Overview & Motivation

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In addition to our business report, we created a marketing pitch which could potentially be used to convince investors and backers of the importance and potential of our project. You can see the full pitch HERE.

Summary

The purpose of this report is to provide an analysis of the development and commercial economic viability and likelihood of the MycoMimic product. This product was produced by the University of Sydney 2015 international Genetically Engineered Machine (iGEM) team. This report was established by the 2015 Sydney_Australia iGEM team, building on the work of Dr Nicholas Coleman at the University of Sydney.

Our project aims to produce ethylene oxide in a more efficient, environmentally friendly, and timely manner. Ethylene oxide is a prominent and versatile substance that is found in a plethora of products we all use daily. However, at present the production method of ethylene oxide is environmentally detrimental, expensive, and uses non-renewable resources as its reagents. A major focus of governments, industries, companies, and universities over the last few decades is to create a more sustainable future. Indeed, in University of Sydney’s Environmental Sustainability Policy, there is an emphasis on encouraging and “integrating relevant environmental sustainability themes into courses and research” (15.B). Furthermore, numerous environmental and sustainability organisations, including the United Nations Environmental Program, have highlighted the need for greener and more sustainable alternative procedures for production of common compounds.

We believe that the product developed in our project can significantly assist in decreasing carbon emissions and in creating a more sustainable future for all. The future demand for ethylene oxide is only going to increase as global population rises and countries such as China and India expand in wealth and industry. Consequently, in a time where the demand for ethylene oxide is increasing we simply cannot afford to continue using inefficient, environmentally damaging, and expensive methods of chemical synthesis.

Our Pitch

Current Situation

Figure 1 - Breakdown of present ethylene oxide usage.

Ethylene oxide, one of the most abundantly produced compounds, is derived from ethylene via an oxidation process. The industries in which this compound is utilised include, but are not limited to, pharmaceutical, medical, and manufacturing industry. The world production of ethylene oxide is \(9.6 x 10^6\) metric tons and it is likely to be increasing, due to the expand economies of countries like China and India.

Due to the abundant use and versatility of ethylene oxide it is evident this compound is essential to our current society. The use of ethylene oxide is not going to decrease, and thus it is imperative we find a way to produce this critical substance in an environmentally friendly and cost efficient way.

Figure 2 - The advantage and disadvantages of production of ethylene oxide through the complete oxidation process.

Current Method of Production

Traditionally, ethylene oxide has been produced through a chlorohydrin process. This process was replaced by the complete oxidation process, pioneered by Theodore Lefort in 1931. Since the production of ethylene oxide, chemists have successfully synthesised other epoxides including propylene oxide. Epoxides are incredibly versatile due to the addition of the oxygen atom on the tetrahedral ring making the compound strained and unstable. Consequently the ring can be easily opened up, releasing abundant amounts of energy that can be used in further reactions, such as nucleophilic addition, hydrolysis and reduction.

Given these numerous disadvantages of the complete oxidation process, which far outweigh the advantages – it is evident a new, clean, green process needs to be created and implemented on an industrial scale.

Case Study: Production of Insulin by e. coli

Figure 3 - A worker tends to fermentation tanks in an Insulin production plant. Image from ²

Though MycoMimic hopes to break new ground in industrial synthesis, the application of bacteria for large-volume chemical synthesis of chemicals is not without precedent: One of the early breakthroughs in this field came in the development of an e. coli strain able to produce Insulin. This venture draws many parallels with our own, and thus serves as a case study against which we can compare².

Prior to the application of bacterial synthesis Insulin was, much like ethylene oxide, produced by an inefficient and environmentally degrading method. This involved the processing of huge volumes of pig pancreases, with more than 8 tons necessary for the production of each kilogram of purified insulin.² Due to this inefficiency researchers sought, as we do with MycoMimic, an alternative production pathway. This eventuated in the late 1970s, when after much research and development scientists finally demonstrated the ability to express a gene in e. coli which enabled it to produce human-viable insulin. This product, Humulin, represented the first recombinant DNA product the world had ever seen:² An amazing breakthrough.

Production was rapidly scaled up to industrial levels, resulting in manufacturing plants containing thousands of 50,000 liter fermentation tanks. The processes these early pioneers developed inspired those used in our project: Antibiotic resistance is utilised to select out appropriate e. coli strains, which are then left to reproduce in an incubator over the course of some days. Once this reaches maximal capacity it is time to begin production, which is kick-started by the addition of an inducer to the broth. Without the means to break through the cellular membrane the Insulin accumulates inside the body of each bacterium, and thus maximal production is reached within the span of a few hours. Following this the bacteria are separated from their broth using a centrifuge, before being broken open to retrieve precursor compounds to Insulin. Though the bacteria's work is done, chemical adjustment and purification processes follow, eventually resulting in Insulin products perfect for human consumption.

Aside from the core industrial processes involved, many aspects of bacterial Insulin production may provide important lessons for our project. For example, in the Insulin process once fermentation tanks are populated (but before inducers are added), the bacterial population is sampled and checked for unexpected mutations:² Discarding mutants at this stage not only minimises the possibility of undesireable end-products, but also reduces the time and material cost the overall process incurs due to these occasional mutations (which in biological systems are inevitable). Similar safety checkpoints will be necessary in any ethylene oxide synthesis process: Via their inclusion the risk of any physical threats (such as explosion of synthesised products) can be monitored, and financial and time costs due to errors in production may be minimised.

A Solution: MycoMimic

Monooxygenase enzymes are capable of performing this epoxide reaction, thus converting alkenes to epoxides safely and efficiently. These biological catalysts are renewable, non-toxic, biodegradable, and can be scaled up for large-volume production. Our product, MycoMimic is a genetically engineered Pseudomonas Putida bacterium which contains the enzyme ethene monooxygenase. With the insertion and successful expression of this enzyme, the bacterium can produce ethylene oxide.

