Production Of Fungal Metabolites Research Articles

The hidden treasures in endophytic fungi: a comprehensive review on the diversity of fungal bioactive metabolites, usual analytical methodologies, and applications.

This review provides a comprehensive overview of the key aspects of the natural metabolite production by endophytic fungi, which has attracted significant attention due to its diverse biological activities and wide range of applications. Synthesized by various fungal species, these metabolites encompass compounds with therapeutic, agricultural, and commercial significance. We delved into strategies and advancements aimed at optimizing fungal metabolite production. Fungal cultivation, especially by Aspergillus, Penicillium, and Fusarium, plays a pivotal role in metabolite biosynthesis, and researchers have explored both submerged and solid-state cultivation processes to harness the full potential of fungal species. Nutrient optimization, pH, and temperature control are critical factors in ensuring high yields of the targeted bioactive metabolites especially for scaling up processes. Analytical methods that includes High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), Gas Chromatography-Mass Spectrometry (GC-MS), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS), are indispensable for the identification and quantification of the compounds. Moreover, genetic engineering and metabolic pathway manipulation have emerged as powerful tools to enhance metabolite production and develop novel fungal strains with increased yields. Regulation and control mechanisms at the genetic, epigenetic, and metabolic levels are explored to fine-tune the biosynthesis of fungal metabolites. Ongoing research aims to overcome the complexity of the steps involved to ensure the efficient production and utilization of fungal metabolites.

Optimizing solid-state fermentation for metabolite enrichment by Aspergillus tamarii on rice bran and wheat

Globalization has led to an increase in the global population and drug resistance, necessitating the development of novel pharmaceuticals. Fungi represent a promising source for new drugs, producing a diverse array of secondary metabolites, some of which are integral to clinically essential drugs. This study aimed to determine the optimal conditions for enhancing metabolite production by the Indonesian fungal strain Aspergillus tamarii (IPBCC 880066) when cultivated on solid media, specifically rice bran and wheat. The optimal fermentation conditions, including moisture content, growth temperature, and incubation duration, were determined based on the number of spots observed on thin-layer chromatography plates. Results indicated that the optimum conditions for metabolite production were a 40% moisture content, a growth temperature of 25°C, and a 14-day incubation period. Subsequent UPLC-MS/MS analysis identified 22 metabolites produced by A. tamarii on rice bran and 20 metabolites on wheat. Notably, two compounds, N-(2-hydroxypropyl)-2-methylacrylamide and N6-[(benzyloxy)carbonyl]-L-lysine, were detected in both rice bran and wheat fermented by A. tamarii. Some of the identified metabolites have the potential to be applied in the pharmaceutical industry. In conclusion, our study emphasizes A. tamarii’s efficacy in diverse metabolite production under optimized conditions, providing valuable insights for maximizing fungal metabolite production in potential pharmaceutical applications.

Valorization of Agro-Industrial Wastes and Residues through the Production of Bioactive Compounds by Macrofungi in Liquid State Cultures: Growing Circular Economy

Vast quantities of side streams produced worldwide by the agricultural and food industry present an environmental challenge and an opportunity for waste upcycling in the frame of the circular bioeconomy. Fungi are capable of transforming lignocellulosic residues and wastes into a variety of added-value compounds with applications in functional food products, pharmaceuticals, chemicals, enzymes, proteins, and the emerging sector of nutraceuticals. The liquid state culture of fungi is an efficient and potentially scalable and reproducible biotechnological tool that allows the optimized production of fungal metabolites. Particularly, the utilization of agro-industrial by-products, residues, and wastes as a substrate for the liquid culture of macrofungi is suggested as an attainable solution in the management of these streams, contributing to climate change mitigation. This review presents recently published literature in the field of liquid state cultures of macrofungi using agro-industrial side streams, the different substrates, methods, and factors affecting their growth and metabolite production, as well as their applications, focusing on the variety of natural valuable compounds produced.

