Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles

Nat Chem. 2023 Jan;15(1):61-69. doi: 10.1038/s41557-022-01082-0. Epub 2022 Dec 22.


The direct genetically encoded cell-based synthesis of non-natural peptide and depsipeptide macrocycles is an outstanding challenge. Here we programme the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each containing two non-canonical amino acids, in Syn61Δ3-derived cells; these cells contain a synthetic Escherichia coli genome in which the annotated occurrences of two sense codons and a stop codon, and the cognate transfer RNAs (tRNAs) and release factor that normally decode these codons, have been removed. We further demonstrate that pyrrolysyl-tRNA synthetase/tRNA pairs from distinct classes can be engineered to direct the co-translational incorporation of diverse alpha hydroxy acids, with both aliphatic and aromatic side chains. We define 49 engineered mutually orthogonal pairs that recognize distinct non-canonical amino acids or alpha hydroxy acids and decode distinct codons. Finally, we combine our advances to programme Syn61Δ3-derived cells for the encoded synthesis of 12 diverse non-natural depsipeptide macrocycles, which contain two non-canonical side chains and either one or two ester bonds.

MeSH terms

  • Amino Acids / metabolism
  • Amino Acyl-tRNA Synthetases* / chemistry
  • Codon
  • Depsipeptides*
  • Hydroxy Acids
  • RNA, Transfer / genetics


  • Codon
  • Amino Acids
  • RNA, Transfer
  • Amino Acyl-tRNA Synthetases
  • Depsipeptides
  • Hydroxy Acids