156 research outputs found
A Type III Polyketide Synthase Specific for Sporulating Negativicutes is Responsible for Alkylpyrone Biosynthesis
Genomic analyses indicate that anaerobic bacteria represent a neglected source of natural products. Whereas a limited number of polyketides have been reported from anaerobes, products of type III polyketide synthases (PKSs) have remained unknown. We found a highly conserved biosynthetic gene cluster (BGC) comprising genes putatively encoding a type III PKS and a methyltransferase in genomes of the Negativicutes, strictly anaerobic, diderm bacteria. By inâ
vivo and inâ
vitro expression of a type III PKS gene, dquA from the oakâassociated Dendrosporobacter quercicolus in E. coli we show production of longâchain alkylpyrones. Intriguingly, this BGC is specific for sporulating Sporomusaceae but absent in related Negativicutes that do not sporulate, thus suggesting a physiological role
Multifactorial Induction of an Orphan PKS-NRPS Gene Cluster in Aspergillus terreus
SummaryMining the genome of the pathogenic fungus Aspergillus terreus revealed the presence of an orphan polyketide-nonribosomal-peptide synthetase (PKS-NRPS) gene cluster. Induced expression of the transcriptional activator gene adjacent to the PKS-NRPS gene was not sufficient for the activation of the silent pathway. Monitoring gene expression, metabolic profiling, and using a lacZ reporter strain allowed for the systematic investigation of physiological conditions that eventually led to the discovery of isoflavipucine and dihydroisoflavipucine. Phytotoxin formation is only activated in the presence of certain amino acids, stimulated at alkaline pH, but strictly repressed in the presence of glucose. Global carbon catabolite repression by CreA cannot be abolished by positive-acting factors such as PacC and overrides the pathway activator. Gene inactivation and stable isotope labeling experiments unveiled the molecular basis for flavipucine/fruit rot toxin biosynthesis
Phytotoxin production in Aspergillus terreus is regulated by independent environmental signals
Secondary metabolites have a great potential as pharmaceuticals, but there are only a few examples where regulation of gene cluster expression has been correlated with ecological and physiological relevance for the producer. Here, signals, mediators, and biological effects of terrein production were studied in the fungus Aspergillus terreus to elucidate the contribution of terrein to ecological competition. Terrein causes fruit surface lesions and inhibits plant seed germination. Additionally, terrein is moderately antifungal and reduces ferric iron, thereby supporting growth of A. terreus under iron starvation. In accordance, the lack of nitrogen or iron or elevated methionine levels induced terrein production and was dependent on either the nitrogen response regulators AreA and AtfA or the iron response regulator HapX. Independent signal transduction allows complex sensing of the environment and, combined with its broad spectrum of biological activities, terrein provides a prominent example of adapted secondary metabolite production in response to environmental competition
Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate âStreptomyces maritimusâ: evidence for the derailment of an aromatic polyketide synthase
AbstractBackground: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-ÎČ-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain âStreptomyces maritimusâ deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides.Results: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from âS. maritimusâ has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis.Conclusions: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery
Context-Dependent Behavior of the Enterocin Iterative Polyketide Synthase A New Model for Ketoreduction
AbstractHeterologous expression and mutagenesis of the enterocin type II polyketide synthase (PKS) system suggest for the first time that the association of an extended set of proteins and substrates is needed for the effective production of the enterocin-wailupemycin polyketides. In the absence of its endogenous ketoreductase (KR) EncD in either the enterocin producer âStreptomyces maritimusâ or the engineered host S. lividans K4-114, the enterocin minimal PKS is unable to produce benzoate-primed polyketides, even when complemented with the homologous actinorhodin KR ActIII or with EncD active site mutants. These data suggest that the enterocin PKS requires EncD to serve a catalytic and not just a structural role in the functional PKS enzyme complex. This strongly implies that EncD reduces the polyketide chain during elongation rather than after its complete assembly, as suggested for most type II PKSs
Induced production, synthesis, and immunomodulatory action of clostrisulfone, a diarylsulfone from clostridium acetobutylicum
