Mining biosynthetic gene clusters in Paenibacillus genomes to discover novel antibiotics

Abstract

Background: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied. Results: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin. Conclusions: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.