Metabolic Engineering of Aminocoumarins: Inactivation of the Methyltransferase Gene <i>cloP</i> and Generation of New Clorobiocin Derivatives in a Heterologous Host

Freitag, Anja; Rapp, Heike; Heide, Lutz; Li, Shu-Ming

doi:10.1002/cbic.200500019

Cited by 23 publications

(20 citation statements)

References 46 publications

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“…This showed that in compound 2 the 5-chloropyrrole-2-carboxylic acid moiety was attached to the 2''-OH group rather than to the 3''-OH group (Schemes 1 and 2). Similar 2''-acylated compounds have been found in earlier studies [13,25,26] and showed very similar NMR spectroscopic signals of H2'' and H3'' as compound 2. Therefore, the structure shown in Scheme 2 was assigned to compound 2, and this second new clorobiocin derivatives was designated as novclobiocin 125.…”

Section: Analysis Of Secondary Metabolitessupporting

confidence: 71%

Use of a Halogenase of Hormaomycin Biosynthesis for Formation of New Clorobiocin Analogues with 5‐Chloropyrrole Moieties

et al. 2008

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The depsipeptide antibiotic hormaomycin, which is produced by Streptomyces griseoflavus W-384, contains a 5-chloropyrrole moiety. In the producer strain we identified the gene hrmQ that shows sequence similarity to FADH(2)-dependent halogenases. This gene was cloned and heterologously expressed in Streptomyces roseochromogenes var. oscitans DS12.976, which is the producer of the aminocoumarin antibiotic clorobiocin, which contains a 5-methylpyrrole moiety. For the present experiment, we used a mutant of this strain in which the respective pyrrole-5-methyltransferase had been inactivated. Expression of the halogenase hrmQ in this mutant strain led to the formation of two new clorobiocin derivatives that carried a 5-chloropyrrole moiety. These compounds were isolated on a preparative scale, their structures were elucidated by (1)H NMR spectroscopy and mass spectrometry, and their antibacterial activity was determined. The substrate of HrmQ is likely to be a pyrrole-2-carboxyl-S-[acyl carrier protein] thioester. If this assumption is true, this study presents the first experiment in combinatorial biosynthesis that uses a halogenase that acts on an acyl carrier protein-bound substrate.

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Section: Analysis Of Secondary Metabolitessupporting

confidence: 71%

Use of a Halogenase of Hormaomycin Biosynthesis for Formation of New Clorobiocin Analogues with 5‐Chloropyrrole Moieties

et al. 2008

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“…The current study completes the functional identification of the three methyltransferases contained in the clorobiocin cluster, i.e. CloP (Freitag et al, 2005), CloN6 (Westrich et al, 2003) and CloU (present study).…”

Section: Discussionsupporting

confidence: 81%

“…In contrast, the signal for 20-H is shifted strongly downfield to 5?36 p.p.m. This shows that the acyl moiety is attached to 20-OH rather than to 30-OH, similar to observations in previous studies (Freitag et al, 2005;Galm et al, 2004). The negative mass spectra showed the same molar ion as observed for novclobiocin 122.…”

Section: Structural Elucidationsupporting

confidence: 90%

Biosynthesis of the unusual 5,5-gem-dimethyl-deoxysugar noviose: investigation of the C-methyltransferase gene cloU

Freitag

Heide

2006

Microbiology

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The aminocoumarin antibiotic clorobiocin contains an unusual branched deoxysugar with a 5,5-gem-dimethyl structure. Inactivation of the putative C-methyltransferase gene cloU was carried out, which led to the loss of the axial methyl group at C-5 of this deoxysugar moiety. This result establishes the function of cloU, and at the same time it proves that the biosynthesis of the deoxysugar moiety of clorobiocin proceeds via a 3,5-epimerization of the dTDP-4-keto-6-deoxyglucose intermediate. The inactivation was carried out on a cosmid which contained the entire clorobiocin biosynthetic gene cluster. Expression of the modified cluster in a heterologous host led to the formation of desmethyl-clorobiocin and a structural isomer thereof. Both compounds were isolated on a preparative scale, their structures were elucidated by 1 H-NMR and mass spectroscopy and their antibacterial activity was assayed.

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“…The fractions after separation on Sephadex LH 20 were analyzed by high-performance liquid chromatography. Fractions containing novclobiocin 103 were pooled and further purified by high-performance liquid chromatography on a Multosphere RP18-5 column (250 by 4 mm; 5-m particle size; CϩS Chromatographie Service, Düren, Germany) at a flow rate of 1 ml/min, using a linear gradient Novclobiocins 101, 103, 104, 105, 107, 112, 113, 114, 117, and 120 were produced as described previously (3)(4)(5)24). A summary of the lineage of the novclobiocins is given in Fig.…”

Section: Methodsmentioning

confidence: 99%

Structure-Activity Relationships of Aminocoumarin-Type Gyrase and Topoisomerase IV Inhibitors Obtained by Combinatorial Biosynthesis

Flatman

Eustáquio²,

Li³

et al. 2006

Antimicrob Agents Chemother

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Novobiocin and clorobiocin are gyrase inhibitors produced by Streptomyces strains. Structurally, the two compounds differ only by substitution at two positions: CH 3 versus Cl at position 8 of the aminocoumarin ring and carbamoyl versus 5-methyl-pyrrol-2-carbonyl (MePC) at the 3؆-OH of noviose. Using genetic engineering, we generated a series of analogs carrying H, CH 3 , or Cl at 8 and H, carbamoyl, or MePC at 3؆-OH. Comparison of the gyrase inhibitory activities of all nine structural permutations confirmed that acylation of 3؆-OH is essential for activity, with MePC being more effective than carbamoyl. Substitution at 8 further enhanced activity, but the effect of CH 3 or Cl depended on the nature of the acyl group at 3؆: in the presence of carbamoyl at 3؆, CH 3 resulted in higher activity; in the presence of MePC at 3؆, Cl resulted in higher activity. This suggests that the structures of both natural compounds are highly evolved for optimal interaction with gyrase. In a second series of experiments, clorobiocin derivatives with and without the methyl group at 4؆-OH of noviose, and with different positions of the MePC group of noviose, were tested. Again clorobiocin was superior to all of its analogs. The activities of all compounds were also tested against topoisomerase IV (topo IV). Clorobiocin stood out as a remarkably effective topo IV inhibitor. The relative activities of the different compounds toward topo IV showed a pattern similar to that of the relative gyrase-inhibitory activities. This is the first report of a systematic evaluation of a series of aminocoumarins against both gyrase and topo IV. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics.

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Metabolic Engineering of Aminocoumarins: Inactivation of the Methyltransferase Gene cloP and Generation of New Clorobiocin Derivatives in a Heterologous Host

Cited by 23 publications

References 46 publications

Use of a Halogenase of Hormaomycin Biosynthesis for Formation of New Clorobiocin Analogues with 5‐Chloropyrrole Moieties

Use of a Halogenase of Hormaomycin Biosynthesis for Formation of New Clorobiocin Analogues with 5‐Chloropyrrole Moieties

Biosynthesis of the unusual 5,5-gem-dimethyl-deoxysugar noviose: investigation of the C-methyltransferase gene cloU

Structure-Activity Relationships of Aminocoumarin-Type Gyrase and Topoisomerase IV Inhibitors Obtained by Combinatorial Biosynthesis

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