Designer Glycolysomes: Colocalisation of Glycolytic Enzymes on a Cellulosome-Based Synthetic Protein Scaffold.

In systems biocatalysis, combining pathway enzymes in vitro allows for the conversion of basic substrates into more complex, valuable chemicals. However, in vitro enzyme cascades are not yet economically viable for large-scale bio-based chemical production. Enhancing pathway efficiency through enzyme colocalization on synthetic protein scaffolds is a proposed solution, though still debated. We constructed a synthetic protein scaffold that colocalises the first three glycolytic enzymes using cohesin-dockerin interactions. Initially, we converted wild-type enzymes to the docking enzyme mode and evaluated their activity. Next, we demonstrate how the colocalisation of the three docking enzymes on distinct scaffolds enhances the enzyme cascade's production. Starting from glucose, the multi-enzyme complexes produced fructose-1,6-bisphosphate, confirming the activity of each enzyme. PfkA, which converts fructose-6-phosphate and ATP to fructose-1,6-bisphosphate and ADP, was identified as the rate-limiting enzyme. We demonstrated that scaffolding proximity effects lead to higher product output than free docking enzymes, particularly at lower enzyme densities. Further research is needed to determine the relevance of enzyme colocalisation under industrial production settings. In addition, optimising an enzyme cascade demands a thorough understanding of reaction mechanisms and kinetics. The VersaTile method streamlines optimisation studies of modular proteins and complexes, enabling analysis of a broader design space by bypassing technical preparatory hurdles.