Secondary metabolites with fungicide potentials from the deep-sea seamount-derived fungus Talaromyces scorteus AS-242.

Marine natural products play an important role in biopesticides. Seven new secondary metabolites with different structural classes, including two cycloheptapeptides, scortide A (1) and scortide B (2), two 19-nor-diterpenoids, talascortene H (3) and talascortene I (4), two diterpenoid acids, talascortene J (5) and talascortene K (6), and one triterpenoid, talascortene L (7) were isolated and identified from the sea-anemone-derived endozoic fungus Talaromyces scorteus AS-242. Their structures were comprehensively assigned by spectroscopic data analysis, single-crystal X-ray diffraction, tandem mass spectrometry, and electronic circular dichroism (ECD) calculations. The result of the antimicrobial assay demonstrated that compounds 1 - 6 have inhibitory activity against several human, aquatic, and plant pathogens with minimum inhibitory concentration (MIC) values ranging from 1 to 64 µg/mL. Specially, compounds 2 and 4 showed significant activities against the pathogenic fungus Curvularia spicifera with the MIC value of 1 µg/mL, providing an experimental basis of 2 and 4 with the potential as lead compounds to be developed into biopesticides.

Antibacterial Polyketides from the Deep-Sea Cold-Seep-Derived Fungus Talaromyces sp. CS-258.

Thirty-two fungal polyketide derivatives, including eleven new compounds, namely (3R,5'R)-5-hydroxytalaroflavone (1), talaroisochromenols A-C (3, 5, and 11), (8R,9R,10aR)-5-hydroxyaltenuene (13), (8R,9R,10aS)-5-hydroxyaltenuene (14), (8R,9S,10aR)-5-hydroxyaltenuene (15), nemanecins D and E (25 and 26), 2,5-dimethyl-8-iodochromone (27), and talarofurolactone A (29), together with one new naturally occurring but previously synthesized metabolite, 6-hydroxy-4-methoxycoumarin (28), were isolated and identified from the deep-sea cold-seep-derived fungus Talaromyces sp. CS-258. Among them, racemic ((±)-11) or epimeric (13-15, 25, and 26) mixtures were successfully separated by chiral or gradient elution HPLC. Meanwhile, compound 27 represents a rarely reported naturally occurring iodinated compound. Their planar structures as well as absolute configurations were determined by extensive analysis via NMR, MS, single-crystal X-ray diffraction, Mosher's method, and ECD or NMR calculation (with DP4+ probability analysis). Possible biosynthetic routes of some isolated compounds, which are related to chromone or isochromone biosynthetic pathways, were put forward. The biological analysis results revealed that compounds 7, 9, 10, 18-22, 24, 30, and 31 showed broad-spectrum antibacterial activities against several human and aquatic pathogens with MIC ranges of 0.5-64 µg/mL.

Pullenvalenes A-D: Nitric Oxide-Mediated Transcriptional Activation (NOMETA) Enables Discovery of Triterpene Aminoglycosides from Australian Termite Nest-Derived Fungi.

We report on the use of nitric oxide-mediated transcriptional activation (NOMETA) as an innovative means to detect and access new classes of microbial natural products encoded within silent biosynthetic gene clusters. A small library of termite nest- and mangrove-derived fungi and actinomyces was subjected to cultivation profiling using a miniaturized 24-well format approach (MATRIX) in the presence and absence of nitric oxide, with the resulting metabolomes subjected to comparative chemical analysis using UPLC-DAD and GNPS molecular networking. This strategy prompted study of Talaromyces sp. CMB-TN6F and Coccidiodes sp. CMB-TN39F, leading to discovery of the triterpene glycoside pullenvalenes A-D (1-4), featuring an unprecedented triterpene carbon skeleton and rare 6-O-methyl-N-acetyl-d-glucosaminyl glycoside residues. Structure elucidation of 1-4 was achieved by a combination of detailed spectroscopic analysis, chemical degradation, derivatization and synthesis, and biosynthetic considerations.

