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One of the aspects that motivated our team throughout the project is the fact that iGEM is more than a lab study, it is an opportunity to be immersed fully: all the way from brainstorming a seed idea to looking at extrapolating our findings entrepreneurially. This year, for the first time in iGEM-Groningen history, we tackled the challenge of commercializing our research. We: Investigated how our research could be upscaled from the lab to the industry, designed a bioreactor setup to determine scalability opportunities and set out to protect our intellectual property by meeting with an IP-lawyer and the patent office to find out which parts of our project we could patent. Furthermore, we received interest from multiple companies, pitched to VC’s, and acquired a substantial investment from an industrial styrene-processing giant who produces 90.000 tons of expanded polystyrene annually, aiming to enhance the sustainability of their feedstock.

Value proposition

An Inconvenient Truth, The Paris Agreement, the ambition of the Netherlands to be climate neutral is 2050, record high crude oil prices... There is an increasing interest in a biobased economy from the industry, as well as the government who has the power to stimulate through fiscal policy as well as passing laws.
The interest in bioplastics is also growing. Currently, polylactic acid, polyhydroxy urethane and polyethylene are being produced as bioplastics. However, there’s an ever-increasing demand for durable biobased plastics. Styrene, one of the most used plastic monomers in the world, is not being produced in a biobased way. Humanity’s hunger for styrene is big as hard plastics, precision electronics and bike tires are not likely to become obsolete anytime soon.

From 2018, the price of oil is expected to begin increasing more rapidly, driven by higher demand than supply. Emerging markets, where high population and GDP growth are fuelling higher demand, will also push up prices

We, the iGEM team of Groningen, have developed an s. cerevisiae strain that is able to grow on cellulose as only carbon source while producing styrene. Cellulose is ubiquitous and abundant as it represents ~30 % of the global annual biomass production. Industrial usage of this material is however currently limited by its chemical and physical properties which for many cellulose streams don’t allow for valorization of cellulose into a valuable product. Hence it is commonly burned for energy.

“The time for this research is now, you want to be ready -not getting started- in 2030”

Linda Dijkshoorn, CEO EV Biotech

Our s.cerevisiae strain utilizes the fact that cellulose is nothing but a highly complexed glucose polymer with glucose being the favorite food for most microorganisms. Rather than burning the cellulose, releasing the tediously fixated CO2 back into the environment, our strain can break it down to glucose and grow on it. The second part of our project uses the fact that the chemical structure of styrene is very similar to phenylalanine, a common amino acid in most lifeforms. Conversion of phenylalanine to styrene can therefore be achieved with only little cloning. Due to our enzymatic approach, we do not need big amounts of acids, and will not use energy expensive heating techniques. We aim to develop StyGreen and expand its impact beyond the scope of this iGEM project.

Scalability

“Optimizing scale and reliable production are of the utmost importance when producing a successful bioplastic”

Vice-President Materials, Toy Multinational

Throughout our entire project, we have thought about the commercialization of aspects of it and strengthening our entrepreneurial skills in the process. We organized meetings with biotech entrepreneurs, professors and business people alike. We got valuable feedback for both the upscaling of our strain into a bioreactor as well as the upscaling of said bioreactor into a company.
One important implication of our approach is that the fermenters should be closely located to e.g. paper factories or sugar refineries (places where cellulosic waste os abundant). Cellulose is heavy and transporting it over long distances increases the carbon footprint and price of StyGreen considerably. Therefore, we decided to look into the scalability opportunities to see how our research project could be extrapolated from the lab to an industrial setting and be commercialized. To implement our microorganism in a working technology we need to find the best way of upscaling the production. For this we designed a bioreactor that enables our yeast to grow under ideal circumstances while optimizing styrene yield.

The two main challenges we tackled through our design are difficulties of bringing cellulose into solution and extracting styrene from the system.
Cellulose consists of many long chains of cellobiose polymers held together hydrogen bonds. Apolar stacking interactions also contribute to insolubility. Both the polar and apolar interactions of cellulose strains can be decreased through phosphorylation of the 6 position or methylation of the 2, 3 or 6 position. However, this requires chemicals and preparations steps that greatly reduce profitability and increases the carbon footprint of StyGreen. Hence, we decided to keep these alterations to a minimum. The solubility of cellulose can be increased significantly when only 30% of the 6 positions are phosphorylated. On top of that we aim to employ dry ball milling to reduce particle size and crystallinity of the cellulose while increasing the amount of amorphous regions. This is important as amorphous cellulose doesn’t form strong apolar interactions, which increases the solubility while also being the site where the cellulose binding domain of our enzyme scaffold binds to cellulose.