The anaerobe Clostridium acetobutylicum belongs to the most important industrially used bacteria. Whereas genome mining points to a high potential for secondary metabolism in C. acetobutylicum , the functions of most biosynthetic gene clusters are cryptic. We report that the addition of supraâphysiological concentrations of cysteine triggered the formation of a novel natural product, clostrisulfone ( 1 ). Its structure was fully elucidated by NMR, MS and the chemical synthesis of a reference compound. Clostrisulfone is the first reported natural product with a diphenylsulfone scaffold. A biomimetic synthesis suggests that pentamethylchromanolâderived radicals capture sulfur dioxide to form 1 . In a cellâbased assay using murine macrophages a biphasic and doseâdependent regulation of the LPSâinduced release of nitric oxide was observed in the presence of 1 .Induction of the industrial anaerobe Clostridium acetobutylicum with cysteine led to the discovery of an unprecedented diarylsulfone natural product named clostrisulfone that likely results from sulfur dioxide capture by chromaneâderived radicals. Its structure was elucidated by NMR and confirmed by synthesis. The tocopherolârelated molecule exerts immunomodulatory activities (see figure). imag
Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor
Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA
A Specialized PolythioamideâBinding Protein Confers Antibiotic SelfâResistance in Anaerobic Bacteria
Understanding antibiotic resistance mechanisms is central to the development of antiâinfective therapies and genomicsâbased drug discovery. Yet, many knowledge gaps remain regarding the resistance strategies employed against novel types of antibiotics from lessâexplored producers such as anaerobic bacteria, among them the Clostridia. Through the use of genome editing and functional assays, we found that CtaZ confers selfâresistance against the copper chelator and gyrase inhibitor closthioamide (CTA) in Ruminiclostridium cellulolyticum . Bioinformatics, biochemical analyses, and Xâray crystallography revealed CtaZ as a founding member of a new group of GyrIâlike proteins. CtaZ is unique in binding a polythioamide scaffold in a ligandâoptimized hydrophobic pocket, thereby confining CTA. By genome mining using CtaZ as a handle, we discovered previously overlooked homologs encoded by diverse members of the phylum Firmicutes, including many pathogens. In addition to characterizing both a new role for a GyrIâlike domain in selfâresistance and unprecedented thioamide binding, this work aids in uncovering related drugâresistance mechanisms
The MAP kinase MpkA controls cell wall integrity, oxidative stress response, gliotoxin production and iron adaptation in Aspergillus fumigatus
The saprophytic fungus Aspergillus fumigatus is the most important air-borne fungal pathogen. The cell wall of A. fumigatus has been studied intensively as a potential target for development of effective antifungal agents. A major role in maintaining cell wall integrity is played by the mitogen-activated protein kinase (MAPK) MpkA. To gain a comprehensive insight into this central signal transduction pathway, we performed a transcriptome analysis of the ÎmpkA mutant under standard and cell wall stress conditions. Besides genes involved in cell wall remodelling, protection against ROS and secondary metabolism such as gliotoxin, pyomelanin and pseurotin A, also genes involved in siderophore biosynthesis were regulated by MpkA. Consistently, northern and western blot analyses indicated that iron starvation triggers phosphorylation and thus activation of MpkA. Furthermore, localization studies indicated that MpkA accumulates in the nucleus under iron depletion. Hence, we report the first connection between a MAPK pathway and siderophore biosynthesis. The measurement of amino acid pools and of the pools of polyamines indicated that arginine was continuously converted into ornithine to fuel the siderophore pool in the ÎmpkA mutant strain. Based on our data, we propose that MpkA fine-tunes the balance between stress response and energy consuming cellular processes
Insectâassociated bacteria assemble the antifungal butenolide gladiofungin by nonâcanonical polyketide chain termination
Genome mining of one of the protective symbionts ( Burkholderia gladioli ) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previouslyâoverlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum . By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerolâderived C3 building block. The key role of an Aâfactor synthase (AfsA)âlike offloading domain was corroborated by CRISPRâCasâmediated gene editing, which facilitated precise excision within a PKS domain
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