Project overview

Methane is a potent greenhouse gas, and is leaked into the atmosphere at different natural and industrial places. A big part of the industrial methane emission is in the agricultural sector in places like barns¹, Bacteria in the rumen of cows and other cattle produce methane. ⁠or paddy fields^2,3 Bacteria in the soil that produce methane. (rice fields). Natural methane emission places are for example wetlands⁴ land areas saturated with water in which methane producing bacteria reside. or gas hydrates⁵ Gas hydrates are trapped ice-like crystals of gas that are only stable in a specific temperature and pressure range. Found on continental shelves and under permafrost. To minimize the leakage of methane in these or other places one would want to breakdown methane locally. Or even better, one could convert methane to methanol or biomass so it can be more easily transported and used as a bio-fuel instead of being discarded. The current technology doesn't allow this kind of small scale local breakdown of methane, because this process requires high pressure and very high temperatures to break the strong bonds within one methane molecule.⁶⁠ An attractive alternative is bio-conversion of methane. Methanotrophs single-cell organisms that metabolise methane. can naturally breakdown methane and use it as their sole carbon and energy source. Even better, the enzyme methane monooxygenase (MMO) link to more info that these methanotrophs use can breakdown methane at ambient temperatures and pressure.^6-9⁠

Thus if we could understand and use this enzyme it would be possible to develop tools to minimize the methane leakage at many places like barns and gas hydrates. However our knowledge about methanotrophs is limited and culturing them is relatively difficult and slow. That is why we want to implement MMO into Escherichia coli (E. coli) so that it can breakdown methane. Since methanol, the breakdown product of methane, is poisonous, we will also implement the Ribulose-Monophosphate (RuMP)- pathway from Bacillus methanolicus to convert methanol to biomass in three steps. To hold the bacteria we want to design an air-filter that could be used practically anywhere for this purpose. Part of our project is based on the iGEM 2014 team from Braunschweig, Germany

Project goal

To reach our goal, or come as close as possible, we divided our project in three sub-goals

1. Breakdown of methane to methanol in E. coli: Using the enzyme complex soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath).


2. Conversion of methanol to biomass in E. coli (based on Müller et. Al 2015) Using the following enzymes from Bacillus methanolicus MGA3;
   * Methanol to formaldehyde with the enzyme methanol dehydrogenase (medh2)
   * Formaldehyde to hexulose-6-phosphate with the enzyme 3-hexulose-6-phosphate synthase (hxlA)
   * Hexulose-6-phosphate to fructose-6-phosphate with the enzyme 6-phospho-3-hexuloisomerase (hxlB)


3. Air-filter containing the engineered E. coli A closed container to restrict the genetically modified E. coli. The surrounding air will be pumped through the filter so the methane gas can be taken up by E. coli and be converted to biomass.

References

1. US EPA, C. C. D. U.S. Greenhouse Gas Inventory Report: 1990 - 2013. at


2. US EPA, C. C. D. Agriculture. at
   
3. Bodelier, P. L. E. Sustainability: Bypassing the methane cycle. Nature 523, 534–5 (2015).


4. Liikanen, A., Silvennoinen, H. & Karvo, A. Methane and nitrous oxide fluxes in two coastal wetlands in the northeastern Gulf of Bothnia, Baltic Sea. Boreal Environ. Res. 14, 351–368 (2009).


5. US EPA, C. C. D. Methane Emissions. at


6. Rosenzweig, A. C. Biochemistry: Breaking methane. Nature 518, 309–10 (2015).


7. Sirajuddin, S. & Rosenzweig, A. C. Enzymatic Oxidation of Methane. Biochemistry 54, 2283–94 (2015).


8. Sazinsky, M. H. & Lippard, S. J. Methane monooxygenase: functionalizing methane at iron and copper. Met. Ions Life Sci. 15, 205–56 (2015).


9. Zhang, Y., Xin, J., Chen, L. & Xia, C. The methane monooxygenase intrinsic activity of kinds of methanotrophs. Appl. Biochem. Biotechnol. 157, 431–41 (2009).