When a bioprocess is developed in the lab, glucose is a popular choice for a carbon source. Even for industrial processes, sugars in general remain the number one substrate.
While some processes will be adapted to methanol as the carbon source, most existing processes will still rely on sugars as these are well established and laborous to change.
By converting renewable methanol to glycogen, the bacterial equivalent to starch, we will provide a universal carbon source, connecting many existing bioprocesses to a sustainable substrate.
To pave the way for an industrial process, we need to modify E. coli to produce high concentrations of glycogen. It has previously been shown that a knockout of one glycogen degradation enzyme leads to the accumulation of glycogen in the cells (Fig. 2) . To further improve the production of glycogen in E. coli, we approached this problem in our project in two ways:
Investigating and overexpressing the synthesis enzymes, GlgA, GlgB and GlgC - Learn more about Synthesis
Knocking out the glycogen degradation enzymes, GlgX and GlgP - Learn more about Knockouts
|Figure 1 - Glycogen enzymes in E. coli|
GlgC forms ADP-glucose from ATP and glucose-1-phosphate. The ADP-glucose is then used by GlgA which also serves as the starting particle through autophosphorylation. GlgB adds branches to the existing chains forming α-1,6-glycosidic bonds. GlgX degrades glycogen by cleaving α-1,6-glycosidic bonds whereas GlgP removes glucose units from the end of linear chains.
|Figure 2 - ∆glgP E. coli cells|
E. coli cells lacking glgP are shown. They accumulated glycogen in granules. By Alonso-Casaju´s Nora et al. 2006 
- Created and characterized single knockouts of glgX and glgP in Escherichia coli BL21 Gold (DE3)
- Achieved a double knockout ∆glgP/glgX in E.coli NEB10β
- Assembled and characterized a functional glycogen synthesis operon
- Combined and characterized synthesis operon (glgCAB) and knockout of glgP in one organism
The glycogen synthesis genes, glgA, glgB and glgC as well as the polycistronic constructs, glgAB and glgCAB, were cloned into BioBrick standard, submitted to the registry, and the expression of all genes was shown on a SDS gel.
Moreover, we successfully knocked out the genes glgP and glgX in BL21 Gold (DE3) using Cas9 and inserted our iGEM Aachen MultiStop. In NEB10β not only the single knockouts but also a glgX/P double knockout was accomplished.
After generating many constructs for glycogen accumulation, we used iodine staining to qualitatively characterize our cells. With this method we showed that the ΔglgP knockout as well as the overexpression of GlgA, GlgC and GlgCAB in Bl21 Gold (DE3) produce considerably more glycogen than the BL21 wild type.
- ↑ Liu S. 2013. Bioprocess Engineering: Kinetics, Sustainability, and Reactor Design.
- ↑ 2.0 2.1 Alonso-Casaju´s Nora et al. 2006. Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli