Abstract
In bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo.
Highlights
In bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth
PACE exploits the rapid M13 bacteriophage lifecycle and couples the production of plasmid-borne gIII, encoding the minor coat protein pIII necessary for both bacterial infection and membrane extrusion[17], to the activity of the evolving biomolecule encoded on a pIII-deficient phage genome
To elucidate the physiological cost of kinetically enhanced ribosomal RNAs (rRNAs) variants, we introduced the wildtype antiRBS sequence into evolved orthogonal rRNAs (o-rRNAs) and assayed their ability to complement the rRNA efficiency of SQ171 E. coli cells and translate all cellular proteins (Fig. 6a and Supplementary Fig. 5a)[25]
Summary
Ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Orthogonal translation systems, which create dedicated pools of researcher-controlled ribosomes that are decoupled from cellular viability[8], have enabled the exploration of sequence-function relationships en route to unnatural bioactivities[1], permitted investigations into a mutation of sequence essential for cell function, and enabled the discovery of augmented ribosomal activities[9] Bolstered by this decoupled translation framework, we developed an orthogonal ribosome-dependent phage-assisted continuous evolution (oRibo-PACE) methodology that enables rapid directed evolution of rRNAs towards researcher-defined activities. Evolved rRNAs furnish ribosomes capable of greatly increasing the yield of proteins bearing noncanonical amino acids in an orthogonal translation system We extend these findings to generate cells harboring only evolved rRNA variants, showcasing elevated proteome-wide translation rates as compared to wild-type E. coli rRNA, with only minor reductions in translational fidelity. Our findings showcase that ribosomes can be evolved for improved protein yield, enhanced genetic code expansion, and faster translation rates in living cells
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