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 bioactivities. Secondary metabolism in actinomycetes is regulated by signalling molecules, often termed "bacterial hormones." In , the γ-butyrolactone (GBL) A-factor (1) plays a key role in regulating secondary metabolism, including streptomycin production. The widespread presence of , the gene encoding A-factor synthase, suggests that GBLs are a major class of signalling molecules in actinomycetes. However, their identification is hindered by the requirement for large-scale cultures. This study presents two methodologies for identifying 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 involving one-pot three enzymatic reactions was developed, allowing the production of GBL standards (10a-10l). Using these standards, HR-LCMS analysis of 31 strains across 10 actinomycetes genera identified GBLs in nearly half of the tested strains, including genera where GBLs were detected for the first time. Chiral HPLC analysis further revealed the presence of the (3)-isomer of GBL (11), an enantiomer of known GBLs. This study uncovers the widespread distribution and structural diversity of GBLs among actinomycetes, providing insights into their regulatory roles and potential for activating secondary metabolism, which may facilitate the discovery of new natural products.