ACYLTRANSFERASE DOMAIN SUBSTITUTIONS IN ERYTHROMYCIN POLYKETIDE SYNTHASE YIELD NOVEL ERYTHROMYCIN DERIVATIVES

The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransfer ase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synt hase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with t hree heterologous AT domains that are believed, based on sequence comp arisons, to be specific for malonyl-CoA. The three substituted AT doma ins were ''Hyg'' An from module 2 of a type I polyketide synthase (PKS )-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, ''Ven'' AT isolated from a PKS-like gene clu ster of the pikromycin producer Streptomyces venezuelae ATCC 15439, an d RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hyg roscopicus ATCC 29253, These changes led to the production of novel er ythromycin derivatives by the engineered strains of S. erythraea ER720 , Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the m ethyl group at C-12 of the macrolactone ring, was produced by the stra ins in which the resident AT1 domain was replaced, and 10-desmethylery thromycin A and 10 desmethyl-12-deoxyerythromycin A, both of which lac k the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activi ty against Staphylococcus aureus. The production of the erythromycin d erivatives through AT replacements confirms the computer predicted sub strate specificities of ''Hyg'' AT2 and ''Ven'' AT and the substrate s pecificity of RAPS AT14 deduced from the structure of rapamycin, Moreo ver, these experiments demonstrate that at least some AT domains of th e complete 6-deoxyerythronolide B synthase of S. erythraea can be repl aced by functionally related domains from different organisms to make novel, bioactive compounds.