Abstract
Antibiotic abuse and soil salinization are two world-wide problems. Inspired by the probiotic and acid producing characteristics of Clostridium tyrobutyricum (C. tyrobutyricum), we aim to make C. tyrobutyricum facultative anaerobic and add it to animal feed to tackle the two issues. The butyric acid produced by the bacteria is good for both biological intestinal tract and alkaline soil. By making C. tyrobutyricum facultative anaerobic, it can function well under anaerobic condition in gastrointestinal tract as a probiotics and under aerobic condition in alkaline soil as soil-improving bacteria. This goal can be achieved by introducing the dps and aceE genes into C. tyrobutyricum. dps protein can enhance the viability of the bacteria in aerobic environment mainly by protecting DNA. PDH expressed by aceE can substitute the main metabolic enzyme PFOR in the bacteria which is inactivated in the presence of oxygen.
1. Project inspiration and background
Soil Salinization:
Ever since subsistence agriculture took root along the banks of the Tigris-Euphrates rivers, soil salinization started haunting generations of farmers to come. In semi-arid and arid lands, soil salinization results in saline-alkali soil and could directly lead to the loss of arable lands and thus bring significant damage to the agriculture. It is currently one of the two main threats to agricultural sustainability worldwide.
Nowadays, more than 6% of topsoil around the world is saline-alkali soil [1]. Jointly, they cost around 27.3 billion USD every year due to declined crop production in irrigated land [2]. Globally, of all the cultivated land, around 23% is saline-alkali [3]. Moreover, scholars estimate that, if left unintended, the total area of saline-alkali soil can quickly rise to taking up 50% of the world’s arable land by the year 2050 [4].
Antibiotic abuse:
In addition, we are also concerned about antibiotic abuse, which refers to the misuse and overuse of antibiotics. Due to antibiotic overuse, antibiotic residue not only biomagnifies through the food chain and bioaccumulate in human body, but also causes bacteria that were previously susceptible to antibiotics to evolve antibiotic resistance. When infecting humans and animals, these resistant strains of bacteria cause infections that are harder to treat than those caused by non-resistant bacteria [5]. Antibiotic resistance caused by antibiotic abuse leads to higher medical costs, prolonged hospital stays, and increased mortality [6].
The phenomenon is seen more commonly in today’s agriculture and aquaculture industries. As adding antibiotics and hormonal drugs helps poultry and domestic animals mature at a higher rate, many breeders use antibiotics in large quantities to avoid potential diseases.
2. Our host organism C. tyrobutyricum
Clostridium tyrobutyricum is an obligate anaerobe. It has a long history of being used in Asia as a probiotic [7]. Its primary metabolite, butyric acid, has multiple benefits that we wish to utilize to concurrently provide solution to the two issues stated above.
On the one hand, butyric acid, as studies have shown, can repair and help regenerate epithelial tissue in the intestine. Researchers found that, compared to the control group, weaned piglets whose basal diet was supplemented with C. tyrobutyricum had higher final body weight, average daily gain, feed conversion rate, and lowered diarrhea rate [8]. This feature makes C. tyrobutyricum a promising candidate for substituting antibiotics.
On the other hand, butyric acid’s acidic nature makes it capable of neutralizing saline soil. As butyric acid will be present in the feces of animals whose feed was supplemented with C. tyrobutyricum, feed additive that contains the bacterium will be able to improve first an animal’s immunity and then soil function at various saline-alkaline fields. We hope to make C. tyrobutyricum survive external to animal body, thus allowing it to grow and reproduce in the wild and continue its salt-affected soil transformation journey.
3. The obstacles and our goal
The main metabolic pathway of C. tyrobutyricum, an obligate anaerobe, will be inactivated instantly in the presence of oxygen as the iron-sulfur site of PFOR, one of the central enzymes of the pathway, oxidizes. Therefore, we direct our focus to creating a whole new metabolic pathway for C. tyrobutyricum under aerobic environments by introducing the genes encoding the Ace-E protein into C. tyrobutyricum.
