Presented by Team Manchester 2020
Miguel González-Valdés Tejero, Jana Hidalgo Hopson, Maria Kesic, Sandrina Mocartaite, William Smith, Ivan Tivodar, Madeleine Webster-Harris
The University of Manchester
Abstract
Regular unprotected exposure to the sun can result in multiple skin disorders, including skin cancer. Skin cancer is the fifth most common type of cancer in the UK, yet 60% of cases are preventable. Sunscreens are therefore important to use, but when used while swimming in the sea, conventional sunscreen ingredients cause oxidative stress on coral reefs, resulting in serious coral bleaching. Our team aims to produce a novel non-toxic, bactericidal broad-spectrum UV filter from hippopotamus sweat to address this problem. Our target compound polymerises naturally into an unreactive molecule minimising environmental effects.
Our goal is to produce the novel sunscreen in bacteria, as a reef-safe alternative to commercially available sunscreens. We use innovative retrosynthesis methods to design a biosynthetic pathway to our target compound and utilise techniques like entrepreneurship, stakeholder engagement, and media analysis to ensure that our product meets an urgent real-world need in a safe and responsible way.
Project advisors
Professor Eriko Takano
Professor Rainer Breitling
Project supervisors
Dr Daniel Schindler
Team:Manchester/Poster
HippoSol – The Sunscreen for the Future
Introduction & Inspiration
Our team set out to create a project involving skin cancer, the 5th most common type of cancer in the United Kingdom. Preliminary research included common challenges faced when preventing and treating the disease. This led us to research sunscreen, our main line of defence against cancer-causing sun damage. It turns out, mainstream chemical sunscreens contain a plethora of toxins which have been found to damage coral reefs around the globe. This has a domino effect of damage to the marine ecosystem as a whole, risking the survival of our oceans. We decided to tackle both problems by aiming to create a sunscreen that’s safe for the body and for the marine environment.
We discovered a novel compound that can act as a UV filter, without toxic effects on the environment. The compound comes from the hippopotamus, which has evolved to combat the sun’s rays by secreting mucus which contains specific anti-UV acids, appropriately named hipposudoric and norhipposudorc acid. Its organic origins give our team hope to utilise it for a sunscreen to prevent the destruction of coral reefs. Hipposudoric acid is a natural molecule, but its biosynthetic pathway remains a mystery. We resorted to computational retrosynthesis and proposed seven possible pathways that could synthesize this molecule from precursors in the E. coli metabolome. Flux balance analysis helped us predict conditions that would optimise production of our target in engineered E. coli cells.
We want to develop hipposudoric acid into a successful product for the consumer market. Extensive market research was carried out on the suncare market. This inspired us to develop our own learning resource for other teams to enhance their entrepreneurial skills. The market research and scientific achievements were informed by our extensive human practices work: collaborations, surveys, and online creative platforms provided us with invaluable feedback and information from stakeholders, ranging from dermatologists to the general public.
We discovered a novel compound that can act as a UV filter, without toxic effects on the environment. The compound comes from the hippopotamus, which has evolved to combat the sun’s rays by secreting mucus which contains specific anti-UV acids, appropriately named hipposudoric and norhipposudorc acid. Its organic origins give our team hope to utilise it for a sunscreen to prevent the destruction of coral reefs. Hipposudoric acid is a natural molecule, but its biosynthetic pathway remains a mystery. We resorted to computational retrosynthesis and proposed seven possible pathways that could synthesize this molecule from precursors in the E. coli metabolome. Flux balance analysis helped us predict conditions that would optimise production of our target in engineered E. coli cells.
We want to develop hipposudoric acid into a successful product for the consumer market. Extensive market research was carried out on the suncare market. This inspired us to develop our own learning resource for other teams to enhance their entrepreneurial skills. The market research and scientific achievements were informed by our extensive human practices work: collaborations, surveys, and online creative platforms provided us with invaluable feedback and information from stakeholders, ranging from dermatologists to the general public.
Project Values
Our core values mainly stem from two sources, our interviews with Lousie Laing, co-founder of people4ocean and the extensive YouTube analysis of sunscreen-related videos. Key core values we have acquired through our project are:
- Finding a reef-safe, broad-spectrum sunscreen is difficult on a budget. We aim to keep our product affordable, aiming at a retail cost around 10 pound per 500ml; thus, we may need to adapt our experimental design in the future to achieve this.
- It is difficult to find sunscreens that are suitable for darker toned skin. We hope to create a product mimicking several skin shades in order to avoid white-cast problems commonly affecting people of color.
