Biological Approach
After it became clear that we wanted to enable Escherichia coli to convert methanol into glycogen we had to find a suitable pathway for methanol assimilation. We found the Methanol Condensation Cycle (MCC) to be the most promising one. For implementing the MCC in E. coli, four additional genes are required:
- methanol dehydrogenase 2 from Bacillus methanolicus (Mdh)
- 3-hexulose-6-phosphate synthase from Bacillus methanolicus (Hps)
- 6-phospho 3-hexuloisomerase from Bacillus methanolicus (Phi)
- phosphoketolase from Bifidobacterium adolescentis (Xpk)
In parallel we developed a strategy for our second module: glycogen accumulation. To enhance glycogen formation, the whole synthesis pathway needs to be upregulated. The three enzymes involved in synthesis are:
- GlgC, ADP-glucose pyrophosphorylase
- GlgA, the glycogen synthase
- GlgB, the branching enzyme
On top of that accumulation can be considerably increased by knocking out glycogen degradation enzymes. Those are GlgP, the glycogen phosphorylase and GlgX, the debranching enyzme.