“Science is well-ordered when the inquiries it pursues are those that accord with the agenda that would have been set by a group of discussants fully informed of the scientific opportunities, fully informed of one another's needs, and dedicated to doing the best they can to accommodate the needs of all” [1]
- Philip Stuart Kitcher, philosopher of science
The quote by Kitcher describes how science is serving its purpose when scientists prioritize inquiries that consider diverse perspectives, address shared needs, and aim for the collective benefit. Among multiple questions which the ideal of well-ordered science prompts us to consider, a fundamental one is about setting research priorities. Considering the vast array of potential topics and the inherent limitation of resources, what should scientists investigate, and how should the resources be distributed between different lines of investigation?
These questions remained important throughout our project and guided us on our way to ensure that our project is meaningful and beneficial for the world.
From the initial days of brainstorming, we welcomed input in the form of meetings, literature reviews, and reflections within the team. This allowed to decide on impactful and innovative aspects our project should contain. For us, the goal became contributing to foundational science in a way that has an immediate application beyond academia. Accordingly, when designing our project, we decided to address problems of high environmental and social relevance, such as enabling large-scale, continuous microbial bioproduction and sustainable vitamin B12 biosynthesis.
Consideration of the questions Kitcher posed requires a certain kind of epistemic humility: openly acknowledging that we might be wrong in what we consider a valuable contribution to science and society. Therefore, continuous engagement with trustworthy sources informed project development and was instrumental in evaluating whether we were addressing the prioritized problems in the most effective way.
INTEGRATED HUMAN PRACTICES
Here, you can follow our journey from the initial days of brainstorming the project idea to its final shape. You can read more on:
- individual meetings and in-person engagment with relevant stakeholders, experts and public (click on the “Additional meeting notes” button for more!)
- the reflective process based on meetings and literature reviews throughout the project in the “Reflection” sections
- a given meeting's influence on the project under “Integrated feedback”
Integrated feedback
To gather inspiring and impactful project ideas, we followed iGEM Freiburg 2022 team member suggestion and reached out to various regional companies and organizations, including Fraunhofer Institute for Solar Energy Systems; dairy manufacturer Schwarzwaldmilch; plastic manufacturer DOMO, the major of Freiburg city, Freiburg University Hospital, Freiburg-based brewery Ganter, to global organizations including The Good Food Institute; Effective Thesis; New Harvest, ALLFED - Alliance to Feed the Earth in Disasters. We connected with researchers at the University of Freiburg, ETH Zürich, University of Tübingen, Reutlingen University, University of Jena, TU Munich and got in touch with other iGEMers from St Andrews iGEM 2012, iGEM TU-Braunschweig 2023, UCSC iGEM 2017. In parallel, we continuously reviewed the literature on the newest developments in the synthetic biology field and looked into cyanobacteria deployment specifically.
Reflection: the potential we see in cyanobacteria
Why did cyanobacteria catch our attention?
From an environmental standpoint, the use of cyanobacteria as more sustainable microbial biofactories holds immense potential since these organisms can efficiently convert atmospheric CO2 into valuable products, utilizing light as an energy source [2, 3]. Additionally, due to their ability to produce oxygen and sustain DNA damage from increased radiation [4], some cyanobacteria species can provide vital support for Mars exploration and eventual habitation [5]. By further leveraging cyanobacteria's abilities, we can establish sustainable bioproduction systems for essential nutrients (like vitamin B12) which would both enhance the feasibility of long-duration space missions and boost sustainable bioproduction on Earth.
Clearly, these bacteria were worth further exploration, but none of us, nor our supervisors had previous experience working with thes organisms. Therefore, we reached out to cyanobacteria-focused research group leaders at the University of Freiburg.
Integrated feedback
Inspired by the idea of coupling cell survival to the presence of (any) compound, we decided to look into the literature to explore current efforts in this direction. The idea of creating an autoregulatory system sounded intriguing and we started brainstorming possible applications of such a system.
Reflection: on choosing a project idea that combines foundational science with real-life applicability
We were intrigued by the idea of creating an autoregulatory system with a wide variety of potential applications, like the one Prof. Annegret Wilde proposed. Upon further literature review, we found no reports of such a universally applicable, dynamic control system design. Also the success of foundational advance-focused project of last year’s iGEM team from Freiburg inspired us to develop our project into foundational science direction. At the same time, we wanted to see its applicability beyond the lab bench. Question arose: can we combine both?
