Tolypocladium Inflatum Research Articles

Structural characterization and bioactivity profiling of the fungal peptaibiotic tolypin reveal protective effects against influenza viruses.

In a bioprospection for new antivirals, we tested nonribosomally biosynthesized polypeptide antibiotics in MDCK II cells for their actions on influenza A and B viruses (IAV/IBV). Only tolypin, a mixture of closely related 16-residue peptaibiotics from the fungus Tolypocladium inflatum IE 1897, showed promising activity. It was selected for further investigation and structural characterization byultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HR-MS/MS) and ultrahigh performance liquid chromatography coupled to in-source collision-induced dissociation tandem mass spectrometry (UHPLC-isCID-HR-MS/MS), revealing 12 partially co-eluting individual peptides that were fully sequenced. Since tolypin-related efrapeptins are potent inhibitors of F1/Fo-ATPase, we screened tolypin for its toxicity against MDCK II cells and larvae of the greater wax moth Galleria mellonella. We found that a nontoxic concentration of tolypin (1 µg/mL) reduced the titer of two IBV strains by 4-5 log values, and that of an H3N2 strain by 1-2 log values, but the H1N1pdm strain was not affected. The higher concentrations of tolypin were cytostatic to MDCK II cells, shifted their metabolism from oxidative phosphorylation to glycolysis, and induced paralysis in G. mellonella, supporting the inhibition of F1/Fo-ATPase as the mode of action. Our results lay the foundations for future work to investigate the interplay between viral replication and cellular energy metabolism, as well as the development of drugs that target host factors.

Light enhances the production of conidia and influences their hydrophobicity in Tolypocladium inflatum

Insect fungal pathogens such as Beauveria bassiana, Metarhizium robertsii, and Tolypocladium inflatum have been used as insect biocontrol agents. Their infection mechanism involves non-specific adhesion to the host cuticle, which is controlled by hydrophobins, small proteins that form an amphipathic monolayer with rodlet morphology on diverse fungal structures. Light is an abiotic factor that may influence a wide range of cellular processes, including conidiogenesis, stress tolerance, and metabolite biosynthesis. Although its effects have been studied in many fungi, little is known about the effects of light on the hydrophobic properties of conidia. The aim of this work was to investigate the influence of visible light on the conidial hydrophobicity of three entomopathogenic fungal species. For this, conidia of B. bassiana, M. robertsii, and T. inflatum were grown either under light or in the dark, drop profiles of water and diiodomethane on conidial surfaces were analyzed, and conidial hydrophobicity was estimated from contact angle measurements. Moreover, conidial production was determined, and their genome was screened with sequences for hydrophobins. Conidia of B. bassiana and M. robertsii are more hydrophobic than conidia of T. inflatum. The light modified the surface tension of T. inflatum; therefore, conidia of T. inflatum became hydrophilic. However, light did not affect the conidial hydrophobicity of B. bassiana and M. robertsii. In addition, light modified the conidial production of B. bassiana and T. inflatum cultures, but it had no effect on the conidial production of M. robertsii. The T. inflatum genome contains two predicted proteins whose sequence is akin to that of proven class II hydrophobins from other ascomycetes. Presumably, these proteins are responsible for the conidial hydrophobicity properties in this fungus. Our study helps elucidate how light affects the conidial hydrophobicity of entomopathogenic fungi.

Effect of Hydrocarbon Pollution on the Fungal Communities of the White and Barents Sea Littoral Sediments

The effect of hydrocarbon pollution on the fungal communities of littoral sediments of the cold-water White and Barents seas was investigated. The samples were collected at locations with different levels of pollution with oil products, from ports to relatively undisturbed areas. Using the diesel fuel-containing medium resulted in detection of hydrocarbon-degrading fungi in almost all studied samples, although in all cases they were less diverse than sugar-degrading fungi. In this relatively small group, Penicillium chrysogenum and Penicillium brevicompactum were the most common organisms. Fungal communities isolated on a sugar-containing medium exhibited higher diversity and abundance, with being the most common sugar degraders. The major factors affecting the structure of the fungal communities were the percentage of hydrocarbons in the total mass of organic carbon in the samples in the case of hydrocarbon-degrading fungi and location, for sugar degraders. In the experiment, the highest hydrocarbon-degrading activity was shown for Penicillium chrysogenum (the loss of residual hydrocarbons was 77.4%), Cadophora fastigiata (72%), and Tolypocladium inflatum (67.2%).

