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 enzymes 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)
Simultaneously, 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, the 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.
By modeling both pathways, we found out that the combination of both modules is possible and can result in remarkable glycogen accumunlation.