scholarly journals A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Eileen Brandenburger ◽  
Markus Gressler ◽  
Robin Leonhardt ◽  
Gerald Lackner ◽  
Andreas Habel ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Nonribosomal Peptide Synthetase ◽  
Full Length ◽  
Amide Bond ◽  
White Rot ◽  
Mass Spectrometry Analysis ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase

ABSTRACT The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subvermispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT1C1T2C2T3C3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N 5-acetyl-N 5-hydroxy-l-ornithine (l-AHO) and N 5-cis-anhydromevalonyl-N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [32P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography–high-resolution electrospray ionization–mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes. IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SM-Xpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form.

scholarly journals Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI

2017 ◽  
Vol 199 (21) ◽  
Author(s):  
Lauren M. Petersen ◽  
Kaitlyn LaCourse ◽  
Tim A. Schöner ◽  
Helge Bode ◽  
Louis S. Tisa
Keyword(s):  
Antimicrobial Activity ◽  
Serratia Marcescens ◽  
Virulence Factors ◽  
Nonribosomal Peptide Synthetase ◽  
Mrna Levels ◽  
Insect Pathogen ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Hemolysin Gene ◽  
Peptide Synthetase

ABSTRACT Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens. The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA, which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli, swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA, these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI. IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential differences in regulation of common virulence mechanisms between these two species. With the emergence of antibiotic-resistant microorganisms, there is a widespread need to understand the regulation of pathogenesis. The significance of this study is the presentation of evidence for cross-pathway regulation of virulence factors and how the elimination of one mechanism may be compensated for by the upregulation of others.

scholarly journals Nonribosomal Peptide Synthetase GenespesLandpes1Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

2012 ◽  
Vol 78 (9) ◽  
pp. 3166-3176 ◽  
Author(s):  
Karen A. O'Hanlon ◽  
Lorna Gallagher ◽  
Markus Schrettl ◽  
Christoph Jöchl ◽  
Kevin Kavanagh ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Opportunistic Pathogen ◽  
Mass Spectrometry Analysis ◽  
Infection Model ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Peptide Synthetases ◽  
Increased Sensitivity

ABSTRACTThe identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen,Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway inA. fumigatus. Deletion of eitherpesL(ΔpesL) orpes1(Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion ofpesLresulted in severely reduced virulence in an invertebrate infection model (P< 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster forA. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in bothA. fumigatuswild-type and ΔpesLstrains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesisin vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.

scholarly journals New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

2012 ◽  
Vol 78 (23) ◽  
pp. 8208-8218 ◽  
Author(s):  
Antonia Gallo ◽  
Kenneth S. Bruno ◽  
Michele Solfrizzo ◽  
Giancarlo Perrone ◽  
Giuseppina Mulè ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Biosynthetic Pathway ◽  
Nonribosomal Peptide Synthetase ◽  
Gene Encoding ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase ◽  
Ochratoxin Α ◽  
Hydrolysis Of ◽  
Insight Into

ABSTRACTOchratoxin A (OTA), a mycotoxin produced byAspergillusandPenicilliumspecies, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated inPenicilliumspecies. InAspergillusspecies, onlypksgenes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producingA. carbonariusITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of annrpsgene product in OTA biosynthetic pathway in anAspergillusspecies. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA inA. carbonariusand that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

scholarly journals Draft Genome Sequence of a Porcine Commensal, Rothia nasimurium, Encoding a Nonribosomal Peptide Synthetase Predicted To Produce the Ionophore Antibiotic Valinomycin

2017 ◽  
Vol 5 (22) ◽  
Author(s):  
Rogier A. Gaiser ◽  
Marnix H. Medema ◽  
Michiel Kleerebezem ◽  
Peter van Baarlen ◽  
Jerry M. Wells
Keyword(s):  
Genome Sequence ◽  
Bacillus Pumilus ◽  
Draft Genome ◽  
Nonribosomal Peptide Synthetase ◽  
Draft Genome Sequence ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase

ABSTRACT We report the draft whole-genome sequence of Rothia nasimurium isolated from a porcine tonsil. The genome encodes a nonribosomal peptide synthetase predicted to produce valinomycin, a cyclic dodecadepsipeptide ionophore. Previously, valinomycin was known to be produced only by Streptomyces species and isolates belonging to the Bacillus pumilus group.

