Exploration of the profiles of steroidal saponins from Rhizoma Paridis and their metabolites in rats by UPLC-Q-TOF-MS/MS.

INTRODUCTION: Steroidal saponins characterised by intricate chemical structures are the main active components of a well-known traditional Chinese medicine (TCM) Rhizoma Paridis. The metabolic profiles of steroidal saponins in vivo remain largely unexplored, despite their renowned antitumor, immunostimulating, and haemostatic activity. OBJECTIVE: To perform a comprehensive analysis of the chemical constituents of Rhizoma Paridis total saponins (RPTS) and their metabolites in rats after oral administration. METHOD: The chemical constituents of RPTS and their metabolites were analysed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). RESULTS: A reliable UPLC-Q-TOF-MS/MS method was established, and a total of 142 compounds were identified in RPTS. Specifically, diosgenin-type saponins showed the diagnostic ions at m/z 415.32, 397.31, 283.25, 271.21, and 253.20, whereas pennogenin-type saponins exhibited the diagnostic ions at m/z 413.31, 395.30, and 251.20. Based on the characteristic fragments and standard substances, 15 specific metabolites were further identified in the faeces, urine, plasma, and bile of rats. The metabolic pathways of RPTS, including phase I reactions (de-glycosylation and oxidation) and phase II reactions (glucuronidation), were explored and summarised, and the enrichment of metabolites was characterised by multivariate statistical analysis. CONCLUSION: The intricate RPTS could be transformed into relatively simple metabolites in rats through de-glycosylation, which provides a reference for further metabolic studies and screening of active ingredients for TCM.

Arthrocolins Synergizing with Fluconazole Inhibit Fluconazole-Resistant Candida albicans by Increasing Riboflavin Metabolism and Causing Mitochondrial Dysfunction and Autophagy.

Our previous study reported that seminaturally occurring arthrocolins A to C with unprecedented carbon skeletons could restore the antifungal activity of fluconazole against fluconazole-resistant Candida albicans. Here, we showed that arthrocolins synergized with fluconazole, reducing the fluconazole minimum and dramatically augmenting the survivals of 293T human cells and nematode Caenorhabditis elegans infected with fluconazole-resistant C. albicans. Mechanistically, fluconazole can induce fungal membrane permeability to arthrocolins, leading to the intracellular arthrocolins that were critical to the antifungal activity of the combination therapy by inducing abnormal cell membranes and mitochondrial dysfunctions in the fungus. Transcriptomics and reverse transcription-quantitative PCR (qRT-PCR) analysis indicated that the intracellular arthrocolins induced the strongest upregulated genes that were involved in membrane transports while the downregulated genes were responsible for fungal pathogenesis. Moreover, riboflavin metabolism and proteasomes were the most upregulated pathways, which were accompanied by inhibition of protein biosynthesis and increased levels of reactive oxygen species (ROS), lipids, and autophagy. Our results suggested that arthrocolins should be a novel class of synergistic antifungal compounds by inducing mitochondrial dysfunctions in combination with fluconazole and provided a new perspective for the design of new bioactive antifungal compounds with potential pharmacological properties. IMPORTANCE The prevalence of antifungal-resistant Candida albicans, which is a common human fungal pathogen causing life-threatening systemic infections, has become a challenge in the treatment of fungal infections. Arthrocolins are a new type of xanthene obtained from Escherichia coli fed with a key fungal precursor toluquinol. Different from those artificially synthesized xanthenes used as important medications, arthrocolins can synergize with fluconazole against fluconazole-resistant Candida albicans. Fluconazole can induce the fungal permeability of arthrocolins into fungal cells, and then the intracellular arthrocolins exerted detrimental effects on the fungus by inducing fungal mitochondrial dysfunctions, leading to dramatically reduced fungal pathogenicity. Importantly, the combination of arthrocolins and fluconazole are effective against C. albicans in two models, including human cell line 293T and nematode Caenorhabditis elegans. Arthrocolins should be a novel class of antifungal compounds with potential pharmacological properties.

The Multifaceted Gene 275 Embedded in the PKS-PTS Gene Cluster Was Involved in the Regulation of Arthrobotrisin Biosynthesis, TCA Cycle, and Septa Formation in Nematode-Trapping Fungus Arthrobotrys oligospora.

