Team:UiOslo Norway/Description

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 fields2,3 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. The first 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. (Click on a goal to directly scroll down).

  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.
  2. US EPA, C. C. D. Agriculture.
  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.
  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).


Project Description


1. Methane to methanol

Since the C-H bond in methane is very strong and requires expensive high tech equipment1⁠ we want to explore the possibilities of bioconversion of methane. Methanotrophs are single-cell organisms that can oxidize methane and use it as their sole carbon and energy source2⁠. To date there are two enzyme complexes known that can do the task of breaking methane; soluble methane monooxygenase (sMMO), and the membrane bound particulate methane monooxygenase (pMMO)1–3⁠. Both enzymes break methane with the following reaction:

CH4 + O2 + NADH + H+ → CH3OH + H2O + NAD+


Other than that they both can convert methane to methanol and require oxygen for the process, are they structurally very different. Most methanotrophs express pMMO, whereas sMMO is less often present. pMMO is expressed at high copper levels, which makes sense as it uses copper in the core of the enzyme to break the strong C-H bond in methane. At low copper levels however, sMMO is expressed which uses iron-ions in the enzyme core for breaking methane.2–4⁠ The methanotroph Methylococcus capsulatus (Bath) (M. capsulatus (Bath)) is one of the most studied methanotrophs that has both pMMO and sMMO. In our project we used the sMMO operon of (M. capsulatus (Bath)), more information about sMMO (insert link to scroll down).

Last years iGEM (2014) team Braunschweig, Germany cloned the sMMO genes of the methanotroph M. capsulatus (Bath) for the purpose of expressing them in Escherichia coli (E. coli). We chose to build on to their project and got their six cloned sMMO genes Bba_K1390001 (mmoB)
Bba_K1390002 (mmoC)
Bba_K1390003 (mmoD)
Bba_K1390004 (mmoX)
Bba_K1390005 (mmoY)
Bba_K1390006 (mmoZ) , which were not available (yet) via the BioBrick system. In addition will we clone the mmoG gene of the sMMO operon which is thought to encode a chaperone (MMOG) involved in folding of the other MMO proteins5,6⁠. MMOG might also be involved in regulating the sMMO operon by binding to a regulatory protein called MMOR4,5⁠. The Braunschweig team used a plasmid with the chaperones GroES, GroEL and TF to help fold the different MMO proteins.

Summary:

  • Our team wants to engineer E. coli so that it can break down methane by cloning and expressing the sMMO of the methanotroph M. capsulatus (Bath).

  • We got the sMMO genes, mmoX, mmoY, mmoZ, mmoB, mmoC, and, mmoD, from iGEM 2014 team Braunschweig, Germany.

  • We will clone the gene mmoG from genomic M. capsulatus (Bath) DNA ourselves.

Soluble methane monooxygenase (sMMO)

The sMMO operon of M. capsulatus (Bath), figure 1, has ten known protein encoding genes, summarized in Table 1. Two genes, mmoQ, and, mmoS, coding for the proteins MMOQ and MMOS can sense copper and play a role in regulating transcription of the sMMO operon3–5⁠. A third gene, mmoR, encoding the protein 'MMOR', is a transcriptional activator of the whole sMMO operon3–5⁠. Since we want to clone the genes that form the enzyme complex sMMO and use them to construct our own sMMO operon for expression in E. coli, the genes mmoQ, mmoS, and mmoR are not relevant and thus excluded from our project.


Figure 1: sMMO operon of Methylococcus capsulatus (Bath)adapted from Scarlan et. al 2009



Six of the other seven genes, mmoX, mmoY, mmoZ, mmoB, mmoC, and, mmoD, encode the proteins of the sMMO complex. Three of these proteins come together and form one big protein, called MMO hydroxylase (MMOH). Hydroxylation means the adding of an -OH group, in this case the change of methane to methanol (CH4 to CH3-OH) which happens inside MMOH. This reaction is assisted by MMOB, encoded by mmoB. MMOC, encoded by mmoC, is the sMMO reductase by providing MMOH with two electrons (by oxidizing NADH). Or to say is more simple, MMOC will reset MMOH so it can break another methane molecule. MMOD, encoded by mmoD, is thought to inhibit the process by blocking the binding of either MMOB or MMOC to MMOH.

The last gene, mmoG, encodes for the protein MMOG, is not previously been uses in a iGEM project. MMOG is thought to be a chaperone which will properly fold the sMMO proteins.

More technical information about the specific functions of each sMMO subproteins after the summary.

Summary:

  • sMMO is build out of 5 proteins, whereof MMOX, MMOY, MMOZ form one protein complex called MMOH.

  • MMOH, MMOB and MMOC are needed to convert methane to methanol.

  • MMOD inhibits the methane to methanol reaction.

  • MMOG is a chaperone folding the other sMMO proteins correctly.