Figure 3 - The pBBR1MCS2 plasmid showing the ethene monooxygenase insert. The “EtnA” “EtnB”, “EtnC”, and “EtnD” are the four subunits of the enzyme.

Given MycoMimic is an easy product to work with, it can be used on a large and small scale. The only requirements for this product are the bacteria MycoMimic itself and a source of ethylene. Thus, both large and small industrial companies can use this product – creating a more accessible, transparent, and open market. We believe this is important as it creates competition in the market, which will lead to a more diverse and fair environment.

Risk analysis

Technical

It is imperative MycoMimic is closely monitored when it is scaled up to an industry level: Ethylene oxide is a highly flammable and reactive compounds. When working with it on a small scale in the laboratory, it poses minimal and controllable threats. However, when utilised at an industry and commercial scale it has the potential to be dangerous and harmful. Thus, it is imperative it is closely monitored and that the producer ensures there are regular safety and security checks to prevent accidents.

This serves to illustrate another major technical challenge of MycoMimic's application on an industrial scale, which is that the necessary logistical and regulatory framework does not already exists. This would therefore require development, which poses a substantially costly engineering project, in which unforeseen challenges may provide time-consuming road blocks. Though the need for logistical development is mainstay in almost all industrial synthesis, as MycoMimic departs considerably from existing synthesis procedures, this will likely provide a major challenge.

Financial

Financial risks include failing to manage cost restraints and changes. In an unstable market with increasing competition from other countries in the Asia-Pacific region, great effort needs to be taken to ensure the financial aspect of the product is under control, Furthermore, the major rival to this product is the pre-existing method of production form crude oil. In the short term, competition from oil-companies may prove to be a risk. However, in the long-term, the financial advantages of MycoMimic are greater. This is because companies are moving away from non-renewable resources (the investment in coal has dropped dramatically over the last 5 years).

Safety and society

The community reaction to products made from a synthetic and genetically modified organism may prove a risk. In the unlikely (but still possible) event the community does not approve of a product made from a genetically modified organism, then this could be a risk. To combat this potential risk, it will be necessary to firstly establish the different views on this issue and secondly to explain or market the product so that the consumers understand the benefits of MycoMimic both to themselves and the environment.

Product Analysis and Development

A SWOT analysis is used to assess the strengths, weaknesses, opportunities, and threats of this project. Building upon this analysis we can proactively plan an effective strategy that will focus on the strengths of this product while catering for weaknesses and possible threats.

A major advantage of MycoMimic is its capacity to be utilised on a large and small scale. Large companies can produce the thousands of tonnes of ethylene oxide necessary to supply an entire nation. Alternatively, smaller companies could produce ethylene oxide to supply only a state or a few communities. This is beneficial for a number of reasons: Firstly, it allows MycoMimic's utilisation in a flexible and changing market, a market which suits both large scale corporations and small organisations. This creates a safety net, allowing production to be varied in response to a changing unstable market. Secondly, the reduced barrier-to-entry and comparative ease of production provided by MycoMimic creates a more accessible market, preventing monopolies from forming in which the control of this important compound might fall into the hands of a few.

Figure 4 - SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis of MycoMimic.

By considering our SWOT analysis (particularly the weaknesses identified), throughout project we made proactive changes to improve MycoMimic:

• To address the identified weakness of toxicity due to excessively high levels of protein production, we co-expressed the operon LacI which will stop the production of ethylene oxide before these levels are reached.

• To take advantage of the opportunity identified for governmental support, we engaged with the New South Wales state government, in return receiving funding for part of our project (see Attributions).

• To investigate the challenges and possibilities of scaling up production of MycoMimic, we discussed what would be involved, and what this might require, with a number of our sponsoring BioTechnology firms (see Attributions). By sharing their experiences of challenges faced in major BioTech projects they were able to help us develop an appreciation for threats and weaknesses that we might have otherwise not perceived. Encouraged by these discussions we set expression in e. coli as the eventual aim of our project, since this is the most common and effective host for production on industrial scales.

• As public concern and opposition plays a major part in all Synthetic Biological ventures, we were able to tailor our approach to Human Practices to address this. By sharing details of our business plans, particularly how they can lessen the environmental impact of industrial processes, we were able to garner considerable interest, and help to allay common (but often misinformed) fears held by the public.

• By identifying opportunities for expansion of our methodology to biological synthesis of other chemicals, our business plan played an important part in motivating our modelling research: Since many hurdles will need to be overcome with each synthesis pathway developed in the future, we designed adaptable tools for codon optimisation. These can help to open up new opportunities by facilitating the expression of a diversity of genetic circuits in industry-suitable bacteria.

Conclusion

As outlined, MycoMimic has the potential to be an incredibly effective, versatile and necessary product in a time where the world is striving to be more environmentally sustainable and friendly.

Disclaimer – This proposal was written by the Sydney_Australia team as part of the International Genetically Engineered Machine competition. Its aim was to demonstrate the capacity and the MycoMimic project has to be applied to the real world and developed into a working product. However, it is not indicative of a final proposal that should be considered by businesses.

¹ United States Environmental Protection Agency, 1986, Locating and estimating air emission from sources of ethylene oxide, viewed 20th of July 2015, http://www.epa.gov/ttnchie1/le/ethoxy.pdf (p. 11).

² Diabetes Forecast, 2015, Making Insulin: A behind-the-scenes look at producing a lifesaving medication, viewed 30th of August 2015, http://www.diabetesforecast.org/2013/jul/making-insulin.html