Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

AbstractRipostatin ist ein vielversprechendes Antibiotikum, welches die RNA‐Polymerase an einer neuen Bindestelle hemmt. Im Mittelpunkt dieser Studie steht die Charakterisierung der Ripostatin‐Biosynthese, die durch eine ungewöhnliche, sowohl auf trans‐ als auch cis‐Acyltransferasen beruhenden hybriden Polyketidsynthase getrieben wird. Im Besonderen beschreiben wir einen bisher unbekannten Mechanismus zur Bildung einer Phenylessigsäure‐Starteinheit, welche durch die Decarboxylierung von Phenylpyruvat gebildet wird. Die Entstehung dieser Starteinheit konnte in vitro rekonstituiert werden, was einen Mechanismus offenlegte, der dem des Pyruvatdehydrogenase‐Komplexes ähnlich ist. Dieser enyzmatische Prozess entfernt ein einzelnes Kohlenstoffatom aus Phenylpyruvat in einer Thiaminpyrophosphat‐abhängigen Decarboxylierung. Die dabei letztlich gebildete Phenylacetyl‐S‐acyl‐Spezies dient als Starteinheit der Ripostatin‐Biosynthese.

In the search for new secondary metabolites from myxobacteria, a strain from the genus Pyxidicoccus was investigated. This led to the identification of a new class of natural products showing structural novelty and interesting biological activity. Isolation and structure elucidation of two analogues led to the identification of pyxipyrrolone A and B, harboring the novel 3-methylene-2,3,4,5,6,7,8,9-octahydro-1H-benzo[e]isoindol-1-one scaffold. Mosher's ester analysis combined with NMR studies allowed the determination of all stereocenters but one. Genome sequencing of the producer strain led to the identification of a putative biosynthetic gene cluster for the pyxipyrrolones. The compounds showed activity against several cancer cell lines (μm range) with pyxipyrrolone B having 2- to 11-fold higher activity than A, although they differ only by one methylene group.

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis-and trans-acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl-C2 unit is derived from phenylpyruvate (phenyl-C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl-C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl-S-acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin.

AbstractAuf der Suche nach neuen Sekundärmetaboliten wurde ein Stamm des Genus Pyxidicoccus untersucht. Es gelang die Identifizierung und Strukturaufklärung einer strukturell neuartigen Naturstoffklasse mit interessanter Bioaktivität. Isolation und Strukturaufklärung führte zu zwei neuen Naturstoffen, Pyxipyrrolon A und B, die eine neuartige 3‐Methylen‐2,3,4,5,6,7,8,9‐octahydro‐1H‐benzo[e]isoindol‐1‐on Einheit enthalten. Kombinationen aus Moshers Methode und NMR‐Experimenten wurden genutzt, um die Konfiguration aller Stereozentren bis auf eines aufzuklären. Nach Genomsequenzierung konnte ein Genclusterkandidat für die Pyxipyrrolon‐Biosynthese gefunden und interpretiert werden. Die Verbindungen wirken gegen verschiedene Krebszelllinien zytotoxisch, wobei Pyxipyrrolon B um das 2‐ bis 11‐fache aktiver ist, obwohl es sich nur durch eine Methyleneinheit vom A‐Derivat unterscheidet.

In our search for new secondary metabolites from myxobacteria, a strain from the genus Pyxidicoccus was investigated. This led to the identification of a new natural product class showing structural novelty and interesting biological activity. Isolation and structure elucidation of two analogs led to the identification of pyxipyrrolone A and B, harboring the novel 3-methylene-2,3,4,5,6,7,8,9-octahydro-1H-benzo[e]isoindol-1-one scaffold. Mosheru0027s ester analysis combined with NMR studies allowed the determination of all stereocenters but one. Genome sequencing of the producer strain led to the identification of a putative biosynthetic gene cluster for the pyxipyrrolones. The compounds showed activity against several cancer cell lines (µM range) with pyxipyrrolone B having 2- to 11-fold higher activity than A, although they differ only by one methylene group.