Genome Mining For Secondary Metabolites Biosynthetic Gene Clusters Of Marine Aspergillus Sp.Strain And Constructing Its Genetic Transformation System

The natural products derived from marine microbes have attracted more attention because of their unique structure and biological activities.Some reports from the recent past suspect that a number of metabolites obtained from marine animals and plants maybe produced by their associated microorganisms.Marine animal-Ligia oceanic is always used for the treatment of coastal fishermen's traumatic bruises in southern China.However,there are few studies about the intestinal microbes of Ligia oceanic and their secondary metabolites(SMs),which might be novel compounds with biological activities.In this study,the marine fungus Z5 isolated from the Ligia oceanic intestine was identified as Aspergillus sp.through rDNA sequencing.Based on the whole genome sequencing and analysis of Aspergillus sp.Z5,the strain had 33.8 Mbp genome size and fell within the A.versicolor clade.A total of 89 SMs biosynthetic gene clusters were predicted by genome mining,while a few compounds were detected by One-Strain-Many-Compounds(OSMAC)analysis.These result showed that Z5 strain potentially synthesize many kinds of SMs.However,most of them were silent under the currently culture condition.If they could express by activating,there will be many compounds with novel structure and biological activity produced by strain Z5.In order to obtain these novel compounds of strain Z5 by its biosynthetic research,we also constructed its genetic transformation system in this work.The results showed that pretreating hyphae by ?-mercaptoethanol and then purifying protoplasts by sucrose fractionation could obtain high quantity of regenerative protoplasts.And Double-Joint PCR was used to construct the gene cassettes instead of the traditional restriction enzyme digestion method.Finally,auxotroph of strain Z5 was obtained by PEG/CaCl2 transformation method for further biosynthetic research.This study is the essential basis of biosynthetic research and application for the marine fungi Z5,and also provides an important resource of novel natural products derived from marine fungi.And it helps to elucidate the evolution mechanisms between marine microorganisms and their host system.