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Aminocoumarin

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Lutz Heide – 1st expert on this subject based on the ideXlab platform

  • Complex Enzymes in Microbial Natural Product Biosynthesis, Part B: Polyketides, Aminocoumarins and Carbohydrates – Chapter 18 Aminocoumarins
    Methods in Enzymology, 2020
    Co-Authors: Lutz Heide

    Abstract:

    Abstract The Aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A 1 are formed by different Streptomyces strains and are potent inhibitors of bacterial gyrase. Their biosynthetic gene clusters have been analyzed in detail by genetic and biochemical investigations. Heterologous expression of these gene clusters by site-specific integration into the genome of the fully sequenced host Streptomyces coelicolor A3(2) readily results in an accumulation of the antibiotics in yields similar to the wildtype strains. In recent years, the Aminocoumarins have developed into a model system for the generation of new antibiotics by genetic methods. Prior to heterologous expression in S. coelicolor , cosmids containing the complete biosynthetic clusters can be manipulated in Escherichia coli by λ RED–mediated recombination, creating single or multiple gene replacements or gene deletions. Thereby, mutant strains are generated which are blocked in the synthesis of certain intermediates or in specific tailoring reactions. For instance, mutasynthetic experiments can subsequently be carried out to generate Aminocoumarin antibiotics that contain modified acyl moieties attached to the Aminocoumarin core, and chemoenzymatic synthesis can be employed for the acylation of the deoxysugar moiety of structural analogues of the Aminocoumarin antibiotics. Metabolic engineering—the combination of gene deletions and foreign gene expression via replicative expression vectors—can be used to generate further structural variants of these antibiotics. These methods can be combined, allowing the generation of a wide variety of new compounds. This chapter may provide general pointers for the use of genetic methods in the generation of new antibiotics.

  • draft genome sequence of streptomyces niveus ncimb 11891 producer of the Aminocoumarin antibiotic novobiocin
    Genome Announcements, 2014
    Co-Authors: Katrin Flinspach, Lutz Heide, Christian Ruckert, Jorn Kalinowski, Alexander Kristian Apel

    Abstract:

    Streptomyces niveus NCIMB 11891 is the producer of the gyrase inhibitor novobiocin, which belongs to the Aminocoumarin class of antibiotics. The genome sequence of this strain was found to contain, besides the gene cluster for novobiocin, a putative gene cluster for the macrolactam antibiotic BE-14106 and further secondary metabolite gene clusters.

  • draft genome sequence of streptomyces roseochromogenes subsp oscitans ds 12 976 producer of the Aminocoumarin antibiotic clorobiocin
    Genome Announcements, 2014
    Co-Authors: Christian Ruckert, Lutz Heide, Jorn Kalinowski, Alexander Kristian Apel

    Abstract:

    ABSTRACT Streptomyces roseochromogenes subsp. oscitans DS 12.976 is the producer of the gyrase-inhibiting Aminocoumarin antibiotic clorobiocin. Here, we present a draft genome sequence of this strain, in which we identified the clorobiocin gene cluster as well as an unusually high number (43) of further putative secondary metabolite clusters.

Shuming Li – 2nd expert on this subject based on the ideXlab platform

  • combinatorial biosynthesis metabolic engineering and mutasynthesis for the generation of new Aminocoumarin antibiotics
    Current Topics in Medicinal Chemistry, 2008
    Co-Authors: Lutz Heide, Christine Anderle, Bertolt Gust, Shuming Li

    Abstract:

    The Aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase and topoisomerase IV, and novobiocin has been licensed as antibiotic for clinical use (Albamycin®). They also have potential applications in oncology. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of nearly all genes contained therein has been elucidated. Rapid and versatile methods have been developed for the heterologous expression of these biosynthetic gene clusters, and in Streptomyces coelicolor M512 as heterologous host these antibiotics were produced in yields comparable to those in the natural producer strains. λ RED-mediated homologous recombination was used for genetic modification of the gene clusters in Escherichia coli. The phage φC31 attachment site and integrase functions were introduced into the cosmid backbones and employed for stable integration of the clusters into the genome of the heterologous hosts. Modification of the clusters by single or multiple gene replacements or gene deletions resulted in the formation of numerous new Aminocoumarin derivatives, providing an efficient tool for the rational generation of antibiotics with modified structure. Additionally, many new antibiotics were generated by mutasynthesis experiments, i.e. the targeted deletion of genes required for the biosynthesis of a certain structural moiety of the antibiotic, and the replacement of this moiety by structural analogs which were added to the culture broth. The diversity of new structures obtained by this approach could be expanded by further genetic modifications of the gene deletion mutants, especially by expression of heterologous biosynthetic enzymes with appropriate substrate specificity.