Apart from that, we also introduced another protein called DNA-binding protein from starved cells (Dps), which belongs to the ferritin superfamily. There are two mechanisms by which Dps protein achieves its purpose of protecting DNA from damage due to oxidization. The first of the two is the prevention of Fe2+ ions from reacting with H 2 O2 by combining with the Fe 2+ ion so that the toxic hydroxyl radical is eliminated. The second is related to the protein’s ability to store and release manganese, which plays an important role in maintaining manganese homeostasis. When Dps protein forms a polymer, the His and Asp amino acids on it form a chelate with excessive Fe 2+, thus preventing the emergence of hydroxyl radical. Dps knock-out bacteria are less viable under oxidative stress compared to the wild type[9].
With a clear goal in mind, we experimented with two genes encoding the Dps and Ace-E proteins. By introducing the two proteins with a pmtl82151 plasmid from E. coli, we inserted the two genes into C. tyrobutyricum so that under aerobic environments, it is free from oxygen damages and achieves a similar metabolic function.
In addition to these, we added a lactose inducible promotor Plac to monitor the gene expression of the two proteins in order to alleviate the bacteria’s burden in anaerobic environments. When the bacteria are in aerobic environment, such as when they are being prepared for feed additive and transportation, lactose will be added so that aerobic respiration and normal growth are possible.
As our genetically modified C. tyrobutyricum is processed into power and added into animal feed, it is expected to stay in the animal bowels and produce butyric acid there. Then, when excreted to the wild, it is expected to survive and continue its metabolic reactions of converting organic matter in the feces and the wild into butyric acid, thus neutralizing salt-affected soils.
References
[1] FAO. Global Map of Salt-affected Soils (GSASmap). GSASmap | Global Soil Partnership | Food and Agriculture Organization of the United Nations. (n.d.). Retrieved September 10, 2022, from https://www.fao.org/global-soil-partnership/gsasmap/en
[2] Qadir, M., Qureshi, R. H., & Ahmad, N. (1997). Nutrient availability in a calcareous saline‐sodic soil during vegetative bioremediation. Arid Soil Research and Rehabilitation, 11(4), 343–352. https://doi.org/10.1080/15324989709381487
[3] Massoud FI. (1981). Salt affected soils at a global scale for control. FAO Land and Water Development Division Technical Paper, Rome, Italy.
[4] Jamil, A., Riaz, S., Ashraf, M., & Foolad, M. R. (2011). Gene expression profiling of plants under salt stress. Critical Reviews in Plant Sciences, 30(5), 435–458. https://doi.org/10.1080/07352689.2011.605739
[5] Duan, X. (2012). The harm of antibiotic abuse and the scientific use of antibiotics. Contemporary medicine, 18(24):19-20. DOI:10.3969/j.issn.1009-4393.2012.24.010.
[6] World Health Organization. (n.d.). Antibiotic resistance. World Health Organization. Retrieved September 10, 2022, from https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance
[7] Wang, K., Cao, G., Zhang, H., Li, Q., & Yang, C. (2019). Effects of clostridium butyricum and enterococcus faecalis on growth performance, immune function, intestinal morphology, volatile fatty acids, and intestinal flora in a piglet model. Food & Function, 10(12), 7844–7854.
[8] Zhao, X., Yang, J., Wang, L., Lin, H., & Sun, S. (2017). Protection mechanism of clostridium butyricum against salmonella enteritidis infection in broilers. Frontiers in Microbiology, 8. https://doi.org/10.3389/fmicb.2017.01523
[9] Suzuki, T., Kobayashi, S., Miyahira, K., Sugiyama, M., Katsuki, K., & Ishikawa, M. (2021). DNA-binding protein from starvation cells traps intracellular free-divalent iron and plays an important role in oxidative stress resistance in Acetobacter pasteurianus NBRC 3283. Journal of Bioscience and Bioengineering, 131(3), 256-263.