Since cyanobacteria remained of great interest, we considered making it a side branch of our project while we do the initial testing of the autoregulatory system design in a faster-growing organism, E. coli. Specifically, cyanobacteria were considered for sustainable vitamin B12 production and, once coupled with the autoregulatory system, could serve as the applied contribution of our project.
Keeping both the autoregulatory system idea and cyanobacteria as sustainable chassis exploration meant work had to be divided to cover both: therefore, we split into subgroups to research the single components for the project.
We also realized that for a project with a lasting impact, continuous engagement with experts and potential stakeholders is crucial: already the first meetings had enriched our perspectives on possibilities synthetic biology holds and shaped the direction project was taking.
Integrated feedback
Until the meeting with David Russo, we still considered A. platensis as a potential chassis for testing B12 production. Yet, given the challenging transformation David Russo mentioned, we decide to focus on one of the cyanobacteria model strains instead.
Integrated feedback
Initially, the design we had considered was based on arranging the genes on two plasmids, which would be dependent on each other for the retention of the whole system. Considering the feedback we got, we decided to rethink this design to ultimately fit it on one plasmid. While we had only planned lab-scale experiments for the system, we now realized that the final testing in a bioreactor might be needed as well to assess its industrial potential.
Reflection: why engagement with industry is important
We noted Max Mundt’s suggestion to test our construct in a bioreactor setting and were interested in exploring the importance of this step through further literature research. Microbial bioproduction, the field we see as an immediate application of our autoregulatory system, suffers greatly from lab-to-bioreactor transition challenges [7]. Industrial strain performance can not be extrapolated from lab-scale experiment unless factors like bioreactor-specific stresses (e.g. pressure, shear, pH), overexpression of production genes and prolonged cultivation times are taken into account [7, 8, 9]. For example, the same microbial population can consist of <3% non-productive cells at generation 37 and shift to 96% non-producers as it reaches generation 60, which corresponds to the time required to grow a sufficient culture in a small bioreactor for scale-up [8].
The apparent gap between lab and industry means that potentially impactful synthetic biology innovations remain in their nascent state [7]. Therefore, decisions were made to engage with strain optimization and industrial bioproduction experts at enGENES Biotech and BRAIN Biotech (see meeting notes below) to increase the applicability of the autoregulatory system we are creating, while also noting potential improvements.
Integrated feedback
Before, we considered a couple of cyanobacteria model strains for B12 production and chose to work with S. elongatus PCC 7942 given Hicks mention of better data availability. Following her advice to look for the origin of replication compatibility, we selected a shuttle vector designed specifically for use in S. elongatus PCC 7942 (developed by iGEM Marburg 2019 team). Lastly, we employed the 2 genes, ssuE and bluB, for B12 de novo biosynthesis based on the theory Hicks’s developed in her thesis.
Integrated feedback
From the meeting we learned that our initial idea of using the E. coli riboswitch in the cyanobacteria would likely not be successful due to its possible activation by natively-produced pseudocobalamin. Therefore, to adjust the autoregulatory system to cyanobacteria, we decided to test another, B12 specific, riboswitch derived from P. freudenreichii. This riboswitch is supposedly not activated by pseudocobalamin and hence suits our objective to detect exclusively B12.
Reflection: combining it all
Engaging with experts from industry and academia to validate and improve the concept of our project was the next important step for the development of our idea towards real-world applicability.
Consultation with Max Mundt, someone with expertise in the biotechnology industry, allowed us to consider adjustments in system design that would be necessary for application in large-scale fermentation processes. David Russo’s advice drove us to carry out our experiments in relatively well researched model organisms. This matches the approach of McKenna Hicks, who started with B12 production in S. elongatus PCC 7942 despite her ultimate goal to establish B12 production in A. platensis. Direct exchange with her gave us valuable insights into potential challenges we could be facing along the way, learning from her experience.
