Investigating the biochemical and genetic basis for the production of specialized metabolites in Streptomyces pratensis ATCC 33331

Abstract

Streptomyces are recognized for producing 70% of clinically used antibiotics, including β- lactam antibiotics and β-lactamase inhibitors. However, most specialized metabolite (SM) synthesizing biosynthetic gene clusters (BGCs) are silent or cryptic. Previous studies in the Tahlan Lab have revealed that Streptomyces pratensis ATCC 33331 synthesize a β-lactamase inhibitor active against Klebsiella pneumoniae in the presence of penicillin G (PenG). However, the metabolite remains unknown. The objective of this study was to identify potential BGCs and SMs contributing to this bioactivity. Genomics analysis of S. pratensis predicted the presence of 30 BGCs, two of which have similarities to the clavulanic acid (CA) and carbapenem MM4550 BGCs in Streptomyces clavuligerus and Streptomyces argenteolus, respectively. CA is a potent β- lactamase inhibitor, and carbapenem MM4550 possesses both β-lactam antibiotic and β-lactamase inhibitory properties. Furthermore, the One Strain MAny Compounds (OSMAC) approach revealed that S. pratensis produced the β-lactamase inhibitor in Maltose-Yeast extract medium (MYM) and soy media. S. pratensis like environmental isolate, JAC18, exhibited both β-lactamase inhibitory and β-lactam antibiotic activities. Transcriptomics analysis of S. pratensis showed the potential role of NRPS1.1, carbapenem MM4550-like and CA-like BGCs in producing β- lactamase inhibitors. Gene disruption analysis confirmed that NRPS1.1 and CA-like BGC in S. pratensis and both carbapenem MM4550-like and CA-like BGC in JAC18, contribute to β- lactamase inhibitor production. Additionally, metabolomics analysis of mutants defective in carbapenem MM4550-like and CA-like BGCs in S. pratensis and wt JAC18 indicated that only early intermediates of the CA pathway, deoxyguanidinoproclavaminic acid and guanidinoproclavaminic acid, were detected in the bioactive samples. Furthermore, this study reports the complete metabolomics analysis of S. pratensis and JAC18.