Isolation And Identification Of Natural Products By Genome Mining From Streptomyces Noboritoensis

Natural products are the main source of drug discovery for their various bioactivity of antibacterial,antivirus,and anticancer and so on.Meanwhile,the pigment natural products are always preferred to the synthetic dyes for their degradable and eco-friendly character.As the abuse of antibiotics,multi-drug resistant pathogens have been widely emerged in hospitals and communities recently.Multi-drug resistant pathogens have become a serious threat to human health and there is an urgent call for the introduction of novel antibiotics to treat the superbugsStreptomyces are gram positive bacteria with high G+C content in their genome,and they are famous for producing many important secondary metabolites as antibiotics,anticancer agents,immunosuppressants and pigments.The genome size of Streptomyces is about 8?10 Mb,and tens of gene clusters like PKS,NRPS related to biosynthesis of secondary metabolites are embedded in the genome,most of which are cryptic or silent in the standard laboratory culture conditions.Heterologous expression is an efficient way to activate those cryptic gene clusters.In this study,we constructed the bacterial artificial chromosome(BAC)genomic library of ten Streptomyces at first and then a heterologous expression strategy was combined to mine novel natural products.We chose a nontalent strain,Streptomyces noboriloensis-24(Snt 24),which could not produce either antibiotics or pigments to undergo the high throughput heterologous conjugation and expression analysis.Finally,six exconjugants which can produce antibacterial activity against Staphylococcus aureus and Bacillus mycoides and a red pigment were discovered.Then we isolated and determined the two red pigments,hybrubin A and hybrubin B,the isolation and determination of the bioactive compounds were also discussed.? Construction of BAC libraryIf we need to clone a specific gene cluster,we need to construct a genomic library at first and then to screen the gene cluster with PCR or Southern blotting.To a specific genome,the smaller the insert size is,the more library clones are needed and the more complicated of the screening process will be and vice versa.The norally used cosmid vectors can bear 35?45 kb exogenous DNA which is not large enough to cover most secondary metabolites gene clusters.The high copy number of cosmids would also make them unstable in Escherichia coli and deletions of DNA fragments on cosmids could be observed.BAC vector can bear exogenous DNA up to 300 kb and it can obtain stable replication in numbered copy in Escherichia coli which makes BAC library a better choice for secondary metabolites screening.It is still a big challenge to construct high quality genomic libraries(with an insert size over 100 kb)for heterologous expression and screening.In this study,we constructed 10 BAC genomic libraries with high quality of 8 Streptomyces strains,?Saccharopolyspora spinosa strain and 1 Nocardia strain.? Screening of Snt24 BAC libraryS.noboritoensis 24 was isolated from the forest soli of Shengnongjia and it was a novel Streptonyces strain from its 16s rDNA sequence.Snt24 could not produce either pigment or antibacterial(E.coli DH10B,Sta.aureus,B.mycoides,Mycobacteria smegmatis mc2155 and Saccharomyces sake as indicators)compounds on several test media(MS,R3,YBP,GYM,YPD,and 18#)which indicated that its secondary metabolites would be silenced in the culture conditions.The BAC library of Snt24 contained 600 clones with an average insert size of 108 kb and insertion rate of 87%which obtained a c.5 genome equivalents.Through the high throughput heterologous expression of Snt24 BAC library coupled with phenotype observation and antibacterial screening,6 exconjugants exhibited antibacterial activity against Sta.aureus and B.mycoides were obtained.Interestingly,a red pigment was also observed around the 6 exconjugants.The 6 clones belong to the same contig according to the Pvu? restriction digestion map and paired-end sequencing.On the other hand,we also discovered a BAC clone producing a