Irritable bowel syndrome (IBS) is relatively unknown, even though the disorder affects approximately one in seven people and has a big impact on their quality of life. There are no medications or molecular diagnostic tools available so far. This is why we, the Viennese iGEM team of 2021, want to help.
We had two important goals for our project: First, we wanted to develop an effective and convenient therapy for fructan sensitivity. For this, we planned to engineer lactobacilli to secrete fructan-degrading enzymes. But the questions remained of how to best deliver the enzymes and how to make sure that a constant amount of these enzymes is present at the right place. Which brought us to the second part of our project: creating a novel enzyme delivery platform by not just encapsulating the enzymes, but the bacteria itself. This offers the advantage of overcoming many issues of standard oral enzyme therapy.
During our iGEM year, we designed over 70 novel composite parts and tested several encapsulation methods and materials. This page will guide you from our initial idea to the final Friendzyme design.
It all started with a presentation on food intolerances held by one of our friends. The prevalence of those and the lack of treatments sparked our project idea, motivated us to begin our literature research, and to contact experts in the field. Especially helpful to us was Prof. Dr. Vogelsang, specialist in the field of gastroenterology, who introduced us to the recent success in the treatment of celiac disease using enzyme therapy. He recommended us to concentrate on one of the gastrointestinal disorders that get less medical attention such as IBS and non-celiac gluten sensitivity (NCGS) if we really wanted to make a change.
We did further research on those topics and found a common denominator in literature: FODMAPs (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols). IBS has been associated with them for some time (1) and low-FODMAP diets have been shown to alleviate symptoms of many patients.(2) Furthermore, even though non-celiac gluten sensitivity, as the name suggests, has been thought to be caused by gluten, recent findings point towards fructans as the root cause.(3)(4)
The mechanism of the disorder is not completely understood yet. However, researchers assume that bacteria ferment FODMAPs, which are indigestible for humans, in a pathophysiological fashion resulting in the excessive formation of gases and other metabolic products, causing adverse effects. This leads to symptoms like bloating, abdominal pain, and headaches.(5) An unbalanced microbiome has been associated with FODMAP sensitivity, but individual problematic bacterial strains could not be identified so far.(6)
The only treatment available for IBS is a low-FODMAP diet, which is highly restrictive, and should not be tried without a professional guidance – and even then they should be kept short to not cause other health problems due to malnutrition.(7) We discovered that fructans are the most problematic type of FODMAPs. They cause the majority of symptoms and are the most prevalent FODMAPs in our diet.(8) Thus, we decided to focus on targeting those.
To confirm our findings, we talked to Prof. Jane Muir from the Monash University in Australia, who is specialized in low-FODMAP diets and guides. She encouraged us to follow this path and substantiated our feeling that an enzyme therapy would benefit people with IBS.
Fructans are polymers with a sucrose core and fructose units attached. They can be separated into three major groups: inulins, levans and graminins.(9) While a levan is characterized by β-2,6-linked fructosyl residues and an inulin by β-2,1-linked fructosyl residues, a graminin contains both of these linkages.
We quickly found enzymes which are suitable for the task of safely digesting each major fructan type, and chose a levanase, an endo-inulinase and an invertase for this purpose. A more detailed description of our selection process and the enzyme sequences can be found here.
Next level oral therapy?
There has been a lot of investment into the development of enzyme therapies going on in the last couple of years but only a few drugs have reached the market so far. This discrepancy can be explained by three major problems which current enzyme therapies face: short in-vivo half-life, lack of tissue specificity, and immunogenicity.(10)
To control the short in-vivo half-life, we decided to go to the next level and not only deliver the enzymes but full-live expression host cells, which produce the fructan-degrading enzymes. This allows for a constant, high-level release of the pharmaceutically active enzymes over a longer time period.
This, however, raised the following questions and problems:
- How to pass the low pH of the stomach?
- How to ensure that the engineered organism releases the enzymes exactly where we want it?
- How to stop lactobacilli from spreading and overtaking the gut?
To overcome these issues, we decided to encapsulate our host in a biocontainer which can protect cells from the low pH of the stomach. Additionall, it has a variable pore size which can be fine-tuned to allow the escape of enzymes and smaller molecules while trapping cells inside. For the biocontainer material, we needed something that was stable and had good biocompatibility. We decided to go for a scaffold made of cellulose sulfate (negatively charged) and polyDADMAC (positively charged), which upon getting in contact with each other, instantly form a polyelectrolyte complex and, thus, very stable polymeric structures.
Both polymers fulfilled our requirements of being safe and stable. Additionally, cellulose sulfate is a renewable resource that can be synthesized from cellulose, the main component of wood. After developing our plan for the scaffold, we got in contact with Prof. Andreas Bernkop-Schnürch, one of the leading experts in the field of thiomers, who confirmed that our scaffold materials will be suitable for the encapsulation of live bacteria and that thiolation will give the scaffold mucoadhesive properties.(11)
Another major aspect of our scaffold was that we wanted it to be mucoadhesive. There are several ways of achieving this. As discussed with Prof. Bernkop-Schnürch, we planned to introduce thiol-groups to the polymers of the scaffold, a process which is called thiolation. These so-called “Thiomers” (thiolated polymers) are currently a very hot topic in research as they are able to form very stable, covalent bonds with the surfaces of the mucosa which itself is heavily interconnected by this type of bond, namely disulfide bridges. The interaction of the thiol-groups with the mucosal surface makes the scaffold stick to it and, thus, allows the release of the enzymes at the same spot over a larger timespan.(12) This also helps us to control the tissue specificity, as the first mucosa after passing the stomach is the beginning of the small intestine, which is exactly where we would like to degrade the fructans: shortly after their release from ingested food in the stomach, so that microorganisms do not have the chance to turn them into gas excessively. In our case we used the biocompatible thiomer chitosan, which already posseses mucoadhesive properties in it's native state. A more in-depth description of our bio container can be found here.