Molecular and biological characterization of a novel partitivirus from Talaromyces pinophilus.

Talaromyces spp. have a worldwide distribution, are ecologically diverse and have been isolated from numerous different substrates. Talaromyces spp. are considered biotechnologically important due to their ability to produce a range of enzymes and pigments. Talaromyces pinophilus, belonging to genus Talaromyces and family Trichocomaceae, is known for producing several important bioactive metabolites. Here we report the isolation and characterisation of a partitivirus from T. pinophilus which we have nominated Talaromyces pinophilus partitivirus-1 (TpPV-1). TpPV-1 possesses a genome consisting of three double stranded (ds) RNA segments i.e., dsRNAs1-3, 1824 bp, 1638 bp and 1451 bp respectively, which are encapsidated in icosahedral particles 35 nm in diameter. Both dsRNA1 and dsRNA2 contain a single open reading frame (ORF) encoding respectively a 572 amino acid (aa) protein of 65 kDa and a 504 aa protein of 50 kDa. The third segment (dsRNA3) is potentially a satellite RNA. Phylogenetic analysis revealed that the TpPV-1 belongs to the family Partitiviridae in the proposed genus Zetapartitivirus. TpPV-1 infection decreases the mycelial growth rate of the host fungus and alters pigmentation as indicated by time course experiments performed on a range of different solid media comparing virus-infected and virus-free isogenic lines. This is the first report of mycovirus infection in T. pinophilus and may provide insights into understanding the effect of the mycovirus on the production of enzymes and pigments by the host fungus.

Characterization and Effect of a Nematophagous Fungus Talaromyces cystophila sp. nov. for the Biological Control of Corn Cyst Nematode.

The dynamic of plant-parasitic nematode populations in soil is closely related to soil microorganisms. Fungi from Heterodera zeae cysts were isolated to explore the phenomenon of decline in the H. zeae population in the soil. Phylogenetic study of partial ITS, BenA, CaM, and RPB2 gene sequences, in addition to morphological investigations, was utilized to identify a nematode-destroying fungus. The nematicidal activity of a novel strain GX1 against H. zeae was assessed in vitro and in the greenhouse. Our findings revealed that strain GX1 is a new species of Talaromyces, named Talaromyces cystophila. It has a strong parasitic and lethal effect on H. zeae cysts, with 91.11% parasitism on cysts at 3 days after treatment. The contents of second-stage juveniles (J2s) and eggs inside the cysts were degraded and formed dense vacuoles, and the damaged eggs could not hatch normally. The spore suspension exhibited high nematophagous activity against nematodes, and fermentation filtrate exhibited marked inhibition of egg hatching and nematicidal activities on J2s. The hatching inhibition rates of eggs exposed to 1 × 108 CFU/ml spore suspensions or 20% 1-week fermentation filtrate (1-WF) for 15 days were 98.56 and 100%, respectively. The mortality of J2s exposed to 1 × 108 CFU/ml spore suspension reached 100% at 24 h; exposure to 50% 2-WF was 98.65 and 100% at 24 and 48 h, respectively. Greenhouse experiments revealed that the spore suspension and fermentation broth considerably decreased H. zeae reproduction by 56.17 to 78.76%. T. cystophila is a potential biocontrol strain with nematophagous and nematicidal activity that deserves attention and application.

Extending the Structural Diversity of Labdane Diterpenoids from Marine-Derived Fungus Talaromyces sp. HDN151403 Using Heterologous Expression.

