Bacterial non-ribosomally produced peptides (NRPs) form a rich source of antibiotics, including more than 20 of these antibiotics that are used in the clinic, such as penicillin G, colistin, vancomycin, and chloramphenicol. Here we report the identification, purification, and characterization of a novel NRP, i.e., brevibacillin 2V (lipo-tridecapeptide), from Brevibacillus laterosporus DSM 25. Brevibacillin 2V has a strong antimicrobial activity against Gram-positive bacterial pathogens (minimum inhibitory concentration = 2 mg/L), including difficult-to-treat antibiotic-resistant Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Notably, brevibacillin 2V has a much lower hemolytic activity (HC₅₀ > 128 mg/L) and cytotoxicity (CC₅₀ = 45.49 ± 0.24 mg/L) to eukaryotic cells than previously reported NRPs of the lipo-tridecapeptide family, including other brevibacillins, which makes it a promising candidate for antibiotic development. In addition, our results demonstrate that brevibacillins display a synergistic action with established antibiotics against Gram-negative bacterial pathogens. Probably due to the presence of non-canonical amino acids and D-amino acids, brevibacillin 2V showed good stability in human plasma. Thus, we identified and characterized a novel and promising antimicrobial candidate (brevibacillin 2V) with low hemolytic activity and cytotoxicity, which can be used either on its own or as a template for further total synthesis and modification.

Increasing prevalence of antimicrobial resistance (AMR) has posed a major health concern worldwide, and the addition of new antimicrobial agents is diminishing due to overexploitation of plants and microbial resources. Inevitably, alternative sources and new strategies are needed to find novel biomolecules to counter AMR and pandemic circumstances. The association of plants with microorganisms is one basic natural interaction that involves the exchange of biomolecules. Such a symbiotic relationship might affect the respective bio-chemical properties and production of secondary metabolites in the host and microbes. Furthermore, the discovery of taxol and taxane from an endophytic fungus, Taxomyces andreanae from Taxus wallachiana, has stimulated much research on endophytes from medicinal plants. A gram-positive endophytic bacterium, Paenibacillus peoriae IBSD35, was isolated from the stem of Millettia pachycarpa Benth. It is a rod-shaped, motile, gram-positive, and endospore-forming bacteria. It is neutralophilic as per Joint Genome Institute’s (JGI) IMG system analysis. The plant was selected based on its ethnobotany history of traditional uses and highly insecticidal properties. Bioactive molecules were purified from P. peoriae IBSD35 culture broth using 70% ammonium sulfate and column chromatography techniques. The biomolecule was enriched to 151.72-fold and the yield percentage was 0.05. Peoriaerin II, a highly potent and broad-spectrum antimicrobial peptide against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 was isolated. LC-MS sequencing revealed that its N-terminal is methionine. It has four negatively charged residues (Asp + Glu) and a total number of two positively charged residues (Arg + Lys). Its molecular weight is 4,685.13 Da. It is linked to an LC-MS/MS inferred biosynthetic gene cluster with accession number A0A2S6P0H9, and blastp has shown it is 82.4% similar to fusaricidin synthetase of Paenibacillus polymyxa SC2. The 3D structure conformation of the BGC and AMP were predicted using SWISS MODEL homology modeling. Therefore, combining both genomic and proteomic results obtained from P. peoriae IBSD35, associated with M. pachycarpa Benth., will substantially increase the understanding of antimicrobial peptides and assist to uncover novel biological agents.