Bubbly: an edible pearl for intestinal microbiome modulation
Presented by: UGent2_Belgium
Bénédicte Améry1 , Bram Jacobs1 , Milan Van Nuffel1 , Louis Coussement2 , Lien De Wannemaeker2 , Jose Manuel Salvador Lopez2 , Michiel Stock2 , Marjan De Mey3 & Wim Van Criekinge4.
1 iGEM student member, 2 Instructor, 3 Primary PI (Center for Synthetic Biology (CSB), Faculty of Bioscience Engineering, Ghent University, Belgium), 4 Secondary PI (BIOBIX, Faculty of Bioscience Engineering, Ghent University, Belgium).
Project Abstract
The WHO estimates that more than 300 million people worldwide suffer from depression (WHO, 2020).
Research has shown that gut microbiome alternations play a key role in a person’s stress
reactivity and influences anxiety-like and depression-like behaviors. In brief, if we can manage
to alter the gut microbiome’s functionality, the bidirectional communication might shift and
lead to less anxiety and depression-like behaviors. We believe that Bubbly can revert
depression and to bring relief to people suffering from depression. Our pearl, a novel food
supplement, is composed of three major components: naringenin, a flavonoid that works
anti-inflammatory, kojibiose, a prebiotic sugar with beneficial effects on the gut microbiome
and vitamin-B12. Now, we are producing naringenin in the lab and working on the
formulation of the pearl. Our pearl can be sold as an independent product and could also be
added to already existing foods as probiotics.
Team:UGent2 Belgium/Poster
Poster: UGent2_Belgium
Bubbly: an edible pearl for intestinal microbiome modulation
Project Goals
We want to produce an edible bubble which:
1. Tastes and feels good
2. Survives the stomach acid and releases in the small intestines
3. Helps reducing the microbial imbalance due to stress
This way hope to be able to play a supporting role in restoring the microbial balance in the gut and hence restoring the gut-brain axis.
Inspiration
Depression is all around us, it does not distinguish between age, wealth or health. And most important of all, due to COVID-19, we are experiencing a global mental health crisis like never before with some countries reporting twice as many cases.
We decided it was time to look for additional support for the standard treatment and started to look for a solution in our field of expertise.
We got our technical inspiration mainly from the combined feedback of our consulted experts. The main problem with our initial idea was our carrier function. With feedback from our principal investigators and literature research we decided to make a U-turn and tackle this specific carrier problem by making a carrier-bubble.
We decided it was time to look for additional support for the standard treatment and started to look for a solution in our field of expertise.
We got our technical inspiration mainly from the combined feedback of our consulted experts. The main problem with our initial idea was our carrier function. With feedback from our principal investigators and literature research we decided to make a U-turn and tackle this specific carrier problem by making a carrier-bubble.
Problem
The WHO estimates that more than 300 million people worldwide suffer from depression. It is also the world's leading cause of functional disorder. Also, the recent COVID-19 pandemic led to a tremendous increase in the prevalence of depression symptoms in people throughout the world. Since it would take years to reduce the long-term effects of this world-wide crisis, the prevalence of depression will still increase in the coming years(Ettman et al., 2020; Mencaccia & Salvib, 2021).
Patients nowadays get drugs like antidepressants prescribed to combat these feelings, whereas only one-third of all patients get the desired release of depression and anxiety. Research has shown that the intestinal microbiome alterations play a crucial role in a person's stress reactivity and influences depression-like behaviors. It is called the (microbiome-)gut-brain axis. The relative abundance of Bacteroidetes species (e.g. Prevotella) is increased, and the relative abundance of Firmicutes (e.g. Faecalibacterium and Ruminococcus) is decreased in patients with depression (Liang et al., 2018). This leads to a disbalance in the homeostasis of gut bacteria, also known as dysbiosis. Furthermore, dysbiosis occurs together with a state of inflammation in the gut. Hence leading to increased physical discomfort.
Patients nowadays get drugs like antidepressants prescribed to combat these feelings, whereas only one-third of all patients get the desired release of depression and anxiety. Research has shown that the intestinal microbiome alterations play a crucial role in a person's stress reactivity and influences depression-like behaviors. It is called the (microbiome-)gut-brain axis. The relative abundance of Bacteroidetes species (e.g. Prevotella) is increased, and the relative abundance of Firmicutes (e.g. Faecalibacterium and Ruminococcus) is decreased in patients with depression (Liang et al., 2018). This leads to a disbalance in the homeostasis of gut bacteria, also known as dysbiosis. Furthermore, dysbiosis occurs together with a state of inflammation in the gut. Hence leading to increased physical discomfort.
