibiotics in farm animals in several countries and regions in Asia, Oceania, and South America exceeded the internationally recommended standard of 50 mg/PCU [2].
Among them, China's use reached 208 mg/PCU, which is about four times the international standard (As shown in Figure 1) [2].
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Figure 3: Antibiotic usage in livestock, 2020 [2]. (Milligrams of total antibiotic use per kilogram of livestock. This is adjusted for differences in livestock numbers and species by standardizing to apopulation-corrected unit ‘PCU’.) |
Livestock husbandry is crucial for ensuring food security. The Food and Agriculture Organization of the United Nations indicates that livestock contributes 40% of global agricultural value and supports the livelihoods and food security of 1.3 billion people [3]. Livestock husbandry is a vast industry, and global demand for livestock products continues to rise overall.
Over the past 50 years, global meat production has rapidly increased, more than quadrupling since 1961 (As shown in Figure 2).
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Figure 4: Global meat production, 1961 to 2022[4]. |
According to the study by Komarek et al. (2021), if income and population trends continue along the medium trajectory, the global average demand for protein from red meat (beef, sheep, goat, and pork), poultry, milk, and eggs is expected to increase by 38% from 2020 to 2050, with per capita protein demand also rising by 38% [5].
China, with its large population, has significant demand for animal products and is a major livestock husbandry country. In 2022, China ranked first in the production of pigs and poultry and third in the number of cattle globally (As shown in Figure 5-7).
As of now, the World Health Organization has declared antimicrobial resistance as one of the top ten global public health threats facing humanity [9]. Particularly concerning is that nearly all antibiotics used clinically have developed resistance [10]. The lack of new antibiotics or antimicrobial agents under development poses significant pressure on human medical systems due to bacterial infections.
In 2017, the WHO recommended that farmers and the food industry cease the routine use of antibiotics to promote healthy animal growth and prevent disease. This new recommendation aims to preserve the effectiveness of antibiotics critical to human medicine by reducing unnecessary use in animals [11]. China has also been taking action to manage antibiotic use. According to the Ministry of Agriculture and Rural Affairs of the People's Republic of China Announcement No. 194, to ensure the safety of animal-derived food and public health, China has fully banned the addition of antibiotics to animal feed [12].
Regarding the control of antibiotics in feed, EU member states had previously imposed a strict ban on all antibiotic growth promoters. However, this ban has led to unexpected consequences for the EU's animal production industry, such as increased animal infections and decreased productivity. Additionally, the higher incidence of disease due to the ban has significantly increased the use of therapeutic antibiotics and disinfectants, leading to an overall increase in antibiotic use in animals [13].
The European experience indicates that controlling antibiotic use in feed is not simply a matter of restricting or banning their use. After the removal of antibiotics from feed, livestock husbandry urgently needs alternatives to address the rise in animal mortality and morbidity. Options being considered include antimicrobial vaccines, immunomodulators, phage therapy, lysine, antimicrobial peptides, and feed enzymes.
We aimed to design an animal immunomodulator that enables animals to leverage their enhanced immune systems to combat various microbial infections. After reviewing a substantial amount of relevant literature, we found that β-glucans show considerable promise. Animal studies indicate that using β-1,3/β-1,6 glucans as a feed component to protect animals from microbial harm demonstrates significant health benefits [14]. Additionally, research suggests that β-glucans can also serve as an additive in aquatic environments to enhance the immune function of aquaculture animals.
β-Glucans are polymers linked by linear glycosidic bonds and are important bioactive polysaccharides widely distributed in plants, bacteria, and fungi, possessing significant nutritional value and biological functions. Depending on the purification methods, extracted β-glucans may contain various impurities, such as mannoproteins, pullulan, melanin, or other cell wall components. β-Glucans extracted from different sources often exhibit varying molecular structures, solubilities, and other properties [15].
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Figure 8: β-Glucan structure–activity relationship [15]. |
β-Glucans can act as immunomodulators, enhancing immunity by training the immune mechanisms of the animal's immune system.
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Figure 9: Trained immunity and tolerance: two opposite functional programmes of innate immunity [16]. |
Dietary β-glucans, after internalization, activate the oxidative burst response in PBMC phagolysosomes through the Dectin-1 structure or the CR3 receptor, thereby conducting immune training [15].
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Figure 10: Immune response overview [15]. |
Increasing research indicates that consuming β-glucans can have beneficial effects on animal immune health, making it a promising alternative to antibiotics for protecting livestock health. However, as our investigation deepened, we discovered that only β-glucans with certain mass and branching degrees can serve as effective immunomodulators to enhance immunity. These specific β-glucans are often found in higher concentrations in yeast and fungi. This implies that chemically producing β-glucans with the required branching is quite challenging.