Bio-synthesis, purification and structural analysis of Cyclosporine-A produced by Tolypocladium inflatum with valorization of agro-industrial wastes

Cyclosporine A (CyA) holds significant importance as a strategic immunosuppressive drug for organ transplant patients. In this study, we aimed to produce pure and cost-effective Cyclosporine A (CyA) by fermenting a culture medium containing dairy sludge, using Tolypocladium inflatum PTCC 5253. Following the fermentation stage, ethyl acetate extraction and fast protein liquid chromatography were employed for sample purification. The initial evaluation of the effectiveness of CyA obtained from these processes was performed through bioassay, wherein the antimicrobial clear zone diameter was found to be larger compared to the sample obtained from the fermentation culture. The concentration of CyA was determined using high-performance liquid chromatography, yielding values of 334 mg/L, 456 mg/L, and 578 mg/L for the fermented, extracted, and purified samples, respectively. Further analysis utilizing liquid chromatography tandem mass spectrometry (LC/MS/MS) confirmed a purity of 91.9% and proper agreement with the standard sample based on the ion intensity of Z/m 1205. To validate the structure of CyA, nuclear magnetic resonance spectroscopy, Fourier-transform infrared (FT-IR), and Raman spectroscopy were employed. X-ray diffraction and differential scanning calorimetry analyses demonstrated that the purified CyA exhibited a crystal structure similar to the standard sample, characterized by two broad peaks at 2θ = 9° and 20°, and comparable glass transition temperatures (57–68 °C for the purified sample; 53–64 °C for the standard sample). Dynamic light scattering analysis confirmed a uniform particle size distribution in both the purified and standard samples. The zeta potentials of the purified and standard samples were determined to be − 25.8 ± 0.16 and − 23.63 ± 0.12 mV, respectively. Our results demonstrate that dairy sludge can serve as a suitable culture medium for the production of (CyA).

Biochemical characterization of a multiple prenyltransferase from Tolypocladium inflatum

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, β-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, β-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals.Key points• The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum.• TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF.• The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.Graphical abstract