The predominant nematode-trapping fungus Arthrobotrys oligospora harbors a unique polyketide synthase-prenyltransferase (PKS-PTS) gene cluster AOL_s00215g responsible for the biosynthesis of sesquiterpenyl epoxy-cyclohexenoids (SECs) that are involved in the regulation of fungal growth, adhesive trap formation, antibacterial activity, and soil colonization. However, the function of one rare gene (AOL_s00215g275 (275)) embedded in the cluster has remained cryptic. Here, we constructed two mutants with the disruption of 275 and the overexpression of 275, respectively, and compared their fungal growth, morphology, resistance to chemical stress, nematicidal activity, transcriptomic and metabolic profiles, and infrastructures, together with binding affinity analysis. Both mutants displayed distinct differences in their TCA cycles, SEC biosynthesis, and endocytosis, combined with abnormal mitochondria, vacuoles, septa formation, and decreased nematicidal activity. Our results suggest that gene 275 might function as a separator and as an integrated gene with multiple potential functions related to three distinct genes encoding the retinoic acid induced-1, cortactin, and vacuolar iron transporter 1 proteins in this nematode-trapping fungus. Our unexpected findings provide insight into the intriguing organization and functions of a rare non-biosynthetic gene in a biosynthetic gene cluster.

Modulating Activity Evaluation of Gut Microbiota with Versatile Toluquinol.

Gut microbiota have important implications for health by affecting the metabolism of diet and drugs. However, the specific microbial mediators and their mechanisms in modulating specific key intermediate metabolites from fungal origins still remain largely unclear. Toluquinol, as a key versatile precursor metabolite, is commonly distributed in many fungi, including Penicillium species and their strains for food production. The common 17 gut microbes were cultivated and fed with and without toluquinol. Metabolic analysis revealed that four strains, including the predominant Enterococcus species, could metabolize toluquinol and produce different metabolites. Chemical investigation on large-scale cultures led to isolation of four targeted metabolites and their structures were characterized with NMR, MS, and X-ray diffraction analysis, as four toluquinol derivatives (1-4) through O1/O4-acetyl and C5/C6-methylsulfonyl substitutions, respectively. The four metabolites were first synthesized in living organisms. Further experiments suggested that the rare methylsulfonyl groups in 3-4 were donated from solvent DMSO through Fenton's reaction. Metabolite 1 displayed the strongest inhibitory effect on cancer cells A549, A2780, and G401 with IC50 values at 0.224, 0.204, and 0.597 µM, respectively, while metabolite 3 displayed no effect. Our results suggest that the dominant Enterococcus species could modulate potential precursors of fungal origin and change their biological activity.

Acetylation of Sesquiterpenyl Epoxy-Cyclohexenoids Regulates Fungal Growth, Stress Resistance, Endocytosis, and Pathogenicity of Nematode-Trapping Fungus Arthrobotrys oligospora via Metabolism and Transcription.

Sesquiterpenyl epoxy-cyclohexenoids (SECs) that depend on a polyketide synthase-terpenoid synthase (PKS-TPS) pathway are widely distributed in plant pathogenic fungi. However, the biosynthesis and function of the acetylated SECs still remained cryptic. Here, we identified that AOL_s00215g 273 (273) was responsible for the acetylation of SECs in Arthrobotrys oligospora via the construction of Δ273, in which the acetylated SECs were absent and major antibacterial nonacetylated SECs accumulated. Mutant Δ273 displayed increased trap formation, and nematicidal and antibacterial activities but decreased fungal growth and soil colonization. Glutamine, a key precursor for NH3 as a trap inducer, was highly accumulated, and biologically active phenylpropanoids and antibiotics were highly enriched in Δ273. The decreased endocytosis and increased autophagosomes, with the most upregulated genes involved in maintaining DNA and transcriptional stability and pathways related to coronavirus disease and exosome, suggested that lack of 273 might result in increased virus infection and the acetylation of SECs played a key role in fungal diverse antagonistic ability.

Polyketide Synthase-Terpenoid Synthase Hybrid Pathway Regulation of Trap Formation through Ammonia Metabolism Controls Soil Colonization of Predominant Nematode-Trapping Fungus.

