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:

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, which were not available (yet) via the BioBrick system. (link to used parts; Bba_K1390001 (MMOB), Bba_K1390002 (MMOC), Bba_K1390003 (MMOD), Bba_K1390004 (MMOX), Bba_K1390005 (MMOY), Bba_K1390006 (MMOZ)). 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.