Biosynthetic\nIncorporation of Non-native Aryl Acid\nBuilding Blocks into Peptide Products Using Engineered Adenylation\nDomains

Nonribosomal\npeptides (NRPs), one of the most widespread\nsecondary\nmetabolites in nature, with therapeutically significant activities,\nare biosynthesized by modular nonribosomal peptide synthetases (NRPSs).\nAryl acids contribute to the structural diversity of NRPs as well\nas nonproteinogenic amino acids and keto acids. We previously confirmed\nthat a single Asn-to-Gly substitution in the 2,3-dihydroxybenzoic\nacid-activating adenylation (A) domain EntE involved in enterobactin\nbiosynthesis accepts monosubstituted benzoic acid derivatives with\nnitro, cyano, bromo, and iodo functionalities at the 2 or 3 positions.\nHere, we showed that the mutant EntE (N235G) accommodates various\ndisubstituted benzoic acid derivatives with halogen, methyl, methoxy,\nnitro, and cyano functionalities at the 2 and 3 positions and monosubstituted\nbenzoic acid with an alkyne at the 3 position. Structural analysis\nof the mutant EntE (N235G) with nonhydrolyzable aryl-AMP analogues\nusing 3-chloro-2-methylbenzoic acid and 3-prop-2-ynoxybenzoic acid\nrevealed how bulky 3-chloro-2-methylbenzoic acid and clickable 3-prop-2-ynoxybenzoic\nacid are recognized by enlarging the substrate-binding pocket of the\nenzyme. When engineered EntE mutants were coupled with enterobactin\nand vibriobactin biosynthetic enzymes, 3-hydroxybenzoic acid-, salicylic\nacid-, and 3-bromo-2-fluorobenzoic acid-containing peptides were produced\nas early stage intermediates, highlighting the potential of NRP biosynthetic\npathway engineering for constructing diverse aryl acid-containing\nmetabolites.