Chemical modulation of the metabolism of an endophytic fungal strain of Cophinforma mamane using epigenetic modifiers and amino-acids

Endophytic fungi are capable of producing a great diversity of bioactive metabolites. However, the presence of silent and lowly expressed genes represents a main challenge for the discovery of novel secondary metabolites with different potential uses. Epigenetic modifiers have shown to perturb the production of fungal metabolites through the induction of silent biosynthetic pathways leading to an enhanced chemical diversity. Moreover, the addition of bioprecursors to the culture medium has been described as a useful strategy to induce specific biosynthetic pathways. The aim of this study was to assess the effects of different chemical modulators on the metabolic profiles of an endophytic fungal strain of Cophinforma mamane (Botryosphaeriaceae), known to produce 3 thiodiketopiperazine (TDKP) alkaloids (botryosulfuranols A-C), previously isolated and characterized by our team. Four epigenetic modifiers, 5-azacytidine (AZA), sodium butyrate (SB), nicotinamide (NIC), homoserine lactone (HSL) as well as 2 amino acids, l-phenylalanine and l-tryptophan, as bioprecursors of TDKPs, were used. The metabolic profiles were analysed by UHPLC-HRMS/MS under an untargeted metabolomics approach. Our results show that the addition of the two amino acids in C. mamane culture and the treatment with AZA significantly reduced the production of the TDKPs botryosulfuranols A, B and C. Interestingly, the treatment with HSL significantly induced the production of different classes of diketopiperazines (DKPs). The treatment with AZA resulted as the most effective epigenetic modifier for the alteration of the secondary metabolite profile of C. mamane by promoting the expression of cryptic genes.

Effect of Crude Drug Extracts on Trypacidin Production in <i>Aspergillus fumigatus</i>

We examined the production of fungal metabolites as biological responses to 120 crude drugs by culturing the filamentous fungus Aspergillus fumigatus CBS101355 with crude drugs and analyzing the culture extracts by HPLC. Nine crude drug extracts [Kyokatsu (Notopterygium), Kyonin (apricot kernel), Kujin (Sophora root), Goboshi (Burdock fruit), Goma (sesame), Shokyo (ginger), Shin’i (magnolia flower), Togashi (Benincasa seed), and Bukuryo (Poria sclerotium)] induced the production of trypacidin, which was not produced by culturing in potato dextrose broth without crude drugs.

Toward Engineered Biosynthesis of Drugs in Human Cells.

Biosynthetic genes are not only responsible for the formation of bioactive substances but also suited for other applications including gene therapy. To test the feasibility of human cells producing antibiotics in situ when provided with a heterologous biosynthetic gene, we focused on cytochrome P450, the class of enzymes important in conferring bioactivity to natural product precursors. We selected Fma-P450 that plays a central role in the fumagillin antimicrobial biosynthesis in Aspergillus fumigatus to examine fungal metabolite production by HeLa cells that express fma-P450 heterologously. Here we show that HeLa cells harboring fma-P450 can biosynthesize 5-hydroxyl-β-trans-bergamoten and cytotoxic 5-epi-demethoxyfumagillol when supplemented with the nontoxic precursor β-trans-bergamotene. While the production level was insufficient to effect cell death, we demonstrate that programming human cells to autogenerate antibiotics by introducing a heterologous biosynthetic gene is feasible.

Aspergillus flavus originated pure compound as a potential antibacterial

Problem BackgroundPenicillin was the first and most famous fungal secondary metabolite used as broad spectrum antibiotic that revolutionarised pharmaceutical research and also saved millions of lives. The over optimistic belief in 1967 that sufficient antibiotics had been discovered to defeat infectious diseases was quickly crashed with the appearance of multidrug resistant (MDR) bacteria in 1990s. This has posed a serious threat to mankind. Although scientists are making efforts to synthesize and discover new antibiotics there are not enough new drugs in pharmaceutical pipeline to beat the pace at which MDR bacteria are emerging. In view of this there is an urgent and serious medical need for new bioactive compounds to be discovered to treat infections caused by MDR pathogens. The present study is aimed to investigate the antibacterial potential of Aspergillus flavus originated compounds that may act as drug leads to treat future infections.MethodologyAmong the 6 isolated fungal strains from the rhizosphere of Mentha piperetta, one was processed for isolation of secondary metabolites on the basis of preliminary antibacterial testing. Observation of morphological and microscopic features helped in identification of the fungal strain as Aspergillus flavus. Potato Dextrose Agar (PDA) medium was used for fungal growth while Czapec Yeast Broth (CYB) medium was used for production of fungal metabolites. Column chromatography technique was utilized for purification of compound from crude fungal extract and the mass of the compound was determined using Liquid Chromatography Mass Spectrometry (LCMS) method. Structure elucidation of the pure compound was performed using 500 Varian Nuclear Magnetic Resonance (NMR) machine. Docking was performed using Glide SP algorithm. Agar well diffusion method was used to determine the invitro antibacterial potential of the compound against two MDR bacterial strains i.e. Staphylococcus aureus and Proteus vulgaris. For this a total of 4 dose concentrations i.e. (100, 250, 500, 1000 μg mL− 1) of the compound were prepared and applied to bacterial strains on Mueller Hinton agar using tetracycline as control.ResultsThe chemical name of the purified compound from A. flavus was determined as (2E)-3-[(3S, 4R)-8-hydroxy-3, 4-dimethyl-1-oxo-3, 4-dihydro-1H-2- benzopyran-7-yl] prop-2-enoic acid with the formula C14H14O5 and exact mass of 262.08. The in-Silico analysis showed that this compound has the potential to inhibit the binding pocket of S. aureus TyrRS (1JII) with docking score of − 8.67 Kcal mole− 1. The results obtained from invitro experiments were encouraging as at 1000 μg mL− 1 the compound showed 58.8% inhibition against S. aureus and 28% inhibition against P. vulgaris.ConclusionsThe pure compound with formula C14H14O5 and exact mass of 262 exhibited antibacterial potential both insilico and invitro against both Gram negative and Gram positive bacteria. The compound was more active against S. aureus in comparison to P. vulgaris. From the obtained results it is concluded that this compound can be used as potent antibacterial candidate but further studies will be needed prior to its use as antibiotic.

