The Discovery Of Genomic - Derived Natural Product And Its Biosynthetic Gene Cluster In Streptomyces Sp

With the development of high-throughput sequencing technology and the biosynthetic mechanism of natural products (NPs), genome mining has become a new stratage to find novel NPs. Genome mining may help to break through the bottlenecks of novel NPs discovery and make the research of NPs undergoing the second golden era. In our research, this method was applied to Streptomyces sp. SS to discover novel gene clusters or natural products.Streptomyces sp. SS, isolated from a soil sample collected from Guizhou province, produces sansanmycin and its analogs which are uridyl peptide inhibitors targeting MraY translocase. Draft genome of streptomyce sp. SS has been sequenced and submitted to the GenBank (No. NZ_AKXV00000000.1). The results of bioinformatics analysis with antiSMASH displayed that the genome of Streptomyces sp.SS contains 21 biosynthetic gene clusters. In addtition to the gene cluster of sansanmycin, function of other gene clusters are still unclear. Products prediction of 21 gene clusters had completed via bioinformatics analysis. We focused on the gene cluster 8 and 2 in this study.We sepculated that the cluster 8 may be invovlved in biosynthesis of foroxymithine by bioinformatics analysis and Norine database search. Foroxymithine is not only an ACE inhibitor with antihypertensive activity, but also a typical siderophore about hydroxamate. It has vital physiological significance but its biosynthetic gene cluster has not been reported yet. According to the characteristics of siderophore, we activated cluster 8 by iron-deficient medium, isolated the target compound from the fermentation broth and confirmed the compound using HPLC-MS, NMR and other analysis methods. We found 5 iron box motifs in cluster 8 and the Streptomyces sp. SS contained highly homologous sequences with DmdR protein when we scanned the whole genome. Both of those indicated that cluster 8 invovled in biosynthesis of foroxymithine, which might be regulated by DmdR protein and Fe2+. We further tried to disrupt the 2 A domain in cluster 8 in order to confirm the cluster 8 was the foroxymithine’s biosynthetic gene cluster. We explored the biosynthetic gene cluster of foroxymithine and speculated its biosynthetic pathway firstly.We found that cluster 2 contained highly homologous sequences with foml-4 and fom A-D involved in fosfomycin’s biosynthetic gene cluster and the gene Y19_RS0101925 could encode phosphoenolpyruvate decarboxylase, which is the key enzyme in the biosynthetic pathway of fosfomycin and encoded by fom 2. The cluster 2 and cluster 3 linked in forward direction, so we presumed they may jointly be responsible for the biosynthesis of fosfomycin or analogs. Fosfomycin which has unique structure, a good and broad antibacterial activity can inhibit the multidrug resistance and extensive drug resistance of gram-positive bacteria and gram-negative bacteria including methicillin-resistant and vancomycin-resistant bacteria. It has been used in clinical treatment of urinary tract infection and other diseases. Given the unique antibacterial activity and promising application of fosfomycin, we tried to avtivate the biosynthetic gene cluster of fosfomycin in Streptomyces sp. SS in order to find obtain high-yield strains or new analogues. In this work, we used the conventional culture medium of fosfomycin to activate or overexpress the gene cluster 2 but it was not work. Then we selected 4 regulatory genes in gene cluster 2,3 and constructed their overexpressed strain hoping to activate the gene cluster. Meanwhile, we explored the application of activating cryptic genes via overexpressing regulators by genome mining.