We have two essential parts in our project, the production of naringenin in Escherichia coli and Cupriavidus necator and the design and concept evaluation and testing of our edible pearl. Due to COVID-19, we only got in the lab late, and the production and testing have mostly remained theoretic. To make the first concept of the edible pearl, we worked together with a company, Cosmo Group. But they were also hindered by the effects of COVID-19. We have split the practical work into what we were able to do and what we had prepared to do.
The Centre for Synthetic Biology (CSB) was willing to let us work in their lab and to use their resources. The production of naringenin was under the guidance of Ir. Lien De Wannemaeker.
The versatile health benefits recently discovered in different epidemiological studies led to an increasing interest in research on flavonoids from plant sources (Kumar & Pandey, 2013; Hermenean et al., 2013). Recent research showed that flavonoids have anti-cancer activities, anti-oxidative (Bugianesi et al., 2002), anti-obesity activities. Some flavonoids even have potential anti-viral activities (Goris et al., 2020). Naringenin is harvested from plants via extensive separation and purification efforts, making it not cost-effective (Trantas et al., 2015). An alternative is to produce flavonoids from de novo chemical synthesis, but this requires toxic solvents in unsustainable and non-green chemical reactions (Kamran Khan et al., 2014). In contrast, the production of flavonoids via engineered microbial hosts is a green and more sustainable route to take (Carbonell et al., 2018; Robinson et al., 2020).
Naringenin is composed of a 15-carbon ring skeleton with a three-ring structure. Naringenin is a secondary metabolite produced by plants primarily (Ganesan et al., 2017). Four genes play a crucial activity in the production of naringenin: tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI).
Figure 1: Reaction pathway of naringenin with four key enzymes depicted; tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI). Via TAL, tyrosine undergoes deamination to p-coumaric acid. 4CL executes the addition of a coenzyme A to the carboxy of coumaric acid. This addition activates the molecule and forms coumaroyl-CoA. The CHS enzyme is responsible for decarboxylation and condensation of coumaroyl-CoA with three malonyl-CoA molecules, resulting in naringenin chalcone. The final step, isomerization of naringenin chalcone into naringenin, is done via CHI. Chemdraw_Bénédicte Amery
As seen in Figure 1, three malonyl-CoA precursors contribute to only one chalcone. Hence, we want to produce naringenin in a microorganism that has a high flux of malonyl-CoA. We believe that a higher flux of malonyl-CoA can lead to higher titres of naringenin. A candidate microorganism that is being hypothesized to have a high flux of malonyl-CoA is Cupriavidus necator.
Due to limited time, we will not design new (biobrick) parts but use existing parts of the hosting lab and test these in different organisms, being E. coli K12 MG1655 and C. necator. Naringenin is produced by expressing four heterologous genes, Figure 1:
- 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., 2).
Promotor used for the expression of the four naringenin pathway genes is the constitutive P22 promotor (De Mey et al., 2007). The fluorescent protein mKate2) is used as a reporter to test the functionality of this constitutive promoter in C. necator. Plasmid backbone pBBR1MCS2 is used as it is a mobilizable shuttle and expression vector which can replicate in many Gram-negative bacteria, including E. coli and C. necator. DNA assembly is done by an in-house developed method called SSAP (Coussement et al., 2017).
We will use E. coli TOP10 for cloning purposes. E.coli K12 MG1655 and, C. necator (PHB-4) will be used for naringenin production.
Figure 2: Plasmid design for testing P22 constitutive promotor (De Mey et al., 2007) using fluorescent molecule mKate2 (Shcherbo et al., 2007) as a reporter gene. Kanamycin is used as a selection marker. Benchling_LienDeWannemaeker
Figure 3: Plasmid design for testing production of naringenin pathway genes using P22 constitutive promotor(De Mey et al, 2007). Kanamycin is used as a selection marker. Benchling_LienDeWannemaeker
Step 1: plasmid assembly.
Construction of both plasmids, i.e. pBBR1MCS2_P22_RBS_mKate2 and pBBR1MCS2_NarProduction Pathway, using the assembly method SSAP (Coussement et al., 2017)
Steps 2 and 3
involve the transformation via electroporation of E. coli TOP10, E. coli K12 MG1655 and C. necator. Kanamycin selection and sequencing are used to verify the presence of the plasmid in the bacterial cell.
Figure 4: Schematic overview of plasmid cloning, transformation and sequencing of E. coli TOP10. Inkscape_Bénédicte Amery
Evaluation of P22 functionality in C. necator. Tests allow for a first indication of the functionality of the promotor and is performed at a small scale (96-well plate).