Fungal Diversity and Mycotoxins in Low Moisture Content Ready-To-Eat Foods in Nigeria.

Low moisture content ready-to-eat foods vended in Nigerian markets could be pre-packaged or packaged at point of sale. These foods are widely and frequently consumed across Nigeria as quick foods. Despite their importance in the daily diets of Nigerians, a comprehensive study on the diversity of fungi, fungal metabolite production potential, and mycotoxin contamination in the foods has not yet been reported. Therefore, this study assessed the diversity of fungi in 70 samples of low moisture content ready-to-eat foods [cheese balls, garri (cassava-based), granola (a mix of cereals and nuts) and popcorn] in Nigeria by applying a polyphasic approach including morphological examination, genera/species-specific gene marker sequencing and secondary metabolite profiling of fungal cultures. Additionally, mycotoxin levels in the foods were determined by LC–MS/MS. Fungal strains (n = 148) were recovered only from garri. Molecular analysis of 107 representative isolates revealed 27 species belonging to 12 genera: Acremonium, Allophoma, Aspergillus, Cladosporium, Fusarium, Microdochium, Penicillium, Sarocladium, Talaromyces, and Tolypocladium in the Ascomycota, and Fomitopsis and Trametes in the Basidiomycota. To the best of our knowledge Allophoma, Fomitopsis, Microdochium, Tolypocladium, and Trametes are reported in African food for the first time. A total of 21 uncommon metabolites were found in cultures of the following species: andrastin A and sporogen AO1 in Aspergillus flavus; paspalin in A. brunneoviolaceus; lecanoic acid and rugulusovin in A. sydowii; sclerotin A in P. citrinum and Talaromyces siamensis; barceloneic acid, festuclavine, fumigaclavine, isochromophilons (IV, VI, and IX), ochrephilone, sclerotioramin, and sclerotiorin in P. sclerotium; epoxyagroclavine, infectopyron, methylorsellinic acid and trichodermamide C in P. steckii; moniliformin and sporogen AO1 in P. copticola; and aminodimethyloctadecanol in Tolypocladium. Twenty-four mycotoxins in addition to other 73 fungal and plant toxins were quantified in the foods. In garri, cheeseballs, popcorn and granola were 1, 6, 12, and 23 mycotoxins detected, respectively. Deoxynivalenol, fumonisins, moniliformin, aflatoxins and citrinin contaminated 37, 31, 31, 20, and 14% of all food samples, respectively. Overall, citrinin had the highest mean concentration of 1481 μg/kg in the foods, suggesting high citrinin exposures in the Nigerian populace. Fungal and mycotoxin contamination of the foods depend on pre-food and post-food processing practices.