Report of the Nomenclature Committee for Fungi: 24

SummaryAn addition to the list of names for protection under Art. F.2.1 from the Cordyceps Working Group, containing two names of genera, is approved. The following two family names are recommended for conservation: Physaraceae against Fuliginaceae; and Metschnikowiaceae against Nectaromycetaceae and Torulopsidaceae. The following six generic names are recommended for conservation: Blastomyces Gilchrist & W.R. Stokes against Blastomyces Costantin & Rolland; Cercospora with a conserved type; Didymium against Mucilago and Spumaria; Discula with a conserved type; Metschnikowia against Torulopsis; and Phyllopsora against Triclinum and Crocynia. The proposal concerning the gender of the genus Glomus is referred to the General Committee. The following 15 species names are recommended for conservation: Agaricus cingulatus (Tricholoma cingulatum) against A. ramentaceus; A. psammopus (Tricholoma psammopus) against A. concolor; Apioporthe corni (Aurantioporthe corni, Cryptodiaporthe corni) against Sphaeronaema aurantiacum and Myxosporium nitidum; Ditiola mucida (Holwaya mucida) against Acrospermum caliciiforme (Crinula caliciiformis); Entoloma sericeum against Agaricus pascuus (E. pascuum); Geoglossum uliginosum Hakelier against G. uliginosum (Pers.) P. Crouan & H. Crouan; Microsphaera alphitoides (Erysiphe alphitoides) with a conserved type; Peziza cinnamomea (Pezicula cinnamomea) against Naemaspora grisea; Phyllosticta yuccae against Leptodothiorella notabilis; Puccinia psidii (Austropuccinia psidii) against Caeoma eugeniarum and Uredo neurophila; Tolypocladium inflatum against Cordyceps subsessilis; Tricholoma sciodes against Agaricus hordus; Tuber aestivum Vittad. against T. aestivum (Wulfen) Spreng. and T. blotii; T. magnatum against T. griseum; and T. melanosporum against T. nigrum. The following two species names are recommended for rejection under Art. 56: Ramularia gibba and Tuber cibarium. Proposal 2307 (concerning Hypocrea lutea (Tode) Petch) is no longer relevant, following the removal in the Shenzhen Code (Turland & al. in Regnum Veg. 159. 2018) of Art. 57.2 of the Melbourne Code (McNeill & al. in Regnum Veg. 154. 2012). Proposal 2233 (to conserve Bipolaris against Cochliobolus) and four proposals concerning names in Trichoderma and Hypocrea (2305, 2306, 2308 and 2309) have been withdrawn because the names were included in a list for protection from an approved working group: for Bipolaris, the Dothideomycetes Working Group, and for Trichoderma, the International Subcommission on Trichoderma and Hypocrea. Three works by Paulet, the unpaginated “Index” of Traité des champignons and two editions of Tabula plantarum fungosarum, are recommended for suppression under Art. 34.

Cordyceps fungus-mediated enantioselective synthesis of chiral alcohol and enhanced biocatalytic efficiency in cyclodextrin/surfactant-buffer system

Asymmetric synthesis of chiral alcohols by biocatalysts holds promising prospect. In this study, a medicinal Cordyceps fungus, namely Tolypocladium inflatum, was adopted as a novel biocatalyst for the synthesis of chiral alcohols. Among the assessed prochiral ketone substrates, T. inflatum exhibited the best catalytic activity towards 1-(4-bromophenyl)−2,2,2-trifluoroethanone (BPFO), yielding a valuable pharmaceutical intermediate (S)−1-(4-bromophenyl)−2,2,2-trifluoroethanol ((S)-BPFL) with >99.9 % enantiomeric excesses (ee). For more efficient biosynthesis of (S)-BPFL, cyclodextrins or surfactants were introduced to the reaction system as additives to strengthen this bioprocess. Among which methyl β-cyclodextrin (MCD) improved cell membrane permeability and alleviated product inhibition, while Triton X-100 enhanced BPFO solubility. Furthermore, synergistic promotion for BPFO bioreduction by MCD and Triton X-100 was observed. Under the optimal reaction conditions, 50 mM BPFO was converted into (S)-BPFL with 89.1 % yield and >99.9 % ee in 12 h, showing 16-fold increase of space-time yield in the developed MCD/Triton X-100-buffer system compared to neat buffer solution. This study illustrated the potential of the enantioselective preparation of chiral alcohols mediated by Cordyceps fungus. Additionally, the biocatalytic efficiency was significantly improved in the constructed synergistic reaction system, and the underlying mechanism was also explored.

Characterization of Aziridine-Forming α-Ketoglutarate-Dependent Oxygenase in l-Isovaline Biosynthesis.

l-Isovaline biosynthesis by TqaLFM-ti from Tolypocladium inflatum was demonstrated in vitro. The biochemical analysis of the α-ketoglutarate-dependent oxygenase TqaL-ti revealed that it produces (2S,3S)-3-ethyl-3-methylaziridine-2-carboxylic acid from l-isoleucine, thus exhibiting a stereoselectivity different from those of the reported homologues. Remarkably, a single mutation on I295 in TqaL-ti completely exchanged its stereoselectivity to produce the C-3 stereoisomer. TqaFM-ti generates d-isovaline from (2S,3R)-aziridine-2-carboxylic acid, suggesting that the stereochemistry of the TqaL product defines that of isovaline.

