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. 2000 May;44(5):1266-75.
doi: 10.1128/AAC.44.5.1266-1275.2000.

Identification and expression of genes involved in biosynthesis of L-oleandrose and its intermediate L-olivose in the oleandomycin producer Streptomyces antibioticus

I Aguirrezabalaga  1 , C Olano, N Allende, L Rodriguez, A F Braña, C Méndez, J A Salas
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Identification and expression of genes involved in biosynthesis of L-oleandrose and its intermediate L-olivose in the oleandomycin producer Streptomyces antibioticus

I Aguirrezabalaga et al. Antimicrob Agents Chemother. 2000 May.

Abstract

A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. Sequence analysis revealed the presence of 8 open reading frames encoding different enzyme activities involved in the biosynthesis of one of the two 2, 6-deoxysugars attached to the oleandomycin aglycone: L-oleandrose (the oleW, oleV, oleL, and oleU genes) and D-desosamine (the oleNI and oleT genes), or of both (the oleS and oleE genes). A Streptomyces albus strain harboring the oleG2 glycosyltransferase gene integrated into the chromosome was constructed. This strain was transformed with two different plasmid constructs (pOLV and pOLE) containing a set of genes proposed to be required for the biosynthesis of dTDP-L-olivose and dTDP-L-oleandrose, respectively. Incubation of these recombinant strains with the erythromycin aglycon (erythronolide B) gave rise to two new glycosylated compounds, identified as L-3-O-olivosyl- and L-3-O-oleandrosyl-erythronolide B, indicating that pOLV and pOLE encode all enzyme activities required for the biosynthesis of these two 2,6-dideoxysugars. A pathway is proposed for the biosynthesis of these two deoxysugars in S. antibioticus.

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Figures

FIG. 1
FIG. 1
(A) Structure of oleandomycin. (B) Schematic representation of the oleandomycin gene cluster. Solid bars, DNA regions hybridizing against the strDEM genes from the streptomycin pathway; open bars, locations of the 7.6-kb SstI fragment and the PCR-amplified oleY gene. B, BamHI; S, SstI; PKS, PKS genes.
FIG. 2
FIG. 2
Alignment of OleW and related proteins. LanT, landomycin biosynthetic pathway of S. cyanogenus (59); Oxidored, oxidoreductase from the rifamycin pathway of A. mediterranei (2); GraORF26, granaticin biosynthetic pathway of S. violaceoruber (22); RdmF, rhodomycin biosynthetic pathway of S. purpurascens (32). Amino acids that are identical in at least three of the five proteins compared are shown on a solid background, and conservative substituted amino acids are shown on a shaded background.
FIG. 3
FIG. 3
Analysis of the products of the biotransformation of erythronolide B by strain IAGS1. (A) Thin-layer chromatography analysis of the reaction. EB, erythronolide B; St, standard; + and −, incubation in the presence and absence of EB, respectively. (B) Mass spectrometry analysis of compound M1. (C) Structure of 3-l-olivosyl-erythronolide B.
FIG. 3
FIG. 3
Analysis of the products of the biotransformation of erythronolide B by strain IAGS1. (A) Thin-layer chromatography analysis of the reaction. EB, erythronolide B; St, standard; + and −, incubation in the presence and absence of EB, respectively. (B) Mass spectrometry analysis of compound M1. (C) Structure of 3-l-olivosyl-erythronolide B.
FIG. 4
FIG. 4
Analysis of the products of the biotransformation of erythronolide B by strain IAGS2. (A) Thin-layer chromatography analysis of the reaction. EB, erythronolide B; St, standard; + and −, incubation in the presence and absence of EB, respectively. (B) Mass spectrometry analysis of compound M2. (C) Structure of 3-l-oleandrosyl-erythronolide B.
FIG. 4
FIG. 4
Analysis of the products of the biotransformation of erythronolide B by strain IAGS2. (A) Thin-layer chromatography analysis of the reaction. EB, erythronolide B; St, standard; + and −, incubation in the presence and absence of EB, respectively. (B) Mass spectrometry analysis of compound M2. (C) Structure of 3-l-oleandrosyl-erythronolide B.
FIG. 5
FIG. 5
Proposed pathways for the biosynthesis of d-desosamine and l-oleandrose by S. antibioticus.
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