Urdamycin G

Multi-omic approaches have recently made big strides toward the effective exploration of microorganisms, accelerating the discovery of new bioactive compounds. We combined metabolomic, molecular networking, and genomic-based approaches to investigate the metabolic potential of the Streptomyces sp. RO-S4 strain isolated from the polluted waters of Bejaia Bay in Algeria. Antagonistic assays against methicillin-resistant Staphylococcus aureus with RO-S4 organic extracts showed an inhibition zone of 20 mm by using the agar diffusion method, and its minimum inhibitory concentration was 16 μg/ml. A molecular network was created using GNPS and annotated through the comparison of MS/MS spectra against several databases. The predominant compounds in the RO-S4 extract belonged to the angucycline family. Three compounds were annotated as known metabolites, while all the others were putatively new to Science. Notably, all compounds had fridamycin-like aglycones, and several of them had a lactonized D ring analogous to that of urdamycin L. The whole genome of Streptomyces RO-S4 was sequenced to identify the biosynthetic gene cluster (BGC) linked to these angucyclines, which yielded a draft genome of 7,497,846 bp with 72.4% G+C content. Subsequently, a genome mining analysis revealed 19 putative biosynthetic gene clusters, including a grincamycin-like BGC with high similarity to that of Streptomyces sp. CZN-748, that was previously reported to also produce mostly open fridamycin-like aglycones. As the ring-opening process leading to these compounds is still not defined, we performed a comparative analysis with other angucycline BGCs and advanced some hypotheses to explain the ring-opening and lactonization, possibly linked to the uncoupling between the activity of GcnE and GcnM homologs in the RO-S4 strain. The combination of metabolomic and genomic approaches greatly improved the interpretation of the metabolic potential of the RO-S4 strain.

Background: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. Results: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. Conclusions: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.

Background: Urdamycin A, the principle product of Streptomyces fradiae Tü2717, is an angucycline-type antibiotic. The polyketide-derived aglycone moiety is glycosylated at two positions, but only limited information is available about glycosyltransferases involved in urdamycin biosynthesis. Results: To determine the function of three glycosyltransferase genes in the urdamycin biosynthetic gene cluster, we have carried out gene inactivation and expression experiments. Inactivation of urdGT1a resulted in the predominant accumulation of urdamycin B. A mutant lacking urdGT1b and urdGT1c mainly produced compound 100-2. When urdGT1c was expressed in the urdGT1b/urdGT1c double mutant, urdamycin G and urdamycin A were detected. The mutant lacking all three genes mainly accumulated aquayamycin and urdamycinone B. Expression of urdGT1c in the triple mutant led to the formation of compound 100-1, whereas expression of urdGT1a resulted in the formation of compound 100-2. Co-expression of urdGT1b and urdGT1c resulted in the production of 12b-derhodinosyl-urdamycin A, and co-expression of urdGT1a, urdGT1b and urdGT1c resulted in the formation of urdamycin A. Conclusions: Analysis of glycosyltransferase genes of the urdamycin biosynthetic gene cluster led to an unambiguous assignment of each glycosyltransferase to a certain biosynthetic saccharide attachment step.