Project Description

Devided 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.

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 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.

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.

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.

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 MMOH^7⁠. 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 MMOH^7–9⁠. 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 proteins^5,6⁠, 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-like chaperone4⁠
mmoQ	MMOQ	69.8	two-component signal transduction system	regulator4⁠
mmoS	MMOS	128.6		sensor4⁠
mmoR	MMOR	63.4	--	Transcriptional activator4⁠

2. Methanol to biomass

something

lots of text you know!

3. Airfilter

something more...

lots of text you know!