  • improved mutasynthetic approaches for the production of modified Aminocoumarin antibiotics
    Chemistry & Biology, 2007
    Co-Authors: Christine Anderle, Susanne Hennig, Bernd Kammerer, Shuming Li, Ludger A Wessjohann, Bertolt Gust, Lutz Heide

    Abstract:

    Summary This study reports improved mutasynthetic approaches for the production of Aminocoumarin antibiotics. Previously, the mutasynthetic production of Aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of Aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the Aminocoumarin moiety and that of clorobiocin in the deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five Aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1 H-NMR spectroscopy.

  • New Aminocoumarin Antibiotics Derived from 4-Hydroxycinnamic Acid are Formed after Heterologous Expression of a Modified Clorobiocin Biosynthetic Gene Cluster
    The Journal of Antibiotics, 2007
    Co-Authors: Christine Anderle, Bernd Kammerer, Shuming Li, Bertolt Gust, Lutz Heide

    Abstract:

    Three new Aminocoumarin antibiotics, termed ferulobiocin, 3-chlorocoumarobiocin and 8′-dechloro-3-chlorocoumarobiocin, were isolated from the culture broth of a Streptomyces coelicolor M512 strain expressing a modified clorobiocin biosynthetic gene cluster. Structural analysis showed that these new Aminocoumarins were very similar to clorobiocin, with a substituted 4-hydroxycinnamoyl moieties instead of the prenylated 4-hydroxybenzoyl moiety of clorobiocin. The possible biosynthetic origin of these moieties is discussed.

Christine Anderle – 3rd expert on this subject based on the ideXlab platform

  • biological activities of novel gyrase inhibitors of the Aminocoumarin class
    Antimicrobial Agents and Chemotherapy, 2008
    Co-Authors: Christine Anderle, Martin Stieger, Matthew R Burrell, Stefan Bernhard Reinelt, Anthony Maxwell, Malcolm G P Page, Lutz Heide

    Abstract:

    A major threat to therapy for human infections is the increase in the levels of antibiotic resistance and the continuing spread of nosocomial pathogens into the community (3, 21). Therefore, it is essential that new antibacterial drugs be developed. In this context, the reevaluation of previously discovered, but so far unexploited, classes of antibiotics has come into the focus of antibiotic research (31). The Aminocoumarin antibiotics inhibit a well-validated drug target (DNA gyrase), but in contrast to the widely used fluoroquinolones, the Aminocoumarins bind to the B subunit of the heterotetrameric gyrase enzyme. The binding site of the Aminocoumarins overlaps with the ATP-binding site of gyrase, located on GyrB, and the Aminocoumarins thereby inhibit the ATP hydrolysis required for ATP-dependent DNA supercoiling (25). In the same way, they inhibit DNA topoisomerase IV, which is a type II topoisomerase similar to gyrase and which is involved both in the control of DNA supercoiling and in the decatenation of daughter chromosomes after DNA replication. The Aminocoumarins show strong activities against gram-positive pathogens like Staphylococcus aureus and Staphylococcus epidermidis (14, 15, 30, 37). Novobiocin (Albamycin; Upjohn) is the only Aminocoumarin which has been licensed for the treatment of human infections, and its efficacy has been confirmed in several clinical trials (32, 33, 38). Aminocoumarins also show the potential for use as anticancer drugs (4, 6, 20, 22, 23, 34).

    Among the limitations of the Aminocoumarins are their poor solubility in water and their poor oral absorption. Their low levels of activity against gram-negative bacteria were perceived as an additional drawback at the time of their discovery (15, 30); it may be argued, however, that this could also present an advantage, since gram-negative bacteria in the gut are not affected by these drugs.

    Structurally, novobiocin and the closely related Aminocoumarin clorobiocin (Fig. ​(Fig.1)1) are composed of a 3-dimethylallyl-4-hydroxybenzoyl moiety (ring A), a 3-amino-4,7-dihydroxycoumarin moiety (ring B) substituted with a methyl group and a chlorine atom, respectively, and a substituted deoxysugar (ring C) (Fig. ​(Fig.1).1). The 3″-OH of the deoxysugar is esterified with a carbamoyl group in the case of novobiocin and with a 5-methylpyrrole-2-carboxyl moiety in the case of clorobiocin. In contrast to the carbamoyl group of novobiocin, the 5-methylpyrrole moiety of clorobiocin is able to occupy an additional hydrophobic pocket in the GyrB subunit and to displace two water molecules (18). Thereby, clorobiocin binds more effectively to the GyrB subunit than novobiocin. The ring A moiety interacts only via hydrophobic bonds with the B subunit of gyrase and contributes only weakly to the antibacterial activity (16). However, ring A may influence the uptake of the compound into the bacterial cell (17).

