Atypical Condensation Domains Guide Discovery and Illuminate Biosynthesis of Myxoglucamides Featuring Vinyl‐Substituted, α‐Oxidized γ‐Amino Acids

Condensation (C) domains in nonribosomal peptide synthetase (NRPS) pathways exhibit versatile functions that drive biosynthetic and chemical novelty. Through genome mining for atypical C domains, we identified a hybrid NRPS/polyketide synthase (PKS) biosynthetic gene cluster ( mxg ) from Cystobacterineae sp. MCy9003 and discovered myxoglucamides, a family of glycolipopeptides featuring an unprecedented vinyl‐substituted γ‐amino acid bearing an α‐hydroxy/α‐ketoamide functionality. Heterologous expression of the promoter‐refactored pathway revealed new O ‐acylated myxoglucamides, and subsequent studies unveiled the C domain‐like enzyme MxgH as a promiscuous O ‐acyltransferase decorating the glucose moiety with short‐chain acyl groups. Biosynthetic investigations demonstrated that the unusual γ‐amino acid originates from l ‐glutamate. Completion of the cryptic β‐hydroxylation of peptidyl carrier protein‐tethered glutamate by the α‐ketoglutarate‐dependent dioxygenase Ox MxgA occurs only concomitantly with upstream chain extension, revealing a bidirectional checkpoint for substrate fidelity. Unexpectedly, the C‐domain‐like interface domain I MxgB is dispensable for this coupled transformation. Mutational analysis of the FMN‐dependent monooxygenase encoded by mxgE , together with characterization of a shunt metabolite, supported its role in α‐oxidation for α‐hydroxy/α‐ketoamide formation during γ‐amino acid assembly. Together, these findings uncover an unrecognized biosynthetic logic for generating vinyl‐substituted, α‐oxidized γ‐amino acids and substantially expand the functional repertoire of NRPS/PKS assembly lines.