Heterologous biosynthesis has become an effective means to activate fungal silent biosynthetic gene clusters (BGCs) and efficiently utilize fungal genetic resources. Herein, thirteen labdane diterpene derivatives, including five undescribed ones named talarobicins A-E (3-7), were discovered via heterologous expression of a silent BGC (labd) in Aspergillus nidulans. Their structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as electronic circular dichroism (ECD) calculations. These labdanes belong to four skeleton types, and talarobicin B (4) is the first 3,18-dinor-2,3:4,18-diseco-labdane diterpene with the cleavage of the C2-C3 bond in ring A and the decarboxylation at C-3 and C-18. Talarobicin B (4) represents the key intermediate in the biosynthesis of penioxalicin and compound 13. The combinatorial heterologous expression and feeding experiments revealed that the cytochrome P450 enzymes LabdC, LabdE, and LabdF were responsible for catalyzing various chemical reactions, such as oxidation, decarboxylation, and methylation. All of the compounds are noncytotoxic, and compounds 2 and 8 displayed inhibitory effects against methicillin-resistant coagulase-negative staphylococci (MRCNS) and Bacillus cereus.

The potential of degrading natural chitinous wastes to oligosaccharides by chitinolytic enzymes from two Talaromyces sp. isolated from rotten insects (Hermetia illucens) under solid state fermentation.

It is difficult to produce chitin oligosaccharides by hydrolyzing untreated natural chitinous waste directly. In this study, two fungi Talaromyces allahabadensis Hi-4 and Talaromyces funiculosus Hi-5 from rotten black soldier fly were isolated and identified through multigene phylogenetic and morphological analyses. The chitinolytic enzymes were produced by solid state fermentation, and the growth conditions were optimized by combining single-factor and central composite design. The best carbon sources were powder of molting of mealworms (MMP) and there was no need for additional nitrogen sources in two fungi, then the maximum chitinolytic enzyme production of 46.80 ± 3.30 (Hi-4) and 55.07 ± 2.48 (Hi-5) U/gds were achieved after analyzing the 3D response surface plots. Pure chitin (colloidal chitin) and natural chitinous substrates (represented by MMP) were used to optimize degradation abilities by crude enzymes obtained from the two fungi. The optimum temperature for hydrolyzing MMP (40 °C both in two fungi) were lower and closer to room temperature than colloidal chitin (55 °C for Hi-4 and 45 °C for Hi-5). Then colloidal chitin, MMP and the powder of shrimp shells (SSP) were used for analyzing the products after 5-day degradation. The amounts of chitin oligosaccharides from SSP and MMP were about 1/6 (Hi-4), 1/17 (Hi-5) and 1/8 (Hi-4), 1/10 (Hi-5), respectively, in comparison to colloidal chitin. The main components of the products were GlcNAc for colloidal chitin, (GlcNAc)2 for MMP, and oligosaccharides with higher degree of polymerization (4-6) were obtained when hydrolyzing SSP, which is significant for applications in medicine and health products.

Isolation, Characterization, Antioxidant, and Wound Healing Activities of Extracellular Polysaccharide from Endophytic Fungus Talaromyces purpureogenus.

In this study, two extracellular polysaccharides (TEPS1 and TEPS2) were isolated from the endophytic fungus (Talaromyces purpureogenus) and purified by DEAE-Sepharose Fast Flow column using NaCl as gradient eluent. The HPLC analysis displayed that TEPS1 was composed of mannose (38.70%), ribose (25.02%), glucose (19.34%), and galactose (16.94%) while the TEPS2 composed by mannose (100%). The NMR results indicated that TEPS1 exhibited α-glycosidic configurations. The both polysaccharides, TEPS1 and TEPS2 were exhibited a good antioxidant activity in terms of DPPH, ABTS, and •OH scavenging. However, TEPS1 showed a higher antioxidant activity than TEPS2. The IC50 of TEPS1 were 32.16, 192.57, and 54.67 µg·mL-1, for DPPH, ABTS, and •OH radical scavenging, respectively. Furthermore, TEPS1 showed the high cellular antioxidant and wound healing activity in the human embryonic kidney (HEK293) cell line. Overall, these two polysaccharides were promising in antioxidant activity.

Bioremediation of petroleum-contaminated saline soil by Acinetobacter baumannii and Talaromyces sp. and functional potential analysis using metagenomic sequencing.