- Transparent communication between HippoSol and the customer is a priority. Our Wiki emphasizes information on UV rays’ impact on the skin, explains common sunscreen labels and application to sensitive skin. This honesty with the customer will enhance their trust in our product and minimize improper application of the product.
Human Practices
Attention to Human Practices enabled our team to situate our project in its social and environmental context to better understand the issues that should influence our design and the future use of our technology.
Interviews
YouTube Analysis
Surveys
Reflection
Interviews
- Conducted online interviews with different stakeholder groups including current producers of sunscreen, a potential future consumer, an expert dermatologist and a coral reef researcher.
- Highlighted what people value in a modern sunscreen: broad-spectrum protection and affordability.
- Discussed how our project strengthens marine biodiversity by improving the fitness and productivity of coral ecosystems.
- Examined mechanisms of sunscreen failure, to ensure our product would work efficiently in real-life applications.
YouTube Analysis
- Used the open-source video website YouTube as a database of stakeholder opinions in response to COVID-19 limitations
- Identified a shift in the perception of sunscreen towards becoming a beauty product among younger individuals.
- Noticed people of colour have a reduced presence in the sunscreen media, through engagement in discussions we realised our project must be suitable for any skin tone.
- Identified an online community who share our project values.
Surveys
- We conducted 2 surveys during our iGEM project. The first analysed our participants’ use of sunscreen and sun exposure habits, the second challenged internal biases in a politically stressed modern climate.
- We found a more regular use of sunscreen among younger individuals and that the immediate environment determines what SPF someone uses.
- We found evidence suggesting that internal biases, such as world-view and religion, do not influence support for or against biotechnology.
- Showed that our sample embraced our project suggesting HippoSol will be acceptable to consumers.
Reflection
- Provided information to identify areas of consumer confusion to minimise mechanisms of sunscreen failure.
- Reflected on criticisms by using our project as a platform to promote ethical consumerism to ensure HippoSol will have a positive impact on the world.
Pathway Design
Our pathway discovery method is based on retrosynthesis, performed by the computational tool RetroPath2.0, which finds sets of reactions connecting our target compound to possible precursors in the E. coli metabolome.
The resulting reaction sets are processed with RP2paths, which validates them and organises them into potential biosynthetic pathways.
In a third step we predict enzymes that could catalyse the reactions in our novel pathways to Hipposudoric Acid, using the computation tool Selenzyme, which suggests enzyme candidates and ranks them according to their probability of catalysing our new target reactions.
For the final step of our pathway, we propose an oxidative dimerisation of HGA is the missing step for the production of Hipposudoric Acid. We proposed that this oxidation will occur spontaneously in two steps: first producing benzoquinone acetate, which is then further oxidised and dimerises to form Hipposudoric Acid.
The resulting reaction sets are processed with RP2paths, which validates them and organises them into potential biosynthetic pathways.
In a third step we predict enzymes that could catalyse the reactions in our novel pathways to Hipposudoric Acid, using the computation tool Selenzyme, which suggests enzyme candidates and ranks them according to their probability of catalysing our new target reactions.
For the final step of our pathway, we propose an oxidative dimerisation of HGA is the missing step for the production of Hipposudoric Acid. We proposed that this oxidation will occur spontaneously in two steps: first producing benzoquinone acetate, which is then further oxidised and dimerises to form Hipposudoric Acid.
Entrepreneurship
Introduction
Key work completed
Figure 1. Value proposition for HippoSol
Key Results of Business Report
The likely users of HippoSol are consumers looking for eco-friendly sun care and manufacturers of eco-friendly sun care. It is clear the consumers value products that are eco-conscious and HippoSol qualifies for this category. HippoSol is likely to be able to survive competition because HippoSol is predicted to be more eco-friendly than its competitors.
HippoSol can be adapted to threats from competition by enhancing its additional benefits, such as its antimicrobial activity.
Table 1. TOWS Analysis
Key Results of Business Plan
The most crucial milestone for our business plan is the experimental confirmation that our bacteria can produce Hipposudoric Acid. We plan to generate profits by licensing our technology or by selling the technology to a third party. This minimises the risk to our team and our investors. The key future steps of our enterprise would be further experimental research for the development of HippoSol. Our main partners during initial development of HippoSol could be researchers helping us to further explore the beneficial qualities of HippoSol and confirm its safety for consumers and the environment.