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Figure 11: The structures of β-glucans and chemical modified β-glucans[17]. |
Consequently, isolating and extracting β-glucans after scaling up microbial cultivation appears to be a better approach. However, the cost of commercially extracting and purifying β-glucans from yeast cell walls using existing technologies is very high, and the resulting product purity is generally low (often below 50%), necessitating further purification. Yeasts of the genus Aureobasidium (commonly known as "black yeast") are highly promising β-glucan-producing strains. However, they face purification issues due to contamination with impurities like melanin and pullulan. Kang et al. developed a mutant strain NP1221 by knocking out the pullulan synthase gene in the wild-type strain, which produced β-glucans without generating pullulan during fermentation. Nevertheless, the yield of this mutant strain remains low [18]. Currently, the highest yield of β-glucans achieved is only 9.9 g/L, which poses a significant challenge for the commercialization of glucans [19]. Therefore, to truly make β-glucans a viable alternative, we need a new pathway for synthesizing more economical and practical β-glucan products.
Consequently, OUC-Haide undertook an investigation and developed a novel β-glucan synthesis pathway utilising synthetic biology, based on black yeast Aureobasidium melanogenum, which was isolated from marine mangroves.
Black yeast is notable for its high levels of β-glucan not only in its cell wall components but also in its extracellular secretions. Consequently, black yeast is an optimal chassis strain for production purposes, as it possesses inherent advantages. However, the secretion of melanin and pullulan presents a challenge for the purification and extraction of β-glucan. The objective was to modify the genome of Saccharomyces cerevisiae in order to enhance the oxygen uptake of the strain and increase the β-glucan production while eliminating the secretion of the aforementioned impurities. Following the optimisation of the culture medium and the exploration of culture conditions, an efficient and economical method for the production of β-glucan was developed. Further details on the modifications made to black yeast can be accessed via the link below.
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Figure 12: Separation of extracellular and cell wall β-glucan. |
The two principal factors impeding the widespread utilisation of beta glucan are the synthetic processes employed in its production and the elevated prices that are typically associated with it. The advent of BEST AB-Free has brought about a more economical and environmentally benign process for the production of β-glucan.
At the same time, the creation of novel synthetic pathways is being pursued, alongside an active investigation into the potential for additional applications.
In addition to enhancing immune function, β-glucan is also safe and non-toxic. Derived from natural sources, β-glucan is non-toxic, safe for long-term use, and harmless to the environment and the human body. Furthermore, glucan is being investigated for its potential use as an aquaculture feed additive. The utilisation of beta glucan as an additive in water bodies circumvents the necessity for humans to regulate pollution from the aquatic environment into the great cycle of the biosphere, whilst simultaneously safeguarding the wellbeing of aquatic animals. OUC-Haide has designed an automated, precise, anti-sedimentation, adjustable and automated feeding system that allows for the significant impact of beta glucan in aquaculture (for further details, please click to jump to Hardware).
The antioxidant properties of β-glucan are increasingly being elucidated by a growing body of research, with investigations extending to diverse fields such as cosmetics and food additives. It is likely that β-glucan will continue to play a role in these and other areas in the future.
For further applications, please refer to the Imp & HP section.
