Polyketomycin is a tetracyclic quinone glycoside produced by Streptomyces diastatochromogenes Tü6028. It shows cytotoxic and antibiotic activity, in particular against Gram-positive multi-drug-resistant strains (for example, MRSA). The polyketomycin biosynthetic gene cluster has been sequenced and characterised. Its identity was proven by inactivation of a alpha-ketoacyl synthase gene (pokP1) of the "minimal polyketide synthase II" system. In order to obtain valuable information about tailoring steps, we performed further gene-inactivation experiments. The generation of mutants with deletions in oxygenase genes (pokO1, pokO2, both in parallel and pokO4) and methyltransferase genes (pokMT1, pokMT2 and pokMT3) resulted in new polyketomycin derivatives, and provided information about the organisation of the biosynthetic pathway.

Mensacarcin is a potent cytotoxic agent isolated from Streptomyces bottropensis. It possesses a high content of oxygen atoms and two epoxide groups, and shows cytostatic and cytotoxic activity comparable to that of doxorubicin, a widely used drug for antitumor therapy. Another natural compound, rishirilide A, was also isolated from the fermentation broth of S. bottropensis. Screening a cosmid library of S. bottropensis with minimal PKS-gene-specific primers revealed the presence of three different type II polyketide synthase (PKS) gene clusters in this strain: the msn cluster (mensacarcin biosynthesis), the rsl cluster (rishirilide biosynthesis), and the mec cluster (putative spore pigment biosynthesis). Interestingly, luciferase-like oxygenases, which are very rare in Streptomyces species, are enriched in both the msn cluster and the rsl cluster. Three cosmids, cos2 (containing the major part of the msn cluster), cos3 (harboring the mec cluster), and cos4 (spanning probably the whole rsl cluster) were introduced into the general heterologous host Streptomyces albus by intergeneric conjugation. Expression of cos2 and cos4 in S. albus led to the production of didesmethylmensacarcin (DDMM, a precursor of mensacarcin) and the production of rishirilide A and B (a precursor of rishirilide A), respectively. However, no product was detected from the expression of cos3. In addition, based on the results of isotope-feeding experiments in S. bottropensis, a putative biosynthesis pathway for mensacarcin is proposed.

LanV is involved in the biosynthesis of landomycin A. The exact function of this enzyme was elucidated with combinatorial biosynthesis by using Streptomyces fradiae mutants that produce urdamycin A. After expression of lanV in S. fradiae DeltaurdM, which is a mutant that accumulates rabelomycin, urdamycinon B and urdamycin B were found to be produced by the strain. This result indicates that LanV is involved in the 6-ketoreduction of the angucycline core, which preceeds a 5,6-dehydration reaction. 9-C-D-Olivosyltetrangulol was also produced by this strain; this demonstrates that LanV catalyses the aromatization of ring A of the angucycline structure. Coexpression of lanV and lanGT2 in S. fradiae AO, a mutant that lacks all four urdamycin glycosyltransferases, resulted in the production of tetrangulol and the glycoside landomycin H, both of which have an aromatic ring A. As glycosylated angucyclines were not observed after expression of lanGT2 in the absence of lanV, we conclude that LanGT2 needs an aromatized ring A for substrate recognition.

The soil-borne and marine gram-positive Actinomycetes are a particularly rich source of carbohydrate-containing metabolites. With the advent of molecular tools and recombinant methods applicable to Actinomycetes, it has become feasible to investigate the biosynthesis of glycosylated compounds at genetic and biochemical levels, which has finally set the basis for engineering novel natural product derivatives. Glycosyltransferases (GT) are key enzymes for the biosynthesis of many valuable natural products that contain sugar moieties and they are most important for drug engineering. So far, the direct cloning of unknown glycosyltransferase genes by polymerase chain reaction (PCR) has not been described because glycosyltransferases do not share strongly conserved amino acid regions. In this study, we report a method for cloning of novel so far unidentified glycosyltransferase genes from different Actinomycetes strain. This was achieved by designing primers after a strategy named consensus-degenerate hybrid oligonucleotide primer (CODEHOP). Using this approach, 22 novel glycosyltransferase encoding genes putatively involved in the decoration of polyketides were cloned from the genomes of 10 Actinomycetes. In addition, a phylogenetic analysis of glycosyltransferases from Actinomycetes is shown in this paper.

e Saccharothrix espanaensis is a member of the order Actinomycetales. The genome of the strain has been sequenced recently, revealing 106 glycosyltransferase genes. In this paper, we report the detection of a glycosyltransferase from Saccharothrix espanaensis which is able to rhamnosylate different phenolic compounds targeting different positions of the molecules. The gene encoding the flexible glycosyltransferase is not located close to a natural product biosynthetic gene cluster. Therefore, the native function of this enzyme might be not the biosynthesis of a secondary metabolite but the glycosylation of internal and external natural products as part of a defense mechanism.

Partially reduced aromatic polyketides are bioactive secondary metabolites or intermediates in the biosynthesis of deoxygenated aromatics. For the antibiotic GTRI‐02 (mensalone) in different Streptomyces spp., biosynthesis involving the reduction of a fully aromatized acetyltrihydroxynaphthalene by a naphthol reductase has been proposed and shown in vitro with a fungal enzyme. However, more recently, GTRI‐02 has been identified as a product of the ActIII biosynthetic gene cluster from Streptomyces coelicolor A3(2), for which the reduction of a linear polyketide precursor by ActIII ketoreductase, prior to cyclization and aromatization, has been suggested. We have examined three different ketoreductases from bacterial producer strains of GTRI‐02 for their ability to reduce mono‐, bi‐, and tricyclic aromatic substrates. The enzymes reduced 1‐ and 2‐tetralone but not other aromatic substrates. This strongly suggests a reduction of a cyclized but not yet aromatic polyketide intermediate in the biosynthesis of GTRI‐02. Implications of the results for the biosynthesis of other secondary polyketidic metabolites are discussed.