Table 2. Business development canvas
- Entrepreneurship is the act of creating value for society through an invention and the implementation of these inventions as novel solutions, products and or services.
- It is essential to integrate entrepreneurship in our project to maximise a value that HippoSol can bring to the society and to facilitate the use of HippoSol. To do this we needed to understand consumer needs and satisfy these within our project design. To facilitate the use of HippoSol in the real world we created a business report and subsequent business plan to visualise this.
Key work completed
- Our business report studied how HippoSol can satisfy identified potential consumer needs, whether HippoSol is resistant to current direct and indirect competition, and the strategies we will use to maximise opportunities and minimise threats.
- Our business plan explained how we would bring HippoSol to the sun care market, what the crucial milestones are for the development of HippoSol, and how we could create a value for investors to obtain resources needed to bring HippoSol to a consumer.
Figure 1. Value proposition for HippoSol
Key Results of Business Report
The likely users of HippoSol are consumers looking for eco-friendly sun care and manufacturers of eco-friendly sun care. It is clear the consumers value products that are eco-conscious and HippoSol qualifies for this category. HippoSol is likely to be able to survive competition because HippoSol is predicted to be more eco-friendly than its competitors.
HippoSol can be adapted to threats from competition by enhancing its additional benefits, such as its antimicrobial activity.
Table 1. TOWS Analysis
Key Results of Business Plan
The most crucial milestone for our business plan is the experimental confirmation that our bacteria can produce Hipposudoric Acid. We plan to generate profits by licensing our technology or by selling the technology to a third party. This minimises the risk to our team and our investors. The key future steps of our enterprise would be further experimental research for the development of HippoSol. Our main partners during initial development of HippoSol could be researchers helping us to further explore the beneficial qualities of HippoSol and confirm its safety for consumers and the environment.
Table 2. Business development canvas
Flux Balance Analysis
After defining our novel pathway towards Hipposudoric Acid, we decided to perform a Flux Balance Analysis (FBA), to understand how our pathway would operate in the context of bacterial metabolism. FBA is a method for analyzing the flux of metabolites through a metabolic network. Using the COBRA Toolbox in MATLAB, the main aim of FBA for our project was to predict optimal growth conditions for the production of our target compound.
To simplify the computational work we decided to do this analysis on a model of E. coli metabolism (iJO1366) plus the reactions in our novel pathway up to the reaction from 3-(4-hydroxyphenyl)pyruvate (34hpp) to Homogentisic acid (HGA), the last precursor for Hipposudoric Acid which is produced in an enzyme-catalysed reaction. The insights gained from the FBA will help us in the future to optimise production of our target compound, possibly by engineering the host strain metabolism.
Figure 1. FBA prediction of the maximum growth rate at various glucose uptake rate (GUR) and oxygen uptake rate (OUR).
To simplify the computational work we decided to do this analysis on a model of E. coli metabolism (iJO1366) plus the reactions in our novel pathway up to the reaction from 3-(4-hydroxyphenyl)pyruvate (34hpp) to Homogentisic acid (HGA), the last precursor for Hipposudoric Acid which is produced in an enzyme-catalysed reaction. The insights gained from the FBA will help us in the future to optimise production of our target compound, possibly by engineering the host strain metabolism.
Figure 1. FBA prediction of the maximum growth rate at various glucose uptake rate (GUR) and oxygen uptake rate (OUR).
Proof of Concept
Due to the COVID-19 pandemic and the lockdown and distancing measures imposed as a result in the UK, we have been unable to work in the laboratory ourselves. We have been lucky in that our host institution, the Manchester Institute of Biotechnology of the University of Manchester, did reopen after some months, and some of its members have been able to continue carrying out laboratory work during the pandemic. They generously collaborated with our team to produce the following results.
Figure 1. Mass spectrometry data for the three replicates from the DH5alpha saHPPD recombinant strain, and the standard of HGA.
We cloned and expressed two variants of 4-Hydroxyphenylpyruvate dioxygenase chosen from the output from our Pathway Design enzyme selection tool, Selenzyme. One of them is an enzyme from Pseudomonas aeruginosa (paHPPD), the other from Streptomyces avermitilis (saHPPD). The enzymes were expressed in Escherichia coli strain DH5alpha, and the product (HGA) validated through Mass Spectrometry and through observing the formation of the red-brown pigment indicating polymerisation of HGA to hipposudoric acid and progressively larger polymers in the presence of oxygen, as we had predicted in our pathway design.
