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.
Highlights
Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated
To facilitate characterization of the hierarchical assembly, the cysteines were introduced to the N termini of RIAD and RIDD to covalently lock the RIAD–RIDD interactions by disulfide bonds and to covalently link tri-enzyme units (TU)
Two-dimensional single-particle analysis on Assembly B found that the crystal structure of the tetrameric MenD can perfectly fit in the images (Supplementary Fig. 4), but MenH was not visible, possibly due to the flexible movement of MenH incurred by the linker
Summary
Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories Natural enzymes in their native metabolic pathway are tightly regulated on when and where to exert their activities, and on the concentration of metabolites to produce[1]. Metabolic engineers seek to increase the flux and reduce the cellular stress by deleting or adding enzymes, adjusting DNA copy numbers, and regulating transcription and translation, as well as including metabolic sensors to install gates and valves on the pathways[5]. The following features make them ideal protein tags for enzyme assembly: (1) the small size (44 and 18 amino acids, respectively), which minimizes the disturbance to the structure, location, and activity of the enzymes when fused as tags, (2) the strong binding affinity (with a KD of 1.2 nM between RIDD dimer and RIAD peptide) to ensure the integrity of the enzyme complexes in the cytoplasm, and (3) a 2:1 binding stoichiometry to give branched architectures.
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