Characterization Of Two Marine-derived Streptomycetes And Studies On Their Bioactive Secondary Metabolites

Marine streptomyces are rich source of novel natural products. Isolation and characterization of marine streptomyces, purification and structure elucidation of bioactive secondary metabolites not only benefit the exploration of the unique genetic resources and the potential of biosynthesis of the new species, but also provide novel bioactive compounds for drug discovery. Imaging mass spectrometery (IMS) has been used as a new technology for microbial natural product using microbial interactions. However, related studies using marine actinomyces have been poorly reported. In our previous studies, marine-derived actinomycetes were isolated from various areas in Dalian, but studies on species characterization and their bioactive secondary metabolites were still limited. In this study, two marine-derived actinomycetes S187and M10were investigated on species characterization, purification and structure elucidation of bioactive compounds, and optimization of fermentation. Meanwhile, bioactive secondary metabolites from these two strains were explored. The results listed below were obtained.1. Identification of strain S187by polyphasic taxonomyStrain S187was investigated by polyphasic taxonomy including morphological characteristics, cultural characteristics, cell chemical composition,16S rRNA sequence analysis and genomic DNA-DNA hybridization. Compared with its phylogenetically closed strain S. flavofuscus NRRL B-8036T and S. albiaxialis DSM41799T, S187differed in the characteristics of phenotype, chemotype and genotype, and it was proposed that strain S187represented a novel species of the genus Streptomyces, for which the name S. xinghaiensis sp. nov. is proposed and the type strain is S187.2. Bioactive secondary metabolites from S. xinghaiensisIMS was performed using S. xinghaiensis growing on TSB agar, A1agar and Marine agar media, and the results indicated that no MS signals was in accordance with the inhibition zone. Xinghaiamine A and B were purified from25L S. xinghaiensis fermentation broth by ethyl acetate extraction, silica gel column, flash C18column and semi-preparative HPLC using S. aureus as an indicator. The sturcture elucidation was performed on the basis of ESI-MS, IR, UV and NMR spectrum (’H-NMR,13C-NMR,’H-’H COSY, HSQC, HMBC and DEPT135). Xinghaiamine A was identified as a novel alkaloid with sulfoxide functional group which has never been reported in microorganism. Xinghaiamine A and B exhibited excellent broad-spectrum antibacterial activities, especially to the MRSA isolates, but no inhibition to C. albicans was observed. Xinghaiamine A also showed cytotoxicity to the tested human tumor cell lines.3. Fermentation optimization of xinghaiamine A production from S. xinghaiensisThe optimum fermentation condition was determined to be initial pH7, inoculation rate6%(v/v), temperature28℃with the medium volume of160mL in500mL flask. The optimum organic carbon source, organic and inorganic nitrogen source were starch, soybean powder and (NH4)2SO4. An addition of3%(w/v) adsorbent resin HP-20at72h post-inoculation significantly improved xinghaiamine A production from0.35to3.4mg/L and shortened fermentation time from18to9days. Self-cytotoxicity of xinghaiamine A was verified and MIC value to its producing strain was estimated to be8μ/mL.4. Genome mining, IMS and MS/MS analysis of M10secondary metabolitesStrain M10was demonstrated to be most closely related to S. marokkonensis by16S rRNA sequence analysis, and bioinformatics analysis identified20secondary metabolites associated gene clusters involved in the genome of M10, including three PKS, four NRPS, four hybrid PKS-NRPS, two lantibiotics, four terpenoid and three siderophores biosynthesis gene clusters. Among the gene clusters, pksl exhibited85%sequence similarity to the candicidin gene cluster and selected genes in pksl gene cluster were demonstrated in transcriptional level by RT-PCR experiment. IMS signals of m/z1152and1168produced by M10were found on both TSB, A1agar and were in accordance with the inhibition zone and time-dependent cocultures of M10and Fulva fulva, which demonstrated that they could be the antifungal compounds produced by M10. Analysis of M10crude extract on A1agar by MS/MS revealed two peptide families (MF1and MF2) with the molecular weight ranging from900to1000Da.5. Purification of antifungal compounds produced by M10A total of630A1agar plates of M10were collected and extracted successively with ethyl acetate and butanol. Three compounds named9-03,9-04and9-05with the typical UV adsorption of polyene molecules were purified from the butanol extract by employing Sephadex LH-20column, flash C18column and semi-preparative HPLC. The NMR data of9-04also exhibited several conjugated double bonds and one lactone bond, indicating that it belongs to the polyene macrolides. In addition, the peptide molecule familes MF1and MF2were detected in the Sephadex LH-20column fractions using MALDI-TOF, indicating that M10produce peptide antibiotics on A1agar medium.