Functional insights into Streptomyces isolates containing both clavulanic acid-like and carbapenem biosynthetic gene clusters

ABSTRACT β-Lactam antibiotics and β-lactamase inhibitor combinations are essential for combating antimicrobial resistance, with many β-lactams, including clavulanic acid (CA), themselves being products of specialized metabolic pathways in bacteria. CA is a potent β-lactamase inhibitor, and in known producers such as Streptomyces clavuligerus , it is co-produced with the β-lactam antibiotic cephamycin C, and their biosynthetic gene clusters (BGCs) are always located adjacent on the chromosome. However, CA-like BGCs have also been identified in other bacteria, often without an accompanying cephamycin C BGC. Similarly, carbapenem BGCs (a subclass of β-lactams), such as those responsible for producing MM 4550, a member of the olivanic acid complex with both antibiotic and β-lactamase inhibitory properties, are also found in Streptomyces species. This study investigated antimicrobial and β-lactamase inhibitory activity production in Streptomyces pratensis and 10 environmental Streptomyces isolates (JAC strains) containing CA-like and MM 4550-like BGCs but lacking cephamycin C BGCs. While the examined isolates do not produce CA, they synthesize predicted monocyclic β-lactam precursors of CA, which potentially represent a previously unrecognized, primordial form of β-lactamase inhibitor. Several JAC isolates also exhibited both β-lactamase inhibitory and β-lactam antibiotic activities, indicating that the carbapenem BGC is active in these strains. Gene disruption analysis confirmed that MM 4550-like carbapenem BGCs contribute to both antimicrobial and β-lactamase inhibitory activities, whereas CA-like clavam BGCs only contribute to β-lactamase inhibition. The findings also suggest that both β-lactam BGC types co-occur in nature more frequently than previously recognized, possibly with functional significance and potential applications in the discovery of novel antibiotic-inhibitor combinations. IMPORTANCE The global rise of antimicrobial resistance calls for innovative strategies to preserve the efficacy of existing antibiotics and identify new therapeutic agents. This study explores naturally occurring β-lactamase inhibitors and antibiotics beyond well-characterized systems. Investigation of clavulanic acid (CA)-like and MM 4550-like biosynthetic gene clusters (BGCs) in Streptomyces pratensis and related environmental isolates revealed a broader occurrence of monocyclic β-lactam precursors and dual-function carbapenems in nature. These findings offer new insights into β-lactam co-production and further indicate that unlinked β-lactam BGCs may have functional significance. The study also highlights the importance of exploring silent counterparts of known BGCs as potential sources of bioactive metabolites, enhancing our understanding of β-lactam BGC diversity and evolution. Notably, it identifies β-lactamase inhibitor and antibiotic-producing strains, opening new avenues for discovering antibiotic-inhibitor combinations of relevance.