Synthetic Protein Scaffolds for Biosynthetic Pathway Colocalization on Lipid Droplet Membranes

Eukaryotic biochemistry is organized throughout the cell in and on membrane-bound organelles. When engineering metabolic pathways this organization is often lost, resulting in flux imbalance and a loss of kinetic advantages from enzyme colocalization and substrate channeling. Here, we develop a protein-based scaffold for colocalizing multienzyme pathways on the membranes of intracellular lipid droplets. Scaffolds based on the plant lipid droplet protein oleosin and cohesin-dockerin interaction pairs recruited upstream enzymes in yeast ester biosynthesis to the native localization of the terminal reaction step, alcohol-O-acetyltransferase (Atf1). The native localization is necessary for high activity and pathway assembly in close proximity to Atf1 increased pathway flux. Screening a library of scaffold variants further showed that pathway structure can alter catalysis and revealed an optimized scaffold and pathway expression levels that produced ethyl acetate at a rate nearly 2-fold greater than unstructured pathways. This strategy should prove useful in spatially organizing other metabolic pathways with key lipid droplet-localized and membrane-bound reaction steps.