Unveiling the fungal diversity and associated secondary metabolism on black apples.

Black apples are the result of late-stage microbial decomposition after falling to the ground. This phenomenon is highly comparable from year to year, with the filamentous fungus Monilinia fructigena most commonly being the first invader, followed by Penicillium expansum. Motivated by the fact that only little chemistry has been reported from apple microbiomes, we set out to investigate the chemical diversity and potential ecological roles of secondary metabolites (SMs) in a total of 38 black apples. Metabolomics analyses were conducted on either whole apples or small excisions of fungal biomass derived from black apples. Annotation of fungal SMs in black apple extracts was aided by the cultivation of 15 recently isolated fungal strains on 9 different substrates in a One Strain Many Compounds (OSMAC) approach, leading to the identification of 3,319 unique chemical features. Only 6.4% were attributable to known compounds based on analysis of high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS/MS) data using spectral library matching tools. Of the 1,606 features detected in the black apple extracts, 32% could be assigned as fungal-derived, due to their presence in the OSMAC-based training data set. Notably, the detection of several antifungal compounds indicates the importance of such compounds for the invasion of and control of other microbial competitors on apples. In conclusion, the diversity and abundance of microbial SMs on black apples were found to be much higher than that typically observed for other environmental microbiomes. Detection of SMs known to be produced by the six fungal species tested also highlights a succession of fungal growth following the initial invader M. fructigena.IMPORTANCEMicrobial secondary metabolites constitute a significant reservoir of biologically potent and clinically valuable chemical scaffolds. However, their usefulness is hampered by rapidly developing resistance, resulting in reduced profitability of such research endeavors. Hence, the ecological role of such microbial secondary metabolites must be considered to understand how best to utilize such compounds as chemotherapeutics. Here, we explore an under-investigated environmental microbiome in the case of black apples; a veritable "low-hanging fruit," with relatively high abundances and diversity of microbially produced secondary metabolites. Using both a targeted and untargeted metabolomics approach, the interplay between metabolites, other microbes, and the apple host itself was investigated. This study highlights the surprisingly low incidence of known secondary metabolites in such a system, highlighting the need to study the functionality of secondary metabolites in microbial interactions and complex microbiomes.

New cytotoxic indole derivatives with anti-FADU potential produced by the endophytic fungus Penicillium oxalicum 2021CDF-3 through the OSMAC strategy.

Fungi possess well-developed secondary metabolism pathways that are worthy of in-depth exploration. The One Strain Many Compounds (OSMAC) strategy is a useful method for exploring chemically diverse secondary metabolites. In this study, continued chemical investigations of the marine red algae-derived endophytic fungus Penicillium oxalicum 2021CDF-3 cultured in PDB media yielded six structurally diverse indole derivatives, including two new prenylated indole alkaloids asperinamide B (1) and peniochroloid B (5), as well as four related derivatives (compounds 2-4 and 6). The chemical structures of these compounds, including the absolute configurations of 1 and 5, were determined by extensive analyses of HRESIMS, 1D and 2D NMR spectroscopic data, and TDDFT-ECD calculations. Compound 1 was found to possess an unusual 3-pyrrolidone dimethylbenzopyran fused to the bicyclo[2.2.2]diazaoctane moiety, which was rare in previously reported prenylated indole alkaloids. In vitro cytotoxic experiments against four human tumor cell lines (HeLa, HepG2, FADU, and A549) indicated that 1 strongly inhibited the FADU cell line, with an IC50 value of 0.43 ± 0.03 µM. This study suggested that the new prenylated indole alkaloid 1 is a potential lead compound for anti-FADU drugs.

AflaILVB/G/I and AflaILVD are involved in mycelial production, aflatoxin biosynthesis, and fungal virulence in Aspergillus flavus.