Comparative proteomics reveals the mechanism of cyclosporine production and mycelial growth in Tolypocladium inflatum affected by different carbon sources.

Cyclosporine A (CsA) is a secondary cyclopeptide metabolite produced by Tolypocladium inflatum that is widely used clinically as an immunosuppressant. CsA production and mycelial growth differed when T. inflatum was cultured in different carbon source media. During early fermentation, CsA was preferred to be produced in fructose medium, while the mycelium preferred to accumulate in sucrose medium. On the sixth day, the difference was most pronounced. In this study, high-throughput comparative proteomics methods were applied to analyze differences in protein expression of mycelial samples on day 6, revealing the proteins and mechanisms that positively regulate CsA production related to carbon metabolism. The differences included small molecule acid metabolism, lipid metabolism, organic catabolism, exocrine secretion, CsA substrate Bmt synthesis, and transcriptional regulation processes. The proteins involved in the regulation of mycelial growth related to carbon metabolism were also revealed and were associated with waste reoxidation processes or coenzyme metabolism, small molecule synthesis or metabolism, the stress response, genetic information or epigenetic changes, cell component assembly, cell wall integrity, membrane metabolism, vesicle transport, intramembrane localization, and the regulation of filamentous growth. This study provides a reliable reference for CsA production from high-efficiency fermentation. This study provides key information for obtaining more CsA high-yielding strains through metabolic engineering strategies.

Volatile Compounds Emitted by Plant Growth-Promoting Fungus Tolypocladium inflatum GT22 Alleviate Copper andPathogen Stress.

Previous studies on the intricate interactions between plants and microorganisms have revealed that fungal volatile compounds (VCs) can affect plant growth and development. However, the precise mechanisms underlying these actions remain to be delineated. In this study, we discovered that VCs from the soilborne fungus Tolypocladium inflatum GT22 enhance the growth of Arabidopsis. Remarkably, priming Arabidopsis with GT22 VCs caused the plant to display an enhanced immune response and mitigated the detrimental effects of both pathogenic infections and copper stress. Transcriptomic analyses of Arabidopsis seedlings treated with GT22 VCs for 3, 24 and 48 h revealed that 90, 83 and 137 genes were differentially expressed, respectively. The responsive genes are known to be involved in growth, hormone regulation, defense mechanisms and signaling pathways. Furthermore, we observed the induction of genes related to innate immunity, hypoxia, salicylic acid biosynthesis and camalexin biosynthesis by GT22 VCs. Among the VCs emitted by GT22, exposure of Arabidopsis seedlings to limonene promoted plant growth and attenuated copper stress. Thus, limonene appears to be a key mediator of the interaction between GT22 and plants. Overall, our findings provide evidence that fungal VCs can promote plant growth and enhance both biotic and abiotic tolerance. As such, our study suggests that exposure of seedlings to T. inflatum GT22 VCs may be a means of improving crop productivity. This study describes a beneficial interaction between T. inflatun GT22 and Arabidopsis. Our investigation of microorganism function in terms of VC activities allowed us to overcome the limitations of traditional microbial application methods. The importance of this study lies in the discovery of T. inflatun GT22 as a beneficial microorganism. This soilborne fungus emits VCs with plant growth-promoting effects and the ability to alleviate both copper and pathogenic stress. Furthermore, our study offers a valuable approach to tracking the activities of fungal VC components via transcriptomic analysis and sheds light on the mechanisms through which VCs promote plant growth and induce resistance. This research significantly advances our knowledge of VC applications and provides an example for further investigations within this field.