Microbial remediation is a potential remediation method for petroleum-contaminated soil. In order to explore the petroleum degradation mechanism by microorganisms, the oilfield soil was remedied by Acinetobacter baumannii combined with Talaromyces sp. The degradation mechanism was studied by analyzing soil microbial community and functional genes through metagenomics during the degradation process. The result showed the degradation rate of petroleum was 65.6% after 28 days. The concentration of petroleum decreased from 1220 mg/kg to 420 mg/kg. In the co-culture group, Acinetobacter baumannii became the dominant species, the annotated genes of it at the species level accounted for 7.34% while that of Talaromyces sp. accounted for only 0.34%. Meanwhile, the annotated genes of Bacillus, Halomonas, and Nitriliruptor at the genus level were up-regulated by 1.83%, 0.90%, and 0.71%, respectively. In addition, large functional genes were significantly up-regulated, including the peroxisome, P450 enzyme (CYP53, CYP116, CYP102, CYP645), and biofilm formulation, promoting the oxidation and hydroxylation, and catalyzing the epoxidation of aromatic and aliphatic hydrocarbons. Meanwhile, the degrading genes of alkanes and aromatic hydrocarbons were expressed promotionally, and degradation pathways were deduced. In conclusion, the inoculation of Acinetobacter baumannii combined with Talaromyces sp. accelerated the degradation of petroleum in oilfield soil and improved the growth of indigenous petroleum-degrading bacteria. Many functional genes related to petroleum degradation were promoted significantly. These results proved the co-culture of bacteria-fungi consortium contributes to the bioremediation of petroleum-contaminated soil.

Biotransformation of testosterone by the filamentous fungus Penicillium pinophilum.

The microbial biotransformation is a robust procedure in developing steroids and fungi are practical tools in this process; therefore, the fungal modification of testosterone by Penicillium pinophilum was investigated. The three prominent metabolites, including 14α-hydroxyandrost-4-en-3,17-dione (II), 14α-hydroxytestosterone (III), and 11α-hydroxytestosterone (IV), were isolated and characterized by chromatographic and spectroscopic methods. The time course profile showed that the content of the metabolites II and III began to decrease after 96 and 24 h, respectively. In comparison, the content of the metabolite IV remained stable after 24 h. In silico studies showed that the probability of binding to the androgen receptor remains high for all three metabolites. However, the probability of binding to the estrogen receptors α and ß increased for metabolite IV but decreased for metabolite III. Penicillium pinophilum as a potentially viable biocatalyst could hydroxylate C-11α and C-14α positions and oxidize the C-17ß hydroxyl group to 17-ketone in testosterone molecule.

Putative Biosynthesis of Talarodioxadione & Talarooxime from Talaromyces stipitatus.

Polyesters containing 2,4-dihydroxy-6-(2-hydroxypropyl)benzoate and 3-hydroxybutyrate moieties have been isolated from many fungal species. Talaromyces stipitatus was previously reported to produce a similar polyester, talapolyester G. The complete genome sequence and the development of bioinformatics tools have enabled the discovery of the biosynthetic potential of this microorganism. Here, a putative biosynthetic gene cluster (BGC) of the polyesters encoding a highly reducing polyketide synthase (HR-PKS) and nonreducing polyketide synthase (NR-PKS), a cytochrome P450 and a regulator, was identified. Although talapolyester G does not require an oxidative step for its biosynthesis, further investigation into the secondary metabolite production of T. stipitatus resulted in isolating two new metabolites called talarodioxadione and talarooxime, in addition to three known compounds, namely 6-hydroxymellein, 15G256α and transtorine that have never been reported from this organism. Interestingly, the biosynthesis of the cyclic polyester 15G256α requires hydroxylation of an inactive methyl group and thus could be a product of the identified gene cluster. The two compounds, talarooxime and transtorine, are probably the catabolic metabolites of tryptophan through the kynurenine pathway. Tryptophan metabolism exists in almost all organisms and has been of interest to many researchers. The biosynthesis of the new oxime is proposed to involve two subsequent N-hydroxylation of 2-aminoacetophenone.

