Einblicke in die Pamamycin‐Biosynthese

Sauerstoffheterocyclen sind verbreitete Strukturmotive in Polyketid‐Naturstoffen. Wir berichten über strukturelle und mechanistische Untersuchungen zu AmbDH3, einer Polyketidsynthase‐Domäne mit dualer Aktivität als Dehydratase (DH) und pyranbildende Cyclase in der Ambruticin‐Biosynthese. AmbDH3 ähnelt monofunktionalen DH‐Domänen und verwendet H51 und D215 zur Dehydratisierung. Durch Mutationsstudien und In‐vitro‐Experimente konnte V173 als entscheidende Aminosäure für die Cyclisierungsaktivität identifiziert werden. Vergleichbare Motive wurden zudem in der scheinbar monofunktionalen Domäne AmDH2 gefunden, welche zusätzlich eine unerwartete Cyclisierungsaktivität zeigte. Unsere Ergebnisse ebnen den Weg zur möglichen Entdeckung bisher verborgener Cyclasen in Biosynthesewegen. Sie ermöglichen zudem interessante Aussichten für die Entwicklung neuer Biokatalysatoren für die chemoenzymatische Synthese und die Herstellung pyranhaltiger Polyketide durch kombinatorische Biosynthese.

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Einblicke in die duale Aktivität einer bifunktionalen Dehydratase‐Cyclase‐Domäne

Sung

Berkhan

Hollmann

et al. 2017

Angewandte Chemie

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Insights into the Dual Activity of a Bifunctional Dehydratase‐Cyclase Domain

et al. 2017

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Oxygen-containing heterocycles are a common structural motif in polyketide natural products and contribute significantly to their biological activity. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratase (DH) and pyran-forming cyclase in ambruticin biosynthesis. AmbDH3 is similar to monofunctional DH domains, using H51 and D215 for dehydration. V173 was confirmed as a diagnostic residue for cyclization activity by a mutational study and enzymatic in vitro experiments. Similar motifs were observed in the seemingly monofunctional AmbDH2, which also shows an unexpected cyclase activity. Our results pave the way for mining of hidden cyclases in biosynthetic pathways. They also open interesting prospects for the generation of novel biocatalysts for chemoenzymatic synthesis and pyran-polyketides by combinatorial biosynthesis.

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Microparticles globally reprogram Streptomyces albus toward accelerated morphogenesis, streamlined carbon core metabolism, and enhanced production of the antituberculosis polyketide pamamycin

Kuhl

Gläser

Rebets

et al. 2020

Biotech & Bioengineering

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Streptomyces spp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline the performance of in these microbes. Here, we studied the production of pamamycins, macrodiolide homologs with a high activity against multiresistant pathogenic microbes, using recombinant Streptomyces albus J1074/R2. Talc particles (hydrous magnesium silicate, 3MgO•4SiO 2 •H 2 O) of micrometer size, added to submerged cultures of the recombinant strain, tripled pamamycin production up to 50 mg/L. Furthermore, they strongly affected morphology, reduced the size of cell pellets formed by the filamentous microbe during the process up to sixfold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and precursor (CoA thioester) supply of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3,341 genes (56% of all genes), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to 1,024-fold). Furthermore, the microparticles perturbed genes encoding for CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased fourfold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. The novel insights into the behavior

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Einblicke in die Pamamycin‐Biosynthese

Cited by 3 publications

References 41 publications

Einblicke in die duale Aktivität einer bifunktionalen Dehydratase‐Cyclase‐Domäne

Einblicke in die duale Aktivität einer bifunktionalen Dehydratase‐Cyclase‐Domäne

Insights into the Dual Activity of a Bifunctional Dehydratase‐Cyclase Domain

Microparticles globally reprogram Streptomyces albus toward accelerated morphogenesis, streamlined carbon core metabolism, and enhanced production of the antituberculosis polyketide pamamycin

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