The discussion with Annegret Wilde and Wolfgang Hess revolved around the functionality of specific system parts in cyanobacteria, as they would likely work differently compared to E. coli. The points they made outlined the adjustment that would be necessary to implement our system into different organisms. Depending on the synthesis or degradation pathway, the riboswitch and the toxin/antitoxin system might need adjustment according to respective conditions. Going back to the transfer from E. coli to cyanobacteria, in our case, a different riboswitch than the one we utilize in E. coli is required because of different affinities to B12 precursors. Additionally, the ratio of toxin and antitoxin expression would need to be adjusted because of reduced impact of the toxin on cyanobacteria.
To us, this emphasizes the relevance of a considerate project development process, striving to achieve excellent functionality of an individually designed system according to the desired application.
Integrated feedback
Following the meeting, we welcomed a doctoral student from Fleck’s group, Jonas Pleyer, as a team supervisor and mathematical modeling tutor: that allowed us to learn modeling basics and improve our system.
Given Fleck’s emphasis on having a manageable and feasible project concept, we arranged further meetings with experts from industry and academia to validate different parts of the autoregulatory system.
Integrated feedback
Inspired by iGEM Marburg 2019, we considered the possibility of using fast growing strains, like S. elongatus UTEX 2973, for our project. However, based on the feedback from Khaled Selim, we decided to stay with the model strain S. elongatus PCC 7942. Instead of trying to test the autoregulatory system and B12 production in parallel, we followed his suggestion to start with the latter first, and only once successful, try integrating the whole system.
Integrated feedback
We put emphasis on the advice from enGenes to characterize all individual parts first before combining them. Moreover, we noted the genome integration for later attuning of the system to accommodate microbial bioproduction requirements.
Reflection: on phenotypic instability and CELLECT application
We were well aware of the term “genetic instability”, exemplified by e.g. plasmid loss. There is a more intricate side to it though, which does not involve the loss of genes but still results in the loss-of-function: phenotypic instability. As mentioned by enGENES Biotech, genome integration is a common practice in bioproduction to secure a construct retention. However, no satisfying solutions have been developed so far to ensure persistent gene expression [8, 9]. Hence, the productivity and stability of industrial strains remain severely limited. Currently available options, like growth-coupled production, require genome in silico modeling [2], while multi-layer genetic circuits rely on specific regulatory components and tailored mathematical models to optimize the circuit´s design [11]; both of which are labor-intensive tasks. The challenge remains to create an optimal gene expression control system that can be easily adjusted to various compounds and host organisms, one that is able to integrate multiple signals in a timely manner, assure genetic stability and minimize the metabolic burden [11]. Does CELLECT’s versatile design make it a feasible solution to phenotypic instability? Potentially, as enGENES agreed. Yet, besides ensuring the robustness of the individual CELLECT parts, we have to test the metabolic burden it imposes on the cell and how that impacts the production yield. For that, precise quantification of the compound we produce, vitamin B12, is required.
Reflection: the challenges with vitamin B12 detection
There is an abundance of methods available for the detection of B12, yet most of them are limited in access due to the requirements for large and expensive equipment, complicated reagents, long assay times, low tolerance to complex matrices, poor limits of detection or insufficient specificity to distinguish between the different forms of B12 [12]. To establish a detection method that is suited to our project’s requirements, we first tested the Demeditec B12-ELISA kit for foods, which turned out not to work well for our application. We ended up testing two separate detection methods from one paper, a riboswitch sensor and an ethanolamine utilization assay for E. coli [13]. While we got mixed results when trying to establish the riboswitch sensor, the ethanolamine assay looked promising (Results/Sensing B12), at least for qualitative detection of B12. We therefore developed and tested it for quantitative measurements, seeking and implementing the advice of Matthias Boll in the process. It evolved to the point where we found it to be a valuable detection method to optimize not only for our purpose, but for other researchers to implement in their work. This assay is affordable and simple: it consists of growing E. coli in a specific medium, in which the cell growth is dependent on the presence of B12. Yet, for the autoregulatory system testing while developing this assay and also for its later accuracy assessment, we still needed a reliable, quantitative method for B12 detection. Therefore, we contacted Luciana Hannibal, a researcher at the University Medical Center Freiburg, specialized in B12 metabolism.
Integrated feedback
Hannibal confirmed that we could convert the B12 in given samples to OHCbl by way of deliberate exposure to white light. Therefore, we always included a step of deliberate white light exposure for a time period of 2-3 hours during sample preparation for LC-MS.