New Insights into Chemical and Biological Properties of Funicone-like Compounds.

Funicone-like compounds are a homogeneous group of polyketides that, so far, have only been reported as fungal secondary metabolites. In particular, species in the genus Talaromyces seem to be the most typical producers of this group of secondary metabolites. The molecular structure of funicone, the archetype of these products, is characterized by a γ-pyrone ring linked through a ketone group to a α-resorcylic acid nucleus. This review provides an update on the current knowledge on the chemistry of funicone-like compounds, with special emphasis on their classification, occurrence, and diverse biological activities. In addition, their potential relevance as mycotoxins is discussed.

Talaverrucin A, Heterodimeric Oxaphenalenone from Antarctica Sponge-Derived Fungus Talaromyces sp. HDN151403, Inhibits Wnt/ß-Catenin Signaling Pathway.

The Wnt/ß-catenin signaling pathway is an evolutionarily conserved signaling cascade involved in a broad range of biological roles. Dysregulation of the Wnt/ß-catenin pathway is implicated in congenital malformations and various kinds of cancers. We discovered a novel Wnt/ß-catenin inhibitor, talaverrucin A (1), featuring an unprecedented 6/6/6/5/5/5/6 fused ring system, from an Antarctica sponge-derived fungus Talaromyces sp. HDN151403. Talaverrucin A exhibits inhibitory activity on the Wnt/ß-catenin pathway in both zebrafish embryos in vivo and cultured mammalian cells in vitro, providing a naturally inspired small molecule therapeutic lead to target the Wnt/ß-catenin pathway.

New Secondary Metabolites from the Marine-Derived Fungus Talaromyces mangshanicus BTBU20211089.

Seven new compounds, namely talaromanloid A (1), talaromydene (2), 10-hydroxy-8-demethyltalaromydine and 11-hydroxy-8-demethyltalaromydine (3 and 4), talaromylectone (5), and ditalaromylectones A and B (6 and 7), together with seven known compounds were identified from a marine-derived fungus, Talaromyces mangshanicus BTBU20211089, which was isolated from a sediment sample collected from the South China Sea. Their chemical structures were determined using spectroscopic data, including HRESIMS, 1D, and 2D NMR techniques. The absolute configurations of 1 and 2 were elucidated by comparing experimental and calculated ECD spectra. Compounds 1, 2, 6, and 7 are new compounds possessing a novel carbon skeleton. Compound 6 is a dimeric molecule of 3 and 9. Compound 7 shared a unique structure of the cyclized dimer of 3 and 4. All the compounds were tested for their bioactivities against Staphylococcus aureus, Escherichia coli, and Candida albicans.

Talaromyces amestolkiae uses organic phosphate sources for the treatment of uranium-contaminated water.

Fungi have received particular attention in regards to alternatives for bioremediation of heavy metal contaminated locales. Enzymes produced by filamentous fungi, such as phosphatases, can precipitate heavy metal ions in contaminated environments, forming metal phosphates (insoluble). Thus, this research aimed to analyze fungi for uranium biomineralization capacity. For this, Gongronella butleri, Penicillium piscarium, Rhodotorula sinensis and Talaromyces amestolkiae were evaluated. Phytate and glycerol 2-phosphate were used as the phosphate sources in the culture media at pH 3.5 and 5.5, with and without uranium ions. After 4 weeks of fungal growth, evaluated fungi were able to produce high concentrations of phosphates in the media. T. amestolkiae was the best phosphate producer, using phytate as an organic source. During fungal growth, there was no change in pH level of the culture medium. After 3 weeks of T. amestolkiae growth in medium supplemented with phytate, there was a reduction between 20 and 30% of uranium concentrations, with high precipitation of uranium and phosphate on the fungal biomass. The fungi analyzed in this research can use the phytic acid present in the medium and produce high concentrations of phosphate; which, in the environment, can assist in the heavy metal biomineralization processes, even in acidic environments. Such metabolic capabilities of fungi can be useful in decontaminating uranium-contaminated environments.

