Genome Seqencing And Analyses Of Amycolatopsis Orientalis HCCB10007 And Genome Mining

Amycolatopsis orientalis is the type species of the genus Amycolatopsis and its industrial strain HCCB10007 has been used for large-scale production of the vital antibiotic vancomycin,which is a potent glycopeptide antibiotic that has been used for more than three decades for the treatment of serious methicillin-resistant Staphylococcus aureus(MRSA)infections.In order to study the genetic knowledge of the strain and discover the novel secondary metabolites,the genome of A.orientalis HCCB 10007 was sequenced and analyzed.The research results were obtained as follows:1.Genome sequencing and analyses of Amycolatopsis orietnalis HCCB10007The complete genome of A.orientalis HCCB 10007 sequenced by using Roche 454 systerm comprises a circular chromosome and a plasmid named pXL100.In total,the size of chromosome was 8.95Mbp,and GC content to be 69%.8,121 coding DNA sequences(CDSs)were predicted,and 5518 CDSs resemble known genes(67.9%).Sequence analysis of pXL100 revealed its total length to be 33,499 bp and GC content to be 68.9%.Bioinformatics analysis revealed that the plasmid encodes 49 genes.However,only seven ORFs resemble known genes.A.orientalis shared 50.3%orthologs with A.mediterranei.The species-specific genomic features of A.orientalis were analyzed in comparison with that of A.mediterranei.The chromosomal core/quasi-core and non-core configurations of the A.orientalis and the A.mediterranei genome were analyzed reciprocally,with respect to further understanding both the discriminable criteria and the evolutionary implementation.This study extended the genetic knowledge of the genus Amycolatopsis.The molecular genetic basis responsible for the components of arabinose,glycine,diaminopimelic acids,mycolic acids,phosphorlipids and menaquinones were analyzed and identified in the genome of two species of Amycolatopsis.These studies indicate that the chemotaxonomic characteristics of this genus,which relate to,but differentiate from Streptomyces and Nocardia,are intrinsically determined by the molecular phylogeny of their encoding genes.Twenty-six secondary metabolite biosynthetic gene clusters were predicted in the complete genome of A.orientalis HCCB10007,including nine type I polyketide synthase(PKS)clusters,one type II PKS cluster,ten non-ribosomal peptide synthetase(NRPS)clusters,three hybrid PKS-NRPS clusters,two clusters for terpenoids,one cluster for lycopene(lyc),and one cluster for ?-carotene(car).The total length of these gene clusters was estimated?552 kb,which is 6.2%of the whole genome.In contrast to the essential genes,most of the secondary metabolite biosynthetic gene clusters(18 out of 26)were in the non-core regions.2.Genome mining for natural products in Amycolatopsis orientalis HCCB10007The term "genome mining" has been used to describe the exploitation of genomic information for the discovery of new processes,targets,and products.It is possible to estimate the biosynthetic potential for a given organism by mining the whole genome sequence.An analysis of the A.orietalis HCCB10007 genome revealed new gene clusters involved in natural product biosynthesis that were not associated with the production of known compounds.Reverse-transcription PCR was used to detect the transcription profiles of the gene clusters that might synthesize potential secondary metabolites.The genes of four clusters(nrps4,pks5,n_p2,and vcm)showed significant levels of transcriptionin the F1 fermentation medium.The n_p2 was a NRPS-PKS hydrid cluster,comprising a halogenase gene.In this study,an indole-type halometabolite was identified by whole genome mining.This compound promoted the growthof wheat seedlings,especially root elongation.Another analysis revealed that nrps3 might the biosynthetic gene cluster for the production of siderophore.The ability of producing siderophore in A.orientalis was detected and confirmed by blue agar CAS assay.Meanwhile,some other metabolites were discovered by using OSMAC(one strain many compounds)and their biosynthetic genes were also analyzed.3.Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vectorThree indigenous plasmids,pMEA100,pMEA300,and pA387,found in Amycolatopsis genus have been sequenced.However,only some vectors based on pA387 have been widely applied in Amycolatopsis research.For this purpose,a 2830-bp fragment containing three ORFs has been identified as essential for replication in pXL100 of A.orientalis.And it has no significant similarity to any known replicons.A vector,pLYZW7-3,was constructed based on the pXL100 replicon and could be transferred into A.mediterranei and A.orientalis by electroporation or conjugation with high frequency.A mutant with a disrupted gene was successfully complemented with the pLYZW7-3 vector,indicating that the vector is potentially useful in Amycolatopsis research.