Aflatoxins (AFs) are produced by fungi such as Aspergillus flavus and A. parasiticus and are one of the most toxic mycotoxins found in agricultural products and food. Aflatoxin contamination, which requires the control of A. flavus, remains problematic because of the lack of effective strategies and the exploration of new compounds that can inhibit A. flavus growth and mycotoxin production is urgently required to alleviate potential deleterious effects. Acetohydroxy acid synthase (AHAS) and dihydroxy acid dehydratase are important enzymes in the biosynthetic pathways of branched-chain amino acids (BCAAs), including isoleucine, leucine, and valine. Enzymes involved in BCAA biosynthesis are present in bacteria, plants, and fungi, but not in mammals, and are therefore, attractive targets for antimicrobial and herbicide development. In this study, we characterized AflaILVB/G/I and AflaILVD, which encode the catalytic and regulatory subunits of AHAS and dihydroxy acid dehydratase, from the pathogenic fungus Aspergillus flavus. The AflaILVB/G/I and AflaILVD deletion mutant grew slower and produced smaller colonies than the wild-type strain when grown on glucose minimal medium, potato dextrose agar, and yeast extract medium for three days at 28°C, and disruption of AflaILVB/G/I caused a significant reduction in conidia production when grown on all kinds of media. Cellular stress assays determined that all strains were sensitive to H2O2. Importantly, the pathogenicity and aflatoxin production were affected when AflaILVB/G/I and AflaILVD were knocked out, particularly AflaILVB/G/I. A series of genes that encoded enzymes involved in aflatoxin synthesis were downregulated, meaning that the knockout of AflaILVB/G/I influenced aflatoxin synthesis in A. flavus strain WT. Collectively, our results demonstrate the potential value of antifungals targeting AflaILVB/G/I in A. flavus.

Fungal metabolite 6-pentyl-α-pyrone reduces canine coronavirus infection.

Canine coronavirus (CCoV) can produce a self-limited enteric disease in dogs but, because of notable biological plasticity of coronaviruses (CoVs), numerous mutations as well as recombination events happen leading to the emergence of variants often more dangerous for both animals and humans. Indeed, the emergence of new canine-feline recombinant alphacoronaviruses, recently isolated from humans, highlight the cross-species transmission potential of CoVs. Consequently, new effective antiviral agents are required to treat CoV infections. Among the candidates for the development of drugs against CoVs infection, fungal secondary metabolites (SMs) represent an important source to investigate. Herein, antiviral ability of 6-pentyl-α-pyrone (6 PP), a SM obtained by Trichoderma atroviride, was assessed against CCoV. During in vitro infection, nontoxic concentration of 6 PP significantly increased cell viability, reduced morphological signs of cell death, and inhibited viral replication of CCoV. In addition, we found a noticeable lessening in the expression of aryl hydrocarbon receptor (AhR), a strategic modulator of CoVs infection. Overall, due to the variety of their chemical and biological properties, fungal SMs can decrease the replication of CoVs, thus identifying a suitable in vitro model to screen for potential drugs against CoVs, using a reference strain of CCoV (S/378), non-pathogenic for humans.

Exploring the in vivo anti-cancer potential of Neosetophomone B in leukemic cells using a zebrafish xenograft model.

Neosetophomone B (NSP-B) is a unique meroterpenoid fungal secondary metabolite that has previously demonstrated promising anti-cancer properties against various cancer cell lines in vitro. However, its in vivo anti-cancer potential remaines unexplored. To fill this gap in our knowledge, we tested NSP-B's in vivo anti-cancer activity using a zebrafish model, an organism that has gained significant traction in biomedical research due to its genetic similarities with humans and its transparent nature, allowing real-time tumor growth observation. For our experiments, we employed the K562-injected zebrafish xenograft model. Upon treating these zebrafish with NSP-B, we observed a marked reduction in the size and number of tumor xenografts. Delving deeper, our analyses indicated that NSP-B curtailed tumor growth and proliferation of leukemic grafted xenograft within the zebrafish. These results show that NSP-B possesses potent in vivo anti-cancer properties, making it a potential novel therapeutic agent for addressing hematological malignancies.

Phytotoxic fungal secondary metabolites as herbicides.

