After our molecular strategy was developed, we put it into pratice in the lab.
To implement the MCC, we successfully built and characterized a polycistronic plasmid expressing all four MCC genes. With this construct we modified an E. coli strain so that it can tolerate higher methanol concentrations. Furthermore, we proved the functional heterologous expression of the methanol dehydrogenase which we considered to be the bottleneck of this pathway.
In parallel to establishing methanol as a carbon source for E. coli, we also enabled it to accumulate remarkable amounts of glycogen. We created and characterized single knockouts of glgX and glgP in Escherichia coli BL21 Gold (DE3) and were also able to do a double-knockout of both genes in E. coli NEB10β. This was done with CRISPR/Cas9 genome editing which we found to be a very efficient method for genome modifications. In addition we assembled and characterized a functional glycogen synthesis operon.
By combining the characterized synthesis operon (glgCAB) and the knockout of glgP in one organism, we further enhanced glycogen formation.
Finally, we fused our two modules by introducing the methanol operon into our BL21 Gold (DE3) ΔglgP strain. We consider this to be the first step taken to make methanol available as a carbon source for many existing bioprocesses.