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Bengamide, aus marinen Schwämmen stammende und als Inhibitoren der Methionin-Aminopeptidase (MetAP) charakterisierte Naturstoffe, wurden intensiv als Wirkstoffe gegen Krebs erforscht. In einem multidisziplinären Forschungsprojekt haben wir die Bereitstellung von Bengamiden îber Fermentation des terrestrischen Myxobakteriums M. virescens, die Aufklärung ihrer Biosynthese und die Optimierung ihrer Eigenschaften als Arzneistoff-Leitstrukturen untersucht. Die Charakterisierung des Biosyntheseweges zeigte auf, dass bakterielle Resistenz gegenüber Bengamiden durch Leu154 des myxobakteriellen MetAP-Proteins vermittelt wird, und ermöglichte den Transfer des gesamten Biosynthesegenclusters in den geeigneteren Produktionsstamm M. xanthus DK1622. Eine Kombination aus Semisynthese mikrobiell gewonnener Bengamide und Totalsynthese führte zum optimierten Derivat 8a. Die nanomolare zellulä•re Wirksamkeit und die hohe metabolische Stabilitä•t waren mit einer verbesserten Halbwertszeit inM•äusen sowie mit Antitumor-Effizienz in einem Melanom-Mausmodell verbunden. [ABSTRACT FROM AUTHOR]

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 ona 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 Leu154 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. [ABSTRACT FROM AUTHOR]

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of ‘glycolate’ units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the ‘glycolate’ unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). ᴅ-[1,2-13C2]Glycerate is also incorporated specifically into the ‘glycolate’ units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-13C2]glycerol were developed as part of this work. [ABSTRACT FROM AUTHOR]

Abstract: Recently, Pseudomonas putida revealed excellent properties as production host for the expression of a natural product gene cluster from myxobacteria, encoding the myxochromide biosynthetic pathway. Here we present a physiological study of the mutant P. putida::CMch37a for the production of this compound. Feeding of alanine, leucine, glutamine, and threonine as building blocks of the cyclic peptide of myxochromides S led to 5-fold increase of production. This improvement was due to increased intracellular levels of these amino acids, as response to the feeding, thus optimizing their availability for the non-ribosomal peptide synthetases from the myxochromide assembly line. As shown by 13C labelling studies, amino acids incorporated into myxochromides S originated predominantly from the externally added pools, whereas de novo synthesis from glucose was very low. Alanine, leucine and glutamine were, however, catabolized to a large extent, which reduces the efficiency of their feeding. Acetate and malonate, as building blocks of the polyketide chain of myxochromide, however, inhibited growth of P. putida::CMch37a and partly reduced myxochromide production. The effective co-utilization of acetate in a mixture with glucose indicated the presence of a highly active acetyl-CoA synthase. The fact that this was not reflected by an increased myxochromide production speaks against a limitation of production, caused by this enzyme. Malonate was not co-consumed with glucose so that malonyl-CoA synthase remains as a potential bottleneck for myxochromide production in P. putida. [Copyright &y& Elsevier]

Abstract: A potent novel analogue of the natural macrolide antibiotic etnangien, a structurally unique RNA polymerase inhibitor from myxobacteria, is reported. It may be readily obtained from fermentation broths of Sorangium cellulosum and shows high antibiotic activity, comparable to that of etnangien. However, it is much more readily available than the notoriously labile authentic natural product itself. Importantly, it is stable under neutral conditions, allowing for elaborate NMR measurements for assignment of the 12 hydroxyland methyl-bearing stereogenic centers. The full absolute and relative stereochemistries of these complex polyketides were determined by a combination of extensive high-field NMIR studies, including J-based configuration analysis, molecular modeling, and synthetic derivatization in combination with an innovative method based on biosynthetic studies of this polyketide which is also presented here. A first look into the solution conformation and 3D structure of these promising macrolide antibiotics is reported. Finally, the complete biosynthetic gene cluster was analyzed in detail, revealing a highly unusual and complex transAT type polyketide biosynthesis, which does not follow colinearity rules, most likely performs programmed iteration as well as module skipping, and exhibits HMG-CoA box-directed methylation. [ABSTRACT FROM AUTHOR]