[1] Ricke, S., Jarquin, R., & Hanning, I. (2012b). Antimicrobials in animal feed: benefits and limitations. In Elsevier eBooks (pp. 411–431). https://doi.org/10.1533/9780857093615.4.411
[2] Mulchandani et al. (2023) – processed by Our World in Data. “Antimicrobial usage in livestock (mg per population corrected units)” [dataset]. Mulchandani et al. (2023) [original data]. https://ourworldindata.org/grapher/antibiotic-usage-in-livestock
[3] Bilotto, F., Harrison, M. T., Vibart, R., Mackay, A., Christie-Whitehead, K. M., Ferreira, C. S., Cottrell, R. S., Forster, D., & Chang, J. (2024b). Towards resilient, inclusive, sustainable livestock farming systems. Trends in Food Science & Technology, 104668. https://doi.org/10.1016/j.tifs.2024.104668
[4] “Data Page: Total meat production”, part of the following publication: Hannah Ritchie, Pablo Rosado and Max Roser (2023) - “Agricultural Production”. Data adapted from Food and Agriculture Organization of the United Nations. Retrieved from https://ourworldindata.org/grapher/global-meat-production [online resource]
[5] Komarek, A. M., Dunston, S., Enahoro, D., Godfray, H. C. J., Herrero, M., Mason-D’Croz, D., Rich, K. M., Scarborough, P., Springmann, M., Sulser, T. B., Wiebe, K., & Willenbockel, D. (2021b). Income, consumer preferences, and the future of livestock-derived food demand. Global Environmental Change, 70, 102343. https://doi.org/10.1016/j.gloenvcha.2021.102343
[6] “Data Page: Pig meat production”, part of the following publication: Hannah Ritchie, Pablo Rosado and Max Roser (2023) - “Agricultural Production”. Data adapted from Food and Agriculture Organization of the United Nations. Retrieved from https://ourworldindata.org/grapher/pigmeat-production-tonnes [online resource]
[7] “Data Page: Poultry meat production”, part of the following publication: Hannah Ritchie, Pablo Rosado and Max Roser (2023) - “Agricultural Production”. Data adapted from Food and Agriculture Organization of the United Nations. Retrieved from https://ourworldindata.org/grapher/poultry-production-tonnes [online resource]
[8] “Data Page: Beef and buffalo meat production”, part of the following publication: Hannah Ritchie, Pablo Rosado and Max Roser (2023) - “Agricultural Production”. Data adapted from Food and Agriculture Organization of the United Nations. Retrieved from https://ourworldindata.org/grapher/beef-and-buffalo-meat-production-tonnes [online resource]
[9] World Health Organization: WHO. (2023b, November 21). Antimicrobial resistance. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance
[10] Bhardwaj, S., Mehra, P., Dhanjal, D. S., Sharma, P., Sharma, V., Singh, R., Nepovimova, E., Chopra, C., & Kuča, K. (2022b). Antibiotics and antibiotic resistance- flipsides of the same coin. Current Pharmaceutical Design, 28(28), 2312–2329. https://doi.org/10.2174/1381612828666220608120238
[11] World Health Organization: WHO. (2017, November 7). Stop using antibiotics in healthy animals to prevent the spread of antibiotic resistance. News Release. https://www.who.int/news/item/07-11-2017-stop-using-antibiotics-in-healthy-animals-to-prevent-the-spread-of-antibiotic-resistance
[12] 中华人民共和国农业农村部. (2019). 公告第194号 [通知]. https://www.moa.gov.cn/
[13] Cheng, G., Hao, H., Xie, S., Wang, X., Dai, M., Huang, L., & Yuan, Z. (2014b). Antibiotic alternatives: the substitution of antibiotics in animal husbandry? Frontiers in Microbiology, 5. https://doi.org/10.3389/fmicb.2014.00217
[14] Williams, D. L., Mueller, A., & Browder, W. (1996b). Glucan-Based macrophage stimulators. Clinical Immunotherapeutics, 5(5), 392–399. https://doi.org/10.1007/bf03259335
[15] De Marco Castro, E., Calder, P. C., & Roche, H. M. (2020b). β‐1,3/1,6‐Glucans and Immunity: State of the Art and Future Directions. Molecular Nutrition & Food Research, 65(1). https://doi.org/10.1002/mnfr.201901071
[16] Netea, M. G., Domínguez-Andrés, J., Barreiro, L. B., Chavakis, T., Divangahi, M., Fuchs, E., Joosten, L. a. B., Van Der Meer, J. W. M., Mhlanga, M. M., Mulder, W. J. M., Riksen, N. P., Schlitzer, A., Schultze, J. L., Benn, C. S., Sun, J. C., Xavier, R. J., & Latz, E. (2020). Defining trained immunity and its role in health and disease. Nature Reviews. Immunology, 20(6), 375–388. https://doi.org/10.1038/s41577-020-0285-6
[17] Zhu, F., Du, B., & Xu, B. (2015). 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
[18] Kang, B., Yang, H., Choi, N., Ahn, K., Park, C., Yoon, B., & Kim, M. (2009b). Production of pure β-glucan by Aureobasidium pullulans after pullulan synthetase gene disruption. Biotechnology Letters, 32(1), 137–142. https://doi.org/10.1007/s10529-009-0127-x
[19] Moriya, N., Moriya, Y., Nomura, H., Kusano, K., Asada, Y., Uchiyama, H., Park, E. Y., & Okabe, M. (2013). Improved β-glucan yield using an Aureobasidium pullulans M-2 mutant strain in a 200-L pilot scale fermentor targeting industrial mass production. Biotechnology and Bioprocess Engineering, 18(6), 1083–1089. https://doi.org/10.1007/s12257-013-0516-9