Polyketide synthase-terpenoid synthase (PKS-TPS) hybrid pathways for biosynthesis of unique sesquiterpenyl epoxy-cyclohexenoids (SECs) have been found to be widely distributed in plant pathogenic fungi. However, the natural and ecological functions of these pathways and their metabolites still remain cryptic. In this study, the whole PKS-TPS hybrid pathway in the predominant nematode-trapping fungus Arthrobotrys oligospora was first proposed according to all the intermediates and their derivatives from all the A. oligospora mutants with a deficiency in each gene involved in SEC biosynthesis. Most mutants displayed significantly increased trap formation which was correlated with alteration of the ammonia level. Further analysis revealed that the main metabolites involved in ammonia metabolism were largely increased in most mutants. However, significantly retarded colonization in soil were observed in most mutants compared to the wild-type strain due to significantly decreased antibacterial activities. Our results suggested that A. oligospora used the PKS-TPS hybrid pathway for fungal soil colonization via decreasing fungal nematode-capturing ability. This also provided solid evidence that boosting fungal colonization in soil was the secondary metabolite whose biosynthesis depended on a PKS-TPS hybrid pathway.

Novel Polyketide-Terpenoid Hybrid Metabolites from a Potent Nematicidal Arthrobotrys oligospora Mutant ΔAOL_s00215g278.

Here, we reported that detailed investigation on trace targeted metabolites from nematode-trapping fungus Arthrobotrys oligospora mutant with deletion of P450 gene AOL_s00215g278 led to isolation of 9 new polyketide-terpenoid hybrid derivatives, including four new glycosides of the key precursor farnesyl hydrotoluquinol (1) and, surprisingly, four new sesquiterpenyl epoxy-cyclohexenoids (SECs) analogues. Among them, two major target metabolites 1 and 14 displayed moderate nematode inhibitory ability. Moreover, the mutant lacking AOL_s00215g278 could form far more nematode-capturing traps within 6 h in contact with nematodes and show rapid potent nematicidal activity with killing 93.7% preys, though deletion of the P450 gene resulted in dramatic decrease in fungal colony growth and failure to produce fungal conidia. The results unequivocally revealed that gene AOL_s00215g278 should be involved in not only the SEC biosynthetic pathway in the nematode-trapping fungus A. oligospora but also fungal conidiation and nematicidal activity.

Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation.

Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents.IMPORTANCEThermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

Novel Polyketide-Terpenoid Hybrid Metabolites and Increased Fungal Nematocidal Ability by Disruption of Genes 277 and 279 in Nematode-Trapping Fungus Arthrobotrys oligospora.

Nematode-trapping fungus Arthrobotrys oligospora can produce a type of sesquiterpenyl epoxy-cyclohexenoid (SEC) metabolites that are regarded as characteristic chemtaxonomic markers. Here, we reported investigation on the functions of a putatively cupin-like family gene 277 and a dehydrogenase gene 279 by gene engineering, chemical metabolite profiling and phenotype analysis. Ten targeted metabolites were isolated from two mutants Δ277 and Δ279 and four novel metabolites including three polyketide-terpenoid (PK-TP) hybrid ones were characterized. Metabolite C277-1 from mutant Δ277 shared the characteristic feature of the first and simplest PK-TP hybrid precursor, prenyl toluquinol, and metabolites C279-1 and C279-2 from mutant Δ279 shared the basic carbon skeleton of the key PK-TP hybrid precursor, farnesyl toluquinol, for biosynthesis of SEC metabolites. These results suggested that gene 277 should be involved in biosynthesis of the second prenyl unit for farnesyl toluquinol precursor, and gene 279 might be responsible for the diagnostic epoxy formation. Further analysis revealed that genes 277 and 279 might play roles in fungal conidiation, predatory trap formation, and nematode-capturing ability.

Metabolites from Two Dominant Thermophilic Fungal Species Thermomyces lanuginosus and Scytalidium thermophilum.

Thermomyces lanuginosus and Scytalidium thermophilum are among the most ubiquitous thermophilic fungi in compost and soil. Chemical study on these two prevalent strains collected from Yunnan led to isolation of 23 metabolites, including one new metabolite, therlanubutanolide, and 15 known compounds, isolated from the YGP culture broth of Thermomyces lanuginosus and 7 known compounds isolated from Scytalidium thermophilum, respectively. Therlanubutanolide shared the quite similar features of the same carbon skeleton and saturation as natural hexadecanoic acids. This was the first reported discovery of such a lactone as natural occurring metabolite. All the compounds were reported for the first time from thermophilic fungi. Among them, N-[(2S,3R,4E,8E)-1,3-dihydroxy-9-methyloctadeca-4,8-dien-2-yl]acetamide was for the first time reported to be a naturally occurring metabolite and its NMR data was first provided in this study. A type of PKS-derived metabolites, three 3,4-dihydronaphthalen-1(2H)-ones, which were widely found in plant pathogenic fungi as phytotoxins and reported to have antimicrobial activity, were obtained from both dominant thermophilic fungi. The frequent occurrence of such PKS phytotoxins in these two thermophilic fungi might suggest particular ecological interest.

