Difference between revisions of "Team:Virginia/Entrepreneurship"

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Manifold

MANIFOLD

Index:

Entrepreneurship

Kickstarting Virginia iGEM’s First Entrepreneurship Committee

Starting an entrepreneurship committee was a natural calling for the Virginia iGEM 2020 team – with the launch of iGEM’s new EPIC committee and increasing concerns regarding IP protection, the establishment of an entrepreneurship committee was a no brainer. There was a rising necessity for a space where our teammates could hold discussions and debates addressing our countless questions on commercialization, patentability, and more importantly, the implications of our product. Essentially, our team needed a space where we could hone our skills in creative problem solving, negotiation, and networking in the business sector. We needed to develop a culture which carefully balanced the open-source innovative nature of iGEM. With enough research, informational interviews, and inspiration from iGEM EPIC, we were able to justify the creation. At the time of a global pandemic and national public health crisis, we craved to be even more intentional about our work than before, and entrepreneurship offered the perfect opportunity to ignite this passion.

The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of

bacterial microcompartments

Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.

(BMCs) with encapsulated dsDNA scaffolds

[1]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis. The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.

Our Journey with IP Protection

Questions about what constitutes a public disclosure first arose in conversation between the wet lab and human practices committees. Bridging the gap between our team, the university, and the Charlottesville (Cville) community required openly sharing our thoughts and ideas to seek expertise and collaboration on MANIFOLD. However, we had to be aware of the implications that public disclosures would pose if we had any plans on commercializing our foundational advance in the future. Our lack of expertise in the space in combination with our curiosity to learn more led to our reaching out to lawyers at Cville BioHub—Cville’s diverse biotech center.

Nikki Hastings, the Executive Director of Cville BioHub, was fundamental in helping the team understand how to become better connected with the Charlottesville community while simultaneously protecting our technology, while lawyers David Vance and Rahul Keshap helped us to navigate startup law.

Vance helped us to understand the ins and outs patent filing logistics. He shared his expertise about biotech startups and gave us some key insight into the startup landscape in Cville and iGEM. Insights gathered included:

A public disclosure constitutes any non-confidential disclosure of our idea or invention in a public environment. This meant one-on-one Zoom calls with experts and mentors would not be an issue, but it would be smart on our part to file for a Provisional Patent Application (PPA) a few weeks before the iGEM wiki freeze
Can we write the PPA ourselves? Absolutely! We should draw thorough diagrams illustrating our invention and learn from previous biotech parents to map out our PPA. We should also refer to the official Manual of Patent Examining Procedure (MPEP) to ensure our work is lawful

Vance enabled us to take charge of our own IP protection and we could not have done so without his help.

Keshap, on the other hand, was important in helping us to understand corporate law. He gave us great advice on how to run a team like a business, and consider the future impact of the decisions we made. Insights included:

It is important to clearly define leadership roles within the team-- team members wear multiple hats in a startup, but the ultimate responsibility must belong to someone to get the work done
Vested equity is a technique where equity is ‘vested’ or secured over time-- this way, teammates will have to earn their equity over time by adding evident value to the team
We must be smart about our decision making and constantly consider the implications of our decisions. For example, if we file our PPA before the wiki, is 9 months long enough to secure a proof-of-concept? We had to have a plan 9 months ahead of time, and consult with various experts in synthetic biology and startup law to make an informed decision. This also meant considering bioethics-- understanding the good and bad impacts of our device on the end-user-- which led to the publishing of our Code of Ethical Conduct by human practices.
Maintain touch with your stakeholders and constantly networking is integral to keeping our creativity sharp and our opportunities open.

Thanks to Nikki, David, and Rahul, we learnt a lot about what it takes to go from a competitive iGEM team idea to a startup. Putting their advice into practice, we were able to have our PPA reviewed by Vance, and by the end of September, our PPA was accepted by the USPTO.

Fig 1. Figure taken from iGEM Tainan 2019 for demo purposes. Notice how the figure is much longer than it is wide, and two images are coupled together to achive this. Try to do that as well so it looks good.

