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.