Idea
Following the study on depression, we concluded that there are multiple factors involved in causing depression but also in maintaining the state of depression. The first two factors are dysbiosis and inflammation. They are positively related to each other, and it is not yet known what causes what (Vitetta et al., 2014). Is it the inflammation that causes dysbiosis? Or dysbiosis that causes inflammation? So we decided to tackle both factors at the same time.
A third factor is genetics; some people have a predisposition towards depression. This does not mean that we cannot aid in reducing the symptoms associated with the psychopathology of depression. As a last factor, there is the environment. Traumatizing events like losing a job, death of a close relative or a friend can make a person more susceptible to depression (Steenbergen et al., 2015; Axt-Gadermann & Rautenberg, 2017). We want to work on reducing the first two factors actively. As for the other factors, research indicates the bilateral relationship between the severity of the pathology and the gut microbiome, hence decreasing the severity of the symptoms is our goal here.
We want to select components that have anti-inflammatory effects and promote the growth of good gut bacteria. Furthermore, we know from previous research on vitamin deficiencies that people with a higher amount of vitamin B12 have a better mental health (Berk et al., 2013; Dash et al., 2015). Also, inflammation and dysbiosis reduce the uptake of vitamin B12. Hence we hypothesized that it might be a good idea to add vitamin B12.
But how do we get all these compounds towards their site of action? For this, we hypothesized creating a certain carrier system, that contains these three components and shuttles them to their site of action. The design needs to resist the gastric environment of the stomach and has to be released over time of the ingredients it contains.
From literature we know that there are four genes involved in the tyrosine ammonia-lyase (TAL)-pathway to produce naringenin (Ganesan et al., 2017).
We will need to introduce these four genes in E. coli TOP10 if we want to produce naringenin. However, production of naringenin in E. coli is not that successful. Hence, we decided to look for another host that is more suitable to produce naringenin. A host that preferably grows on green and cheap materials. By working with multiple hosts, we will need to work with a plasmid that is suitable for multiple hosts, a selection marker to see if the organism took up the plasmid and a constitutive promoter that works in multiple hosts.
A third factor is genetics; some people have a predisposition towards depression. This does not mean that we cannot aid in reducing the symptoms associated with the psychopathology of depression. As a last factor, there is the environment. Traumatizing events like losing a job, death of a close relative or a friend can make a person more susceptible to depression (Steenbergen et al., 2015; Axt-Gadermann & Rautenberg, 2017). We want to work on reducing the first two factors actively. As for the other factors, research indicates the bilateral relationship between the severity of the pathology and the gut microbiome, hence decreasing the severity of the symptoms is our goal here.
We want to select components that have anti-inflammatory effects and promote the growth of good gut bacteria. Furthermore, we know from previous research on vitamin deficiencies that people with a higher amount of vitamin B12 have a better mental health (Berk et al., 2013; Dash et al., 2015). Also, inflammation and dysbiosis reduce the uptake of vitamin B12. Hence we hypothesized that it might be a good idea to add vitamin B12.
But how do we get all these compounds towards their site of action? For this, we hypothesized creating a certain carrier system, that contains these three components and shuttles them to their site of action. The design needs to resist the gastric environment of the stomach and has to be released over time of the ingredients it contains.
From literature we know that there are four genes involved in the tyrosine ammonia-lyase (TAL)-pathway to produce naringenin (Ganesan et al., 2017).
We will need to introduce these four genes in E. coli TOP10 if we want to produce naringenin. However, production of naringenin in E. coli is not that successful. Hence, we decided to look for another host that is more suitable to produce naringenin. A host that preferably grows on green and cheap materials. By working with multiple hosts, we will need to work with a plasmid that is suitable for multiple hosts, a selection marker to see if the organism took up the plasmid and a constitutive promoter that works in multiple hosts.
Methodology
Our methodology consists of two parts:
We developed a carrier system, a bubble, that transports microbiome modulating components towards specific sites in the gut.
To study release of the components, we used a modeling system.
Next, we wanted to produce one of those microbiome-modulating components. The component of interest is naringenin.
We developed a carrier system, a bubble, that transports microbiome modulating components towards specific sites in the gut.
To study release of the components, we used a modeling system.
Next, we wanted to produce one of those microbiome-modulating components. The component of interest is naringenin.
