Genome Mining Of New Secondary Metabolites From Four Actinomycetes

Natural products from microorganisms are an important source of small molecule drugs.Although the genomic studies reveal that there are large number of unexploited biosynthetic gene clusters in the microbial genomes,most of them is silent or poorly expressed under the standard laboratory culture conditions.Therefore,how to activate these cryptic gene cluster with high efficiency is still one of hotspots in the field of natural product research.Dozens of AHBA(3-amino-5-hydroxybenzoic acid)synthase gene positive actinomycetes,which may have the potential to produce ansamycins or other AHBA-containing compounds,were obtained in our previous searching program.In this thesis,we are attempt to discover new natural products from four selected strainsAlthough more than 80 natural products,grouped into 16 types,were identified from marine Streptomyces sp.LZ35,there are more than 20 biosynthetic pathways still need to be explored.Among them,sm42 is predicted to biosynthesize the cinnamoyl-containing nonribosomal peptides(CCNPs).Coprisamide A(1)and a new seco-derivative(2)were obtained by medium screening,expanded fermentation,and targeted isolation from LZ35 mutant SR?,in which eight known gene clusters for main products were deleted.The identity of sm42 as coprisamide gene cluster was confirmed by large fragment disruption and comparative metabolite profiling.A reasonable biosynthetic pathway of coprisamide was proposed by bioinformatical analysis.In addition,the sm2 gene cluster was accidentally activated when we search for the sporulating condition of SR?.Two new germicidin derivatives,germicidin A-4-O-?-D-glucuronide(3)and germicidin D-4-O-?-D-glucuronide(4),were isolated and identified.None of the above compounds exhibited obvious antibacterial and cytotoxic activities.Genome sequence analysis indicate that Streptomyces sp.XZYN4 contained a gene cluster predicted to biosynthesize a nonansamycin skeleton with AHBA unit.We attempt to mining the product of this gene cluster by heterologous expression strategy.After promoter screening,the gene cluster was reconstructed by combination of Gibson assembly,BioBrick assembly and site-specific recombination strategies,and was introduced into four surrogate hosts,including Streptomyces albus J1074,Streptomyces lividans SBT5,Streptomyces sp.SR? and Streptomyces sp.S001.However,compared with the wild type,no significant difference were observed in the HPLC profile of all the heterologous expression mutant strains.Bioinformatic analysis of Verrucosispora sp.NS0172 genome identified vas as a novel pentaketide ansamycin gene cluster.In our previous study,several biosynthetic shunt products were obtained from the vasR2(SARP family regulatory gene)overexpression mutant.In order to obtain finally product of vas gene cluster,we constructed a vasR1(LAL family regulatory gene)and vasR2 co-overexpression mutant.Compared with the wild type,the mutant with double regulatory gene overexpression showed several extra peaks,though the yield is low.Subsequently,we increased the yield of the target peak by nearly eight times by feeding 250 mg/L AHBA precursors.However,due to unstable yield during expanded fermentation,we failed to obtain sufficient compound for structure elucidation yetS001-sm3 is a type ? PKS gene cluster in Streptomyces sp.S001.There are two peaks with retention time of about 12.5 min disappeared(named as A7 and A8)in the metabolic profiles of the PKS gene knockout mutant,comparing to the wild type,which indicate that sm3 may be responsible for the biosynthesis of these two compounds.Through 5 L YMG fermentation,HPLC guided isolation and structure elucidation,we identified A7 and A8 as known compounds SEK4 and SEK4b,which is spontaneously cyclized products of nascent octaketide chain released from type ?minimal PKS.In order to activate the silent cyclase gene and other post-modification genes,we constitutively expressed the cluster situated SARP family regulatory genes Compared with the wild type strain,HPLC profile of the mutant showed several peaks,which need to further isolation and identification.In addition,SEK4 showed weak cytotoxic activity against HeLa(IC50?14.4±0.25 ?M)in our bioactivity assayIn summary,through genome mining of selected four AHBA-synthase gene positive strains,we identified cyclic depsipeptide compound coprisamide A,two new germicidin glucuronides and their biosynthetic gene clusters in Streptomyces sp.LZ35,reconstructed and heterologously expressed the nonansamycin gene cluster from Streptomyces sp.XZYN4,activated the pentaketide ansamycin gene cluster vas in Verrucosispora sp.NS0172 and the type ? PKS gene cluster sm3 in Streptomyces sp.S001.The above-mentioned studies in this thesis provide useful references for genome mining of natural products from microbes,especially actinobacteria,and gained experience for the development of more effective cryptic gene cluster activation strategies.