Among the alternatives to synthetic plant protection products, biocontrol appears as a promising method. This review reports on the diversity of fungal secondary metabolites phytotoxic to weeds and on the approach generally used to extract, characterize, identify and exploit them for weed management. The 183 phytotoxic fungal secondary metabolites discussed in this review fall into five main classes of molecules: 61 polyketides, 53 terpenoids, 36 nitrogenous metabolites, 18 phenols and phenolic acids, and 15 miscellaneous. They are mainly produced by the genera Drechslera, Fusarium and Alternaria. The phytotoxic effects, more often described by the symptoms they produce on plants than by their mode of action, range from inhibition of germination to inhibition of root and vegetative growth, including tissue and organ alterations. The biochemical characterization of fungal secondary metabolites requires expertise and tools to carry out fungal cultivation and metabolite extraction, phytotoxicity tests, purification and fractionation of the extracts, and chemical identification procedures. Phytotoxicity tests are mainly carried out under controlled laboratory conditions (not always on whole plants), while effectiveness against targeted weeds and environmental impacts must be assessed in greenhouses and open fields. These steps are necessary for the formulation of effective, environment-friendly fungal secondary metabolites-derived bioherbicides using new technologies such as nanomaterials. © 2023 Society of Chemical Industry.

New prenylated indole-benzodiazepine-2,5-diones with α-glucosidase inhibitory activities from the mangrove-derived Aspergillus spinosus.

Mangrove-derived fungi have been demonstrated to be promising source of structurally diverse and widely active secondary metabolites. During our search for new bioactive compounds, eight new indole-benzodiazepine-2,5-dione derivatives asperdinones A-H (1-8) and two known congeners (9 and 10) were isolated from the culture extracts of the mangrove-derived fungus Aspergillus spinosus WHUF0344 guided by one strain many compounds (OSMAC) and the heteronuclear 1H, 13C single-quantum coherence (HSQC) based small molecule accurate recognition technology (SMART) strategies. The structures and absolute configurations of the new compounds were elucidated by detailed spectroscopic analyze and electronic circular dichroism (ECD) calculations. The putative biosynthetic pathway of these compounds was proposed. Compounds 1-10 were evaluated for their antibacterial and α-glucosidase inhibitory activities. None of compounds showed antibacterial activity. Compounds 2-6 and 8 exhibited moderate inhibitory effects against α-glucosidase with IC50 values in the range of 24.65-312.25 µM. Besides, both 3 and 4 inhibited α-glucosidase variedly. Furthermore, the molecular docking study showed that compounds 2-4 were perfectly docking into the active sites of α-glucosidase. This study not only enriched the chemical diversity of secondary metabolites from the mangrove-derived fungi, but also provided potential hit compounds for further development of α-glucosidase inhibitors.

Identifying Fungal Secondary Metabolites and Their Role in Plant Pathogenesis.

Pathogenic fungi are the main infectious agents of plants. Secondary metabolites produced by these fungi, also recognized as natural products, are key mediators of plant-fungal interactions. Knowledge on the biosynthesis of these metabolites, the accessibility to fungal genome sequences, and the development of gene disruption techniques open up opportunities to identify many more of these metabolites both in vitro and in planta. This methodology chapter gives a detailed systematic approach aiming to discover new natural products from phytopathogenic fungi and characterize their role in triggering plant cell death and plant disease. This approach takes advantage of the global regulation of fungal secondary metabolite production by regulatory proteins reported in various fungal species.

Amesia hispanica sp. nov., Producer of the Antifungal Class of Antibiotics Dactylfungins.