Biosynthetic machineries of anthraquinones and bisanthraquinones in Talaromyces islandicus.

Talaromyces islandicus is a unique fungus that produces more than 20 numbers of anthraquinones (AQs) and their dimeric natural products, bisanthraquinones (BQs). These compounds share a 9,10-anthracenedione core derived from emodin. The biosynthetic pathway of emodin has been firmly established, while that of other AQs and BQs is still unclear. In this study, we identified the biosynthetic gene clusters for chrysophanol and skyrin. The function of key modification enzymes was examined by performing biotransformation experiments and in vitro enzymatic reactions with emodin and its derivatives, allowing us to propose a mechanism for the modification reactions. The present study provides insight into the biosynthesis of AQs and BQs in T. islandicus.

A Fungal Versatile GH10 Endoxylanase and Its Glycosynthase Variant: Synthesis of Xylooligosaccharides and Glycosides of Bioactive Phenolic Compounds.

The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 ß-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been heterologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)-beechwood xylan at 70 °C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabinoxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize ß-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological applications.

Identification of the Talaromyces cellulolyticus Gene Encoding an Extracellular Enzyme with ß-galactosidase Activity and Testing it as a Reporter for Gene Expression Assays.

The filamentous fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is currently being intensively studied as a promising industrial producer of a number of secreted cellulolytic enzymes. In this study, the T. cellulolyticus gene lacA, which encodes a protein orthologous to the fungal extracellular ß-galactosidases of family 35, was identified. The substitution of the lacA upstream region with a constitutive promoter demonstrated that the product of this gene is effectively secreted and possesses ß-galactosidase activity. The optimal pH and temperature values for the hydrolysis of o-nitrophenyl-ß-D-galactopyranoside by this enzyme were determined to be pH 4.5-5.5 and 50 °C, respectively. The negligible production of ß-galactosidase activity by strains expressing lacA under native regulation raises the possibility of using lacA as a reporter gene. To test this hypothesis, the native promoter of lacA was replaced with the strong inducible promoter of the T. cellulolyticus cellobiohydrolase I gene. The cultivation of the resulting strain in various media showed that the ß-galactosidase activity depends on cultivation conditions similar to the cellobiohydrolase activity. Thus, the suitability of lacA as a reporter for evaluating promoters with a wide range of expression profiles was demonstrated.

Biosynthesis of the Fungal Organophosphonate Fosfonochlorin Involves an Iron(II) and 2-(Oxo)glutarate Dependent Oxacyclase.

The fungal metabolite Fosfonochlorin features a chloroacetyl moiety that is unusual within known phosphonate natural product biochemistry. Putative biosynthetic genes encoding Fosfonochlorin in Fusarium and Talaromyces spp. were investigated through reactions of encoded enzymes with synthetic substrates and isotope labelling studies. We show that the early biosynthetic steps for Fosfonochlorin involve the reduction of phosphonoacetaldehyde to form 2-hydroxyethylphosphonic acid, followed by oxidative intramolecular cyclization of the resulting alcohol to form (S)-epoxyethylphosphonic acid. The latter reaction is catalyzed by FfnD, a rare example of a non-heme iron/2-(oxo)glutarate dependent oxacyclase. In contrast, FfnD behaves as a more typical oxygenase with ethylphosphonic acid, producing (S)-1-hydroxyethylphosphonic acid. FfnD thus represents a new example of a ferryl generating enzyme that can suppress the typical oxygen rebound reaction that follows abstraction of a substrate hydrogen by a ferryl oxygen, thereby directing the substrate radical towards a fate other than hydroxylation.