Mutation and cloning of eryG, the structural gene for erythromycin O-methyltransferase from Saccharopolyspora erythraea, and expression of eryG in Escherichia coli

Abstract

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.

Functional Analysis of OleY l -Oleandrosyl 3- O -Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

“…In erythromycin biosynthesis by S. erythraea, eryG codes for a 3-O-methyltransferase that converts L-mycarose into L-cladinose (and therefore erythromycin C into erythromycin A). This is the final step in erythromycin biosynthesis and occurs once both sugars, L-mycarose and D-desosamine, are attached to the aglycon (17). The action of the OleY methyltransferase differs from that of these three methyltransferases.…”

Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives

“…These results indicate that EryG, the enzyme that O-methylates the CЉ-3 hydroxyl on the L-mycarose moiety, can use either the 10-or 12-desmethyl derivative efficiently as a substrate. This could not have been predicted since it was previously discovered that some erythromycin derivatives that contained changes in the structure of the lactone ring, e.g., 6-deoxyerythromycin, 2-desmethylerythromycin, and ⌬ 6,7 -anhydroerythromycin C, had reduced activity as substrates for EryG (9,24,27,40).…”

Deoxysugar Methylation during Biosynthesis of the Antitumor Polyketide Elloramycin by Streptomyces olivaceus

“…Several genes from antibiotic-producing organisms have been isolated and proposed to code for enzymes responsible for these methylation steps. These genes are mainly from macrolide producers and include tylE and tylF from the tylosin producer Streptomyces fradiae (5,6), mycF from the mycinamycin producer M. griseorubida (7), oleY from the oleandomycin producer S. antibioticus (8), eryG from the erythromycin producer S. erythraea (10), and novP from the novobiocin producer S. sphaeroides (28). The involvement of some of these genes in O-methylation has been proved.…”

“…The involvement of some of these genes in O-methylation has been proved. However, experimental evidence on their action as methylating enzymes has been provided so far only for eryG (10) and mycF (7). Furthermore, most of the mentioned methyltransferases are the only methylating enzymes acting on the corresponding sugar that therefore only possess a single methoxy group.…”

“…Its product OleY is involved in the conversion of L-olivose into L-oleandrose (9). Finally, eryG encodes an O-methyltransferase that catalyzes the last step in the erythromycin biosynthesis in Saccharopolyspora erythraea, the 3-O-methylation of L-mycarose into L-cladinose, thus also acting on a completely glycosylated intermediate (10). With the exception of tylosin, only a single methylation event modifying a deoxysugar moiety occurs in all of the biosyntheses of these macrolides.…”