Effects Of Light On Biosynthesis Of Metabolites In Marine Actinobacteria And Genome Mining Of Key Strains

Marine actinobacteria are important sources of novel and bioactive natural products due tospecial marine environment where they inhabit.Therefore,studies on marine actinobacteria and their application to drug discovery have received great attention.It is known that many environmental factors affect biosynthesis of the secondary metabolite in actinobacteria,but the related effect of light is rarly investigated.The molecular networking strategy and the development of Global Natural Product Social Molecular Networking(GNPS)make it feasible to efficiently sift target compounds or de-replicate the repetitive metabolites by MS/MS spectra comparison when a large number of samples were tested by HPLC-MS/MS system.The application of genome mining is speeding up the discovery of novel natural products,while the study on the genomes of marine actinobacteria is still limited.The rapid development of genome sequencing technologies has made it more convenient to obtain the genome information than before,and genome mining has revealed much greater biosynthetic potential of microorganisms than previously expected.However,there is still a big gap between the genome information and the discovery of related compounds.The GNPS technology could be combined with genome mining,which would further benefit the exploration of biosynthetic potencies of the marine actinobacteria.In this thesis,the effects of light on the growth and secondary metabolites production of 63 marine actinobacterial strains were investigated using the HPLC-MS/MS and GNPS.It is observed that not only the cell growth,but also the metabolites of the strains were affected by light.The shape,color and size of the colony and the amount of spores were different under light comparing with that under a dark condition.The majority of strains prefer the light condition to produce more unique metabolites and only small amount of strains prefer the dark condition.Also,different strains response to light in different extent.Specifically,strains such as L036,S077 and L014 produced more than 40 unique metabolites at a light condition,whereas some other strains for example,strain L064,showed no response to light.From the results of the identified molecular clusters,most of them are cyclic,including cyclic peptides(surfactin,piperazimycin and surugamide)and macrolides(bafilomycin),and the majority of them are produced under the light condition.It was reported that most of these cyclic metabolites exhibit antibiotic activities and transport functions on ions or nutrition.It is presumable that these metabolites are responsible for the transportation of nutrient to assist cell growth under light.Besides,pigments including medermycin,actinorhodin and streptorubin are assumed to absorb light to reduce light pressure or turn light into energy for growth and metabolism.The studies mentioned above revealed that some special marine streptomycetes strains endow biosynthetic potenty of various metabolites.Therefore,four key strains were focused and secondary metabolites produced by these strains were explored.Strains DUT11,S063 and S.xinghaiensis S187 are with high anti-complement activity,which are associated with a wide range of diseases related with human immune systems and with good medical application prospect.Marine Gordonia alkanivorans strain S104 have great potency to produce pigments.Therefore,biosynthesis of secondary metabolites from these strains was further explored using genome mining and the GNPS platform.The discovery of medermycin,nonactin and tunicamycin analogs,including several novel structures of medermycin and noactic from Streptomyces sp.DUT11 is acheived through the alignment between the genome information and GNPS molecular networking map.The genome mining and the knockout of four gene clusters in Streptomyces sp.S063 indicate that the four gene clusters are not involved in biosynthesis of anti-complement agents.The genome information of 5104 assisted the provision of the light effect on the pigment and indicated that it is a novel marine actinobacteria.Finally,gene cluster related to the biosynthesis of anti-complementary agents in S.xinghaiensis S187was focused by genome mining.Analysis of the genome revealed that glycopeptide biosynthetic gene cluster(BGC)nrpsl shared high similarity with the BGC of the known microbial anti-complement compound complestatin.The nrpsl contains two more genes sinPS and sinAS and is with an absence of one methyl domain.The nrpsl BGC was proved to be associated with anti-complement activity by the knockout of the precursor synthesis gene sinPD.To sum up,combination of molecular networking platform GNPS and genome mining facilitates the identification of secondary metabolites from marine actinobacteria,and the effect of light on specific metabolite production was revealed.The alignment between the anti-complement activities and corresponding BGC in S.xinghaiensis S187 provides more insights for further discovery of novel anti-complement agents from microbial strains.The results in this thesis provide a basis for further exploration of useful natural products from marine actinobacteria.