The anthracycline-like polyketide drug elloramycin is produced by Streptomyces olivaceus T?2353. Elloramycin has antibacterial activity against Gram-positive bacteria and also exhibits antitumor activity. From a cosmid clone (cos16F4) containing part of the elloramycin biosynthesis gene cluster, three genes (elmMI, elmMII, and elmMIII) have been cloned. Sequence analysis and data base comparison showed that their deduced products resembled S-adenosylmethionine-dependent O-methyltransferases. The genes were individually expressed in Streptomyces albus and also coexpressed with genes involved in the biosynthesis of l-rhamnose, the 6-deoxysugar attached to the elloramycin aglycon. The resulting recombinant strains were used to biotransform three different elloramycin-type compounds: l-rhamnosyl-tetracenomycin C, l-olivosyl-tetracenomycin C, and l-oleandrosyl-tetracenomycin, which differ in their 2'-, 3'-, and 4'-substituents of the sugar moieties. When only the three methyltransferase-encoding genes elmMI, elmMII, and elmMIII were individually expressed in S. albus, the methylating activity of the three methyltransferases was also assayed in vitro using various externally added glycosylated substrates. From the combined results of all of these experiments, it is proposed that methyltransferases ElmMI, ElmMII, and ElmMIII are involved in the biosynthesis of the permethylated l-rhamnose moiety of elloramycin. ElmMI, ElmMII, and ElmMIII are responsible for the consecutive methylation of the hydroxy groups at the 2'-, 3'-, and 4'-position, respectively, after the sugar moiety has been attached to the aglycon.

Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus T?2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC). The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC. The ElmGT glycosyltransferase from S. olivaceus T?2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.

Elloramycin A (1) belongs to a small family of naphthacenequinones characterized by a unique highly hydroxylated cyclohexenone moiety. A cosmid clone 16F4, harboring genes for the production of 1 from Streptomyces olivaceus T?2353, has been previously isolated. DNA sequence analysis of a 3.2-kb fragment from 16F4 revealed four open reading frames--the elmGHIJ genes. Heterologous expressions of the elmGHI genes in either Escherichia coli or Streptomyces lividans, followed by biochemical characterizations of the ElmGHI proteins, established ElmG as tetracenomycin B2 oxygenase, ElmH as tetracenomycin F1 monooxygenase, and ElmI as tetracenomycin F2 cyclase. These results provide direct biochemical evidence for the hypothesis that the biosynthesis of 1 in S. olivaceus parallels that of tetracenomycin C (2) in Streptomyces glaucescens and support the notion that the biosynthesis of the highly hydroxylated cyclohexenone moiety in other polyketides most likely follows the same paradigm as the tetracenomycin B2 or A2 oxygenase.

The glycosyltransferase ElmGT from Streptomyces olivaceus is involved in the biosynthesis of the antitumor drug elloramycin, and it has been shown to possess a broad deoxysugar recognition pattern, being able to transfer different l- and d-deoxysugars to 8-demethyl-tetracenomycin C, the elloramycin aglycone. Site-directed mutagenesis in residues L309 and N312, located in the alpha/beta/alpha motif within the nucleoside diphosphate-sugar binding region, can be used to modulate the substrate flexibility of ElmGT, making it more precise for transfer of specific deoxysugars.

Elloramycin is an anthracycline-like antitumour drug produced by Streptomyces olivaceus T?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.

Oligonucleotide primers were designed and successfully applied to amplify DNA fragments of dNDP-glucose dehydratase genes from actinomycete species producing natural compounds which contain deoxysugar moieties. The deduced amino acid sequence of the isolated fragments revealed similarity to known dNDP-glucose dehydratases. A phylogeny for the deduced proteins of the obtained fragments and for dNDP-glucose dehydratases described in the data bases was constructed. dNDP-glucose dehydratases from actinomycetes were more related to each other than to dehydratases from species of other orders. The phylogenetic analysis also revealed a close relation between dehydratases from strains producing natural compounds with similar deoxysugar moieties.

A silent dibenzoxazepinone (DBP) biosynthetic gene cluster (myc) was mutagenically activated in Streptomyces olivaceus SCSIO T05, enabling the discovery of mycemycin C (4) and three new analogues [mycemycins F-H (1-3)]. Gene disruption, complementation experiments, and enzymatic assays unveiled salicylic acid and 5-Cl-kynurenine as biosynthetic precursors and shed significant functional insights into MycO, MycB, MycR, and MycJ, enzymes responsible for fine-tuning of the DBP scaffold.

With the developments in taxonomy, the classically used highly conserved 16S rRNA molecular marker has shown some disadvantages among closely related species. For further taxonomic studies of the prokaryotes, specific PCR primers were designed from two conserved regions in the amino acid sequences of the 70-kDa heat shock protein sourced from 20 different genera in actinomycetes. These were used for the amplification of the hsp70 genes in 16 Streptomyces strains. Then, we investigated the phylogenetic relationships among these Streptomyces strains and compared the tree topology based on the hsp70 gene with those based on the previously used markers (16S rRNA and gyrB). To our knowledge, this is the first use of the hsp70 gene as a molecular marker for the taxonomic identification of Streptomyces.