Figure 2. (A) negative control; (B) paHPPD expression; (C) saHPPD expression after 2 weeks storage at 4 degrees; initially, the cultures turned red, indicating the formation of hipposudoric acid. Further polymerization proceeds slowly, and reduces the environmental harm caused by our product
Conclusions and Achievements
- We carefully analysed the sunscreen market and interacted with YouTube sunscreen user communities to develop our ideas for an eco-friendly and sustainable new sunscreen product
- We successfully used computational retrosynthesis to engineer a novel biosynthetic pathway to a reef-friendly natural sunscreen
- We used constraint-based modelling (FBA) to predict the function of our pathway in the context of bacterial metabolism
- We obtained proof of concept by expression of our engineered novel pathway in E. coli
- We used our newly gained entrepreneurship skills to contribute an Entrepreneurship Learning Resource for other iGEM teams
- We partnered with Team St Andrews to explore stakeholder reactions to our project and adjusted our project plans based on their input
- We collaborated with Team Heidelberg to understand the factors underlying the success of iGEM Wiki designs – providing guidance for future iGEM teams
Contributions
Wiki Study
What makes an iGEM Wiki likely to appeal to the judges and win the Wiki prize? We decided to find out through a collaboration with Team Heidelberg who wrote a script to carry out web scraping for our data collection on a large set of previous iGEM Wikis. We carried out a binomial logistic regression analysis comparing various quantitative features of
Figure 1. Bar chart depicting Variable significance in relation to winning the Wiki prize
Learning resource
What makes an iGEM Wiki likely to appeal to the judges and win the Wiki prize? We decided to find out through a collaboration with Team Heidelberg who wrote a script to carry out web scraping for our data collection on a large set of previous iGEM Wikis. We carried out a binomial logistic regression analysis comparing various quantitative features of
- “winners”, those teams which have won the wiki prize and the runners up, and
- “losers”, teams that did not win the wiki prize
Figure 1. Bar chart depicting Variable significance in relation to winning the Wiki prize
Learning resource
Doing our extensive entrepreneurship work was a step out of our team’s comfort zone, and when looking at previous iGEM projects we noticed a distinct lack of entrepreneurship integrated into the projects. This surprised us because the iGEM criteria fits the definitions of enterprise; the application of novel solutions or ideas to practical solutions or problems. Thus, we decided to help other iGEM teams explore entrepreneurship to increase the value their projects by producing an “Essential Entrepreneurship” learning resource. Our resource consists of 7 modules covering key entrepreneurial considerations such as value creation, market review and acquisition of intellectual property. This work was guided by interviews with members of iGEM’s Entrepreneurship Program Innovation Community (EPIC).
Future Steps
Our results show that Hipposudoric acid is a strong candidate for an organic UV filter. However, this project is far from over; there remains plenty of work to do for future iGEM teams. Before commercialisation, it will be necessary to scale up production, develop protocols to control the degree of polymerisation desired for the final product, set up a pipeline for the purification of our compound, and characterise its properties in more detail. Additionally, it will be essential to carry out a toxicology study and allergenicity tests to confirm our product's safety for human use.
We also expect future teams to build on our entrepreneurial resource by incorporating more aspects of the business field, helping young scientists gain their foothold in the entrepreneurial world, such as: creating an outline for a strong business pitch. This way, more iGEM teams will be able to analyse the commercial feasibility of their own projects. For the future of HippoSol, working with marketing specialists, chemical manufacturers and distributors will be the next key steps in understanding the logistics and financial requirements for a sunscreen start-up company.
Our Wiki study also offers opportunities for future expansion, by collecting data from future and current teams, and incorporating new variables such as: the ratio of original figures vs those taken from other sources. Additionally, it would be of great interest to have a closer look at the change in quality of Wikis over time, and how that affects the standards of the competition. The expansion of our Wiki study will assist future teams to make the most of their Wiki.
We also expect future teams to build on our entrepreneurial resource by incorporating more aspects of the business field, helping young scientists gain their foothold in the entrepreneurial world, such as: creating an outline for a strong business pitch. This way, more iGEM teams will be able to analyse the commercial feasibility of their own projects. For the future of HippoSol, working with marketing specialists, chemical manufacturers and distributors will be the next key steps in understanding the logistics and financial requirements for a sunscreen start-up company.
Our Wiki study also offers opportunities for future expansion, by collecting data from future and current teams, and incorporating new variables such as: the ratio of original figures vs those taken from other sources. Additionally, it would be of great interest to have a closer look at the change in quality of Wikis over time, and how that affects the standards of the competition. The expansion of our Wiki study will assist future teams to make the most of their Wiki.
Acknowledgements and References
Acknowledgements
Dr Mark Dunstan
Dr Andrew Currin
Dr Ruth Stoney
Dr Hong Zeng
Dr Kris Niño Valdehuesa
Jakub Chromy MSc
Prof Philip Shapira
Louise Laing MSc
Nick Matthews
Will Wright MSc
St Andrews Team
Heidelberg Team
Sponsors
References
Dr Mark Dunstan
Dr Andrew Currin
Dr Ruth Stoney
Dr Hong Zeng
Dr Kris Niño Valdehuesa
Jakub Chromy MSc
Prof Philip Shapira
Louise Laing MSc
Nick Matthews
Will Wright MSc
St Andrews Team
Heidelberg Team
Sponsors
References
- Hashimoto K, Saikawa Y, and Nakata M. (2007) Studies on the red sweat of the Hippopotamus amphibius. Pure and Applied Chemistry 79, 4, 507-517
- Delépine B, Duigou T, Carbonell P, Faulon JL. (2018) RetroPath2.0: A retrosynthesis workflow for metabolic engineers. Metabolic Engineering 45, 158-157
- Carbonell P, Wong J, Swainston N, Takano E, Turner NJ, Scrutton, NS, Kell DB, Breitling R, and Faulon JL. (2018) Selenzyme: Enzyme selection tool for pathway design. Bioinformatics 34, 2153-2154
- Orth J, Thiele I, and Palsson, B. (2010) What is flux balance analysis? Nat Biotechnol 28, 245–248
- Heirendt L, Arreckx S, Pfau T, et al. (2019) Creation and analysis of biochemical constraint-based models using the COBRA Toolbox v.3.0. Nat Protoc 14, 639–702
- Vlassis N, Pacheco MP, Sauter T. (2014) Fast reconstruction of compact context-specific metabolic network models. PLoS Comput Biol 10, e1003424
- Orth JD, Conrad TM, Na J, Lerman JA, Nam H, Feist AM, and Palsson BØ. (2011) A comprehensive genome-scale reconstruction of Escherichia coli metabolism. Molecular Systems Biology 7, 535
- Zhang E, Brewer JM, Minor W, Carreira LA, Lebioda L. (1997) Mechanism of enolase: The crystal structure of asymmetric dimer enolase−2-phospho-d-glycerate/enolase−phosphoenolpyruvate at 2.0 Å resolution. Biochemistry, 6, 12526-12534.
- Carbonell P, Jervis AJ, Scrutton NS, Robinson CJ, Yan C, Dunstan M, Swainston N, Vinaixa M, Hollywood KA, Currin A, Rattray NJW, Taylor S, Spiess R, Sung R, Williams AR, Fellows D, Stanford NJ, Mulherin P, Feuvre RL, Barran P, Goodcare R, Turner NJ, Goble C, Chen GG, Kell DB, Micklefield J, Breitling R, Takano E, Faulon JL. (2018) An automated Design-Build-Test-Learn pipeline for enhanced microbial production of fine chemicals, Communications Biology, 1, 1-10
- Danovaro, R, Bongiorni, L, Corinaldesi, C, Giovannelli, D, Damiani, E, Astolfi, P, Greci, L, Pusceddu, A. (2008) Sunscreens cause coral bleaching by promoting viral infections, Environmental Health Perspectives, 116, 441-447
- Galasso V, Pichierri F. (2009) Probing the Molecular and Electronic structure of Norhipposudoric and Hipposudoric acids from the red sweat of Hippopotamus amphibius: A DTF investigation, The Journal of Physical Chemistry, 113, 2534-2543
- Corinaldesi C, Marcellini F, Nepote E, Damiani E, and Danovaro R. (2018) Impact of inorganic UV filters contained in sunscreen products on tropical stony corals (Acropora spp.) Science of The Total Environment, 637, 1279-1285
- Mintel, Romanowski S. (2015) Brands try to increase sunscreen usage by adding more benefits. Available at: s://reports.mintel.com/display/736543/?fromSearch=%3FcontentType%3DInsight%26freetext%3Dsunscreen (Last Access:24/10/2020)
- Mintel, Fisher A. (2017) Sun care UK. Available at: https://reports.mintel.com/display/794403/?fromSearch=%3Ffreetext%3DSuncare (Last Access:24/10/2020)