Discovery Of Bioactivities Of Two Marine-Derived Streptomycetes By Genome Mining And Preliminary Studies Of The Bioactive Agents

Marine actinomycetes have received extensive attentions due to their capability to produce many unique bioactive compounds which is resulted from their special habitats. Microbial genome mining is a novel approach to discover novel secondary metabolites. Marine actinomycetes genome mining may be defined as the process of analyzing genome sequences, followed by predicting the biosynthetic potential and using the information obtained by bioinformatics analysis for subsequent purification and structure elucidation of natural compounds. Genome mining has been a popular topic in microbial natural product studies, however, currently, only few studies are focused on the genome mining of marine actinomycetes.In this study, genome mining of Streptomyces sulphureus L180and S. xinghaiensis S187was performed. Bioinformatics analysis revealed that there is a lantibiotic gene cluster in L180genome which shows the highest similarity to the gene cluster that was isolated from rare actinobacteria Planomonospora alba, and meanwhile, there is a NRPS gene cluster in S187genome which shows the highest similarity to the biosynthetic gene cluster of an anticomplementary compound complestatin. It was thus deduced that L180and S187have the potential to produce antibacterial compounds and anticomplementary compounds, respectively, which was proved in the subsequent experiments using culture broth of these two strains.In the further study, the antimicrobial activities of the culture broth of L180were confirmed, and it was found that the culture supernatant showed strong antagonistic activity against Micrococus luteus. In addition, weak activity against drug-resistant Acinetobacter baumanii was also observed, whereas no obvious antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa was found under the conditions used in this study. The physicochemical properties of the culture supernatant of L180were also investigated, and it was showed that the antimicrobial compounds in the culture supernatant were stable at30℃to60℃and pH4to6, and the antibacterial activities against M. luteus were also stable after digestion by two proteases. The crude active compounds against M. luteus were obtained, and the retention times of the activity compounds in HPLC analysis was preliminarily determined. A putative gene encoding StrR family transcriptional regulator termed as sinR was identified in the biosynthetic gene cluster for the synthesis of compounds with anticomplementary activity. To identify the active compounds, sinR was cloned and the effect of its overexpression on anticomplementary activity was examined. The results showed that overexpression of sinR caused increase of anticomplementary activity compared to wild-type. The anticomplementary activity components were extracted from the culture broth and mycelia using ethyl acetate, and the ethyl acetate phase was further separated by silica gel purification. Four active fractions were obtained from the culture broth, and the activity of Fr-A-7fraction was the highest. In addition, n-butanol extraction phase of the water phase of the mycelia after ethyl acetate extraction was also separated and purified using silica gel. Four active fractions were obtained, and the activity of Fr-B-8component was the highest. The retention time in HPLC analysis was also determined. In the HPLC profile, the retention time of the active compound was deduced to be at8.9min. HPLC-MS detection revealed four signals with molecular weights in the predicted range.The results of this study not only provided basis for genome mining of natural products from marine streptomycetes, but also offered valuable information for systematic studies of related strains and species.