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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.

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.

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 we aim to develop StyGreen and expand its impact beyond the scope of this iGEM project.

Scalability

Throughout our entire project, we have thought about the commercialization of some 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 for example paper factories, sugar refineries and any place where a lot of cellulose waste and bagasse occurs as cellulose is heavy and transporting it over long distances increases the carbon footprint and the price of our styrene 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 complications 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 that from many hydrogen bonds. Apolar stacking interactions also contributed 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. These alterations however require chemical conditions and preparations steps that would greatly reduce the profitability as well as the carbon footprint of our final product. Hence we decided to keep these alterations to a minimum. The solubility of cellulose can already 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 only sites where the cellulose binding domain of our enzyme scaffold can actually bind 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). We considered that styrene might get actively transported out of the cell, using the s.cerevisiae native Pleiotropic Multidrug Resistance system as styrene can also be toxic to the DNA through intercalation and covalent binding. Although the PDR5 system seems to be able to export styrene it cannot be expected to export large quantities of styrene at an efficient rate. Hence 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 was an apparent choice as it 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 which can be introduced simply by stopping of the fermenter stirring. The apolar phase will likely contain many extracellular, apolar impurities which can be removed through reverse extraction with water as styrene is the most apolar compound in the entire mixture. The styrene can be separated from the ethyl acetate through evaporation. The ethyl acetate and the reverse extract can be recycled into the bioreactor system so no fresh apolar phase is required.

Upscaling a company from this tech requires facilities closely located to factories where a lot of cellulose waste occurs, such as paper industry 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 cellulose waste on the other hand is promising but currently difficult as we are working with a genetically modified organism that many companies might not be comfortable handling or might not have suitable facilities for.

SWOT 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") analysis. This analysis is a 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 SWOT analysis is divided in an internal and an external analysis. The internal analysis is done to identify the values and the weaknesses that should be covered to achieve a stronger market position. The external analysis is done to clarify the opportunities we have to grasp and to acknowledge the threats against which we have to protect StyGreen is order to reach its full potential.

• Strengths: characteristics of the business or project that give it an advantage over others.
• Weaknesses: characteristics of the business that place the business or project at a disadvantage relative to others.
• Opportunities: elements in the environment that the business or project could exploit to its advantage.
• Threats: elements in the environment that could cause trouble for the business or project.

Protecting our intellectual property

We set out to protect our intellectual property by meeting with an IP-lawyer and the university’s Intellectual Property office to find out which parts of our project we could patent. 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. 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. (Our method to degrade cellulose and produce styrene. First use Ball Milling to mechanically break down the product, and then use phosphorylation to chemically alter it. Lastly, use the scaffold enzyme to break down the preprocessed cellulose into glucose.)

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 joined quest of exploring the possibility to produce bio based styrene in a scaled-up industrial setting post-iGem.

To conclude, StyGreen is a promising product and although our production cannot 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 outcomes of our value proposition, industry research and SWOT analysis show that the method will have high potential to be commercialized and that the IP position is strong due to the pending patent. This is further supported by the interest we have received from the industry.