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 OxMxgA occurs only concomitantly with upstream chain extension, revealing a bidirectional checkpoint for substrate fidelity. Unexpectedly, the C-domain-like interface domain IMxgB 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.