New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster.
Using a previously developed expression system based on the erythromycin-producing strain of Saccharopolyspora erythraea, O-methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14-membered polyketide macrolactone. The spnI, spnK and spnH methyltransferase genes were expressed individually in the S. erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII. Exogenous 3-O-rhamnosyl-erythronolide B was efficiently converted into 3-O-(2'-O-methylrhamnosyl)-erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3-O-(2'-O-methylrhamnosyl)-erythronolide B was, in turn, fed to a culture of S. erythraea SGT2 (spnK), 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was identified in the culture supernatant, whereas S. erythraea SGT2 (spnH) was without effect. These results confirm the identity of the 2'- and 3'-O-methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was found not to be a substrate for the 4'-O-methyltransferase SpnH. Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl-erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl-erythronolide precursors using the S. erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3-O-(2'-O-methylrhamnosyl)-erythromycin D was tested and found to be significantly active against a strain of erythromycin-sensitive Bacillus subtilis.
Gaisser
S
S
Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Lill
R
R
Wirtz
G
G
Grolle
F
F
Staunton
J
J
Leadlay
P F
PF
D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000737:chemistry; D017353:Gene Deletion; D005798:Genes, Bacterial; D013058:Mass Spectrometry / Q000379:methods; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics; D012210:Rhamnose / Q000378:metabolism*; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003470:Culture Media; D004917:Erythromycin; D008780:Methyltransferases; D012210:Rhamnose