black pigment which was also analyzed and discussed here.? Determination of hybrubin and elucidation of its biosynthetic pathwayStreptomyces lividans SBT18/6D1 was fermented and extracted,followed by column chromatography fractionation and HPLC analysis.We obtained 2 mg A,0.5 mg B,and 1 mg C named hybrubin A-C respectively.HRESI-QTOF analysis showed hybrubin A-C with[M+H]+(m/z)of 297.1191,297.1190,and 285.0831 which indicated that hybrubin A and hybrubin B would be geometric isomers.The structures of hybrubin A and hybrubin B were further determined by NMR spectrometry.Hybrubins were characterized by the bipyrrole and tetramic acid moieties,and hybrubin A and C were differed at the C5 variants on the tetramic acid which is an allyl group for hybrubin A and carbonyl group for hybrubin C.Hybrubin B was not enough for 13C NMR analysis and its chemical structure needs to be confirmed.6D1 was further sequenced and an NRPS spanning-40 kb exhibited low similarity to known NRPSs was discovered.On the base of the sequence of 6D1 and the chemical structure of hybrubin,we proposed that hybrubin should be synthesized by the red pathway for the bipyrrole moiety from the heterologous expression host S.lividans SBT18 and the NRPS for the tetramic acid moiety.To verify the correlation of the NRPS to the biosynthesis of hybrubin,we constructed four large fragment deletions of 6D1 which were pHZZL1,pHZZL2,pHZZL3,and pHZZL4.pH27L1 contained the entire NRPS gene cluster was 60,399 bp,pHZZL2 contained the 16 genes from U to R1 was 34,694 bp,pHZZL3 contained the 10 genes from U to H was 26,668 bp and pHZZL4 contained gene U,A and truncated B was 22,391 bp.pHZZL1-4 were transferred to S.lividans SBT18 by triparental mating.Comparative metabolic profiling of the EtOAc extracts of S.lividans SBT18/pHZZL1,S.lividans SBT18/pHZZL2,S.lividans SBT18/pHZZL3,S.lividans SBT18/pHZZL4,and S.lividans SBT18/6D1 revealed-that all the exconjugants except S.lividans SBT18/pHZZL4 can produce hybrubin A-C.This indicated that the NRPS(hrnB to hrnH)is necessary to the biosynthesis of hybrubin.To confirm the origin of the bipyrrole moiety of hybrubin,we transferred pHZZL3 to a redMN deleted S.lividans SBT5 strain,S.lividans XF2.There were no hybrubin A-C detected in the EtOAc extract of S.lividans XF2/pHZZL3 which indicated that the bipyrrole moiety should be originated from the MBC synthesized by redMN.We deleted the genes between hrnU and hrnH on pHZZL3 and genes contributed to the biosynthesis of hybrubin were determined.Finally,we proposed the biosynthetic pathway of hybrubin on the basis of the deletion experiments.? Isolation of the bioactive compoundsBesides hybrubins,.S.lividans SBT18/6D1 and the other 5 S.lividans SBT18 exconjugants could produce antibacterial compounds against Sta.aureus and B.mycoides.There was a 50 kb overlap among the 6 BAC clones according to the Pvu?restriction digestion map and the paired-end sequencing and a cryptic NRPS about 35 kb mentioned above which was responsible for the antibacterial activity was embedded.There were two regulatory genes R1 which belongs to the TetR family and R2 which belongs to the SARP family in the gene cluster.Deletion of R1 could increase the bioactivity to some extend and so R1 was looked as a negative regulator.S.coelicolor M1154 exhibited a better yield of antibacterial activity out of the tested surrogate hosts apparently different from hybrubin(S.lividans SBT18 was the best)Therefore,to exclude the interference of the redundant genes,we constructed pHZZL5 which contains the entire NRPS gene cluster and then transferred into S.coelicolor M1154/pJTU6728 for fermentation.After column chromatography fractionation traced by bioactivity against Sta.aureus and LC-MS,we discovered that the active compounds exhibited no UV absorbtion.With the help of HRESI-QTOF analysis,we discovered the active fraction is mainly composed of a compound with[M+H]+(m/z)of 342.Further purification of the active compound with the help of LC-MS is still ongoing.The identification of the bioactive compound and reveal of its biosynthetic route will be studied in the near future.