The global rise of antibiotic resistance poses a public health challenge, emphasizing the urgent need for novel antibiotics featuring unique chemical scaffolds and mechanisms of action. Here, we report the discovery and characterization of darumycins, guanidine-containing pentacyclic sesterterpene antibiotics that exhibit potent activity against high-priority Gram-positive pathogens and mycobacteria. The darumycin biosynthetic gene cluster was identified through genome mining in the Actinobacterium Micromonospora rubida and heterologously expressed in Streptomyces chassis strains. Gene cluster engineering facilitated the discovery of novel darumycin derivatives, thereby expanding the chemical diversity within the sesterterpene class of natural products and revealing nuanced variations in their bioactivity. Targeted gene deletions, along with LC–MS and NMR analyses, enabled us to propose a darumycin biosynthetic pathway, further complemented by in vitro biochemical characterization of two O-methyltransferase tailoring enzymes, DarM and DarG. The high-resolution crystal structure of DarM in complex with SAH provided valuable insights into the enzymatic mechanism and revealed a distinct architecture compared to other methyltransferases acting on terpene scaffolds.
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.
Tingting Wang, Alexander Popoff, Maja Hunter et al.· Angewandte Chemie· 2 citations
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