The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster

Background: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosyl ated with four sugar components, 2X D-olivose (first and last sugar of a C- glycosidically bound trisaccharide chain at the g-position), and 2 x L-rhod inose (in the middle of the trisaccharide chain and at the 12b-position). L imited information is available about both the biosynthesis of D-olivose an d L-rhodinose and the influence of the concentration of both sugars on urda mycin biosynthesis. R Results: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open readi ng frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each o ne showing significant homology to deoxysugar biosynthetic genes. We inacti vated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactiv ation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactiv ation 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 urdamy cins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ ac cumulated Various known diketopiperazines. Conclusions: Analysis of deoxysugar biosynthetic genes of the urdamycin bio synthetic gene cluster revealed a widely common biosynthetic pathway leadin g to D-olivose and L-rhodinose. Several enzymes responsible for specific st eps of this pathway could be assigned. The pathway had to be modified compa red to earlier suggestions. Two glycosyltransferases normally involved in t he C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conver sion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity f or their activated deoxysugar co-substrate and their alcohol substrate, res pectively. They can transfer activated D-rhodinose (instead of D-olivose) t o the B-position, and attach L-rhodinose to the 4A-position normally occupi ed by a D-olivose unit, respectively.