Naringenin Production
Current production methods of naringenin are non-sustainable and non-green. New ways of naringenin production using microorganisms are rising but struggle to get high quantities of naringenin. So, we attempted to produce naringenin using Cupriavidus necator PHB-4. This strain of C. necator has enhanced acetyl–CoA flux. A limited flux of acetyl-CoA is the bottleneck to produce high titers of naringenin. Furthermore C. necator is able to use glycerol as a carbon source, which is a waste stream from bioethanol production. Hence, C. necator is a green host that is well-suited to produce naringenin (Trantas et al., 2009, 2015).
The genes we want to use to produce naringenin were selected after a brief literature review and after reviewing which genes are present in-house. These are fjTAL, tyrosine ammonia-lyase from Flavobacterium johnsoniae (Jendresen et al., 2015; Haslinger & Prather, 2019); pc4CL, 4-coumaryl-CoA ligase from Petroselinum crispum (Wu et al., 2014; Zang et al., 2019); phCHS_MUT, chalcone synthase from Petunia hybrida (Wu et al., 2014; Zang et al., 2019); msCHI, chalcone isomerase from Medicago sativa (Wu et al., 2014; Zang et al., 2019).
Multiple hosts are involved in the process of cloning, testing, and producing naringenin, so we need a broad-host-range plasmid and promotor. Hence, we used pBBR1-MCS2 plasmid, which is a broad-host-range-plasmid. For the expression of the genes, we used the constitutive P22 promoter (De Mey et al., 2007).
We first tested the efficiency of the promoter in different hosts by evaluating the expression levels of mKate2, a fluorescent reporter protein, over a period of 24 hours of E.coli and 48 hours for C. necator. One result was obtained for the promoter efficiency in E.coli K12 MG1655 and indicated that there is expression of mKate2 by the P22 promoter.
The genes we want to use to produce naringenin were selected after a brief literature review and after reviewing which genes are present in-house. These are fjTAL, tyrosine ammonia-lyase from Flavobacterium johnsoniae (Jendresen et al., 2015; Haslinger & Prather, 2019); pc4CL, 4-coumaryl-CoA ligase from Petroselinum crispum (Wu et al., 2014; Zang et al., 2019); phCHS_MUT, chalcone synthase from Petunia hybrida (Wu et al., 2014; Zang et al., 2019); msCHI, chalcone isomerase from Medicago sativa (Wu et al., 2014; Zang et al., 2019).
Multiple hosts are involved in the process of cloning, testing, and producing naringenin, so we need a broad-host-range plasmid and promotor. Hence, we used pBBR1-MCS2 plasmid, which is a broad-host-range-plasmid. For the expression of the genes, we used the constitutive P22 promoter (De Mey et al., 2007).
We first tested the efficiency of the promoter in different hosts by evaluating the expression levels of mKate2, a fluorescent reporter protein, over a period of 24 hours of E.coli and 48 hours for C. necator. One result was obtained for the promoter efficiency in E.coli K12 MG1655 and indicated that there is expression of mKate2 by the P22 promoter.
After successful testing of the promoter, we wanted to compare production efficiency of E. coli K12 MG1655 and C. necator PHB-4. First, we wanted to test this in Erlenmeyer flasks and then continue to upscale to fermentation reactors. Sampling would occur over a period of 24 and respectively 48 hours. We also wanted to test production of naringenin when C. necator
uses glycerol as a carbon-source. Next to upscaling the production method, we were also working on the optimization of the downstream processing of naringenin extraction, purification, and detection.
Building the Bubble
To tackle depression, we selected three active components that affect the gut microbiome.
To produce a bubble that selectively delivers components to the gut, we contacted a company called CosmoGroup. They aided in formulating the bubble.
Naringenin, a plant flavonoid, that has anti-inflammatory effects. The reason for this is that dysbiosis is correlated with inflammation and inflammation triggers dysbiosis. Hence, naringenin has the capacity to reduce the inflammation and restore the microbiome balance (Bugianesi et al. 2002).
Kojibiose, a low-calorie sweetener with prebiotic effects, feeds only the good gut bacteria and adds a sweet flavour to our bubble (Beerens et al., 2017).
Vitamin B12 directly influences depression and is related to a higher prevalence of Faecalibacterium prausnitzii
To produce a bubble that selectively delivers components to the gut, we contacted a company called CosmoGroup. They aided in formulating the bubble.
Iterative testing of the bubble is done by using tests that are an in vitro representation of the gut and by using dissolution tests. All of these methods are standardized. Later testing is done using the SHIME, which is the closest representation of in vitro testing possible.
Additional note, for this last paragraph only the theoretical groundwork has been laid.
