Niquille DL Hansen DA Mori T Fercher D Kries H Hilvert D 2018 Biosynthetic modification of nonribosomal peptide backbones represents a potentially powerful strategy to modulate the structure and properties of an important class of therapeutics. Using a high-throughput assay for catalytic activity, we show here that an L-Phe-specific module of an archetypal nonribosomal peptide synthetase can be reprogrammed to accept and process the backbone-modified amino acid (S)-β-Phe with near-native specificity and efficiency. A co-crystal structure with a non-hydrolysable aminoacyl-AMP analogue reveals the origins of the 40,000-fold α/β-specificity switch, illuminating subtle but precise remodelling of the active site. When the engineered catalyst was paired with downstream module(s), (S)-β-Phe-containing peptides were produced at preparative scale in vitro (~1 mmol) and high titres in vivo (~100 mg l-1), highlighting the potential of biosynthetic pathway engineering for the construction of novel nonribosomal β-frameworks. https://sonar.ch/global/documents/40871 eng info:eu-repo/semantics/altIdentifier/doi/10.1038/nchem.2891 info:eu-repo/semantics/altIdentifier/pmid/29461527 Nature chemistry. - 2018 Biocatalysis Molecular Structure Peptide Biosynthesis Peptide Synthases Peptides Protein Engineering Ribosomes Nonribosomal biosynthesis of backbone-modified peptides. http://purl.org/coar/resource_type/c_6501