Nonribosomal Peptide Synthetase Genes
            <i>pesL</i>
            and
            <i>pes1</i>
            Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

O’Hanlon, Karen; Gallagher, Laura; Schrettl, Markus; Jöchl, Christoph; Kavanagh, Kevin; Larsen, Thomas Ostenfeld; Doyle, Seán

doi:10.1128/aem.07249-11

Cited by 44 publications

(41 citation statements)

References 57 publications

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“…The gene encoding calmodulin (AFUA_4G10050), which is constitutively expressed in A. fumigatus, served as a control in reverse transcription-PCR (RT-PCR) experiments (6). Real-time PCR was performed using the LightCycler 480 Sybr green 1 master mix (Roche) on a LightCycler 480 real-time PCR system as previously described (27). For realtime PCRs, a 1/10 dilution of cDNA from each sample was used as a template and each reaction was performed in triplicate.…”

Section: Methodsmentioning

confidence: 99%

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

et al. 2012

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The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H 2 O 2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P ‫؍‬ 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H 2 O 2 (P < 0.01), unexpectedly, exogenous gliotoxin relieved H 2 O 2 -induced growth inhibition in a dose-dependent manner (0 to 10 g/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ⌬gliK 26933 deletion mutant. The A. fumigatus ⌬gliK 26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ⌬gliK 26933 mutant than in those of the wild type (P ‫؍‬ 0.0024 [fold difference, 24] and P ‫؍‬ 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ⌬gliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ⌬gliK 26933 mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P ‫؍‬ 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ⌬gliK 46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus. Gliotoxin was discovered in 1936, and its structure was elucidated in 1943 (19, 38). Much initial interest in the molecule was directed toward exploiting its antifungal activities; however, after observations that gliotoxin exhibited growth-inhibitory effects against animal cells, it was posited as a putative anti-cancer or organ transplant rejection agent, and a large number of subsequent studies investigated the mechanism of action of gliotoxin against mammalian cell types (3, 24, 37).Until recently, the mechanism of gliotoxin biosynthesis had proven to be especially refractory to analysis, despite multiple studies between 1960 and 1985 involving radiolabeled precursor feeding and analysis of proteins produced during gliotoxin biosynthesis (2, 18). Since the discovery of the gliotoxin gene cluster gli (14), now known to contain 13 genes, the function of a number of cluster genes has been established by a combination of targeted gene deletion and phenotypic and biochemical analyses (4, 10, 11, 13, 22, 31-33, 34, 36). gliZ [a Zn(II) 2 -Cys(6) binuclear cluster domain transcription factor] deletio...

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Section: Methodsmentioning

confidence: 99%

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

et al. 2012

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“…With genetic manipulation of fungal genomes becoming available, genetic and chemical pathways leading to NRPS-derived metabolites could be identified through relatively straightforward gene deletions of NRPS-encoding genes coupled with analytical screenings for loss of compound production. Examples of compounds and pathways that have been identified in this traditional fashion include those of cyclosporine [27], HC-toxin [29], AM-toxin [30], peptaibols [31], ergotpeptine [32], fusarin C [33], equisetin [34], peramine [35], sirodesmin [36], gliotoxin [37], fumitremorgin [38], tenellin [39], pseurotin A [40], cytochalasin [41], cyclopiazonic acid [42], aureobasidin A [43], fumiquinazolines [44], apicidin [45], tryptopquialanine [46], ochratoxin A [47], fumigaclavines [48], ardeemin [49], nidulanin A [50], and pneumocandin [51]. Although many of the aforementioned NRPS-derived fungal secondary metabolites display biological activity, the need for discovery of new antibiotics is becoming more urgent [52] and calls for innovative avenues of uncovering cryptic metabolites.…”

Section: Introductionmentioning

confidence: 99%

Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi

Soukup

Keller

Wiemann

2016

Methods in Molecular Biology

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Filamentous fungi are historically known as rich sources for production of biologically active natural products, so-called secondary metabolites. One particularly pharmaceutically relevant chemical group of secondary metabolites is the nonribosomal peptides synthesized by nonribosomal peptide synthetases (NRPSs). As most of the fungal NRPS gene clusters leading to production of the desired molecules are not expressed under laboratory conditions, efforts to overcome this impediment are crucial to unlock the full chemical potential of each fungal species. One way to activate these silent clusters is by overexpressing and deleting global regulators of secondary metabolism. The conserved fungal-specific regulator of secondary metabolism, LaeA, was shown to be a valuable target for sleuthing of novel gene clusters and metabolites. Additionally, modulation of chromatin structures by either chemical or genetic manipulation has been shown to activate cryptic metabolites. Furthermore, NRPS-derived molecules seem to be affected by cross talk between the specific gene clusters and some of these metabolites have a tissue- or developmental-specific regulation. This chapter summarizes how this knowledge of different tiers of regulation can be combined to increase production of NRPS-derived metabolites in fungal species.

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“…32 So far, three members of the fumigaclavine family have been described: fumigaclavine A, B, and C. Fumigaclavine C is the end-product in the biosynthetic pathway of these metabolites, whereas fumigaclavine A and B are the ultimate and penultimate intermediate metabolites, respectively. 33 The structure of fumigaclavine A is the closest to that of the new fumigaclavine; therefore, they probably have a common Figure 4. Structure elucidation of a new fumigaclavine produced by A. fumigatus.…”

Section: ■ Results and Discussionmentioning

confidence: 98%

New Untargeted Metabolic Profiling Combining Mass Spectrometry and Isotopic Labeling: Application on Aspergillus fumigatus Grown on Wheat

et al. 2013

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Characterization of fungal secondary metabolomes has become a challenge due to the industrial applications of many of these molecules, and also due to the emergence of fungal threats to public health and natural ecosystems. Given that, the aim of the present study was to develop an untargeted method to analyze fungal secondary metabolomes by combining high-accuracy mass spectrometry and double isotopic labeling of fungal metabolomes. The strain NRRL 35693 of Aspergillus fumigatus , an important fungal pathogen, was grown on three wheat grain substrates: (1) naturally enriched grains (99% (12)C), (2) grains enriched 96.8% with (13)C, (3) grains enriched with 53.4% with (13)C and 96.8% with (15)N. Twenty-one secondary metabolites were unambiguously identified by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analysis. AntiBase 2012 was used to confirm the identity of these metabolites. Additionally, on the basis of tandem mass spectrometry (MS(n)) experiments, it was possible to identify for the first time the formula and the structure of fumigaclavine D, a new member of the fumigaclavines family. Post biosynthesis degradation of tryptoquivaline F by methanol was also identified during HPLC-HRMS analysis by the detection of a carbon atom of nonfungal origin. The interest of this method lies not only on the unambiguous determination of the exact chemical formulas of fungal secondary metabolites but also on the easy discrimination of nonfungal products. Validation of the method was thus successfully achieved in this study, and it can now be applied to other fungal metabolomes, offering great possibilities for the discovery of new drugs or toxins.

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Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

Cited by 44 publications

References 57 publications

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi

New Untargeted Metabolic Profiling Combining Mass Spectrometry and Isotopic Labeling: Application on Aspergillus fumigatus Grown on Wheat

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