Aanlyses Of The Antibiotic Paenimacrolidin And The Siderophore Paenibactin Based On PKS And NRPS Genes

Polyketides (PKs) and nonribosomal peptides (NRPs), the two important classes of secondary metabolites, have important application in agriculture, medicine and environmental protection. Facing the increasingly severer tendency of bacterial drug resistance, new diseases and worsened enviroment, the identification of novel PKs and NRPSs with new bioactivites has attracts many attentions. Theoretical analyses indicate large numbers of uncovered PKs and NRPs. However, due to the interference from the known compounds, the traditional method for screening natural products is inefficiency. Hence, high throughput screening methods and other high-efficiency screening methods have received a great deal of attentions from scholars throughout the world. In this study, via PCR and genome sequencing, we obtained polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes fragments from Paenibacillus tianmuensis F6-B70and Paenibacillus elgii B69. Through sequence analysis, the two strains were predicted to be novel PK and NRP producers. In accordance with our prediction, a novel antibiotic paenimacrolidin (PAM) and a novel siderophore paenibactin were purified from P. tianmuensis F6-B70and P. elgii B69, respectively. Subsequently, we tested the bioactivities of PAM and paenibactin, and discussed the function and evolution of the paenibactin biosynthetic gene cluster. Furthermore, we provided insights into the mechanism giving rise to product diversity. The results were as following:1. Analysis of the antibiotic PAM based on PKS and NRPS genes. Four partitial NRPS gene sequences and four PKS gene fragments were obtained by using the genome of P. tianmuensis F6-B70as a template for PCR amplication. Through sequence analysis, P. tianmuensis F6-B70was predicted to be a novel PK and NRP producer. Subsequently, the cell extracts of P. tianmuensis F6-B70was purified and proved to contain a novel antibiotic, named PAM. PAM is the first PK isolated from the genus Paenibacillus, with molecular formula C33H50O6. Its molecular weight is542Da. PAM can inhibit Staphylococcus spp. strains, including the methicillin-resistant Staphylococcus aureus and the ampicillin-resistant Staphylococcus epidermidis strains. The minimum inhibitory concentration (MIC) values were in the range of16-32μg/mL. The chemical of PAM was unstable at37℃, and its MIC value increased rapidly with the incubation time elongation. The instability and the bacteriostatic mechanism weaken the antibiotic activity of PAM.2. Isolation, purification and identification of the siderophore paenibactin based on NRPS genes. Through genome analysis, we identified a NRP biosynthetic gene cluster (pae) in P. elgii B69, which comprises6genes, paeGACEBF. On the basis of the sequence analysis, we speculated that the assembled product consisted of DHB (2,3-dihydroxybenzoate), threonine and a building block structurally analogous to glycine, and may represent a hitherto unknown siderophore. Subsequently, P. elgii B69was grown in iron-deficient medium for siderophore expression. A novel siderophore, designated paenibactin, was purifed and characterized by MS analysis and NMR spectroscopy. It structure was determined to be a trimeric ester of DHB-Ala-Thr. The molecular formula is C42H48N6O18. Paenibactin is the first characterized siderophore isolated from the genus Paenibacillus. The iron-binding stoichiometry is1:1.3. The biosynthetic gene cluster of paenibactin. The DNA fragment coding for PaeF-Al domain was cloned from P. elgii B69genomic DNA into plasmid pET-28a, and then transformed into Escherichia coli BL21(DE3) for heterologous expression. The in vitro biochemical assay of PaeF-A1showed a preference for Ala, which supported the assignment of the pae gene cluster as the producer of paenibactin. Additional, we proposed models for paenibactin biosynthesis, ferric-paenibactin uptake and paenibactin-bounded iron release. 4. The evolution of the paenibactin biosynthetic gene cluster. Via phylogenetic analysis, we demonstrated that the pae gene cluster evolved from an ancestral bacillibactin biosynthetic gene cluster. Paenibacillus strains acquired the ancestral pae gene cluster via horizontal gene transfer. Mutation may play an important role in diversifying the product.