The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Doublestranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB r , and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4,6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, in Streptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.Novobiocin is produced by Streptomyces spheroides and Streptomyces niveus and belongs to the aminocoumarin antibiotics. Bacterial DNA gyrase represents the target of these coumarins (41), and novobiocin inhibits this enzyme by interaction with the N-terminal 24-kDa subdomain of the gyrB subunit (27). In addition to its antibacterial action, novobiocin shows synergistic effects with antitumor drugs such as etoposide or teniposide (37,49).Little is known about the biosynthesis of novobiocin. Structurally, it is composed of three moieties, a noviose sugar (ring C), a substituted coumarin (ring B), and a prenylated 4-hydroxybenzoic acid (ring A), and these rings are linked by glycosidic and amide bonds (Fig. 1). Radioactive feeding experiments in the 1960s and 1970s showed that noviose is directly derived from D-glucose, whereas tyrosine serves as a precursor of ring A and ring B (3, 6, 31). This was recently confirmed by a feeding experiment with [1-13 C]glucose (33) which also showed that the dimethylallyl moiety of novobiocin was formed through the nonmevalonate pathway.Molecular biological studies have been restricted to the investigation of novobiocin resistance genes (43, 52), especially gyrB r (61, 62), and the production of novobiocin-deficient mutants (19). Discovery of the genetic basis of the biosynthesis of aminocoumarin antibiotics could provide a useful tool for drug development. "Combinatorial biosynthesis," the interchange of genes involved in antibiotic biosynthesis between different microorganisms or the creation of hybrid genes and, consequently, proteins with new enzymatic properties, allows the production of modified or even novel antibiotics (23). In the past, much effort has been undertaken in the manipulation of the biosynthesis of polyketide antibiotics (25,42,56), and recently, progress has also been made in the construction of hybrid peptide synthetase genes (55, 59). The discovery of gene clusters for other types of secondary metabolites can offer additional possibilities for combinatorial biosynthesis.Here we report on the identification of the novobiocin biosynthetic gene cluster from S. spheroides NCIB 11891. The gene...

The avilamycin A biosynthetic gene cluster represents an interesting system to study the formation and attachment of unusual deoxysugars. Several enzymes putatively responsible for specific steps of this pathway could be assigned. Two genes encoding enzymes involved in post-PKS tailoring reactions were deleted allowing the production of new analogues of avilamycin A.

Research on ribosomally synthesized and posttranslationally modified peptides (RiPPs) has led to an increasing understanding of biosynthetic mechanisms, mostly drawn from bacterial examples. In contrast, reports on RiPPs from fungal producers, apart from the amanitins and phalloidins, are still scarce. The fungal cyclopeptide omphalotin A carries multiple N-methylations on the peptide backbone, a modification previously known only from nonribosomal peptides. Mining the genome of the omphalotin-producing fungus for a precursor peptide led to the identification of two biosynthesis genes, one encoding a methyltransferase OphMA that catalyzes the automethylation of its C-terminus, which is then released and cyclized by the protease OphP. Our findings suggest a novel biosynthesis mechanism for a RiPP in which a modifying enzyme bears its own precursor peptide.

Small wonder: Atrop‐abyssomicin C is a small, yet complex spirotetronate (see scheme) that is active against Gram‐positive bacteria, such as MRSA. Feeding studies and genetic manipulation of its producer, Verrucosispora maris AB‐18‐032, for the first time give insight into its biosynthesis and demonstrate how closely related the members of this important class of molecules are.

The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A 1 , and clorobiocin were investigated. All three clusters contained a gyrB R resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A 1 clusters were found to contain an additional, similar gene, named parY R . Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrB R and likewise of parY R in Streptomyces lividans TK24 resulted in resistance against novobiocin and coumermycin A 1 , suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and of Streptomyces coelicolor contained two additional genes which hybridized with either gyrB R or parY R and which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes, novA and couR5, were found in the novobiocin and the coumermycin A 1 cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin and coumermycin A 1 , suggesting that these genes may be involved in antibiotic transport.The aminocoumarin antibiotics novobiocin, clorobiocin, and coumermycin A 1 (Fig. 1A) are known as potent inhibitors of gyrase (18). Their equilibrium dissociation constants are in the range of 10 nM (10), i.e., their affinity for gyrase is considerably higher than that of modern fluoroquinolones. Novobiocin is licensed as an antibiotic for clinical use (Albamycin; Pharmacia-Upjohn) and is used for the treatment of infections with multiresistant gram-positive bacteria, e.g., Staphylococcus aureus.Novobiocin is produced by Streptomyces spheroides (synonym S. caeruleus [15]) NCIMB 11891, clorobiocin is produced by S. roseochromogenes var. oscitans DS12.976, and coumermycin A 1 is produced by S. rishiriensis DSM 40489 (2). Obviously, these organisms must protect their gyrases from the inhibitory effect of aminocoumarin during antibiotic formation. reported that the principal resistance mechanism of the novobiocin producer S. sphaeroides is the de novo synthesis of a coumarin-resistant gyrase B subunit, which replaces the sensitive GyrB subunit in the active (GyrA) 2 (GyrB) 2 heterotetramer. Thus, this novobiocin producer contains two gyrB genes, a constitutively expressed gyrB S , encoding the coumarin-sensitive protein, and the gyrB R gene, encoding the resistant protein and expressed in the presence of novobiocin. The promoter of gyrB R appears to be regulated by changes in the superhelical density of DNA (30). Mitchell et al. (20) supplied evidence that additional genes may contribute to novobiocin resistance. They used the novobiocin producer S. niveus, which has recently been identified as a subjective synonym for S. spheroides (15).We cloned and sequenced the novobiocin biosynthetic gene cluster (27), depicted in Fig. 1B. On its right border, the...

The bengamides, sponge‐derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M. virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M. xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half‐life in mice and antitumor efficacy in a melanoma mouse model.

Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K(m) values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 microM and 13.8 microM, respectively.

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