Identification of a Polymyxin Synthetase Gene Cluster of Paenibacillus polymyxa and Heterologous Expression of the Gene in Bacillus subtilis

Polymyxin, a long-known peptide antibiotic, has recently been reintroduced in clinical practice because it is sometimes the only available antibiotic for the treatment of multidrug-resistant gram-negative pathogenic bacteria. Lack of information on the biosynthetic genes of polymyxin, however, has limited the study of structure-function relationships and the development of improved polymyxins. During whole genome sequencing of Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium, we identified a gene cluster encoding polymyxin synthetase. Here, we report the complete sequence of the gene cluster and its function in polymyxin biosynthesis. The gene cluster spanning the 40.6-kb region consists of five open reading frames, designated pmxA, pmxB, pmxC, pmxD, and pmxE. The pmxC and pmxD genes are similar to genes that encode transport proteins, while pmxA, pmxB, and pmxE encode polymyxin synthetases. The insertional disruption of pmxE led to a loss of the ability to produce polymyxin. Introduction of the pmx gene cluster into the amyE locus of the Bacillus subtilis chromosome resulted in the production of polymyxin in the presence of extracellularly added l-2,4-diaminobutyric acid. Taken together, our findings demonstrate that the pmx gene cluster is responsible for polymyxin biosynthesis.Since polymyxin was first isolated from Bacillus polymyxa in 1947 (1, 4, 47), at least 15 unique polymyxins have been reported (31, 49). Because of its excellent bactericidal activity against gram-negative bacteria, polymyxin antibiotics (polymyxin B and polymyxin E) were used until early 1970 as therapies against many diseases caused by pathogenic microorganisms. However, because they carried serious side effects, including fever, skin eruption, and pain, and also induced severe nephrotoxicity and neurotoxicity (18, 37), it was rapidly replaced by other, better-tolerated antibiotics. In recent years, its application has been restricted to use as an ointment on local surface wounds.Due to the increased and often unnecessary use of antibiotics, pathogenic microorganisms with resistance to antibiotics have become more widespread (2, 14, 30, 38). Under the limited therapeutic options available to treat multidrug-resistant gram-negative bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, polymyxins are sometimes the only available active antibiotics and have now become important therapeutic agents (13, 25, 28, 29, 55). Many recent reports have shown that patients infected with multidrug-resistant gram-negative pathogens improved upon treatment with polymyxins (19, 27, 44, 48). In addition, polymyxins have been applied to prevent septic shock by removing circulating endotoxin to polystyrene fibers in an immobilized form (8). Therefore, the clinical value of polymyxin, an antibiotic discovered 6 decades ago, is currently being reappraised. However, until now, we have had a very limited understanding of various characteristics of this agent, especially its biosynthetic genes.To analyze structure-function relationships and to develop improved polymyxins with lowered toxicities, novel polymyxin derivatives must be generated. Recently, total or semisynthesis or modifications of polymyxins was performed chemically or enzymatically, and the resulting products were effectively used for structure-function study (6, 20, 36, 45, 50, 52). There is a limitation to obtaining diverse derivatives by using chemical or enzymatic approaches, however, and this limitation is related to the structural complexity of polymyxin. The basic structure of polymyxin is a cyclic heptapeptide with a tripeptide side chain acylated by a fatty acid at the amino terminus (49). Normally, 6-methyloctanoic acid or 6-methylheptanoic acid is attached to the side chain. This structure favors solubility of polymyxin in both water and organic solvent. Unlike other general ribosomally translated peptides, polymyxin is produced by a nonribosomal peptide synthetase (NRPS) (22, 31). NRPSs are multienzyme complexes that have modular structures (35, 46). A module is a distinct section of the multienzyme that is responsible for the incorporation of one or more specific amino acids into the final product. Each module can be divided into different domains, each of which is responsible for a specific biochemical reaction. Three types of domains, the adenylation (A), thiolation (T; also referred to as the peptidyl carrier protein, PCP), and condensation (C) domains, are essential for nonribosomal peptide synthesis. The A-domain plays a role in the selection and activation of an amino acid monomer, the T-domain is responsible for transportation of substrates and elongation intermediates to the catalytic centers, and the C-domain catalyzes peptide bond formation. In addition to these core domains, there are the thioesterase domain (TE-domain), the epimerization domain (E-domain), and some other modification domains. Many NRPS gene clusters have been reported, but no polymyxin biosynthetic gene cluster has been reported to date.During whole genome sequencing of Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium, we found a gene cluster encoding polymyxin synthetase. In this study, the complete sequences of the polymyxin synthetase genes and the function of the gene cluster have been identified and analyzed by domain analysis, insertional mutagenesis, and heterologous expression of the genes, as well as by antibacterial assay and liquid chromatography-mass spectrometry (LC/MS) analysis of the strains and their culture supernatants. The genome information and the heterologous expression of the polymyxin synthetase gene cluster will be useful for further studies of the regulation of pmx genes, their structure-function relationships, and the improvement of polymyxins.