Exploring the molecular mechanisms of Butenolide biosynthesis and Acyl selection in NRPS condensation starter-domains

γ-Butyrolactones (GBLs) act as signaling molecules in Gram-positive bacteria, regulating secondary metabolism and differentiation. Their biosynthesis relies on the α C–H acidity of a 3-keto fatty acid precursor, enabling lactone formation via condensation with dihydroxyacetone phosphate. This study examines Pseudomonas-derived butenolides, their biosynthetic key enzymes, and the distribution of homologous gene clusters across Gram-negative and Gram-positive bacteria. We identified and characterized the acaterin biosynthetic gene cluster (BGC) from Pseudomonas and demonstrated via heterologous expression that a 3-keto fatty acid condenses with glyceraldehyde-3-phosphate followed by a Dieckmann-type cyclization to yield the butenolide core. Phylogenetic analysis of AcaA reveals acaterin-like BGCs in Pseudomonas, Burkholderia, Streptomyces, and Rhodococcus. Structural modelling of AcaA homologues and the GBL enzyme AfsA highlights conserved architectural features relevant to ecological roles. Genetic analysis delineated four gene cluster families (GCFs); expression of one uncharacterized BGC produced antifungal fugomycins, and another GCF holds potential for novel butenolides. Given the centrality of α C–H acidity in both GBL and butenolide formation, we also explored pyreudione biosynthesis—alkaloids produced by a rare monomodular non-ribosomal peptide synthetase (NRPS) with a Cs–A–T–TE architecture. Using Pys (3-keto fatty acid–selective) and Aps (unmodified fatty acid–selective) as models, chimeric and mutational studies identified eight key residues controlling substrate specificity. Substituting Pys’s core motif with Aps’s led to mixed products; further targeted mutations fully converted Pys into an Aps-like enzyme. Bioinformatic classification of 72 unique Cs domains into four substrate-accepting categories, together with crystallographic insights, indicates that steric and electrostatic factors jointly determine substrate specificity.