Summary

As a result of the increased greenhouse effect the Earth’s climate is changing noticeably. Methane being a greenhouse gas like carbon dioxide (CO2) traps heat in the atmosphere increasing the global warming. However, it has to be kept in mind that methane’s impact on the greenhouse effect is 25-fold greater compared to CO2.
One of several natural sources of methane is enteric fermentation in ruminants such as milk cows and cattle. In their rumen, billions of microorganisms release greenhouse gases such as carbon dioxide and methane as they help to digest the animal’s food. This is, in fact, a natural process, but industrial-scale farming and increasing demand intensify the emissions and with that the greenhouse effect. Although more and more people are willing to permanently do without dairy products and beef, the majority does not.
Methane can be utilised as a carbon source by so called methanotrophic bacteria one of them being the modelorganism Methylococcus capsulatus. M. capsulatus uses the the well characterised soluble methane monooxygenase (sMMO), an enzyme complex consisting of three subunits, to convert methane to methanol.
Considering the huge impact of methane in comparison to CO2, methane emissions are a substantial point of action for future climate protection and thus gave us the idea for our project: We are going to enable an engineered bacterium to metabolize methane just at its place of origin - the cow’s rumen.
During this iGEM season we were able to develop a technique to successfully express all components of the methane monooxygenase in E. coli in a soluble form and we furthermore proofed the activity of the complete enzyme complex. After construction and transformation of our final device and optimization of the cultivation conditions, we suceeded to produce one of the sMMO’s subunits, MMOC, solubly and another one, MMOX, in inclusion bodies (see figure 1). In order to achieve effective production of the remaining subunits in the soluble fraction we further analysed cotransformation of our engineered E.coli strain and different combinations of known chaperone proteins. Our findings suggest that the expression of the sMMO works best when coexpressed with the bacterial chaperones GroEL and GroES (see figure 2). For reasons of protection against bacteria consuming ciliates in rumen fluid and thus to enable survival of E.cowli we also tested entrapment and cultivation in alginate beads (figure 3). Using a methane sensor we were also able to show degradation of methane by our engineered bacterium E.cowli.
To evaluate the efficiency of E.cowli in the natural environment of the cow’s rumen, we generated a mathematical model based on experimentally achieved and literature data. Hence, we figured out that a total of 50 dollars per year is needed to reduce the annual methane emission by 110 kg per cow. Our mathematical model clearly indicates that a significant amount of methane can be degraded directly in the cow’s rumen when E.cowli is added to the feed. That implies that the greenhouse gas methane issued from enteric fermentation is no longer released into the Earth’s atmosphere and thus can no longer contribute to the greenhouse effect. Future plans are to obtain purified sMMO in appropriate amounts in order to turn it into an industrially useful product. The new usability of the sMMO conquers three major concerns of the industrialized economy: pollution, shortage of energy and of course global warming.
As the sMMO is able to degrade a broad variety of substrates such as one of the major water pollutants, the industrial solvent trichlorethylene (TCE), our project can serve as a foundation for a new approach of biological water treatment or environmental care in general.
One could also exploit the sMMO’s ability to convert methane to methanol: Methane can serve as a source of energy but due to its gaseous state it is very difficult to transport compared to
the liquid methanol. Up to now methane is chemically converted to methanol for transportation and later again chemically converted to components of diesel fuel and gasoline or to propylene and ethylene, important precursors of important chemical substances. Using the sMMO to oxidize methane is a much more economical and eco-friendly approach to utilize methane as a resource for energy production.
Hence, our project has a huge significance regarding a wide-ranging spectrum of problems and opportunities.

This year's iGEM team of the Technische Universität Braunschweig presents a novel approach towards the reduction of greenhouse gas emissions: We are going to engineer a bacterium able to metabolise methane just at its place of origin - the rumen of the cow. The well characterised and easily manageable model organism Escherichia coli is therefore planned to be equipped with an enzyme complex, the methane monooxygenase (MMO), which turns the greenhouse gas methane into a a natural intermediate of the cellular metabolism. This way, no methane would be released into the atmosphere. The six subunits of the MMO as well as other potentially required proteins will be incorporated into the new E. cowli bacterium. This would allow for the worldwide methane emissions to be reduced and, considering the constant population growth, for future food supply to be rendered more climate friendly.