Cell-Based Synthesis of Non-Natural Peptide and Depsipeptide Macrocycles Using Genetically Programmed ncAA Incorporation

Macrocyclic peptides are ring-shaped molecules that combine the target selectivity of biologics with the cell-permeability advantages of small molecules, making them among the most sought-after scaffolds in drug discovery. This 2023 Nature Chemistry paper demonstrated that non-natural macrocyclic peptides and depsipeptides can be genetically programmed and synthesised directly inside living cells using Syn61-derived strains.

The team used Syn61Δ3 cells, a Syn61 derivative in which two sense codons and a stop codon, along with their cognate tRNAs and the release factor that normally decodes the stop codon, have been removed. This created three blank codons simultaneously available for reassignment. Using these freed codons, the researchers genetically encoded 25 diverse non-natural macrocyclic peptides, each containing two different non-canonical amino acids at defined positions within the ring.

The paper also demonstrated that pyrrolysyl-tRNA synthetase/tRNA pairs can be engineered to incorporate alpha-hydroxy acids, building blocks that create ester bonds rather than amide bonds within the peptide backbone. This enabled the genetically encoded synthesis of depsipeptide macrocycles: cyclic molecules containing one or two ester bonds, a structural motif found in many natural product drugs but difficult to produce via conventional peptide synthesis. The team defined 49 engineered mutually orthogonal pairs that recognise distinct ncAAs or alpha-hydroxy acids and decode distinct codons.

For therapeutic development, this work demonstrates that the structural diversity accessible through genetic code expansion extends well beyond linear peptides. Macrocyclic scaffolds are of particular interest for targets considered difficult to drug by conventional small molecules, including protein-protein interactions. The ability to genetically encode macrocycle synthesis, rather than relying on multi-step chemical cyclisation, means these molecules can be produced by fermentation with the same scalability advantages as linear peptides.