    FIG. 1.

    Structures of novobiocin and clorobiocin.

    Mutasynthesis, i.e., the feeding of synthetic precursor analogs to mutants of microbial producer strains of natural products, is an important and powerful tool for drug discovery and lead optimization. We recently generated a series of 31 new Aminocoumarin compounds by addition of synthetic analogs of the ring A moiety to specific mutants of the novobiocin and clorobiocin producers (1, 2). In the current study, we utilized these compounds to investigate the structure-activity relationships of this class of gyrase inhibitors as a prerequisite for the development of Aminocoumarin antibiotics with improved properties. Different biochemical and reporter gene assays, as well as a computational method and antibacterial assays, were used to examine the biological effects of these compounds, allowing a comparison of the results of these different methods. Specifically, two different biochemical assays were used, i.e., an ATPase assay and a supercoiling assay. The ATPase assay measured the ATP hydrolyzing activity of the B subunit of Escherichia coli DNA gyrase and its inhibition by Aminocoumarin antibiotics (25). The supercoiling assay (24) measured the supercoiling activity of the intact E. coli gyrase heterotetramer; it exploited the fact that the formation of a DNA triplex (an alternative structure to the DNA double helix) is favored by the negative supercoiling of DNA. A plasmid containing a 20-bp insert with triplex-forming potential was partially supercoiled by the action of gyrase and subsequently captured by a biotinylated oligonucleotide which was bound to the streptavidin-coated surface of a micotiter plate. After unbound plasmids were washed out, the nucleic acids in the microtiter well were quantified by the use of a DNA-specific fluorescent dye. This assay allowed the rapid, automated determination of gyrase inhibition by the test compounds.

    The experimentally obtained results were compared with the binding energies calculated from in silico docking studies by using Moloc software (www.moloc.ch). Furthermore, two reporter gene assays with living bacterial cells were used. The gyrA promoter responds to changes in the superhelical density of the DNA in the bacterial cell caused by gyrase inhibition. Therefore, fusions of this promoter to a reporter gene can be used to screen for inhibitors that attack at either subunit of gyrase (GyrA or GyrB). The SOS-inducible sulA promoter responds to agents that ultimately interfere with DNA replication (36). Inhibition of gyrase affects DNA replication and therefore leads to induction of the sulA promoter. The gyrA and sulA promoters were fused to the five-gene luxCDABE operon from Photorhabdus luminescens for facile monitoring of kinetic responses (27).

    The selected compounds were further investigated for their MICs against a panel of bacterial pathogens.

  • combinatorial biosynthesis metabolic engineering and mutasynthesis for the generation of new Aminocoumarin antibiotics
    Current Topics in Medicinal Chemistry, 2008
    Co-Authors: Lutz Heide, Christine Anderle, Bertolt Gust, Shuming Li

    Abstract:

    The Aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase and topoisomerase IV, and novobiocin has been licensed as antibiotic for clinical use (Albamycin®). They also have potential applications in oncology. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of nearly all genes contained therein has been elucidated. Rapid and versatile methods have been developed for the heterologous expression of these biosynthetic gene clusters, and in Streptomyces coelicolor M512 as heterologous host these antibiotics were produced in yields comparable to those in the natural producer strains. λ RED-mediated homologous recombination was used for genetic modification of the gene clusters in Escherichia coli. The phage φC31 attachment site and integrase functions were introduced into the cosmid backbones and employed for stable integration of the clusters into the genome of the heterologous hosts. Modification of the clusters by single or multiple gene replacements or gene deletions resulted in the formation of numerous new Aminocoumarin derivatives, providing an efficient tool for the rational generation of antibiotics with modified structure. Additionally, many new antibiotics were generated by mutasynthesis experiments, i.e. the targeted deletion of genes required for the biosynthesis of a certain structural moiety of the antibiotic, and the replacement of this moiety by structural analogs which were added to the culture broth. The diversity of new structures obtained by this approach could be expanded by further genetic modifications of the gene deletion mutants, especially by expression of heterologous biosynthetic enzymes with appropriate substrate specificity.

  • improved mutasynthetic approaches for the production of modified Aminocoumarin antibiotics
    Chemistry & Biology, 2007
    Co-Authors: Christine Anderle, Susanne Hennig, Bernd Kammerer, Shuming Li, Ludger A Wessjohann, Bertolt Gust, Lutz Heide

    Abstract:

    Summary This study reports improved mutasynthetic approaches for the production of Aminocoumarin antibiotics. Previously, the mutasynthetic production of Aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of Aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the Aminocoumarin moiety and that of clorobiocin in the deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five Aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1 H-NMR spectroscopy.