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A mutant strain derived by chemical mutagenesis of Saccharopolyspora erythraea (formerly known as Streptomyces erythreus) was isolated that accumulated erythromycin C and, to a lesser extent, its precursor, erythromycin D, with little or no production of erythromycin A or erythromycin B (the 3"-O-methylation products of erythromycin C and D, respectively). This mutant lacked detectable erythromycin O-methyltransferase activity with erythromycin C, erythromycin D, or the analogs 2-norerythromycin C and 2-norerythromycin D as substrates. A 4.5-kilobase DNA fragment from S. erythraea originating approximately 5 kilobases from the erythromycin resistance gene ermE was identified that regenerated the parental phenotype and restored erythromycin O-methyltransferase activity when transformed into the erythromycin O-methyltransferase-negative mutant. Erythromycin O-methyltransferase activity was detected when the 4.5-kilobase fragment was fused to the lacZ promoter and introduced into Escherichia coli. The activity was dependent on the orientation of the DNA relative to lacZ. We have designated this genotype eryG in agreement with Weber et al. (J.M. Weber, B. Schoner, and R. Losick, Gene 75:235-241, 1989). It thus appears that a single enzyme catalyzes all of the 3"-O-methylation reactions of the erythromycin biosynthetic pathway in S. erythraea and that eryG codes for the structural gene of this enzyme.

A comparative evaluation of three brands of poly(styrene-divinylbenzene) copolymers, Hamilton PRP-1 (10 micron), Rogel (8 micron) and TSK-Gel (10 micron), as column packing materials for high-performance liquid chromatographic separation of erythromycins is presented. Erythromycins A, B and C, anhydroerythromycin A, erythromycin A enol ether, N-demethylerythromycin A, anhydro N-demethylerythromycin A and N-demethylerythromycin A enol ether were chromatographed. The effects of column temperature, concentration of organic modifier in the mobile phase, concentration of phosphate buffer, the addition of quaternary ammonium salts and pH are described. The best separations were obtained on TSK-Gel with the mobile phase acetonitrile-methanol-0.2 M tetramethylammonium hydroxide pH 8.0-0.2 M phosphate buffer pH 8.0-water (30:15:25:5:25). PRP-1 and Rogel gave equally good separations but with higher retention volumes.

The identification of five novel compounds, pseudo-erythromycin A-6,9-hemiketal, 8,9-anhydro-pseudo-erythromycin A-6,9-hemiketal, 8,9-anhydro-pseudo-N-demethylerythromycin A-6,9-hemiketal, 5-O-beta-D-desosaminylerythronolide A and 15-nor-erythromycin C, in mother liquor concentrates of Streptomyces erythraeus is described. The pseudo-erythromycin derivatives are characterized by a 12-membered macrocyclic ring as a result of C13----C11 trans-lactonization. The five compounds have very little antimicrobial activity.

The process of purification of benzylpenicillin, penicillin V and erythromycin solutions with the use of semi-permeable membranes was studied. Practically complete separation of high-molecular admixtures and microbial colonies and separation of significant amounts of the colored compounds were shown to be possible. The purification process may be used effectively at the early stages of antibiotic production.

The separating capacity of a laboratory cup centrifuge and industrial centrifugal separator was compared. It was shown that the process of emulsion centrifugal separation could be modeled under laboratory conditions: antibiotic fermentation broth filtrate--organic solvent in industrial apparatus under similar conditions of preliminary mixing aqueous and organic phases.

An improved high-performance liquid chromatographic method for analysis of erythromycin is described. The separation can be performed under mild conditions of pH (6.5) and temperature (35 degrees C) on C8 and C18 silica-based reversed-phase materials of different origins. The mobile phase, with a flow-rate of 1.5 ml/min, contained various amounts of acetonitrile (25-40%, v/v), 5% (v/v) 0.2 M ammonium phosphate buffer pH 6.5, 20% (v/v) 0.2 M tetramethylammonium phosphate and water. UV detection at 215 nm allows quantitation of erythromycins A, B and C, N-demethylerythromycin A, erythromycin A enol ether and anhydroerythromycin A. The column history plays a major role, older columns often giving better separations.

(14R)-14-Hydroxy-6-O-methylerythromycin A and (14S)-epimer have been isolated as active metabolites from human urine after oral administration of 6-O-methylerythromycin A (TE-031, A-56268). The structures of these metabolites were determined by means of mass, 1H and 13C NMR spectroscopies. Antimicrobial activities of (14R)-14-hydroxy-6-O-methylerythromycin A, a major metabolite, were comparable to those of the parent drug TE-031, whereas (14S)-14-hydroxy-6-O-methylerythromycin A was 4- to 8-fold less active than TE-031 against bacterial standard strains.