Challenges of Enzymes, Conidia and 6-Pentyl-alpha-pyrone Production from Solid-State-Fermentation of Agroindustrial Wastes Using Experimental Design and T. asperellum Strains

In a context of growing awareness regarding environmental protection, biomass valorization is gaining a lot of attention. The byproducts volumes generated by agro-industry are massive and, left to decay, can constitute environmental pollutions. Use of agro-industrial wastes and solid-state fermentation (SSF) technology offers advantages to produce value-added products such as antibiotics, pigments, aromas and enzymes of industrial interest like cellulases, chitinases, amylases, etc. Several studies have already demonstrated the advantages of SSF for the production of fungal metabolites, yet the optimal conditions for metabolites production strongly depend on the culture conditions and microbial strain utilized. Therefore, the aim of this study was to improve the conidia, lytic enzymes (cellulase, lipase and amylase), and antifungal—6-pentyl-alpha-pyrones (6-PP)—production by three Trichoderma asperellum strains cultivated using SSF. Designs of experiments have been achieved in order to identify influential factors on 6-PP, conidia and enzymes (cellulase, lipase, and amylase) production by the fungal culture. A significantly enzymes activities, conidiation and 6-PP production were observed on mix of substrates: vine shoots, potatoes flour, jatropha, olive pomace and olive oil on high carbon/nitrogen ratio 37 which was used by T. asperellum TV104 as a source of nutrients and also as a matrix.

Exploration of insecticidal potential of an alpha glucosidase enzyme inhibitor from an endophytic Exophiala spinifera.

The present study aimed to isolate and screen endophytes from Trachyspermum ammi with the ability to inhibit alpha glucosidase enzyme and evaluate their insecticidal potential. Endophytic fungi isolated from T. ammi were screened for alpha glucosidase inhibitory activity. Maximum inhibition (96%) was observed in an isolate AZ-9, identified to be Exophiala spinifera on morphological and molecular basis. Production of fungal metabolites was carried out in malt extract broth followed by extraction with ethyl acetate. Brown coloured gummy residue obtained after evaporation of ethyl acetate was partially soluble in water yielding white precipitates. The precipitate exhibiting α-glucosidase inhibitory activity was purified by repeated washing and centrifugation. The insecticidal activity of inhibitor was evaluated on Spodoptera litura (Fab.) by feeding this pest on diet amended with inhibitor. It resulted in significant larval mortality as well as deformities in emerging adults. A reduction invivo digestive enzyme activity was also observed. Nutritional analysis revealed the toxic effect of AZ-9 inhibitor on various food utilization parameters of S. litura. A significant reduction was recorded in relative growth and consumption rate of S. litura. This is the first report on production of an alpha glucosidase inhibitor from E. spinifera with insecticidal activity. The study highlights the importance of endophytes in providing protection against insect pests to the host. It also suggests the insecticidal potential of alpha glucosidase inhibitor from E. spinifera against polyphagous pest S. litura.

Carbon catabolite repression gene creA regulates morphology, aflatoxin biosynthesis and virulence in Aspergillus flavus

Carbon catabolite repression (CCR) is a very important mechanism employed in the utilization of carbon as an energy source, required for the regulation of growth, development and secondary metabolite production in fungi. Despite the wide study of this mechanism in fungi, little is known about the major CCR gene creA in A. flavus. Hence, we report identification of A. flavus carbon catabolite repression gene creA, which is responsible for the repression of secondary carbon sources. Gene deletion and over-expression was employed to explicate the role of creA in the morphology, pathogenicity, and secondary metabolite production in A. flavus. We investigated these factors using three carbon sources including glucose, sucrose and maltose. Gene deletion mutant (ΔcreA) had a significant growth defect on complete medium and minimal medium containing maltose. Conidia production in ΔcreA was significantly impaired irrespective of the carbon source available, while sclerotia production was significantly increased, compared to wild type (WT) and over-expression strain (OE::creA). Importantly, ΔcreA produced insignificant amount of aflatoxin in complete medium, and its ability to colonize hosts was also impaired. Concisely, we showed that creA played an important role in the morphology, pathogenicity and secondary metabolite production of A. flavus.

Production of an emericellin and its analogues as fungal biological responses for Shimbu-to extract.

This research examined the production of fungal metabolites as a biological response to Kampo medicines. Shimbu-to (SMB) is a Kampo medicine composed of five herbal components: peony root (Shakuyaku), ginger (Shokyo), processed aconite root (Bushi), Poria sclerotium (Bukuryo), and Atractylodes lancea rhizomes (Sojutsu). High-performance liquid chromatography (HPLC) analysis of the fungus Aspergillus nidulans CBS 112.46 incubated in potato dextrose broth supplemented with SMB extract revealed emericellin (2) as the major peak and new xanthone analogues 24-hydroxyshamixanthone (1), shamixanthone (3), epishamixanthone (4), pre-shamixanthone (5), and variecoxanthone A (6) as minor peaks. The structure of 1 was determined by detailed analysis of 1D-NMR, 2D-NMR, and MS data. The results suggest that SMB extract regulates the biosynthesis of emericellin and its analogues in A. nidulans. Further investigations revealed that glucose induces the biosynthesis of emericellin and its analogues in A. nidulans in a concentration-dependent manner.

