Manipulating sugar utilization in Escherichia coli through synthetic fusions to membrane transporters

Substrate channelling describes the movement of intermediate between active sites without entering equilibrium with the bulk cell material. The fusion of membrane transport proteins to downstream enzymes may enhance substrate channelling of naturally occurring or synthetic pathways in microbes. This thesis describes the production of a system suitable for assembling libraries of protein fusions, with a focus on enhancing D-xylose metabolism, an industrially relevant metabolite.
The cloning technique Golden Gate assembly was selected to produce libraries of chimeric proteins, capable of producing fusions within a one-pot reaction. Low copy number plasmids suitable for expression of membrane proteins were developed and tested for this work, demonstrating high success rate and easy application. The D-xylose transporter XylE, alongside other MFS homologues, were then fused to GFP to confirm stability towards C-terminal fusion.
As XylE was a suitable target for fusion, attention turned towards the E. coli D-xylose isomerase, XylA. The crystal structure of XylA was obtained to understand oligomerization and organisation of the protein – finding that it formed a tetramer, which was distinct from early research suggesting a dimeric structure. However, direct fusion of the XylE C-terminal to the XylA N-terminal caused a loss of function for the D-xylose isomerase, possibly because of its oligomeric state or the location of the N-terminal with respect to the active site. Scaffolds are an alternative to protein fusion to enhance substrate channelling. The cohesin-dockerin scaffold system was therefore applied to reduce the steric hinderance seen in direct fusion – however while cohesin tagging increased XylA functionality, there was no evidence of scaffold formation, and therefore no substrate channelling occurred.
Overall, while this study did not demonstrate improved substrate channelling in the D-xylose utilization pathway, the systems produced are suitable for generating libraries of protein fusions and scaffolds, that, with time and troubleshooting, should facilitate substrate channelling.