MMOH, MMOC and MMOB

MMOH, the hydroxylase, consist of three subunits α (60.6 kDa), β (45.1 kDa), and γ (19.8kDa), encoded by mmoX, mmoY, and mmoZ2⁠. These bound subunits form again a dimer, resulting in α2β2γ2, with in the middle a 'canyon'. On each of the αβγ subunits is a binding site for either MMOB, the regulator, or MMOC, the reductase2⁠. Binding of MMOB to MMOH is needed for efficient hydrocarbon oxidation (the actual breaking of the C-H to C-OH bonding)2⁠. MMOC transfers the two electrons from NADH to the diiron site in MMOH2⁠. It is in this diiron center of MMOH where the actual conversion of methane to methanol takes place. The two iron ions, the electrons (which change the iron ions), and the oxygen together with methane in the center of MMOH (bound to MMOB) make the magic happen to break one C-H bond in methane to a C-OH. Afterward helps MMOC to release methanol and resets MMOH by providing new electrons1,3⁠.

MMOD

There is not much known about the function of the 12 kDa protein MMOD, formerly known as orfY. The first group showing that MMOD is expressed in M. capsulatus (Bath), also has evidence that MMOD can bind to the hydroxylase MMOH7⁠. It seems that MMOD binds to MMOH in competition with the regulatory protein MMOB, which hints to an inhibitory function of MMOD7⁠. They also showed a possible involvement of MMOD in the assembly of the metal center in MMOH7–9⁠. But all their data is based on heterogeneously expressed MMO parts and purified from E. coli and used in in vitro studies. Further studies are needed to resolve the functionality of MMOD both in vitro and in vivo.

MMOG

The mmoG gene is suggested to encode a GroEL-like chaperone that contributes to the correct folding of the other sMMO proteins5,6⁠, but this is so far not proven. They did show that MMOG (and 'MMOR') are required for protein binding to the promoter region of the sMMO operon in the methanotroph Methylosinus trichosporium OB3b5⁠. In another iron monooxygenase, Bacterial binuclear iron monooxygenases, a similar protein as MMOG called mimG was needed for proper folding of one of the other monooxygenase proteins6⁠.


Table 1: Summary of the sMMO genes and function.

Gene Protein
(subunit)		 Size (kDa) Proteincomplex Function
mmoX MMO X/α 60.6

MMOH (part of sMMO)
(α2β2γ2)

hydroxylase2⁠
mmoY MMO Y/β 45.1
mmoZ MMO Z/γ 19.8
mmoB MMOB 15.9 sMMO Regulatory2⁠
mmoC MMOC 38.5 sMMO Reductase2⁠
mmoD MMOD 12 sMMO Inhibitor?7⁠
mmoG MMOG 59.5 -- GroEL-like
chaperone4⁠
mmoQ MMOQ 69.8 two-component signal
transduction system		 regulator4⁠
mmoS MMOS 128.6 sensor4⁠
mmoR MMOR 63.4 -- Transcriptional
activator4⁠

References

  1. Rosenzweig, A. C. Biochemistry: Breaking methane. Nature 518, 309–10 (2015).
  2. Sirajuddin, S. & Rosenzweig, A. C. Enzymatic Oxidation of Methane. Biochemistry 54, 2283–94 (2015).
  3. Sazinsky, M. H. & Lippard, S. J. Methane monooxygenase: functionalizing methane at iron and copper. Met. Ions Life Sci. 15, 205–56 (2015).
  4. Csáki, R., Bodrossy, L., Klem, J., Murrell, J. C. & Kovács, K. L. Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus (Bath): cloning, sequencing and mutational analysis. Microbiology 149, 1785–95 (2003).
  5. Scanlan, J., Dumont, M. G. & Murrell, J. C. Involvement of MmoR and MmoG in the transcriptional activation of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b. FEMS Microbiol. Lett. 301, 181–7 (2009).
  6. Furuya, T., Hayashi, M. & Kino, K. Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli. Appl. Environ. Microbiol. 79, 6033–9 (2013).
  7. Merkx, M. & Lippard, S. J. Why OrfY? Characterization of MMOD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). J. Biol. Chem. 277, 5858–65 (2002).
  8. Rudd, D. J. et al. Determination by X-ray absorption spectroscopy of the Fe-Fe separation in the oxidized form of the hydroxylase of methane monooxygenase alone and in the presence of MMOD. Inorg. Chem. 43, 4579–89 (2004).
  9. Sazinsky, M. H., Merkx, M., Cadieux, E., Tang, S. & Lippard, S. J. Preparation and X-ray structures of metal-free, dicobalt and dimanganese forms of soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath). Biochemistry 43, 16263–76 (2004).


2. Methanol to biomass

More information coming soon





























More information coming soon


























3. Filter design

The aim of this part of the project is to design and assemble a filter that makes it possible to capture methane from the atmosphere.
The system includes a filter, a methane sensor, a vessel for gas, a pump and a valve.


Figure 2: Draft of the filter system.

Filter

The most important part of the system is the filter. The filter consists of a plastic column, which is shown in the figure below. Porous, plastic pads were introduced to the column to create turbulence and efficient gas-liquid exchange. Its on these plastic pads that the media containing the engineered E. coli bacteria will be applied. The filter was modified by drilling a hole on a side of the column and sealing the top. This was done in order to connect it to the gas output and to create a close system.


Figure 3: pictures coming soon.

In order to show that our system worked a methane sensor was added. The sensor measured the concentration of methane after the methane had passed through the filter. For the measurement the methane sensor MQ4 was used. Its function is based on SnO2 whose low conductivity rises when natural gases are present. For the MQ4 sensor to work it was connected to Arduino board, which was then assembled to the breadboard.


Figure 4: Assembled sensor.

























More information coming soon