Additionally, protein linkers are usually present between this nucleic acid binding domain and the enzyme structure to prevent inhibition of enzyme activity. However the exact linker(s) used, if any, is(are) also dependent on the specific use case of the invention. These pathway enzymes are attached to the nucleic acid scaffolds via their nucleic acid binding domains. The nucleic acid recognition sequences (22) are unique or semi-unique sequences of nucleic acid monomers on the nucleic acid scaffolds to which the utilized nucleic acid binding domains have some degree of molecular complementarity. These nucleic recognition sequences comprise most of the scaffold and mark the locations to which the DNA binding domains of the pathway enzymes attach to the scaffolds. The nucleic acid spacers (32) are relatively short sequences of nucleic acid monomers that are also present on the nucleic acid scaffolds, between the recognition sequences. The linkage between the nucleic acid scaffolds (18) and protein shell (10) provides a means by which the nucleic acid scaffolds are bound to the protein shell through direct or multi-molecule complementarity. This linkage is found between the nucleic acid scaffolds and the protein shell. One example is through the addition of a nucleic acid binding domain (24) to one or more of the shell proteins forming a nucleic acid binding domain, shell protein fusion (14). Like the pathway enzymes, this nucleic acid binding-domain can be either internal to the shell protein structure or at its N or C terminus, where the exact placement depends on the shell protein being utilized. Alternatively, one or more intermediate proteins can be used to adhere the nucleic acid scaffolds to the shell, where the region of the protein interacting with the shell binds the shell via protein-protein complementarity (28) with a given shell protein, and the region of the protein interacting with the nucleic acid scaffold binds another recognition sequence on the nucleic acid scaffold through another nucleic acid binding domain (30). This forms a shell protein binding, nucleic acid domain fusion (26).

Industry Research

Industry research was an integral component in contextualizing the use and applications of MANIFOLD. The chemical manufacturing industry is one of the biggest in the world, producing over $5 trillion worth of chemicals per year. It is a mature market that grows on par with the world economy, at an annual rate of 3%. The key players are established companies from around the world, including DowDuPont in the US; BASF, Evonik and Bayer in Germany; Sinopec in China; Sumitomo, Mitsui, Nippon Kayaku and Mitsubishi in Japan; LG Chem in Korea; and DSM in the Netherlands. The market comprises four subsegments that overlap to differing degrees - diversified chemicals, fine chemicals, specialty chemicals and commodity chemicals.

[1]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

Consolidation within the industry, caused by subpar market expansion and increased globalization, has led to less competition and fewer rivals being able to gain market share from the chemical titans previously mentioned. As key players in the chemical industry continue to innovate, synthetic biology is quickly becoming a go-to for solutions aimed at lowering costs and synthesizing novel products. Many synthetic biology companies are already leading the charge; Amyris has engineered yeast to produce the antimalarial Artemenisin, Geltor is using synthetic biology to produce collagen substitutes, Biossance is biochemically synthesizing squalane, Evolva is producing nootkatone via fermentation, Conagen is using bacteria to produce vanillin, Antheia is biosynthetically manufacturing opioids with yeast, and Renew is using microbes to produce cannabinoids.

[2]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

We, Virginia iGEM, are in the early stages of a paradigm shift towards a sustainable economy, and interest in green chemicals has never been as strong as it is now. Whether this continued push comes from companys’ own initiatives or from government subsidies, a move away from environmentally damaging catalysts and energy intensive reaction conditions is widely regarded as essential.

[1]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

Capital intensity poses a threat to the manufacturing industry. Manufacturers have to analyze long term demand-trends to decide whether to expand or shut down large plants, which can often leave them vulnerable due to short term market volatility. The advantage of synthetic biology is that biosynthetic processes can be scaled up or down with unprecedented ease, allowing for more motility. Another advantage is that biochemical plants can be placed near feedstocks or end-markets, reducing transportation costs and turnaround times.