Antimicrobial properties and the influence of temperature on secondary metabolite production in cold environment soil fungi

The Arctic and Antarctic share environmental extremes. To survive in such environments, microbes such as soil fungi need to compete with or protect themselves effectively from other soil microbiota and to obtain the often scarce nutrients available, and many use secondary metabolites to facilitate this. We therefore (i) screened for antimicrobial properties of cold-environment Arctic and Antarctic soil fungi, and (ii) identified changes in the secreted secondary metabolite profiles of a subset of these strains in response to temperature variation. A total of 40 polar soil fungal strains from King George Island, maritime Antarctic and Hornsund, Svalbard, High Arctic, were obtained from the Malaysian National Antarctic Research Centre culture collections. The plug assay technique was used to screen for antimicrobial potential against Gram-positive and Gram-negative human pathogenic bacteria (Bacillus subtilis, B. cereus, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichia coli). About 45% of the tested fungal strains showed antimicrobial activity against at least one tested microorganism. Three fungal isolates showed good bioactivity and were subjected to secondary metabolite profiling at different temperatures (4, 10, 15 and 28 °C). We observed a range of responses in fungal metabolite production when incubated at varying temperatures, confirming an influence of environmental conditions such as temperature on the production of secondary metabolites.

Induction of apoptosis and ganoderic acid biosynthesis by cAMP signaling in Ganoderma lucidum

Apoptosis is an essential physiological process that controls many important biological functions. However, apoptosis signaling in relation to secondary metabolite biosynthesis in plants and fungi remains a mystery. The fungus Ganoderma lucidum is a popular herbal medicine worldwide, but the biosynthetic regulation of its active ingredients (ganoderic acids, GAs) is poorly understood. We investigated the role of 3′,5′-cyclic adenosine monophosphate (cAMP) signaling in fungal apoptosis and GA biosynthesis in G. lucidum. Two phosphodiesterase inhibitors (caffeine and 3-isobutyl-1-methylxanthine, IBMX) and an adenylate cyclase activator (sodium fluoride, NaF) were used to increase intracellular cAMP levels. Fungal apoptosis was identified by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and a condensed nuclear morphology. Our results showed that GA production and fungal apoptosis were induced when the mycelium was treated with NaF, caffeine, or cAMP/IBMX. Downregulation of squalene synthase and lanosterol synthase gene expression by cAMP was detected in the presence of these chemicals, which indicates that these two genes are not critical for GA induction. Transcriptome analysis indicated that mitochondria might play an important role in cAMP-induced apoptosis and GA biosynthesis. To the best of our knowledge, this is the first report to reveal that cAMP signaling induces apoptosis and secondary metabolite production in fungi.

Reconstitution of biosynthetic machinery of fungal polyketides: unexpected oxidations of biosynthetic intermediates by expression host.

Reconstitution of whole biosynthetic genes in Aspergillus oryzae has successfully applied for total biosynthesis of various fungal natural products. Heterologous production of fungal metabolites sometimes suffers unexpected side reactions by host enzymes. In the studies on fungal polyketides solanapyrone and cytochalasin, unexpected oxidations of terminal olefin of biosynthetic intermediates were found to give one and four by-products by host enzymes of the transformants harboring biosynthetic genes. In this paper, we reported structure determination of by-products and described a simple solution to avoid the undesired reaction by introducing the downstream gene in the heterologous production of solanapyrone C.

Multicomponent Analysis of the Differential Induction of Secondary Metabolite Profiles in Fungal Endophytes.

Small molecule histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors are commonly used to perturb the production of fungal metabolites leading to the induction of the expression of silent biosynthetic pathways. Several reports have described the variable effects observed in natural product profiles in fungi treated with HDAC and DNMT inhibitors, such as enhanced chemical diversity and/or the induction of new molecules previously unknown to be produced by the strain. Fungal endophytes are known to produce a wide variety of secondary metabolites (SMs) involved in their adaptation and survival within higher plants. The plant-microbe interaction may influence the expression of some biosynthetic pathways, otherwise cryptic in these fungi when grown in vitro. The aim of this study was to setup a systematic approach to evaluate and identify the possible effects of HDAC and DNMT inhibitors on the metabolic profiles of wild type fungal endophytes, including the chemical identification and characterization of the most significant SMs induced by these epigenetic modifiers.

