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-kiobase 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.
An alpha-galactosidase from the erythromycin-producing bacterium Saccharopolyspora erythraea was purified to near homogeneity. The enzyme has an apparent molecular mass of 45 kDa as determined by SDS-PAGE. The pH optimum, K(m) for p-nitrophenyl-alpha-D: -glucopyranoside (pNPalphaG), K(m) for melibiose and the V(max) are similar to those of other studied alpha-galactosidase enzymes. The N-terminal amino-acid sequence of this protein was determined. PCR amplification was used to generate a 640-bp product using oligonucleotide primers based on the N-terminal amino-acid sequence and a downstream region that is conserved in other related alpha-galactosidase enzymes. This fragment was used as a probe to clone the alpha-galactosidase gene, designated melA, from a S. erythraea lambda phage chromosomal library. S. erythraea appears to possess an unique alpha-galactosidase enzyme, encoded by melA, that can utilize galactopyranosides as carbon sources. Furthermore, the ability to use the product of melA as a reporter enzyme in S. erythraea has been demonstrated. The alpha-galactosidase uses the substrates 5-bromo-4-chloro-3-indoyl-alpha-D: -galactosidase (X-alpha-gal) on agar media and pNPalphaG in liquid media.
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