The Biosynthesis Study Of The Diisonitrile Chalkophore SF2768 In Streptomyces Thioluteus

Streptomyces thioluteus attracted our research interest due to its ability to produce the polyketide antibiotic aureothin and the dithiopyrone antibiotic thiolutin.Bioinformatics analysis of the draft genome sequence of S.thioluteus displayed that there are more than 40 secondary metabolite biosynthetic gene clusters besides two published biosynthetic systems for aureothin and thiolutin.The products of most clusters could not be assigned by bioinformatics methodology.Our previous study found that a novel NRPS(nonribosomal peptide synthase)biosynthetic gene cluster from S.thioluteus heterologously expressed in a clean host Streptomyces lividans ZX1 and produced bioactive compounds with broad spectrum antimicrobial activity.In this study,the bioactive product was isolated and purified from the large scale fermentation broth of the heterologous expression strain S.lividans::p ZMY13-2cut.The chemical structure of the compound was elucidated by HR-MS and NMR spectroscopy analyses.The novel NRPS biosynthetic gene cluster(named as sfa)catalyzes the biosynthesis of the diisonitrile antibiotic SF2768.For functional study of the SF2768 biosynthetic gene cluster,the fermentation products of each gene deletion mutant strains were detected by bioassay and HR-LC-MS,and revealed that the deletion of sfa A,sfa B,sfa C,sfa D or sfa E abolished the production of SF2768.Two new SF2768 analogues 3 and 4 were isolated from the gene deletion mutant strain ?sfa E,which are considered as the intermediate or shunt products of SF2768.The substrate specificity assay in vitro disclosed that the optimum substrate of the adenylation domain in the NRPS Sfa D is lysine.Functional and bioinformatics analyses of the sfa cluster combined with the chemical structures of SF2768 and two new intermediate or shunt products 3 and 4,allowed the proposal of the biosynthetic pathway of antibiotic SF2768.The SF2768 biosynthetic cluster is homologous to the cluster responsible for the catechol-type siderophore myxochelins from myxobacteria,suggesting that SF2768 might be used as a metallophore by the producers.Bioinformatics based alignment found a putative ATP-binding cassette(ABC)transporter related to iron import within the biosynthetic gene cluster,which implied that the product might be a siderophore.However,characterization of the metal-binding properties by high-resolution electrospray ionization mass spectrometry(HR-ESI-MS),thin-layer chromatography(TLC),chrome azurol S(CAS)assys and metal-ligand-titration experiment showed that SF2768 could chelate copper specifically instead of iron,to form stable complexes.The extracellular/intracellular content ratio of SF2768 was compared etween S.lividans::p ZMY13-2cut and mutant strain ?orf12.This result demonstrated that the major facilitator superfamily(MFS)exporter Orf12 was involved in the export of the proposed chalkphore SF2768.We employed ICP-MS to analysed the ability of S.lividans::p ZMY13-2cut and the mutant strain ?orf19-21 to uptake 65Cu-SF2768 complex and confirmed that the copper-SF2768 complex was internalized by ABC superfamily transporter Orf19-21.These results preliminarily elucidates the uptake and export mechanism of SF2768 and lays the foundation for a reasonable explanation of the transport mode of SF2768.Addition of Cu2+ in the bioassay counteracted the inhibition effect of SF2768.Two possible explanations for the phenomenon were proposed: 1)SF2768 chelated Cu2+ of the medium and the complexes could not be transported into B.subtilis 168.The "copper starvation" would lead an inhibition of the indicator strain growth.The additional Cu2+ could eliminate the "starvation" so that the growth of the indicator bacteria was restored.2)The isonitrile groups of SF2768 are pharmacophore to directly inhibit the growth of the indicator bacteria cells.When they were blocked with Cu2+ by specifically binding,the antibiotic SF2768 could not work.