Biosynthesis of elloramycin in Streptomyces olivaceus requires glycosylation by enzymes encoded outside the aglycon cluster

Abstract

Elloramycin is an anthracycline-like antitumour drug produced by Streptomyces olivaceus Tü 2353. Cosmid cos16F4 has been previously shown to direct the biosynthesis of the elloramycin aglycon 8-demethyltetracenomycin C (8-DMTC), but not elloramycin. Sequencing of the 24.2 kb insert in cos16F4 shows the presence of 17 genes involved in elloramycin biosynthesis (elm genes) together with another additional eight ORFs probably not involved in elloramycin biosynthesis. The 17 genes would code for the biosynthesis of the polyketide moiety, sugar transfer, methylation of the tetracyclic ring and the sugar moiety, and export. Four genes (rhaA, rhaB, rhaC and rhaD) encoding the enzymic activities required for the biosynthesis of the sugar L-rhamnose were also identified in the S. olivaceus chromosome. The involvement of this rhamnose gene cluster in elloramycin biosynthesis was demonstrated by insertional inactivation of the rhaB gene, generating a non-producer mutant that accumulates the 8-DMTC C aglycon. Coexpression of cos16F4 with pEM4RO (expressing the four rhamnose biosynthesis genes) in Streptomyces lividans led to the formation of elloramycin, demonstrating that both subclusters are required for elloramycin biosynthesis. These results demonstrate that, in contrast to most of the biosynthesis gene clusters from actinomycetes, genes involved in the biosynthesis of elloramycin are located in two chromosomal loci.

Identification and Manipulation of the Caprazamycin Gene Cluster Lead to New Simplified Liponucleoside Antibiotics and Give Insights into the Biosynthetic Pathway

“…Analogous to the biosynthesis of elloramycin, L-rhamnose would be synthesized from enzymes encoded elsewhere on the genome (40). The dNDP-L-rhamnose probably constitutes the substrate for a transfer reaction to the CPZ aglycon catalyzed by the putative rhamnosyltransferase Cpz31.…”

Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis

“…In addition, formation of a natural compound often relies on the supply of specific precursors provided by genes encoded outside the gene cluster, e.g., deoxysugars (48), isoprenoid derived moieties (20), or other small subunits (45,58). Therefore, the new assembly approach described in this study may help to establish heterologous expression and genetic engineering of scattered gene clusters.…”

“…However, biosynthetic gene clusters of rhamnosylated compounds often lack the genes for L-rhamnose formation (9,16,38,54). In the case of elloramycin (48) and spinosyn (41), these genes were identified elsewhere in the genome. Inactivation experiments in Saccharopolyspora spinosa suggested that the gtt, gdh, epi, and kre genes provide the rhamnosyl moiety for both spinosyn and cell wall biosynthesis.…”

Organisation of the Biosynthetic Gene Cluster and Tailoring Enzymes in the Biosynthesis of the Tetracyclic Quinone Glycoside Antibiotic Polyketomycin

“…PokMT3 shares 54 % identical aa with FdmN, an Omethyltransferase involved in the fredericamycin biosynthesis in S. griseus, [26] 51 % with TcmN, which is an O-methyltransferase that is involved in the tetracenomycin D3 biosynthesis in S. glaucescens [27,28] and 49 % with ElmNII, which is an O-methyltransferase that is involved in the elloramycin biosynthesis in S. olivaceus. [29] Based on the function of these proteins, PokMT3 is proposed to catalyse the methyl transfer to the phenolic hydroxy group in ring D of POK. In contrast to TcmN, a methyltransferase that also acts as cyclase, a typical cyclase motif could not be found in PokMT3.…”