The overall architecture of the gene cluster responsible for epothilone biosynthesis has been determined. The availability of the cluster should facilitate the generation of designer epothilones by combinatorial biosynthesis approaches, and the heterologous expression of epothilones in surrogate microbial hosts.

The 54-kbp Type I polyketide synthase gene cluster, most probably involved in rifamycin biosynthesis by Amycolatopsis mediterranei, was cloned in E. coli and completely sequenced. The DNA encodes five closely packed, very large open reading frames reading in one direction. As expected from the chemical structure of rifamycins, ten polyketide synthase modules and a CoA ligase domain were identified in the five open reading frames which contain one to three polyketide synthase modules each. The order of the functional domains on the DNA probably reflects the order in which they are used because each of the modules contains the predicted acetate or propionate transferase, dehydratase, and beta-ketoacyl-ACP reductase functions, required for the respective step in rifamycin biosynthesis.

Rifamycin B biosynthesis in Amycolatopsis mediterranei N/813 was inactivated by introducing a small deletion in the rifF gene situated directly downstream of the rifamycin polyketide synthase (PKS) gene cluster. The corresponding mutant strain produced a series of linear intermediates of rifamycin B biosynthesis that are most probably generated by obstruction of the normal release of the end product of the rifamycin PKS. This result provides evidence that the rifF gene product catalyses the release of the completed linear polyketide from module 10 of the PKS and the intramolecular macrocyclic ring closure by formation of an amide bond, as indicated by sequence similarity of this protein to amide synthases. The chemical structures of the new rifamycin polyketide synthase intermediates released from modules 4 to 10 were determined by spectroscopic methods (UV, IR, NMR and MS) and gave insight into the reaction steps of rifamycin ansa chain biosynthesis and the timing of the formation of the naphthoquinone ring. The intermediates released from modules 6 and 8 were isolated as lactones formed by the terminal carboxyl group ; proton NMR double resonance and ROESY(rotated frame nuclear Overhauser enhancement spectroscopy) experiments enabled the deduction of the relative configurations in the linear chain which correspond to the known absolute stereochemistry of rifamycin B.Keywords : antibiotic biosynthesis, ansamycins, amide synthase, gene replacement, pathway engineering INTRODUCTIONRifamycins are clinically important ansamycin antibiotics, composed of a naphthalenic chromophore spanned by a long aliphatic ansa chain. The rifamycins and the semisynthetic drugs derived from them exert their antibiotic activity by specific inhibition of bacterial DNA-dependent RNA polymerase (Wehrli, 1977). At higher concentrations, these antibiotics also inhibit the RNA-dependent DNA polymerase of retroviruses (Szabo et al., 1976 bacterium leprae, causative agents of tuberculosis and leprosy, respectively, they are also active against a variety of other organisms, including bacteria and viruses (Szabo et al., 1976 ;Oppenheim et al., 1986 ;Barakett et al., 1993 ;Bachs et al., 1992). Furthermore, it was shown recently that rifampicin-containing regimens are able to cure staphylococcal implant-related infections (Zimmerli et al., 1998).The identification and sequencing of the rifamycin polyketide synthase (PKS) gene cluster by our group (Schupp et al., 1998) and by August et al. (1998) Our interest in further studying rifamycin B biosynthesis led us to undertake the inactivation of the rifF gene, situated directly downstram of the PKS genes, and to analyse the effect of this mutation on rifamycin biosynthesis. The rifF gene product has been characterized as rifamycin amide synthase by sequence homologies to different arylamine N-acetyltransferases (August et al., 1998) and the putative function of the RifF protein in rifamycin biosynthesis was suggested to be a cyclase, catalysing the formation of an intramolecular amide bond between ...

Desferrioxamine B is the main siderophore of Streptomyces pilosus. Its production is induced in response to iron limitation. Two genes involved in desferrioxamine production have been cloned and were found to be translated from a polycistronic mRNA that is produced only under conditions of iron limitation (T. Schupp, C.Toupet, and M. Divers, Gene 64:179-188, 1988). Here we report the nucleotide sequence of the desferrioxamine (des) operon promoter region. The transcriptional start site was localized by S1 nuclease mapping.Deletion analysis defined a 71-bp region downstream of the -35 region that is sufficient for iron regulation in the original host, S. pilosus, and also in Streptomyces lividans. Site-directed mutagenesis was used to create a mutation that abolishes iron repression. Two iron-independent mutants were obtained by deletion of part of a 19-bp region with dyad symmetry which overlaps the -10 promoter region and the transcriptional start site. The putative repressor-binding site identified by these constitutive mutations is not homologous to the consensus binding site of the Escherichia coil central iron repressor, Fur (ferric uptake regulation), but is similar to the DtxR-binding site in the iron-regulated promoter of the corynebacterial diphtheria toxin gene.Desferrioxamines are low-molecular-weight, iron-chelating compounds (siderophores) produced and secreted by many actinomycetes, including species of Streptomyces, Nocardia, and Micromonospora (50). Together with specific receptors, they serve the high specific iron uptake of these bacteria (5,7,28,29). Desferrioxamines are trihydroxamate type siderophores synthesized from the amino acids lysine and ornithine as precursors (35). Desferrioxamine B is the main siderophore of Streptomyces pilosus (3). The first step of the biosynthetic pathway, decarboxylation of lysine, is catalyzed by lysine decarboxylase DesA (38). desA is also the first gene of the des operon (37). As in other bacteria producing iron chelators, the synthesis of desferrioxamine B has been shown to be under transcriptional control and the genes are turned on only under iron-limiting conditions (37). Iron deficiency induces, besides systems for iron uptake, the production of several exotoxins in pathogenic bacteria, like diphtheria toxin in Corynebacterium diphtheriae, Shiga toxin in Shigella dysenteriae, and Shiga-like toxins in Escherichia coli (6,30,43).In principle, two mechanisms of regulation are possible, namely, activation under low-iron conditions or repression under iron-rich conditions (19). A well studied example of iron-regulated gene expression is the gram-negative E. coli, where transcription of about 40 iron-regulated genes involved in the synthesis and uptake of siderophores is repressed under iron-rich conditions by a complex between the repressor protein Fur (ferric uptake regulation) that has ferrous iron as the corepressor (2,17,28 consensus Fur-binding site (which is based on a comparison of it with other iron-controlled promoter sequences in E. coli [8,16,32]...

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