Evaluation of naringenin production in E. coli K12 MG1655 and C. necator. The evaluation starts at a larger scale compared to testing the promoter.
Upscaling of the naringenin production in C. necator PHB-4 working with fermentation batches.
Performed in the lab:
The functionality of the promotor in the different organisms is tested using the fluorescent reporter mKate2, based on the absence/presence of the fluorescent signal we will be able to assess the functionality of P22 in E. coli K12 MG 1655 and C. necator. Growth trials involve continued measurements of optical density at 600nm (OD600) and mKate2 fluorescence (Excitation 588nm, emission 622 nm and gain 110). Measurements were taken during 24 hours for E. coli K12 MG1655 and during 48 hours for C. necator. The growth test was only performed in E. coli K12 MG1655 cells due to difficulties growing C. necator, see learn and improve for more details.
Figure 5: Overview of the first test; testing functionality of the constitutive P22 promotor in different hosts. Inkscape_Bénédicte Amery
Upscaling of the naringenin production will start with a subculture in test tubes to ensure consistency. A subculture allows for standardization of the inoculum because it will only contain growing cells. When taking cells from a plate, they might be in different growth phases or in a dormant state. Starter cultures are taken from the subculture and divided over the Erlenmeyer flasks. We want to compare growth, so we want all the cells to be in the same growth phase when the experiment starts. Larger amounts of cell densities can be grown in the Erlenmeyer flask, and we might get a first impression of the differences in concentration of naringenin between E. coli and C. necator. E. coli and C. necator grow at a different rate, so endpoint measures are taken at 24 hours and 48 hours, respectively.
There is need for downstream processing for enabling naringenin determination. Cell lysis needs to be conducted first, followed by the extraction of naringenin by an organic solvent like ethyl acetate. The extracted organic compounds, including naringenin, are then filtered. Ultra-Performance Liquid Chromatography (UPLC) is optimized to have good separation and detection of naringenin is conducted via a UV detection system.
5) Learn and improve
The first step in design, cloning the plasmid, did not work out well for the naringenin pathway. So we needed to be creative in the cloning process by using different polymerases, different primers, different PCR settings, etcetera…. Details are written in the SOP and notebook notes. We have learned that it is hard to clone with massive pieces of DNA like the genes involved in the naringenin pathway. Furthermore, using software to predict the behaviour of the PCR is not always accurate since we work with biological material. The cloning of pBBR1MCS2_P22_RBS_mKate2 was very efficient, and the first attempt was successful.
Unfortunately, due to COVID-19, we had limited time in the lab. However, we are sure, if we had more time in the lab, we would have improved our cloning process and would have made this possible for the plasmid pBBR1MCS2_NarProduction Pathway.
Working with different organisms that are easy to handle and of which SOP’s are known, is fun. You can know what to expect. Handling C. necator was a different story. Although we were able to clone the pBBR1MCS2_P22_RBS_mKate2 plasmid in no time, it was hard to obtain representative and reproductive growth of C. necator transformants.
For example, after electroporation of the construct in C. necator, a certain amount of C. necator needs to be plated onto a Lysogeny Broth (LB) agar plate with kanamycin and gentamicin. The quantity needed to be determined experimentally; at first, 50µL and 100µL were tried. It was not possible to take single colonies for the growth trial because the plate was overgrown. As a solution, two things could be done, adding more antibiotics and plate the same amount of C. necator or plate a smaller quantity of C. necator. Eventually, the amount taken for plating out was reduced to 20µL.
Furthermore, storing C. necator for further handling in the fridge is not a good idea. The cells tend to get in a dormant state and have trouble to change back to their normal state. So planning was crucial when growth trials were executed, remember that C. necator needs to grow for 48 hours. It was only after several attempts and contradicting growth on LB agar + kanamycin and gentamicin that this conclusion was formed. Early testing on what are the best cultivation conditions of C. necator is essential to make upscaling easier. An example of improving cultivation conditions is to use glycerol as a carbon source instead of glucose. Glycerol is a waste stream in production of bio-diesel and should give higher yields of C. necator.
The downstream processing methodology for recovering the naringenin is based on a protocol using UPLC. The protocol can be found in the SOP page. C. necator is a gram-negative bacterium, as is E. coli; thus, there should remain only a little optimization. This is trial and error and is hard to predict beforehand.
Depression is one of the most occurring psychiatric illnesses and is the most important cause of health issues and disorders worldwide (WHO, 2020). It can be triggered by a combination of genetic, biological, psychological and environmental factors. The most frequent treatment is drug administration (Steenbergen et al., 2015; Axt-Gadermann & Rautenberg, 2017). The medicines most frequently prescribed are antidepressants, although not all patients react positively to antidepressants. Only 30% of patients get acceptable results; 40% gets intermediate results. The remaining 30% perceives terrible consequences from taking an antidepressant (Vitetta et al., 2014). Furthermore, the actual cause of depression is not treated by taking antidepressants because the hypothesis is that the medicines remove the symptoms of depression by working into neurotransmitters. (Dinan et al., 2013).
