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

• Published in: Nature chemistry Vol. 15; no. 1; pp. 61 - 69
• Main Authors: Spinck, Martin, Piedrafita, Carlos, Robertson, Wesley E. E., Elliott, Thomas S. S., Cervettini, Daniele, de la Torre, Daniel, Chin, Jason W. W.
• Format: Journal Article
• Language: English
• Published: BERLIN NATURE PORTFOLIO 01.01.2023; Nature Publishing Group; Nature Publishing Group UK
• Subjects: Amino acids; Amino Acids - metabolism; Amino Acyl-tRNA Synthetases - chemistry; Chains; Chemistry; Chemistry, Multidisciplinary; Codon; Codons; Depsipeptides; E coli; Genetic code; Genomes; Hydroxy Acids; Peptides; Physical Sciences; RNA, Transfer - genetics; Science & Technology; Stop codon; Synthesis; Transfer RNA; tRNA; LIBRARIES; CODE; TRANSFER-RNA SYNTHETASE; AMINO-ACIDS; LYSINE; LIGATION; LANTHIPEPTIDES; PROTEINS
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/s41557-022-01082-0

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 Syn61 & UDelta;3-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 Syn61 & UDelta;3-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.