Project Description

Please read the project overview first.

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:

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 proteins^5,6⁠. MMOG might also be involved in regulating the sMMO operon by binding to a regulatory protein called MMOR^4,5⁠. The Braunschweig team used a plasmid with the chaperones GroES, GroEL and TF to help fold the different MMO proteins.

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 operon^3–5⁠. A third gene, mmoR, encoding the protein 'MMOR', is a transcriptional activator of the whole sMMO operon^3–5⁠. 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.

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.

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 electrons^1,3⁠.