Abstract
Assembling enzymes into complexes facilitates the transfer of intermediates, insulates intermediate leakage, streamlines the metabolic flux, and increases the production of the desired products during biosynthesis. Here, we report the construction of membrane-bound multienzyme complexes, multienzyme caveolar membranes (MCMs), based on sequential protein assemblies on a membrane scaffold. β-Cav1, an engineered caveolin-1 isoform beta, self-assembles to form caveolar membranes. Enzymes that catalyze the biosynthesis of isopentenyl diphosphate and dimethylallyl pyrophosphate to α-farnesene were assembled on caveolar membranes through noncovalent interactions or covalent protein reactions. Bacterial strains harboring MCMs gave α-farnesene production titers up to 10 times higher than the control strains without enzyme assembly. Isolated MCMs can produce farnesyl diphosphate (FPP) and α-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, α-farnesene, for example.
Published Version
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