The Model
We created three models that replace a planned experiment. In this experiment, we wished to measure the release of naringenin ,one of our three active components, from the bubble to the environment. Due to limited access to the lab, we came up with our models.
We consider three different approaches:
We wish to remark that these models are a simplification of reality where we do not take factors as pH and intestinal motility into account. These are all improvements that will be incorporated in new models and experiments. We postpone them to a more suitable time, when access to the lab is less restricted.
Diffusion within a solid sphere: The naringenin molecules migrate to the pearl's surface and diffuse in the medium. The model is based on a system of partial differential equations.
Bubble as a solid sphere: The naringenin concentration is constant in the pearl. This model is valuable when the pearl is small and/or the diffusion is very rapid. This model is based on a ordinary differential equation.
Bubble as a dissolving sphere: The bubble dissolves; this means that diffusion is negligible since naringenin is released when the bubble dissolves. This model is based on a ordinary differential equation.
We wish to remark that these models are a simplification of reality where we do not take factors as pH and intestinal motility into account. These are all improvements that will be incorporated in new models and experiments. We postpone them to a more suitable time, when access to the lab is less restricted.
Integrated Human Practices
Our integrated human practices are dividable into three main parts: consulting experts, getting in touch with our target audience and reflecting on our project.
First, we contacted experts from the beginning to the end of our project. They guided us through every step and were constantly involved in our project. They gave us feedback regarding technical details like experts in medical fields or experts with a background in pharmaceutical chemistry and so on. Equally as important were the experts who provided us with knowledge about the social context. They helped us with questions like which people are suffering from depression, how do we get in touch with them, how should we ask our questions to remain neutral and get relevant information.
This way, we were able to get in touch with our audience by making for example a survey which gauged their interest in this product and providing us with feedback straight from our target group.
As a last part, we talked with experts about our final idea and how it would still be feasible and functional in a real-world scenario. This way we could refine our product in some domains, however some other issues are still not solved. For example how can we make sure our product will not be used as a mere symptom relief drug which does not work on the external situation which got them there in the first place. It should be considered as a food supplement, used additionally with their treatment.
First, we contacted experts from the beginning to the end of our project. They guided us through every step and were constantly involved in our project. They gave us feedback regarding technical details like experts in medical fields or experts with a background in pharmaceutical chemistry and so on. Equally as important were the experts who provided us with knowledge about the social context. They helped us with questions like which people are suffering from depression, how do we get in touch with them, how should we ask our questions to remain neutral and get relevant information.
This way, we were able to get in touch with our audience by making for example a survey which gauged their interest in this product and providing us with feedback straight from our target group.
As a last part, we talked with experts about our final idea and how it would still be feasible and functional in a real-world scenario. This way we could refine our product in some domains, however some other issues are still not solved. For example how can we make sure our product will not be used as a mere symptom relief drug which does not work on the external situation which got them there in the first place. It should be considered as a food supplement, used additionally with their treatment.
Conclusion
As all teams did, we thought this would be a year full of practical lab work, perfect for practice our theoretical knowledge. However before we set foot in the lab, even before we had our final idea on the drawing board, COVID-19 struck. Frankly at that point we thought it was over, we talked to other teams about how they were going to deliver anything more than ideas and some of them had great tips. However, luckily for us, the labs did eventually reopen albeit with strict limitations. This is the reason that we still managed to take some of our ideas from the drawing board into the real world.
From the two species of bacteria we used, only one of them yielded results. Not surprisingly, this was the E. coli rather than the Cupriavidus necator. However it turned out, after testing if the modification was successful, we possibly did find some naringenin production in these cultures but our tests were not able to pick this up. For E. coli K12 MG1655 however got substantial results, expressing mKate2 by the P22 promoter.
All things considered, we were lucky to be able to spend some time on our practical aspect, however with mixed results. Further research is therefore much needed.
From the two species of bacteria we used, only one of them yielded results. Not surprisingly, this was the E. coli rather than the Cupriavidus necator. However it turned out, after testing if the modification was successful, we possibly did find some naringenin production in these cultures but our tests were not able to pick this up. For E. coli K12 MG1655 however got substantial results, expressing mKate2 by the P22 promoter.
All things considered, we were lucky to be able to spend some time on our practical aspect, however with mixed results. Further research is therefore much needed.
Acknowledgements
Acknowledgements
Prof. Dr. Ir Marjan De Mey
Prof. Dr. Ir Wim Van Crieckinge
Ir. Lien De Wannemaeker
Ir. Louis Coussement
José Manuel Lopez
Dr. Ir. Michiel Stock