Here we report the use of a novel gene delivery and expression system (described by RODRIGUEZ et al. for rapid engineering of recombinant polyketide synthases (PKSs) in an erythromycin-overproducing strain of Saccharopolyspora erythraea K24-1) to introduce DEBS genes harboring a mutagenized acyltransferase domain in module 4 (AT4, mutation 3 in reference 6) into S. erythraea K24-1 for the production of 6-demethyl erythromycins. The genes encoding the engineered PKS were transferred from a Streptomyces lividans expression vector to the pSET152 integrating vector under the control of the native eryAp promoter, resulting in the vector pKOS159-42, which was introduced into S. erythraea K24-1 by conjugation. Culture broths of antibiotic-producing isolates were screened by LC/MS for compounds with mass spectra consistent with 6-demethyl-erythromycin analogues. A compound with m/z 690 (consistent with 6-demethyl-erythromycin D) was selected for scale-up in 10-liter bioreactors and isolated as Compound 1 (7-hydroxy-6-demethyl-6-deoxy erythromycin D). Compound 1 has a molecular formula of C35H63NO12, as determined by 13C NMR and high-resolution mass spectral data. Its structure, which features a hydroxyl group at the 7-position (instead of the expected 6-position) and demethylation at the 6-position, was confirmed by multiplicity-edited HSQC, gsCOSY, and constant time HMBC spectra. The antibacterial activity of Compound 1 was marginal against microorganisms sensitive to erythromycin A, as it lacks the 12-OH and 3'-O-CH3 groups that contribute substantially to antibiotic activity. The unexpected hydroxylation at the 7-position rather than the 6-position is presumably due to an altered binding configuration of the substrate (6-nor-erythronolide B) in the active site of the EryF cytochrome P450 oxidase.