Regarding the host organism, we decided to use Lactobacillus plantarum for several reasons: as natural inhabitant of the human gut, it is considered a GRAS, generally recognized as safe, organisms and has also been shown to be probiotic and beneficial for the human gut.(13) For instance, it is able to degrade amylase trypsin inhibitors, which are also discussed problematic components for IBS patients.(14) We also chose it for practical reasons, as it is a well-established model organism, and our PIs have long-term experience working with it.
Taking all together, our idea of a next-level oral enzyme therapy, featuring an engineered Lactobacillus which degrades fructans inside a biocontainer, was born.
(1) Marsh, A., Eslick, E. M., & Eslick, G. D. (2016). Does a diet low in FODMAPs reduce symptoms associated with functional gastrointestinal disorders? A comprehensive systematic review and meta-analysis. European journal of nutrition, 55(3), 897–906. https://doi.org/10.1007/s00394-015-0922-1
(2) Staudacher, H., Irving, P., Lomer, M. et al. (2014). Mechanisms and efficacy of dietary FODMAP restriction in IBS. Nat Rev Gastroenterol Hepatol 11, 256–266. https://doi.org/10.1038/nrgastro.2013.259
(3) Skodje, G. I., Sarna, V. K., Minelle, I. H., Rolfsen, K. L., Muir, J. G., Gibson, P. R., Veierød, M. B., Henriksen, C., & Lundin, K. (2018). Fructan, Rather Than Gluten, Induces Symptoms in Patients With Self-Reported Non-Celiac Gluten Sensitivity. Gastroenterology, 154(3), 529–539.e2. https://doi.org/10.1053/j.gastro.2017.10.040
(4) Priyanka, P., Gayam, S., & Kupec, J. T. (2018). The Role of a Low Fermentable Oligosaccharides, Disaccharides, Monosaccharides, and Polyol Diet in Nonceliac Gluten Sensitivity. Gastroenterology research and practice, 2018, 1561476. https://doi.org/10.1155/2018/1561476
(5) Central Clinical School, Monash University. (2015). IBS symptoms, the low FODMAP diet and the Monash app that can help
(6) Serena, G., Kelly, C. P., & Fasano, A. (2019). Nondietary Therapies for Celiac Disease. Gastroenterology clinics of North America, 48(1), 145–163. https://doi.org/10.1016/j.gtc.2018.09.011
(7) Barrett J. S. (2017). How to institute the low-FODMAP diet. Journal of gastroenterology and hepatology, 32 Suppl 1, 8–10. https://doi.org/10.1111/jgh.13686
(8) Tuck, C., Ly, E., Bogatyrev, A., Costetsou, I., Gibson, P., Barrett, J., & Muir, J. (2018). Fermentable short chain carbohydrate (FODMAP) content of common plant-based foods and processed foods suitable for vegetarian- and vegan-based eating patterns. Journal of human nutrition and dietetics : the official journal of the British Dietetic Association, 31(3), 422–435. https://doi.org/10.1111/jhn.12546
(9) Chibbar, R. N., Jaiswal, S., Gangola, M., Båga, M. (2016). Carbohydrate Metabolism. Reference Module in Food Science, Elsevier. ISBN 9780081005965. https://doi.org/10.1016/B978-0-08-100596-5.00089-5
(10) De la Fuente, M., Lombardero, L., Gómez-González, A., Solari, C., Angulo-Barturen, I., Acera, A., Vecino, E., Astigarraga, E., and Barreda-Gómez, G. (2021). Enzyme Therapy: Current Challenges and Future Perspectives. International Journal of Molecular Sciences 22, 9181.
(11) Gunzburg, W. H., Aung, M. M., Toa, P., Ng, S., Read, E., Tan, W. J., Brandtner, E. M., Dangerfield, J., & Salmons, B. (2020). Efficient protection of microorganisms for delivery to the intestinal tract by cellulose sulfate encapsulation. Microbial cell factories, 19(1), 216. https://doi.org/10.1186/s12934-020-01465-3
(12) Leichner, C., Jelkmann, M., & Bernkop-Schnürch, A. (2019). Thiolated polymers: Bioinspired polymers utilizing one of the most important bridging structures in nature. Advanced drug delivery reviews, 151-152, 191–221. https://doi.org/10.1016/j.addr.2019.04.007
(13) Chiang, S. S., & Pan, T. M. (2012). Beneficial effects of Lactobacillus paracasei subsp. paracasei NTU 101 and its fermented products. Applied microbiology and biotechnology, 93(3), 903–916. https://doi.org/10.1007/s00253-011-3753-x
(14) Caminero, A., McCarville, J. L., Zevallos, V. F., Pigrau, M., Yu, X. B., Jury, J., Galipeau, H. J., Clarizio, A. V., Casqueiro, J., Murray, J. A., Collins, S. M., Alaedini, A., Bercik, P., Schuppan, D., & Verdu, E. F. (2019). Lactobacilli Degrade Wheat Amylase Trypsin Inhibitors to Reduce Intestinal Dysfunction Induced by Immunogenic Wheat Proteins. Gastroenterology, 156(8), 2266–2280. https://doi.org/10.1053/j.gastro.2019.02.028