Mining Of Secondary Metabolites In Deep-sea Actinomycetes Streptomyces Koyangensis SCSIO 5802 Based On Genomic Information

Objective: Streptomyces koyangensis SCSIO 5802 was isolated from deep sea sediment from the South China Sea.In the preliminary in vitro screening the isolate showed strong inhibitory activity against Methicillin-resistant Staphylococcus aureus(MRSA)and potent cytotoxic activities against the various cancer cell lines screened.The active strain was to exploit for the identification of more compounds by gene knockout,fermentation optimization and activation of silent biosynthetic gene clusters.Methods: The whole genome of S.koyangensis SCSIO 5802 was sequenced using Illumina Miseq combined with the third generation sequencing technology.Thus,the obtained sequence was annotated using bioinformatics tools and the gene cluster responsible for secondary metabolites biosynthesis was analyzed by antiSMASH(http://antismash.secondarymetabolites.org/)online analysis tool.PCR-targeting system in-frame deletion of the polyketone synthase genes abmB1 and canD,which are responsible for the biosynthesis of abyssomicin and candicidin obtained a double exchange mutant strains S.koyangensis SCSIO 5802.Secondary metabolites of S.koyangensis SCSIO 5802 AC were excavated by means of fermentation optimization.Finally,the silent biosynthesis gene clusters in the 5802 genome were further activated based on the engineering strain of S.koyangensis SCSIO 5802 AC.Results: 1.The whole genome of S.koyangensis SCSIO 5802 was scanned and displayed 6.86 Mb in length with the GC content of 73.4% containing 6419 encoding genes,75 tRNA and 3119 rRNA.A total of 21 secondary metabolite biosynthetic gene clusters were predicted by using online tool antiSMASH.2.Polyketone synthase genes abmB1,which is responsible for the biosynthesis of abyssomicins/neoabyssomicins was in-frame deleted using gene interdict method to obtain a double exchange mutant strain S.koyangensis SCSIO 5802 A which was successfully activated the over production of candicidin.Further,the biosynthetic gene cluster of candicidins was identified and the biosynthetic pathway for candicidins was proposed using bioinformatics tools.The mutant strain SCSIO 5802 A was used as the starting material and the polyketosynthase gene canD which was responsible for the production of candicidin was also successfully deleted using the same method.Finally,neither production of abyssomicins/neoabyssomicins compounds nor the background of the clean production candicidin compound mutant strain of S.koyangensis SCSIO 5802 AC was obtained in order to dig for subsequent secondary metabolites.3.The engineered strain S.koyangensis SCSIO 5802 AC was selected for medium optimization using 11 different fermentation mediums with a hope for the production of new compounds.Among the medium screened R5 medium,Hmt medium,P2 medium and A1 medium showed new peaks over 19 min with strong UV absorption at 500 nm.4.The engineering strain S.koyangensis SCSIO 5802 AC with clear genetic background and clean metabolism background was selected as an initial strain.Six promoter engineering mutants were successfully constructed by targeted insertion of the strong promoter kasOp* in front of structure genes by PCR-targeting and interspecies conjugation methodologies.The fermentation of mutant strains and HPLC-DAD analysis founded that the expression of compounds that may encode herboxidiene was activated.Conclusion: This thesis by using knockout,taking advantage of fermentation optimization and addition strong promoter to mining the strain of S.koyangensis SCSIO 5802 geneome.