Reflection: science literacy and what we did about it
In the introduction, we touched upon Philip Kitcher’s view of well-ordered science which conceives “scientists [..] as artisans working for the public good” [1], a view we embrace. Yet, an important question remains: how does the public know that we do? Why would anyone beyond the lab doors trust that we are putting the resources into good use? As historian of science Naomi Oreskes writes, trust is an important part of effective science communication- and one way to create it is by building connections from the research institutions into the local communities [14]. CIBSS Open Door day surely allowed us to connect with the neighborhood, but it also highlighted the importance of science literacy. Why? Because it empowers people to have a closer engagement with the newest developments in fields like synthetic biology and consequently provides bases for a better dialogue with scientists; one which is enriching for both sides. Already during the Open Door days, we observed keen interest from visitors to learn about foundational synthetic biology principles. Making biology more accessible in an engaging way became an inspiration for us, encouraging more education-related projects to come (Education).
Integrated feedback
We learned that the risk of the mutation of the toxin could be a severe hindrance for the success of our project and came up with a second design of our system, which would allow us to address the instability of the toxin (Design).
Integrated feedback
Following Hess’s advice to focus on securing the promoter, which was suggested to be the part most prone to mutations, we came up with an idea to employ a selection marker gene downstream of the promoter, which would only be expressed if the promoter was working. As Hess confirmed, this re-design of the system could work to limit the risk of toxin loss-of-function. Therefore, we continued with the cloning of this construct.
Integrated feedback
The B.R.A.I.N. feedback confirmed our approach of securing the promoter controlling toxin expression. Additionally, following up on their suggestion, we collected and sent for LC-MS measurements supernatant samples from the production cultures to determine how much B12 is released by lysing cells.
Reflection: on the good, the bad and the future
Presenting CELLECT to various people from academia and industry allowed us to gather perspectives on both its potential and shortcomings. The most notable advantage remained the versatile, comparatively simple design of the system, with minimal mathematical modeling requirements and applicability across different fields. Among the needed improvements was stabilization of the most vulnerable part of CELLECT, the toxin gene and its promoter, to minimize the occurrence of mutations. In response, we developed new versions of CELLECT, which, while not tested during this project due to time constraints, encompass toxin-stabilizing elements. From the industry meetings, we noted that an industrially-applicable version of the system would also need to be genome-integrated to avoid antibiotic-based selection and the financial and environmental burden it implies on large scale. Lastly, replacement of chemically inducible promoters with constant or heat-inducible ones would make CELLECT even more applicable for bioproduction and degradation purposes. Nearing the end of the project, we came across a recent publication on a novel multi-control system which employed a mechanism similar to one we propose. Utilizing ligand-dependent riboswitch, tightly controlled inducible promoter and low-copy number strain, the system is designed to enable cloning of toxic or unstable genes [15]. The study supports some of the observations during our project (such as low toxicity of MazF toxin in E.coli strain harboring native antitoxin) and serves as an intriguing outlook on yet another potential application for CELLECT.
Reflection: importance of vitamin B12, from health to bioproduction
Only microorganisms can synthesize vitamin B12 by themselves. Other animals, including mammals, cannot, yet they depend on it. Consequently, sufficient availability of B12 is a basic requirement for human health.
Although there are certain gut microbiota that can produce the compound, B12 from these bacteria is not bioavailable to humans, meaning that sufficient amounts of the vitamin derived from nutrition can only be achieved by consumption of animal foods. It is worth mentioning however that even in farm animals B12 levels have dropped over the last 30 years. Many times, these animals are injected with essential micronutrients themselves as they do not get them from the food they are provided with [16].
Independent of that, plant-based diets gain more and more popularity. While this shift can eventually alleviate the threat to the environment that factory farming is, it could cause another basic concern for human health instead. Sufficient amounts of an essential micronutrient might not be guaranteed, with potentially increasing demand for B12 to the point where there is more demand than supply. Exemplary for the shift in this direction, children day-care centers and elementary schools in Freiburg switch to an exclusively vegetarian meal-plan with the start of the new semester, which is observed as a potential example for the entirety of Baden-Württemberg to follow [17].
All of this speaks to the fact that the availability of B12 in the present and in the future needs to be addressed.
