Directed evolution of rRNA improves translation kinetics and recombinant protein yield

Nat Commun. 2021 Sep 24;12(1):5638. doi: 10.1038/s41467-021-25852-5.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Directed Molecular Evolution / methods
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Kinetics
  • Mass Spectrometry / methods
  • Models, Molecular
  • Mutation
  • Nucleic Acid Conformation
  • Protein Biosynthesis*
  • Proteome / metabolism
  • RNA, Ribosomal / chemistry
  • RNA, Ribosomal / genetics
  • RNA, Ribosomal / metabolism*
  • RNA, Ribosomal, 16S / chemistry
  • RNA, Ribosomal, 16S / genetics
  • RNA, Ribosomal, 16S / metabolism
  • Recombinant Proteins / metabolism*
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism*
  • Ribosomes / genetics
  • Ribosomes / metabolism*


  • Proteome
  • RNA, Ribosomal
  • RNA, Ribosomal, 16S
  • Recombinant Proteins
  • Ribosomal Proteins