During a study of the diversity of soilborne fungi from Spain, a strain belonging to the family Chaetomiaceae (Sordariales) was isolated. The multigene phylogenetic inference using five DNA loci showed that this strain represents an undescribed species of the genus Amesia, herein introduced as A. hispanica sp. nov. Investigation of its secondary metabolome led to the isolation of two new derivatives (2 and 3) of the known antifungal antibiotic dactylfungin A (1), together with the known compound cochliodinol (4). The planar structures of 1-4 were determined by ultrahigh performance liquid chromatography coupled with diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS) and extensive 1D and 2D nuclear magnetic resonance (NMR) spectroscopy after isolation by HPLC. All isolated secondary metabolites were tested for their antimicrobial and cytotoxic activities. Dactylfungin A (1) showed selective and strong antifungal activity against some of the tested human pathogens (Aspergillus fumigatus and Cryptococcus neoformans). The additional hydroxyl group in 2 resulted in the loss of activity against C. neoformans but still retained the inhibition of As. fumigatus in a lower concentration than that of the respective control, without showing any cytotoxic effects. In contrast, 25″-dehydroxy-dactylfungin A (3) exhibited improved activity against yeasts (Schizosaccharomyces pombe and Rhodotorula glutinis) than 1 and 2, but resulted in the appearance of slight cytotoxicity. The present study exemplifies how even in a well-studied taxonomic group such as the Chaetomiaceae, the investigation of novel taxa still brings chemistry novelty, as demonstrated in this first report of this antibiotic class for chaetomiaceous and sordarialean taxa.

Characterization and metabolomic profiling of two pigment producing fungi from infected fruits of Indian Gooseberry.

Two pigment producing fungi, Talaromyces atroroseus and Penicillium choerospondiatis, were isolated and identified from infected fruits of Phyllanthus emblica L. based on amplification and sequencing of internal transcribed spacer region and beta-tubulin gene. This is the first occurrence report of these two fungi from fruits of P. emblica. Culture extract containing metabolites of T. atroroseus and P. choerospondiatis contained phenolics of 26.35 mg and 30.89 mg GAE/g dry extract respectively; whereas no significant amount of flavonoids and tannins were detected. P. choerospondiatis metabolites extract showed higher DPPH and ABTS activity with IC50 values of 21.94 mg/ml and 27.03 mg/ml respectively than T. atroroseus. LC-HRMS analysis of metabolites extract of T. atroroseus revealed presence of trimethyl-isopropyl-butanamide, perlolyrine, N-hexadecanoylpyrrolidine etc. whereas P. choerospondiatis displayed presence of tangeraxanthin, ugaxanthone, daphniphylline, etc. Therefore, fungal metabolites are rich natural sources of diversified compounds that can be utilized in dyeing industries, cosmetics and novel drug development.

Mycotoxin Contamination in Hazelnut: Current Status, Analytical Strategies, and Future Prospects.

Hazelnuts represent a potential source of mycotoxins that pose a public health issue due to their increasing consumption as food ingredients worldwide. Hazelnuts contamination by mycotoxins may derive from fungal infections occurring during fruit development, or in postharvest. The present review considers the available data on mycotoxins detected in hazelnuts, on fungal species reported as infecting hazelnut fruit, and general analytical approaches adopted for mycotoxin investigation. Prompted by the European safety regulation concerning hazelnuts, many analytical methods have focused on the determination of levels of aflatoxin B1 (AFB1) and total aflatoxins. An overview of the available data shows that a multiplicity of fungal species and further mycotoxins have been detected in hazelnuts, including anthraquinones, cyclodepsipeptides, ochratoxins, sterigmatocystins, trichothecenes, and more. Hence, the importance is highlighted in developing suitable methods for the concurrent detection of a broad spectrum of these mycotoxins. Moreover, control strategies to be employed before and after harvest in the aim of controlling the fungal contamination, and in reducing or inactivating mycotoxins in hazelnuts, are discussed.

Diagnosis of Aspergillosis in Horses.

Invasive pulmonary aspergillosis (IPA) may be a rare cause of granulomatous pneumonia in horses. The mortality of IPA is almost 100%; direct diagnostic tools in horses are needed. Bronchoalveolar lavage fluid (BALF) and serum samples were collected from 18 horses, including individuals suffering from IPA (n = 1), equine asthma (EA, n = 12), and 5 healthy controls. Serum samples were collected from another 6 healthy controls. Samples of BALF (n = 18) were analyzed for Aspergillus spp. DNA, fungal galactomannan (GM), ferricrocin (Fc), triacetylfusarinin C (TafC), and gliotoxin (Gtx). Analysis of 24 serum samples for (1,3)-ß-D-glucan (BDG) and GM was performed. Median serum BDG levels were 131 pg/mL in controls and 1142 pg/mL in IPA. Similar trends were observed in BALF samples for GM (Area under the Curve (AUC) = 0.941) and DNA (AUC = 0.941). The fungal secondary metabolite Gtx was detected in IPA BALF and lung tissue samples (86 ng/mL and 2.17 ng/mg, AUC = 1).

