Production of Insoluble Starch-Like Granules in Escherichia coli by Modification of the Glycogen Synthesis Pathway

While investigating the conversion of cellulosic biomass to starch-like materials for industrial use, it was observed that the overexpression of native ADP-glucose pyrophosphorylase GlgC in Escherichia coli led to the formation of insoluble polysaccharide granules within the cytoplasm, occupying a large fraction of the cell volume, as well as causing an overall increase in cellular polysaccharide content. TEM microscopy revealed that the granules did not have the lamellar structure of starch, but rather an irregular, clustered structure. On starvation, cells overexpressing GlgC appeared unable to fully degrade their polysaccharide material and granules were still clearly visible in cultures after 8 days of starvation. Interestingly, the additional overexpression of the branching enzyme GlgB eliminated the production of granules and led to a further increase in cellular polysaccharides. GlgC is generally thought to be responsible for the rate-limiting step of glycogen synthesis. Our interpretation of these results is that excess GlgC activity may cause the elongation of glycogen chains to outpace the addition of side branches, allowing the chains of adjacent glycogen molecules to reach lengths at which they spontaneously intertwine, forming dense clusters that are largely inaccessible to the host. However, upon additional upregulation of the GlgB branching enzyme, the branching of the polysaccharide is able to keep speed with the synthesis of linear chains, eliminating the granule phenotype. This study suggests potential avenues for increasing bacterial polysaccharide production and recovery.