The second problem our strain faces is, that the apolar styrene is likely to localize into the lipid bilayer which will eventually destroy the membrane, limiting the amount of styrene that can be produced in a single batch before the cells die (modeled here). Styrene may get actively transported out of the cell, using the s.cerevisiae native Pleiotropic Multidrug Resistance system as styrene can be toxic to the DNA through intercalation and covalent binding. The PDR5 system is be able to export styrene, although it cannot yet be expected to efficiently export large quantities. Therefore, we decided to employ a biphasic medium in our bioreactor. In a biphasic medium an insoluble, organic phase is added to the aqueous medium. Ethyl acetate can donate hydrogen bonds while not accepting them, making it immiscible in water while not denaturing extracellular proteins. Styrene has a high preference for the apolar phase (log P = 2,8) and hence will likely localize into the ethyl acetate phase which can then be siphoned off the system after phase separation. This can simply be introduced by terminating fermenter stirring. The apolar phase contains extracellular, apolar impurities which can be removed through reverse extraction with water: styrene is the most apolar compound in the entire mixture. The styrene is separated from the ethyl acetate through evaporation. The ethyl acetate and the reverse extract are recycled into the bioreactor system: no fresh apolar phase is required.

Through use of our metabolic modelling we discovered that trading culture growth for styrene production was not beneficial. Therefore, we aim to limit growth and biomass by introducing tryptophan deficiency thorough a knock out which is beneficial as tryptophan competes with phenylalanine synthesis for the ARO5 substrates chorismate, thereby increasing the flow to phenylalanine. Continuous fermenting has many advantages over a batch or fed-batch approach. Because of the relatively slow growth on cellulose, the tryptophan knockout and most importantly the biphasic medium with easy styrene removal. Running the bioreactor system therefore requires continuous cooling, stirring, phosphorylated and ball milled cellulose, tryptophan and other essential chemicals for medium. The system produces heat, yeast biomass and styrene.

Upscaling a company from our technology requires facilities closely located to cellulosic waste, such as paper factories or sugar refineries. Contracts with both cellulose suppliers as well as styrene customers, that value sustainability, are essential as well. Licensing our system to companies that have a lot of cellulosic waste on the other hand is also promising. When licensing, current reservations in the field of GMOs need to be taken into account: we are working with a genetically modified organism that companies currently may not be comfortable handling or have suitable facilities for.

SWOT and Porter analysis

For the successful commercialisation of StyGreen, we aimed to realistically map our technologies opportunities and limitations using a SWOT ("Strengths Weaknesses, Opportunities and Threats") and a Porter analysis. The SWOT and Porter analysis are frequently used strategic planning technique used to help a venture identify strengths, weaknesses, opportunities, and threats. In this way it is possible to determine how its objectives can be accomplished, and what obstacles must be overcome (or minimized) to reach the desired potential. The analysis is done to identify strengths and weaknesses, in order to strengthen market position. Furthermore, to identify opportunities and threats, to protect StyGreen is order to reach its full potential. We mapped the strengths, weaknesses, opportunities and limitations of StyGreen during our TRIZ masterclass.

Protecting our intellectual property

To challenge ourselves and maximize StyGreen’s potential, as an iGem Groningen first, we set out to protect our intellectual property by meeting with an IP-lawyer and the university’s Intellectual Property council to find out which parts of our project we could patent, what goes into filling a patent, and how IP is divided among stakeholders. We discovered that in order to acquire a patent, our findings on the patented topic may not have been disclosed yet, and have to be novel and inventive. Furthermore, we learnt that patenting a sub-step of an invention can have many benefits over patenting the entire process. While researching we decided that the technology most suitable to patent was our novel way to transport trans-cinnamate outside of the cell and while also outsourcing the PAL2 and FDC1, using multiple export tags. Read into our intellectual property adventure here.

Business Model Canvas

Industry interest

Economics 101 is the theory of supply and demand. Commercial success is not just dependent on the quality of our own research, the ‘supply.’ Demand from the industry is necessary to successfully commercialize our project. Throughout our project we received interest from multiple parties including feedstock suppliers, biotech- and styrene processing companies including EPS producer Unipol and toy-giant LEGO. Interviewing these parties allowed us to understand the important aspects to keep in mind when exporting our research to an industrial setting. Furthermore, we pitched to VC’s, and acquired a substantial investment from Unipol, an industrial styrene-processing giant who produces 90.000 tons of expanded polystyrene annually, aiming to enhance the sustainability of their feedstock. Their financial investment as well as interest in our technology as a large industrial player is highly valuable to the entrepreneurial success of our project. We plan to continue our quest of exploring the possibility to produce bio based styrene in a scaled-up industrial setting post-iGem.

StyGreen is a promising product and although our production cannot currently compete to petrochemically produced styrene by a considerable financial margin our product will become more competitive as the availability of crude oil decreases while prices rise and customers value carbon footprint higher than manufacturing costs. The outcome of our value proposition, industry research, expert interviews and SWOT analysis show that the method has high potential to be commercialized and that the IP position is strong, operating in a market with a high barrier to entry. This high potential is further supported by the interest we have received from the industry.