[1]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

The synthetic biology industry’s rapid expansion has been forecasted by many established financial companies and economic institutions. McKinsey and Company, a global consulting firm, recently reported that biological approaches could end up producing 60% of physical inputs to the world economy.

[3]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

Boston Consulting Group predicted the synthetic biology industry to grow at an annual rate of 34% from $6 billion in 2018 to $20 billion in 2022.

[4]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

A report published by the European Commission suggested that biotechnological approaches could replace 30% of petroleum derived products in the EU by 2030.

[5]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

The Organization of Economic Development published a report in 2009, named “The Bioeconomy to 2030”, that estimated the bioeconomy market at $1.6 trillion by the end of the decade. The report also stated that 39% of the bioeconomy would consist of using smart cells for industrial production of sustainable biomaterials such as PET bottles, synthetic rubber, dietary supplements, cosmetics and others.

[6]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of

bacterial microcompartments

Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.

(BMCs) with encapsulated dsDNA scaffolds

[1]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis. The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.

Market Research

We then decided to narrow down the scope of our research to the market we would first be targeting. The market size for resveratrol was about $62 million in 2017, and is expected to grow to around $140 million by 2026 with a forecasted compound annual growth rate of 9.7%.

[7]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

China is the largest manufacturer of resveratrol, followed by Europe, while North America is the largest consumer. The majority of resveratrol production in Europe comes in the form of grape skin extract, while the Chinese market focuses on knotweed extraction. These extraction processes utilize many environmentally harmful chemicals, such as methanol, ethanol, hydrochloric acid and methyl tert butyl ether.

[8,9]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

The main companies comprising 80% of the resveratrol market are: DSM, Evolva, Sabinsa, InterHealth, Maypro, Laurus Labs, JF-NATURAL, Great Forest Biomedical, Shaanxi Ciyuan Biotech, Chengdu Yazhong, Changsha Huir Biological-tech, Xi'an Gaoyuan Bio-Chem and Xi'an.

[10]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

Our team reached out to each individual manufacturer listed above, however we were not able to identify a production cost for extracted resveratrol, due to exact numbers being guarded as trade secrets. According to Sigma Aldrich, the bulk price of p-coumaric acid— the main substrate in the resveratrol pathway— is $0.004 per mg, while that of trans-resveratrol— our product— is around $0.80 per mg. The 200-fold increase in price is a promising metric for understanding the economic viability of our product, because once our system is optimized it is expected to produce a 1:1 conversion between the two molecules.

In chapter 5 of ‘Peanuts: Processing Technology and Product Development’ (2016), published by the Institute of Food Science and Technology at the Chinese Academy of Agricultural Sciences in Beijing, the price for a kilogram of 95%+ pure resveratrol is stated as being between 8,000-10,000$US. If our system was able to reach near perfect yields and we simply purchased p-coumaric acid in bulk from Sigma-Aldrich, then it would be able to produce a kilogram of pure resveratrol for half the price - around $4,000. The chapter also indicated that global resveratrol production in 2016 was at 50-60 tonnes, while the demand for the same time period was over 100 tonnes, underlining the great future prospects for the resveratrol market and the possibility for new manufacturers to enter the market.

[11]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

After having spoken to the CEO of RevGenetics, another prominent distributor in North America, the team was left with an understanding that there is still a slight stigma around synthetic resveratrol. However, the sentiment is slowly shifting after it has been found that herbicides, pesticides, heavy metals and emodin are present in many extracted resveratrol products, especially those coming from China. Evolva is currently using yeast fermentation to make its signature Veri-te™ resveratrol, touting high purity and consistent batch production. DSM has patented a chemical process that allows it to make its resVida™ resveratrol from 1-(3,5-diacetoxyphenyl)-bromoethane. Ultimately, the sustainable advantages presented by our biosynthetic solution to drug, specifically resveratrol, production evidences its utility as a more environmentally-friendly and cost-effective means of synthesis.