Characterization Of Lipopeptide Pelgipeptins Produced By Paenibacillus Elgii B69and The Biosynthetic Gene Cluster

Lipopeptide antibiotic is one important kind of antibiotics with broad spectrum and good antibacterial effect, and usually it is not easy to make pathogens resistant. As the launch of daptomycin and echinocandin recently, much more attention was focused on the development of this kind of compounds. The strain, isolated and identified as Paenibacillus elgii B69in our lab, showed potent antimicrobial activity to the tested indicator strains, including gram-positive, gram-negative bacteria, and fungi, which attracted our attention for further study. In this study, the work was mainly carried out on the separation, purification, structure elucidation and biological activity of the bioactive compounds isolated from this strain. Meanwhile, according to the genome information, we obtained the gene cluster possibly responsible for these bioactive antibiotics. Further study on the functional relationship between the compounds and the gene cluster was performed, including gene knock-out and heterologous expression The results were shown as follows:1. The purification and identification of lipopeptide antibiotics-pelgipeptins. Under the appropriate medium composition and culture conditions, four unobserved lipopeptide antibiotics, named pelgipeptins A, B, C and D, were isolated by macroporous resin adsorption and RP-HPLC separation in the B69fermentation culture. The molecular mass of these four compounds were1072Da,1100Da,1086Da,1086Da, respectively. Mass-spectrometry and NMR spectroscopy indicated that all these active compounds belonged to the polypeptin family, and pelgipeptins A and D were the novel antibiotics to date.2. The biological characterization of pelgipeptins. The biological activities of pelgipeptins were tested as follows:pelgipeptins A-D showed potent antibacterial activity to the tested gram-negative bacteria and gram-positive bacteria, including MRSA, suggests application of such compounds in the potential field of medicine. In addition, pelgipeptins also showed strong inhibition on the fungi, such as Candida albicans, Fusarium oxysporium, Fusarium graminearum. Fusarium maniliforme, Rhizoctonia solani. Colletotrichum lini. Also, in plant experiments, Rhizoctonia solani, Botrytis cinerea. Sphaerotheca fuliginea, Blumeria graminis and Pseudoperonospora cubensis were effectively inhibited during its growth under the pelgipeptin with the concentration of250μg/mL. Among these tests, the biocontrol property in Rhizoctonia solani was the best. In vitro time kill curve, the bactericidal effect of these antibiotics performed in a concentration-dependent manner. Through the outer membrane permeability test, we found that pelgipeptin B might be with the similar bactericidal action of polymyxin B, suggesting that the targets of the class of antibiotics may be associated with the inner and outer membrane of bacteria to achieve rapid bactericidal effect.3. The pip gene cluster responsible for pelgipeptins biosynthesis. In order to analyze pelgipeptins biosynthetic pathway in strain B69, we obtained the draft genome sequence through the Solexa paired-end sequencing technology. Several unknown giant gene clusters were found in B69genome, including two nonribosomal peptide synthetases (NRPS), one polyketide synthetase (PKS) and three hybrid NRPS-PKS synthetases gene. Further inspection revealed that one NRPS gene cluster (plp) harboring8open reading frames (plp A-H) could be assigned to pelgipeptins biosynthesis. According to the gene blast and functional perdition, the proposed structure of this secondary metabolite was in agreement with that of pelgipeptins, indicating that pelgipeptins might be catalyzed through plp gene cluster in NRPS synthesis pathway.4. The functional analysis of plp gene cluster. Three A domains in plp gene cluster were cloned by PCR. Then, the genes were cloned into plasmid pET-28a and expressed in E.coli BL21(DE3). In vitro biochemical analysis showed that the measured substrate specificities of tested A domain proteins were consistent with the corresponding amino acids in pelgipeptins. These results provided the indirect evidence that the plp gene cluster was responsible for the catalytic synthesis of pelgipeptins.