Pantoea Natural Product 3 is encoded by an eight-gene biosynthetic gene cluster and exhibits antimicrobial activity against multi-drug resistant Acinetobacter baumannii and Pseudomonas aeruginosa.

Multi-drug resistant Acinetobacter baumannii and Pseudomonas aeruginosa continue to pose a serious health threat worldwide. Two Pantoea agglomerans strains, 3581 and SN01080, produce an antibiotic effective against these pathogens. To identify the antibiotic biosynthetic gene clusters, independent genetic screens were conducted for each strain using a mini-Tn5 transposon, which resulted in the identification of the same conserved eight-gene cluster. We have named this antibiotic Pantoea Natural Product 3 (PNP-3). The PNP-3 biosynthetic cluster is composed of genes encoding two Major Facilitator Superfamily (MFS) transporters, an ArsR family regulator, and five predicted enzymes. The biosynthetic gene cluster is found in only a few Pantoea strains and is not present within the antiSMASH and BAGEL4 databases, suggesting it may be novel. In strain 3581, PNP-3 production is linked to pantocin A production, where loss of pantocin A production results in a larger PNP-3 zone of inhibition. To evaluate the spectrum of activity, PNP-3 producers, including several PNP-3 mutants and pantocin A site-directed mutants, were tested against a collection of clinical, drug-resistant strains of A. baumannii and P. aeruginosa, as well as, Klebsiella, Escherichia coli, Enterobacter, Staphylococcus aureus, and Streptococcus mutans. PNP-3 was found to be effective against all strains except vancomycin-resistant Enterococcus under the tested conditions. Heterologous expression of the four predicted biosynthetic genes in Erwinia amylovora resulted in antibiotic production, providing a means for future overexpression and purification. PNP-3 is a natural product that is effective against drug-resistant A. baumannii, P. aeruginosa, and enteric species for which there are currently few treatment options.