Multienzyme Catalysis in Phase-Separated Protein Condensates

Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, and understanding how phase separation regulates multienzyme catalysis may need the help of in vitro investigations. Recently we have constructed synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. Here, we describe the methods for checking the enzyme assembly using fluorescent microscopy and centrifugation assay. We further provide steps for analysis of the cascade enzyme catalytic efficiencies inside the condensates, using enzymes from terpene biosynthesis pathway.