Nonribosomal biosynthesis of backbone-modified peptides

• Published in: Nature chemistry 2018-03, Vol.10 (3), p.282-287
• Main Authors: Niquille, David L., Hansen, Douglas A., Mori, Takahiro, Fercher, David, Kries, Hajo, Hilvert, Donald
• Format: Article
• Language: English
• Subjects: 631/181/735; 631/45/607/1167; 631/92/60; 639/638/92/552; Amino acids; Analytical Chemistry; Backbone; Biochemistry; Biosynthesis; Catalysis; Catalytic activity; Chemistry; Chemistry/Food Science; Crystal structure; Inorganic Chemistry; Organic Chemistry; Peptides; Physical Chemistry
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/nchem.2891

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. Nonribosomal peptide synthetases (NRPSs) produce vital natural products but have proven recalcitrant to biosynthetic engineering. Now, a combination of yeast surface display and fluorescence-activated cell sorting (FACS) has been used to reprogram an L -Phe-incorporating module for β-Phe. The resulting module is highly selective and functions efficiently in NRPS pathways.