发现一种迭代终端缩合结构域，可催化真菌多酮合成酶-非核糖体肽合成酶生物合成过程中连续形成酯键和酰胺键的反应

Abstract

Fungal polyketide–nonribosomal peptide (PK–NRP) hybrids are typically assembled by condensation (C) domains that form an amide bond between a polyketide and an amino acid unit. Here, we describe a family of bis-anthranilated PK–NRP hybrids, Neodiabzacids A and B, in which a linear polyketide is sequentially linked to two anthranilic acid (Ant) units through an ester and an amide bond. Their structures and absolute configurations were fully determined by NMR analysis, chemical derivatization, and computational approaches. Through heterologous reconstitution experiments, in vivo feeding, and in vitro assays, we propose a biosynthetic model in which the single-module nonribosomal peptide synthetase NeoB, in collaboration with the α/β-hydrolase NeoD, executes a stepwise “esterification–hydrolysis–amidation” process to sequentially incorporate two Ant units. Our findings support a dual catalytic role for the C T domain in performing two chemically distinct condensations (ester and amide) iteratively, while recognizing both carrier protein-bound and free substrates. This study expands the catalytic repertoire of fungal PK–NRP assembly lines by revealing the recruitment of a C T domain for iterative, chemically divergent condensations, thereby broadening the biosynthetic logic available for PK–NRP natural product diversification.