As chemical synthesis of B12 is extraordinarily ineffective, commercial B12 is commonly produced through large-scale fermentation using the microorganisms Propionobacterium freudenreichii or Pseudomonas denitrificans [18, 19].
The cobalamin forms extracted from the bacteria are then treated with cyanide for conversion to cyanocobalamin (CNCbl). If needed, conversion to the desired B12-form is accomplished by way of chemical synthesis. This could be done to, e.g., produce hydroxocobalamin (OHCbl) for therapeutic use, as this is the preferred version of B12 for therapeutic injections, or for production of OHCbl for cyanide detoxification [20].
The majority of B12 on the market is synthesized in China [21]. This opens up a geopolitical conversation. With a B12 world market share of about 90%, the distribution of B12 around the globe is highly dependent on this one country. Establishment of B12 production outside of China would greatly benefit the worldwide distribution of power over this aspect of human health.
Another point is waste. The free cobalt that is required in culture media as a basis for the bacteria to produce the vitamin is never converted to B12 efficiently, causing a lot of cobalt waste that eventually ends up in the environment. Bioproduction in many western countries is subject to more strict regulations, implying the reduction of such waste. For example, researchers around Prof. Martin Warren at Quadram Institute (UK) do already confront this challenge by creating a sustainable bioproduction method of B12 in E. coli [22].
Martin Warren’s research group furthermore explores the influence of B12 on the gut microbiome. This falls in line with an emerging interest in the effect that the vitamin has on bacteria in the gastrointestinal (GI) tract. An exemplary cascade of action, explained to us by Luciana Hannibal, would be this: The gut microbiome of Inflammatory Bowel Disease (IBD) patients may inherit a comparatively higher population of microbes that consume vitamin B12 to produce B12 analogs in turn. On one hand, more B12 entering the GI tract is taken up by these bacteria, leaving less of it to be absorbed by the patient. On the other hand, those microbiota can produce additional B12 analogs, which are taken up into the bloodstream as well. The B12 analogs then compete with B12, meaning they bind to the enzymes to which the B12 molecules would normally bind, but do not help catalyze the corresponding reaction. This thereby causes insensitivity to B12 in the respective human. Moreover, oral supplementation with high doses of B12 might mitigate the shift towards higher B12 consuming microbe populations, thereby increasing the risk for B12 deficiency once lower doses of the vitamin are administered [23].
References
- [1] Kitcher P. Responsible biology. BioScience. 2004 Apr 1;54(4):331-6.
- [2] Guillaume MC, Santos FBD. Assessing and reducing phenotypic instability in cyanobacteria. Current Opinion in Biotechnology [Internet]. 2023 Apr 1;80:102899. Available from: https://doi.org/10.1016/j.copbio.2023.102899
- [3] Knoot CJ, Ungerer J, Wangikar PP, Pakrasi HB. Cyanobacteria: Promising biocatalysts for sustainable chemical production. Journal of Biological Chemistry [Internet]. 2018 Apr 1;293(14):5044–52. Available from: https://doi.org/10.1074/jbc.r117.815886
- [4] Verseux C, Baqué M, Lehto K, De Vera JP, Rothschild LJ, Billi D. Sustainable life support on Mars – the potential roles of cyanobacteria. International Journal of Astrobiology [Internet]. 2015 Aug 3;15(1):65–92. Available from: https://doi.org/10.1017/s147355041500021x
- [5] Mapstone LJ, Leite MN, Purton S, Crawford I, Dartnell L. Cyanobacteria and microalgae in supporting human habitation on Mars. Biotechnology Advances [Internet]. 2022 Oct 1;59:107946. Available from: https://doi.org/10.1016/j.biotechadv.2022.107946
- [6] Cengic I, Cañadas IC, Minton NP, Hudson EP. Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803. ACS Synthetic Biology [Internet]. 2022 Aug 15;11(9):3100–13. Available from: https://doi.org/10.1021/acssynbio.2c00375
