Team:UiOslo Norway/Description
Project Description
Devided in three sub-goals
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 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 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 (LINK??). 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
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.
| 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-likechaperone4 |
| mmoQ | MMOQ | 69.8 | two-component signaltransduction system | regulator4 |
| mmoS | MMOS | 128.6 | sensor4 | |
| mmoR | MMOR | 63.4 | -- | Transcriptionalactivator4 |
2. Methanol to biomass
something
lots of text you know!
3. Airfilter
something more...
lots of text you know!