In the period from 2005-2015, the WHO registered 18% more cases of depression (WHO, 2020). With antidepressants as the treatment that does not work as expected in 70% of the cases. A treatment that only treats symptoms and the number of depressions on the rise, there is a need to find new alternative treatments for depression.
“All disease starts in the gut” Hippocrates 460-375 BC
Recent research points out that gastrointestinal bacteria have strong communication with the brain via the ‘gut-brain-axis’(Lima-Ojeda et al., 2017, 2018). This understanding led to the extension of ‘the gut-brain axis’ to the ‘microbiota-gut-brain-axis’ (Borre et al., 2014). More importantly, these bacteria present in the gut might aid in the emergence of depression, but also in sustaining the depression (Liang et al., 2018). An error in the balance of the gut bacteria, more specifically, a reduced level of Faecalibacterium prausnitzii is a marker for depression and other disorders and diseases (Jiang et al., 2015). Another feature correlated with depression is local inflammation, and inflammation also has its influence on dysbiosis. Dysbiosis triggers inflammation and vice versa; one cannot be seen without the other (Vitetta et al., 2014).
However, some natural product already exists that help in reducing the symptoms of depression. For example, vitamin B12 and vitamin D enhances mental health (Berk et al., 2013; Dash et al., 2015). Prebiotics are a source for ‘good’ gut bacteria, the kind Faecalibacterium praustnitzii belongs to. Prebiotics feed the ‘good’ bacteria that help reduce symptoms of depression. The prebiotic itself is also transmitted into short-chain-fatty-acids (SCF) which are anti-inflammatory agents. (Kelly et al., 2017; Burokas et al., 2017)
We were determined to find an alternative treatment for depression by making a combination of these exciting findings.
From our extensive research, we found that there are multiple factors involved in maintaining a state of depression:
- Dysbiosis, with lower Faecalibacterium prausnitzii
And there were already some leads that can help reduce the depression disorder:
- vitamin B12
- Faecalibacterium prausnitzii grows on media containing vitamin B-12
- anti-inflammatory agents
Furthermore, there is an overlap between the functionalities of the components that can reduce depression symptoms. This overlap might create an extra potent cocktail.
Figure 6: Overview of the different factors influencing depression. Inkscape Bénédicte Amery
If we could transport prebiotics, anti-inflammatory agents and vitamin B12 to the site of inflammation in the gut, we might be targeting one of the causes of depression. An edible pearl which releases these compounds in the gut could be an ideal deliverable vehicle.
Our edible pearl or bubble “bubbly” contains an anti-inflammatory agent, a prebiotic and vitamin B12. As an anti-inflammatory agent naringenin is used, this flavonoid has many benefits to human health and is approved for consumption by EFSA. Furthermore, it can be sustainably produced using microorganisms (see section “Naringenin production”). As a prebiotic, we will add kojibiose (Beerens et al., 2017)It has a dual function in being a sweetener and a prebiotic. Kojibiose is currently not approved for consumption yet, but once it is approved for consumption, we can add it. Vitamin B12 is added as a direct influencer on depression, but it is also related to enhanced growth of Faecalibacterium prausnitzii.
Vitamin B12 is added as (cyano)cobalamin and should be transported past the second segment of the duodenum, only at this point will the vitamin B12 bind the intrinsic factor. Binding to the intrinsic factor is essential for active uptake in the small intestine (Busti & Herrington, 2015). When not being bound to the intrinsic factor, vitamin B12 can be taken up by passive absorption, although this is not remarkably effective.
Kojibiose is ideally released in the colon, but this is a long route to stay in the bubble. However, one aspect of prebiotics is that they are fermented in the colon. Hence, we assume that kojibiose will be transported intact to the colon, even if it is released earlier in the small intestine. Endogenous enzymes present in the small intestine cannot hydrolyze fibres. (Davani-Davari et al., 2019).
Polyphenols in general exhibit a prebiotic and immunomodulating effect in the gut. The flavone naringenin can stay for a more extended period in the human gut. Its release is ideally in close vicinity to the colon, but this is not mandatory (Kawabata et al., 2019; Pei et al., 2020).
