Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring

Abstract

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3' end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P450 monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P450-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.

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

“…These reactions are catalyzed by a modular type I polyketide synthase comprising three large polypeptides, named OleA1, OleA2, and OleA3 (24,25). The oleandomycin biosynthetic gene cluster has been fully sequenced and characterized (1,15,16,18,19,24,25) (Fig. 1B).…”

“…1B). Two DNA regions flanking the genes encoding the three multifunctional polypeptides of the polyketide synthase contain a number of genes that code for other enzymatic functions required for the biosynthesis of the two sugars and their transfer to the aglycon (1,16,18), genes encoding an ABC transporter system responsible for secretion (15), genes encoding an inactivating-reactivating enzymatic system that confers self-resistance to the producer organism (18,26), and a gene encoding a cytochrome P-450 oxygenase involved in epoxidation of the macrolactone ring at carbon 8 (19,24). The oleandomycin macrolactone ring is glycosylated by two 6-deoxysugars (L-oleandrose and D-desosamine).…”

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

“…The fact that elloramycin (containing a permethylated L-rhamnose) was synthesized by this recombinant strain strongly suggested that cos16F4 should contain the methyltransferase genes required for methylating the hydroxy groups at carbons 2, 3, and 4 of the L-rhamnose moiety. In order to isolate these genes, we used as probe a 1.1-kb EcoRI-XhoI fragment from S. antibioticus ATCC 11891 (oleandomycin producer) that contains most of the oleY gene (only the last 74 base pairs are absent), which codes for a methyltransferase involved in L-oleandrose biosynthesis in S. antibioticus (8). With this probe we initially restricted the hybridizing region to a 10-kb BamHI fragment located at the right side of cos16F4 and within this to three restriction fragments: 0.4-kb BamHI-PstI, 2.8-kb PstI, and 3-kb PstI (Fig.…”

“…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.…”

“…From the gene cluster of the structurally closely related macrolide antibiotic mycinamycin produced by Micromonospora griseorubida, another gene, mycF, was isolated and proposed to encode a macrocin O-methyltransferase (7). Another O-methyltransferase gene, oleY, was reported in Streptomyces antibioticus, the oleandomycin producer (8). Its product OleY is involved in the conversion of L-olivose into L-oleandrose (9).…”