Investigation of the Thrombolytic Potential of Exoproteinases Produced by the Micromycete <i>Tolypocladium inflatum</i> 62A Isolated from the Soils of the White Sea

Thrombolytic properties of the micromycete Tolypocladium inflatum 62a isolated from the White Sea soils exoproteinases preparation, as well as its individual fractions, obtained after isoelectric focusing, were studied in comparison to the closest analogue, the producer of thrombolytic enzymes T. inflatum k1. A pronounced thrombolytic potential, the presence of fibrinolytic and plasminogen activator activity, specific proteolytic activity with respect to certain substrates (plasmin-like and subtilisin-like) as well as a prolonged thrombolytic effect in connection to fibrin clots were demonstrated, which can be used in the development of new drugs for the thrombotic conditions treatment, the development of anti-hematoma drugs for external use as well as diagnostic kits for the pathology of the human hemostasis system.

Production of cyclosporin A by Tolypocladium inflatum using dairy waste medium: optimization and investigation of the effect of ultrasound, high hydrostatic pressure, and pulsed electric field treatments on the morphology of fungus

Production of cyclosporin A by Tolypocladium inflatum using dairy waste medium: optimization and investigation of the effect of ultrasound, high hydrostatic pressure, and pulsed electric field treatments on the morphology of fungus

Natural infection of Chiromyzinae larvae (Diptera: Stratiomyidae) in southern Chile by Tolypocladium valdiviae sp. nov.

A new species from the fungal genus Tolypocladium (Hypocreales: Ophiocordycipitaceae) that infects Stratiomyidae larva from the genus Hylorops is described: Tolypocladium valdiviae Gallardo-Pillancari, Montalva & González. The description is based on both genomic data and morphological characteristics. The sexual stage of T. valdiviae presents fleshy and visible stromata; unlike Tolypocladium ophioglossoides, it is smaller and emerges directly from its host and resembles Tolypocladium longisegmentis and Tolypocladium capitatum, both of which are parasites of deer truffle fungi of the genus Elaphomyces (Ascomycota: Eurotiales). In the anamorphic state, T. valdiviae presents conidiogenous cells similar in shape and arrangement to those of Tolypocladium inflatum, however T. valdiviae produces larger conidiogenous cells and, occasionally, produces chlamydospores. Phylogenetic evidence suggested that T. valdiviae is in a clade close to T. longisegmentis, T. inflatum and T. ophioglossoides, species also recognized to be parasites of fungi of the genus Elaphomyces. The new species is known so far only from Valdivia, southern Chile.

Valorization of Cheese Whey as a Feedstock for Production of Cyclosporin A by Tolypocladium inflatum

Food waste-based biorefineries are considered an essential concept for the implementation of a sustainable circular economy. In this study, cheese whey powder (CWP), a dairy industry waste, was utilized to produce cyclosporin A (CsA). As it is difficult to valorize CWP because its components vary depending on the origin, a process for sugar conversion via acid hydrolysis was designed to obtain reproducible results using refined whey powder (WP) of a consistent quality. Acid hydrolysis was carried out using 2% (w/w) HCl and biomass loading of 50 g/L at 121 °C for 20 min. CWP hydrolysates were utilized to ferment Tolypocladium inflatum ATCC 34921. CsA production was found to be 51.3 mg/L at 12 days, a 1.4-fold increase compared to the control (commercial glucose, 36.3 mg/L). Our results showed that 100 g CWP can be converted to 81.8 mg of CsA. This finding demonstrated that CWP can be used as a sustainable feedstock for biorefineries.

Optimization of the production of roasted-nutty aroma by a newly isolated fungus Tolypocladium inflatum SRH81 and impact of encapsulation on its quality

