A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea

The biological activity of polyketide antibiotics is often strongly depende nt on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin-producing stra in of Saccharopolyspora erythraea, for detection of hybrid glycoside format ion, and this system has been used to demonstrate that an amino sugar chara cteristic of 14-membered macrolides (d-desosamine) can be efficiently attac hed to a 16-membered aglycone substrate. First, the S. erythraea mutant str ain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 fr om Streptomyces antibioticus, which transfers l-oleandrose, has recently be en shown to transfer rhamnose to the oxygen at C-3 of erythronolide B and 6 -deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3-O-rhamnosyl-erythronolide B and 3-O-rhamno syl-6-deoxyerythronolide B were produced. Having thus validated the express ion system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 fr om Streptomyces fradiae to utilise a different amino sugar, tylM2 was integ rated into S. erythraea SGT2, and the resulting strain was fed with the 16- membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glyco side was isolated in good yield and characterized as 5-O-desosaminyl-tylact one, indicating that TylM2 may be a useful glycosyltransferase for combinat orial biosynthesis. 5-O-glucosyl-tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone i n these cells.