Rapid Identification of γ-Butyrolactone Signalling Molecules in Diverse Actinomycetes Using Resin-Assisted Isolation and Chemoenzymatic Synthesis

Actinomycetes are prolific producers of secondary metabolites with diverse chemical structures and bioactivities. Secondary metabolism in actinomycetes is regulated by signalling molecules, often termed “bacterial hormones.” In Streptomyces griseus, the γ-butyrolactone (GBL) A-factor (1) acts as a critical regulator of secondary metabolism, including antibiotic streptomycin production. The widespread presence of afsA, the gene encoding A-factor synthase, suggests that GBL compounds constitute a major class of signalling molecules in actinomycetes. However, the identification of GBLs has been limited by the requirement for large-scale actinomycete cultures. This study introduces two methodologies for the rapid identification of natural GBLs. First, a resin-assisted culture method combined with MS-guided screening enabled the isolation and structural determination of GBLs (2–5) from smaller-scale cultures. Second, a chemoenzymatic synthesis method for GBLs was developed, involving a one-pot system with three in vitro enzymatic reactions. Using isolated and synthesized GBLs (10a–10l), HR-LCMS analysis was conducted on 31 strains across 10 genera of actinomycetes. Synthesized GBLs were identified in nearly half of the tested strains, including genera where GBLs were detected for the first time. The absolute configurations of the GBLs were analysed using chiral HPLC, leading to the discovery of a (3S)-form GBL (11), an enantiomer of known GBLs. This study revealed the widespread distribution and structural diversity of GBLs among actinomycetes, providing insights into their regulatory roles and potential for activating secondary metabolism that support the discovery of new natural products.