Taking together the release sites of the three compounds, the first release should take place in the small intestine. This is where vitamin B 12 should be released for efficient uptake. The other molecules, kojibiose and naringenin, can be transported passively to their sites of action. Digestion time varies among individuals, and this can influence the efficiency of the components in the gut. As a general guideline, we apply the following criteria:
- The bubble needs to survive the stomach meaning there is no/minimal diffusion of the components in during the passage through the stomach.
- All components in the bubble should be released after 8 hours. This is the time point when the bubble enters the colon. All vitamin B12 should be released from the bubble before this stage.
To transport the components in the pearl, we need a vehicle behaving according to the predetermined criteria for optimal delivery to the target site of action. For the formulation of our bubble, we worked together with Petra Van Gucht from Cosmo Group. Specific tests are created for the formulation of the pearl; it ensures optimal release of the compounds near their site of action. Different testing methods are used to decide upon the concentrations of the compounds in the pearl.
Cosmo Group designs the first concept of the pearl because they have extensive experience in nutrition. The Minekus et al., 2014 standardized protocol for food is the basis for preliminary testing after receiving the first concept pearls in Cosmo Group. The minekus protocol is a static in vitro digestion method that resembles the gastrointestinal tract by dividing it into an oral, gastric, and intestinal phase. The oral stage is the mouth, the gastric is the stomach, and the intestinal resembles the small and large colon passage.
Different systems that are observed:
- See how long the pearls exist within each different tube or a batch system. This allows setting the limits of the pearl within each of the digestive phases.
- Follow the ‘path of digestion’ and take new samples in a close interval or continuous system. This allows some prediction of the pearl’s behaviour along the digestive tract.
Figure 7:Minekus protocol without dissolution testing. To add dissolution testing the tubes are simply replaced by dissolution device. The main steps remain the same. Inkscape Bénédicte Amery
The results from the first preliminary testing lead to the first optimization cycle. Another standardized method, dissolution testing, delivers complementary results to the static Minekus protocol.
The SHIME is used to optimize the concentration of components in the pearl.
Figure 8: Illustration of the SHIME from Prodigest. Sampling occurs at specific time intervals during the whole digestion tract. Inkscape Bénédicte Amery
Evaluation of the output of the SHIME and detection of naringenin is performed via Chen et al., 2019. Chen et al. created rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS) based method for the evaluation of naringin and its gut microbial metabolites naringenin. After incorporating the results from the SHIME, we are as close as we can get in in vitro observations.
Saturation of vitamin B12 uptake mechanisms needs to be added as a variable when calculating/modelling the optimal concentrations of vitamin B12 (O’Leary & Samman, 2010; Adams et al., 1971).
Learn And Improve
The first concept pearls, designed by Cosmo Group, are subjected to different tests, and the results of the tests are input for the release/diffusion modelling system. The model then aids in providing information about the formulation. After adapting the formulation, testing starts again to see if the formulation gives improvement. This iterative process continues until the formulation of the pearl meets the criteria set in the design.
Figure 9: Illustration of the engineering cycle. Data feeds our model when the model turns out to be better compared to the previous model, and we adapt our formulation or component concentration accordingly. Inkscape Bénédicte Amery
Because we had not the time to implement the results of CosmoGroup, we constructed three different diffusion models that simulate the release of the naringenin from pearl to the small intestine. We used three initial situations for the models. A vital remark that we must make is that we did not take all factors into account. We did encounter pH differences and intestinal motility in our models.
The most significant limitation of the Minekus protocol is that it is static. A way to circumvent this is by adding dissolution experiments in addition to the data from the Minekus protocol. The dissolution experiments give a more dynamic input. Dissolution is a highly standardized method that needs specialized equipment. This was not present in the lab; hence it was advised to contact the Eurofins group or the pharmacological department of Ghent University. It would have been even better to combine both methods, using the fluids described by minekus and with the standardized dissolution settings. Trial and error and discussions with the involved parties (Eurofins or the pharmacological department) ought to help in the decision making of the exact experimental setup and parameters.
The different methods will remain in the experimental setup because it is possible to distinguish the main dissolving factors in the digestion phase by looking at the batch system. The continuous systems indicate how the pearl will act when it encounters the different stages of digestion in chronological order. For example, by only remaining 2 hours in the gastric phase (stomach) cracking of the pearls can occur. This cracking will lead in the early release of some components in the pearl in the intestine. It might be that without passage through the gastric phase, the pearl will barely dissolve in the intestinal phase. This evaluation is only possible when combining a batch and a continuous system.
The SHIME will give more details about the fate of the prebiotic kojibiose and naringenin as the SHIME contains complex and stable microbial communities. These microbial communities are similar in structure and function compared to those in the human colon. The SHIME is used to give a complete study about the metabolic fate of our pearl and is as realistic as possible. (https://www.prodigest.eu)
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