scholarly journals Characterization of the Saframycin A Gene Cluster from Streptomyces lavendulae NRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

2007 ◽  
Vol 190 (1) ◽  
pp. 251-263 ◽  
Author(s):  
Lei Li ◽  
Wei Deng ◽  
Jie Song ◽  
Wei Ding ◽  
Qun-Fei Zhao ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Combinatorial Biosynthesis ◽  
Common Mechanism ◽  
Nonribosomal Peptide ◽  
Streptomyces Lavendulae ◽  
Peptide Synthetase

ABSTRACT Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.

scholarly journals Identification of a Gene Involved in the Synthesis of a Dipeptidyl Peptidase IV Inhibitor in Aspergillus oryzae

2012 ◽  
Vol 78 (19) ◽  
pp. 6996-7002 ◽  
Author(s):  
Kazuhiko Imamura ◽  
Yoshihito Tsuyama ◽  
Terukage Hirata ◽  
Sumihiro Shiraishi ◽  
Kazutoshi Sakamoto ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Expression Patterns ◽  
Biosynthetic Gene Cluster ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase ◽  
Isoquinoline Derivative

ABSTRACTWYK-1 is a dipeptidyl peptidase IV inhibitor produced byAspergillus oryzaestrain AO-1. Because WYK-1 is an isoquinoline derivative consisting of threel-amino acids, we hypothesized that a nonribosomal peptide synthetase was involved in its biosynthesis. We identified 28 nonribosomal peptide synthetase genes in the sequenced genome ofA. oryzaeRIB40. These genes were also identified in AO-1. Among them, AO090001000009 (wykN) was specifically expressed under WYK-1-producing conditions in AO-1. Therefore, we constructedwykNgene disruptants of AO-1 after nonhomologous recombination was suppressed by RNA interference to promote homologous recombination. Our results demonstrated that the disruptants did not produce WYK-1. Furthermore, the expression patterns of 10 genes downstream ofwykNwere similar to the expression pattern ofwykNunder several conditions. Additionally, homology searches revealed that some of these genes were predicted to be involved in WYK-1 biosynthesis. Therefore, we propose thatwykNand the 10 genes identified in this study constitute the WYK-1 biosynthetic gene cluster.

scholarly journals Cryptic Production oftrans-3-Hydroxyproline in Echinocandin B Biosynthesis

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Johanna Mattay ◽  
Stefanie Houwaart ◽  
Wolfgang Hüttel
Keyword(s):  
Amino Acids ◽  
Nonribosomal Peptide Synthetase ◽  
Peptide Antibiotic ◽  
Prolonged Incubation ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Echinocandin B ◽  
Peptide Synthetase ◽  
Proline Hydroxylation ◽  
Proline Hydroxylase

ABSTRACTEchinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines:trans-4-hydroxy-l-proline and, in most echinocandin structures, (trans-2,3)-3-hydroxy-(trans-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis byGlarea lozoyensis, both amino acids are found in pneumocandin A0, while in pneumocandin B0the latter residue is replaced bytrans-3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis byAspergillusspecies, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster inAspergillus pachycristatus. Surprisingly,l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced byCryptosporiopsis.IMPORTANCESecondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A0and B0are produced by the fungusGlarea lozoyensis. They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A0and B0, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis inAspergillus pachycristatusproduces the same hydroxyprolines, with an increased proportion oftrans-3-hydroxyproline. However, echinocandin B biosynthesis does not requiretrans-3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation inG. lozoyensisandA. pachycristatusprovides an unusual insight into peptide antibiotic biosynthesis—namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.

scholarly journals Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor

2008 ◽  
Vol 74 (14) ◽  
pp. 4366-4380 ◽  
Author(s):  
Yong-Sun Moon ◽  
Bruno G. G. Donzelli ◽  
Stuart B. Krasnoff ◽  
Heather McLane ◽  
Mike H. Griggs ◽  
...  
Keyword(s):  
Metarhizium Anisopliae ◽  
Developmental Stages ◽  
Cyclic Peptide ◽  
Pathogenic Fungus ◽  
Nonribosomal Peptide Synthetase ◽  
Dna Polymorphisms ◽  
Beet Armyworm ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

ABSTRACT Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in ΔManps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in ΔManps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. ΔManps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the ΔManps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor.

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