Sesquiterpenyl Epoxy-Cyclohexenoids and their Signaling Functions in Nematode-Trapping Fungus Arthrobotrys oligospora.

In this study, we purified three new sesquiterpenyl epoxy-cyclohexenoid (SEC) analogues, arthrobotrisin D (11) and its two derivatives, from nematode-trapping fungus Arthrobotrys oligospora. Our results revealed that arthrobotrisin type SEC metabolites could be detected in all the test fungal strains from geographically distinct regions grown on different nutrient media, indicative of unique diagnostic character as chemical indicators for A. oligospora. The time course designs over short-term intervals of the fungus under direct contact and indirect contact with living or dead nematodes revealed that arthrobotrisin B and D (6 and 11) displayed significant relationships (positive or negative correlation) with fungal saprophytic and pathogenic stages during a nematode predation event. Interestingly, fungus on nutrient-limiting medium conducive to fungal trap formation could rapidly drop the concentration levels of arthrobotrisins B and D within 6 h when dead nematodes were around, in great contrast to that for living nematodes. Moreover, only in the fungal strain under direct contact with living dominant soil bacteria, arthrobotrisins B and D exhibited significant increase in amounts. Among them, the new SEC, arthrobotrisin D (11) was found to be a key unique metabolic signal for fungal colony growth and fungal interaction with prey and bacteria. Our study suggested that chemical analysis of SEC metabolites in A. oligospora provides a window into the fungal growth status and much valuable information about ecological environments associated with the nematode infections.

Unexpected Biosynthesis of Fluorescein-Like Arthrocolins against Resistant Strains in an Engineered Escherichia coli.

Here we provide an unprecedented biofactory where fluorescent dye-like complex xanthenes could be produced in an engineered Escherichia coli. Feeding the strain with toluquinol or hydroquinones resulted in production of novel "unnatural" natural products including four arthrocolins embedded with indolyltriphenyl quaternary carbons. Arthrocolins A-C potently inhibited various human cancer cell lines including paclitaxel-resistant cell line A549/Taxol and methicillin-resistant Staphylococcus aureus and immensely restored the sensitivity of intractable fluconazole-resistant human pathogen Candida albicans to fluconazole.

Integrated Metabolomics and Morphogenesis Reveal Volatile Signaling of the Nematode-Trapping Fungus Arthrobotrys oligospora.

The adjustment of metabolic patterns is fundamental to fungal biology and plays vital roles in adaptation to diverse ecological challenges. Nematode-trapping fungi can switch their lifestyle from saprophytic to pathogenic by developing specific trapping devices induced by nematodes to infect their prey as a response to nutrient depletion in nature. However, the chemical identity of the specific fungal metabolites used during the switch remains poorly understood. We hypothesized that these important signal molecules might be volatile in nature. Gas chromatography-mass spectrometry was used to carry out comparative analysis of fungal metabolomics during the saprophytic and pathogenic lifestyles of the model species Arthrobotrys oligospora Two media commonly used in research on this species, cornmeal agar (CMA) and potato dextrose agar (PDA), were chosen for use in this study. The fungus produced a small group of volatile furanone and pyrone metabolites that were associated with the switch from the saprophytic to the pathogenic stage. A. oligospora fungi grown on CMA tended to produce more traps and employ attractive furanones to improve the utilization of traps, while fungi grown on PDA developed fewer traps and used nematode-toxic furanone metabolites to compensate for insufficient traps. Another volatile pyrone metabolite, maltol, was identified as a morphological regulator for enhancing trap formation. Deletion of the gene AOL_s00079g496 in A. oligospora led to increased amounts of the furanone attractant (2-fold) in mutants and enhanced the attractive activity (1.5-fold) of the fungus, while it resulted in decreased trap formation. This investigation provides new insights regarding the comprehensive tactics of fungal adaptation to environmental stress, integrating both morphological and metabolomic mechanisms.IMPORTANCE Nematode-trapping fungi are a unique group of soil-living fungi that can switch from the saprophytic to the pathogenic lifestyle once they come into contact with nematodes as a response to nutrient depletion. In this study, we investigated the metabolic response during the switch and the key types of metabolites involved in the interaction between fungi and nematodes. Our findings indicate that A. oligospora develops multiple and flexible metabolic tactics corresponding to different morphological responses to nematodes. A. oligospora can use similar volatile furanone and pyrone metabolites with different ecological functions to help capture nematodes in the fungal switch from the saprophytic to the pathogenic lifestyle. Furthermore, studies with A. oligospora mutants with increased furanone and pyrone metabolites confirmed the results. This investigation reveals the importance of volatile signaling in the comprehensive tactics used by nematode-trapping fungi, integrating both morphological and metabolomic mechanisms.