Antimicrobial Potential of Crude Extract of Curvularia lunata, an Endophytic Fungi Isolated from Cymbopogon caesius

Cymbopogon caesius grass is an aromatic tall grass species which grows in waste field and along the edges of crop fields in Karnataka. From this grass fungal endophytes were isolated on PDA medium and identified as Curvularia lunata. The fungus was cultured on Potato Dextrose broth for production of fungal metabolites. Ethyl acetate extract of the C. lunata was obtained by liquid-liquid partition of broth of endophyte and evaporation. Dried crude extract was tested for antimicrobial activity by agar well diffusion method. The extract showed antimicrobial nature against all the test pathogens. Crude extract of C. lunata is highly active against Staphylococcus aureus with the inhibition percentage of 92 and Candida albicans with 81 percent inhibition.

Bioremediation of TNT contaminated soil with fungi under laboratory and pilot scale conditions

Bioremediation of contaminated soil involves the utilization of innovative technology such as mycoremediation. The capability of fungi to grow in 2,4,6-trinitrotoluene (TNT) contaminated soil, to produce lignin modifying enzymes and to degrade TNT in soil was studied. White-rot fungi, namely Gymnopilus luteofolius, Kuehneromyces mutabilis, and Phanerochaete velutina, were incubated with TNT contaminated non-sterile soil (1000 mg kg−1). All the fungi produced high amounts of manganese peroxidase (MnP) in TNT contaminated soil, but no laccase. The most efficient fungus, P. velutina, degraded 80% of TNT in 2.5 months and was selected for further scale-up experiment with 0.3 t (0.56 m3) of soil and inoculum soil ratio of 1:30. The degradation of TNT was 70% in 49 days, and production of fungal metabolites, namely 4-amino-2,6-diaminotoluene and 2-amino-4,6-diaminotoluene was only 1% of the original TNT concentration. These metabolites were degraded to less than 0.5% during the following 58 days incubation. Fungal remediation process was scaled-up with P. velutina, which tolerated high concentration of TNT and was able to invade the whole mass of soil with only 10 kg of fungal inoculum growing on pine bark. With these parameters the process should be easy to scale-up for soil treatment in field.

Exploring the chemodiversity and biological activities of the secondary metabolites from the marine fungus Neosartorya pseudofischeri.

The production of fungal metabolites can be remarkably influenced by various cultivation parameters. To explore the biosynthetic potentials of the marine fungus, Neosartorya pseudofischeri, which was isolated from the inner tissue of starfish Acanthaster planci, glycerol-peptone-yeast extract (GlyPY) and glucose-peptone-yeast extract (GluPY) media were used to culture this fungus. When cultured in GlyPY medium, this fungus produced two novel diketopiperazines, neosartins A and B (1 and 2), together with six biogenetically-related known diketopiperazines,1,2,3,4-tetrahydro-2,3-dimethyl-1,4-dioxopyrazino[1,2-a]indole (3), 1,2,3,4-tetrahydro-2-methyl-3-methylene-1,4-dioxopyrazino[1,2-a]indole (4), 1,2,3,4-tetrahydro-2-methyl-1,3,4-trioxopyrazino[1,2-a] indole (5), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio)gliotoxin (11), didehydrobisdethiobis(methylthio)gliotoxin (12) and N-methyl-1H-indole-2-carboxamide (6). However, a novel tetracyclic-fused alkaloid, neosartin C (14), a meroterpenoid, pyripyropene A (15), gliotoxin (7) and five known gliotoxin analogues, acetylgliotoxin (8), reduced gliotoxin (9), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio) gliotoxin (11) and bis-N-norgliovictin (13), were obtained when grown in glucose-containing medium (GluPY medium). This is the first report of compounds 3, 4, 6, 9, 10 and 12 as naturally occurring. Their structures were determined mainly by MS, 1D and 2D NMR data. The possible biosynthetic pathways of gliotoxin-related analogues and neosartin C were proposed. The antibacterial activity of compounds 2–14 and the cytotoxic activity of compounds 4, 5 and 7–13 were evaluated. Their structure-activity relationships are also preliminarily discussed.

Activation of dormant secondary metabolite production by introducing neomycin resistance into the deep-sea fungus, Aspergillus versicolor ZBY-3.

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.