Multienzyme Assembly on Caveolar Membranes In Cellulo

Assembling enzymes into complexes facilitates the transfer of intermediates, insulates intermediate leakage, stream-lines the metabolic flux, and increases the production of the desired products during biosynthesis. Here, we report the construction of membrane-bound multienzyme complexes, multi -enzyme caveolar membranes (MCMs), based on sequential protein assemblies on a membrane scaffold. beta-Cav1, an engineered caveolin-1 isoform beta, self-assembles to form caveolar mem-branes. Enzymes that catalyze the biosynthesis of isopentenyl diphosphate and dimethylallyl pyrophosphate to alpha-farnesene were assembled on caveolar membranes through noncovalent inter-actions or covalent protein reactions. Bacterial strains harboring MCMs gave alpha-farnesene production titers up to 10 times higher than the control strains without enzyme assembly. Isolated MCMs can produce farnesyl diphosphate (FPP) and alpha-farnesene ex vivo, indicating the structural and functional independence of MCMs in vitro, in cellulo, and ex vivo. This work shows an under-reported direction of multienzyme assembly: creating a hydrophobic microenvironment for the biosynthetic enzymes at nanoscales significantly increases the titer of the hydrophobic product, alpha- farnesene, for example.