New Bioactive Macrocyclic Diterpenoids from Euphorbia helioscopia.

Three new macrocyclic diterpenoids, euphoscopoids A - C (1 - 3), including two new jatrophanes and a new lathyrane, were isolated from the whole plant of Euphorbia helioscopia. Their structures were elucidated by spectroscopic methods. Antifeedant and cytotoxic activities of these isolates were evaluated. All compounds showed significant antifeedant activity against a generalist plant-feeding insect, Helicoverpa armigera, with EC50 values ranging from 2.05 to 4.34 µg/cm2 . In addition, compound 2 showed moderate cytotoxicity against tumor cell lines NCI-H1975, HepG2, and MCF-2, while compounds 1 and 3 were not active at 80 µm. The results suggested not only the defensive function of macrocyclic diterpenoids in E. helioscopia against insect herbivores, but also their potential applications as new natural insect antifeedants.

Selected Mutations Revealed Intermediates and Key Precursors in the Biosynthesis of Polyketide-Terpenoid Hybrid Sesquiterpenyl Epoxy-cyclohexenoids.

Sesquiterpenyl epoxy-cyclohexenoids (SECs) show impressive biological activities. However, the key pathways for SECs still remain unambiguous. Unexpectedly, 11 new SECs and derivatives with diverse oxidation patterns were isolated after the deletion of gene 274. A high accumulation of toluquinol and its new glycosides in mutant Δ276 and further isolation of the most crucial precursors farnesyl hydroquinone, farnesyl quinone, and three new derivatives from mutant Δ278 confirm that farnesylation at toluquinol is the key step for SECs.

Potent Nematicidal Activity and New Hybrid Metabolite Production by Disruption of a Cytochrome P450 Gene Involved in the Biosynthesis of Morphological Regulatory Arthrosporols in Nematode-Trapping Fungus Arthrobotrys oligospora.

Types of polyketide synthase-terpenoid synthase (PKS-TPS) hybrid metabolites, including arthrosporols with significant morphological regulatory activity, have been elucidated from nematode-trapping fungus Arthrobotrys oligospora. A previous study suggested that the gene cluster AOL_s00215 in A. oligospora was involved in the production of arthrosporols. Here, we report that disruption of one cytochrome P450 monooxygenase gene AOL_s00215g280 in the cluster resulted in significant phenotypic difference and much aerial hyphae. A further bioassay indicated that the mutant showed a dramatic decrease in the conidial formation but developed numerous traps and killed 85% nematodes within 6 h in contact with prey, in sharp contrast to the wild-type strain with no obvious response. Chemical investigation revealed huge accumulation of three new PKS-TPS epoxycyclohexone derivatives with different oxygenated patterns around the epoxycyclohexone moiety and the absence of arthrosporols in the cultural broth of the mutant ΔAOL_s00215g280. These findings suggested that a study on the biosynthetic pathway for morphological regulatory metabolites in nematode-trapping fungus would provide an efficient way to develop new fungal biocontrol agents.

Nematicidal Key Precursors for the Biosynthesis of Morphological Regulatory Arthrosporols in the Nematode-Trapping Fungus Arthrobotrys oligospora.

