Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux

TLDR

Enzymatic reactions in living cells are highly dynamic yet tightly regulated, prompting enzyme engineers to build multienzyme complexes that prevent intermediate diffusion, improve product yield, and control metabolic flux. The authors present a simple scaffold‑free strategy that uses short peptide tags RIAD and RIDD to assemble enzymes and impose metabolic control in microbial biosynthetic factories. They achieve this by exploiting RIAD‑RIDD interactions to create enzyme assemblies without scaffolds, enabling tunable stoichiometry and geometry. In vitro, these assemblies form protein nanoparticles with adjustable stoichiometry, size, geometry, and catalytic efficiency, and in *E.

Abstract

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD-RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.