- [7] Wehrs M, Tanjore D, Eng T, Lievense J, Pray T, Mukhopadhyay A. Engineering robust production microbes for Large-Scale cultivation. Trends in Microbiology [Internet]. 2019 Jun 1;27(6):524–37. Available from: https://doi.org/10.1016/j.tim.2019.01.006
- [8] Rugbjerg P, Myling-Petersen N, Porse A, Sarup-Lytzen K, Sommer MOA. Diverse genetic error modes constrain large-scale bio-based production. Nature Communications [Internet]. 2018 Feb 20;9(1). Available from: https://doi.org/10.1038/s41467-018-03232-w
- [9] Czajka JJ, Okumuş B, Koffas M, Blenner M, Tang YJ. Mitigation of host cell mutations and regime shift during microbial fermentation: a perspective from flux memory. Current Opinion in Biotechnology [Internet]. 2020 Dec 1;66:227–35. Available from: https://doi.org/10.1016/j.copbio.2020.08.003
- [10] Hicks, M. (2019). Engineering photosynthetic bacteria as factories for the sustainable manufacturing of vitamins and medications. [Master's thesis, University of California, Santa Cruz]. https://escholarship.org/uc/item/6mr6j0zk
- [11] Cui S, Lv X, Xu X, Chen T, Zhang H, Liu Y, et al. Multilayer genetic circuits for dynamic regulation of metabolic pathways. ACS Synthetic Biology [Internet]. 2021 Jul 2;10(7):1587–97. Available from: https://doi.org/10.1021/acssynbio.1c00073
- [12] Haro JJQ, Wegner SV. An adenosylcobalamin specific Whole‐Cell biosensor. Advanced Healthcare Materials [Internet]. 2023 May 1; Available from: https://doi.org/10.1002/adhm.202300835
- [13] Fowler CC, Brown ED, Li Y. Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in E. coli. Chemistry & Biology [Internet]. 2010 Jul 1;17(7):756–65. Available from: https://doi.org/10.1016/j.chembiol.2010.05.025
- [14] Science communication and scientific judgment [Internet]. CIRSD. Available from: https://www.cirsd.org/en/horizons/horizons-winter-issue-20/science-communication--and-scientific-judgment
- [15] Vandierendonck J, Girardin Y, De Bruyn P, De Greve H, Loris R. A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli. Toxins [Internet]. 2023 Aug 18;15(8):508. Available from: https://doi.org/10.3390/toxins15080508
- [16] Farm animal B12 deficiency & supplementation for meat dairy product consumption [Internet]. www.EatingOurFuture.com. 2018. Available from: https://eatingourfuture.wordpress.com/eating-meat-raises-risks-of-cancer-heart-disease-early-death-shorter-life/farm-animal-b12-deficiency-supplementation-for-meat-dairy-product-consumption/
- [17] Aktuell S. Vegetarisches Schulessen für Kinder: Grünen-Fraktion im Landtag sieht Freiburg als Vorbild für ganz BW [Internet]. swr.online. 2023. Available from: https://www.swr.de/swraktuell/baden-wuerttemberg/vegetarisches-essen-fuer-kinder-reaktionen-bw-100.html
- [18] Eschenmoser A, Wintner CE. Natural Product Synthesis and Vitamin B12. Science [Internet]. 1977 Jun 24;196(4297):1410–20. Available from: https://doi.org/10.1126/science.867037
- [19] Balabanova L, Averianova L, Марченок МВ, Son O, Tekutyeva L. Microbial and genetic resources for cobalamin (Vitamin B12) biosynthesis: From ecosystems to industrial biotechnology. International Journal of Molecular Sciences [Internet]. 2021 Apr 26;22(9):4522. Available from: https://doi.org/10.3390/ijms22094522
- [20] Hydroxocobalamin - Medical Countermeasures Database - CHEMM [Internet]. Available from: https://chemm.hhs.gov/countermeasure_hydroxocobalamin.htm
- [21] Ukri. GTR [Internet]. Available from: https://gtr.ukri.org/projects?ref=BB%2FS002197%2F1
- [22] An environmentally friendly vitamin B12 production method that makes manufacture more affordable - Quadram Institute [Internet]. Quadram Institute. 2023. Available from: https://quadram.ac.uk/case_studies/an-environmentally-friendly-vitamin-b12-production-method-that-makes-manufacture-more-affordable/
- [23] Lurz E, Horne R, Määttänen P, Wu R, Botts SR, Li B, et al. Vitamin B12 deficiency alters the gut microbiota in a murine model of colitis. Frontiers in Nutrition [Internet]. 2020 Jun 5;7. Available from: https://doi.org/10.3389/fnut.2020.00083