Arthrobotrys oligospora is the first recognized nematode-trapping fungus and by far the most abundant in the environment. Our recent study revealed the polyketide synthase (PKS) gene AOL_s00215g283 in A. oligospora involved in the production of many secondary metabolites and the trap formation of the fungus. Here we report that the disruption of two genes in the upstream flanking region of the gene AOL_s00215g283, AOL_s00215g281 and AOL_s00215g282, which putatively encoded one amidohydrolase and one cytochrome P450 monooxygenase, respectively, both resulted in significant nematicidal activity of the cultural broths of the mutants and loss of morphological regulatory arthrosporols. Chemical investigation revealed the huge accumulation of 6-methylsalicylic acid in the cultural broth of the mutant ΔAOL_s00215g281 and the high production of m-cresol in the mutant ΔAOL_s00215g282, respectively. Further bioassay revealed that 6-methylsalicylic acid and m-cresol displayed significant nematicidal activity toward root-knot nematodes Meloidogyne incognita with IC90 values of 300 and 100 µg/mL, respectively. The mutant ΔAOL_s00215g282 displayed a more complex metabolite profile than the mutant ΔAOL_s00215g281, suggesting that m-cresol was a more versatile key precursor than 6-methylsalicylic acid. These findings not only demonstrated that the gene AOL_s00215g283 encodes the 6-methylsalicylic acid synthase and the gene AOL_s00215g281 encodes the decarboxylase for 6-methylsalicylic acid but also provided evidence for the potential functions of the precursors in fungal complex biosynthetic pathways and had more implications for the establishment of efficient fungal biocontrol agents.

High Trap Formation and Low Metabolite Production by Disruption of the Polyketide Synthase Gene Involved in the Biosynthesis of Arthrosporols from Nematode-Trapping Fungus Arthrobotrys oligospora.

A group of morphology regulatory arthrosporol metabolites have been recently characterized from carnivorous fungus Arthrobotrys oligospora that can develop trapping networks to capture their prey. A combination of genetic manipulation and chemical analyses was applied to characterize the function of one polyketide synthase (PKS) gene AOL_s00215g283 in A. oligospora, which was putatively involved in the production of 6-methylsalicylic acid. High-performance liquid chromatography analysis showed that the disruption of the PKS gene not only led to the total loss of the arthrosporol A but also resulted in significant reduction in the production of secondary metabolites in the cultural broth of the mutant ΔAOL_s00215g283 strain. Interestingly, the mutant strain displayed significant increases in the trap formation and the nematicidal activity by 10 and 2 times, respectively, higher than the wild-type strain. These findings revealed a pathogenicity-related biosynthetic gene of this agriculturally important biological agent and have implications for establishment of efficient fungal biocontrol agents.

Bacteria can mobilize nematode-trapping fungi to kill nematodes.

In their natural habitat, bacteria are consumed by bacterivorous nematodes; however, they are not simply passive preys. Here we report a defensive mechanism used by certain bacteria to mobilize nematode-trapping fungi to kill nematodes. These bacteria release urea, which triggers a lifestyle switch in the fungus Arthrobotrys oligospora from saprophytic to nematode-predatory form; this predacious form is characterized by formation of specialized cellular structures or 'traps'. The bacteria significantly promote the elimination of nematodes by A. oligospora. Disruption of genes involved in urea transport and metabolism in A. oligospora abolishes the urea-induced trap formation. Furthermore, the urea metabolite ammonia functions as a signal molecule in the fungus to initiate the lifestyle switch to form trap structures. Our findings highlight the importance of multiple predator-prey interactions in prey defense mechanisms.

New insights into the echinocandins and other fungal non-ribosomal peptides and peptaibiotics.

Non-ribosomal peptide synthetases (NRPSs) are a primary modality for fungal peptidic natural product assembly and are responsible for some of the best known, most useful, and most destructive fungal metabolites. Through genome sequencing and computer-assisted recognition of modular motifs of catalytic domains, one can now confidently identify most NRPS biosynthetic genes of a fungal strain. The biosynthetic gene clusters responsible for two of the most important classes of NRP fungal derived drugs, cyclosporine and the echinocandins, have been recently characterized by genomic sequencing and annotation. Complete biosynthetic gene clusters for the pneumocandins and echinocandins have been mapped at the genetic level and functionally characterized to some extent. Genomic sequencing of representative strains of most of the variants in the echinocandin family, including the wild-type of the three fungal strains employed for industrial-scale production of caspofungin, micafungin and anidulofungin, has enabled characterization of the basic architecture of the echinocandin NRPS pathways. A comparative analysis of how pathway genes cause variations in lipoinitiation, biosynthesis of the non-proteinogenic amino acids, amino acid substitutions, and hydroxylations and sulfonations of the core peptide and contribute to the molecular diversity of the family is presented. We also review new information on the natural functions of NRPs, the differences between fungal and bacterial NRPSs, and functional characterization of selected NRPS gene clusters. Continuing discovery of the new fungal nonribosomal peptides has contributed new structural diversity and potential insights into their biological functions among other natural peptides and peptaibiotics. We therefore provide an update on new peptides, depsipeptides and peptaibols discovered in the Fungi since 2009.