BackgroundPyrazines are used in food industry to impart the foods nutty-roasted flavor. However, their extraction from natural sources is difficult and expensive. At the same time, there is awareness against the chemical food additives. Microorganisms are approved as natural producers of flavors. The aim of the present study was to assess the ability of the newly isolated fungus Tolypocladium inflatum SRH81 to produce pyrazines and studying the effect of encapsulation in gum Arabic on the quality of the biogenerated volatiles. The parameters affecting the biogeneration of pyrazines were optimized. The headspace volatiles of each culture were isolated and identified by solid phase microextraction (HS-SPME) and gas chromatography coupled with mass spectrometry (GC-MS). The volatiles showed the highest pyrazines content and best nutty-roasty flavor was subjected to encapsulation. ResultsThe selected fungus was identified as Tolypocladium inflatum SRH81. A high correlation was found between the consumed sugar and dry matter content of each culture. Incubation of the fungus culture enriched with 0.5 g amino acids/50 mL medium for 12 days at pH 8 showed the highest generation of pyrazines and best odor sensory quality. Nine pyrazines were identified among them 2-methylpyrazine was the major compound after incubation for 12 days. A positive correlation was found between the total pyrazines and intensity of roasty-nutty aroma. Encapsulation gave rise to a significant decrease in the total volatiles, while the odor intensity showed insignificant decrease. ConclusionsThe results of the present study revealed the potential ability of Tolypocladium inflatum SRH81, that was isolated from Egyptian soil, to produce pyrazines having roasted- nutty aroma.

Thrombolytic Potential of Micromycetes from the Genus Tolypocladium, Obtained from White Sea Soils: Screening of Producers and Exoproteinases Properties

Thrombotherapy is an important approach in treatment of various diseases associated with pathologies of the cardiovascular and human hemostasis systems. Screening for producers of modern, specific, and safe thrombolytic substances is an important task for medicine and biotechnology. The aim of this study was to characterize thrombolytic potential of seven strains of micromycete belonging to the genus Tolypocladium, which was obtained from White Sea soils. The Tolypocladium inflatum 62a strain was considered the most promising producer of thrombolytic agent activities suitable for possible use in thrombotherapy or diagnostics of hemostasis pathologies. It demonstrated a high radial growth rate and was characterized not only by a sufficiently high value of enzymatic index in media with fibrin and fibrinogen but also by the highest specificity for fibrillar proteins among all strains. The preparation obtained from it demonstrated pronounced thrombolytic effectiveness and substrate specificity.

Minos and Restless transposon insertion mutagenesis of psychrotrophic fungus for red pigment synthesis adaptive to normal temperature

The polar psychrotrophic fungus Geomyces sp. WNF-15A can produce high-quality natural red pigment for the potential use as edible pigment. However, it shows low-temperature-dependent synthesis of red pigment, which limits its large-scale industrial applications due to the difficult and high-cost bioprocess control. This study aims to develop transposon-mediated mutagenesis methods to generate mutants that are able to synthesize red pigment at normal temperature. Four transposable systems, including single and dual transposable systems, were established in this fungus based on the Minos from Drosophila hydei and the Restless from Tolypocladium inflatum. A total of 23 production-dominant mutants and 12 growth-dominant mutants were thus obtained by constructed transposable systems. At 14 °C and 20 °C, the MPS1 mutant strain achieved the highest level of red pigment (OD520 of 43.3 and 29.7, respectively), which was increased by 78.4% and 128.7% compared to the wild-type, respectively. Of note, 4 mutants (MPS1, MPS3, MPS4 and MPD1) successfully synthesized red pigment (OD520 of 5.0, 5.3, 4.7 and 4.9, respectively) at 25 °C, which broke the limit of the wild-type production under normal temperature. Generally, the dual transposable systems of Minos and Restless were more efficient than their single transposable systems for mutagenesis in this fungus. However, the positive mutation ratios were similar between the dual and single transposable systems for either Minos or Restless. This study provides alternative tools for genetic mutagenesis breeding of fungi from extreme environments.Graphical

Chemical mutagenesis and high throughput media optimization in Tolypocladium inflatum MTCC-3538 leads to enhanced production of cyclosporine A.

The online version contains supplementary material available at 10.1007/s13205-022-03219-x.

Tolypocladamides A-G: Cytotoxic Peptaibols from Tolypocladium inflatum.

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 μM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.

Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species.

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey's analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.