Antibacterial spirooxindole alkaloids from Penicillium brefeldianum inhibit dimorphism of pathogenic smut fungi.

Three new antibacterial spirooxindole alkaloids, spirobrefeldins A-C (1-3), together with four known analogs, spirotryprostatin M (4), spirotryprostatin G (5), 12ß-hydroxyverruculogen TR-2 (6), and 12α-hydroxyverruculogen TR-2 (7), were isolated from terrestrial fungus Penicillium brefeldianum. All the new compounds were elucidated extensively by the interpretation of their NMR (1D and 2D) spectra and high-resolution mass data, and their absolute configurations were determined by computational chemistry and CD spectra. The absolute configurations of spiro carbon C-2 in spirotryprostatin G (5) and spirotryprostatin C in literature were reported as S, which were revised to R based on experimental and calculated CD spectra. All the compounds were evaluated for their antimicrobial activities toward Pseudomonas aeruginosa PAO1, Dickeya zeae EC1, Staphylococcus epidermidis, Escherichia coli, and Sporisorium scitamineum. Compound 7 displayed moderate inhibitory activity toward dimorphic switch of pathogenic smut fungi Sporisorium scitamineum at 25 µM. Compounds 3 and 6 showed weak antibacterial activities against phytopathogenic bacterial Dickeya zeae EC1 at 100 µM.

Terrien, a metabolite made by Aspergillus terreus, has activity against Cryptococcus neoformans.

Antimicrobial compounds, including antibiotics, have been a cornerstone of modern medicine being able to both treat infections and prevent infections in at-risk people, including those who are immune-compromised and those undergoing routine surgical procedures. Their intense use, including in people, animals, and plants, has led to an increase in the incidence of resistant bacteria and fungi, resulting in a desperate need for novel antimicrobial compounds with new mechanisms of action. Many antimicrobial compounds in current use originate from microbial sources, such as penicillin from the fungus Penicillium chrysogenum (renamed by some as P. rubens). Through a collaboration with Aotearoa New Zealand Crown Research Institute Manaaki Whenua-Landcare Research we have access to a collection of thousands of fungal cultures known as the International Collection of Microorganisms from Plants (ICMP). The ICMP contains both known and novel species which have not been extensively tested for their antimicrobial activity. Initial screening of ICMP isolates for activity against Escherichia coli and Staphylococcus aureus directed our interest towards ICMP 477, an isolate of the soil-inhabiting fungus, Aspergillus terreus. In our investigation of the secondary metabolites of A. terreus, through extraction, fractionation, and purification, we isolated nine known natural products. We evaluated the biological activity of selected compounds against various bacteria and fungi and discovered that terrein (1) has potent activity against the important human pathogen Cryptococcus neoformans.

Optimization of the production process for the anticancer lead compound illudin M: downstream processing.

BACKGROUND: Secondary metabolites have played a key role as starting points for drug development programs due to their often unique features compared with synthetically derived molecules. However, limitations related to the discovery and supply of these molecules by biotechnological means led to the retraction of big pharmaceutical companies from this field. The reasons included problems associated with strain culturing, screening, re-discovery, purification and characterization of novel molecules from natural sources. Nevertheless, recent reports have described technical developments that tackle such issues. While many of these reports focus on the identification and characterization of such molecules to enable subsequent chemical synthesis, a biotechnological supply strategy is rarely reported. This may be because production processes usually fall under proprietary research and/or few processes may meet the requirements of a pharmaceutical development campaign. We aimed to bridge this gap for illudin M-a fungal sesquiterpene used for the development of anticancer agents-with the intention to show that biotechnology can be a vital alternative to synthetic processes dealing with small molecules. RESULTS: We used µL-scale models to develop an adsorption and extraction strategy for illudin M recovery from culture supernatant of Omphalotus nidiformis and these findings were successfully transferred into lab-scale. By adsorbing and eluting the product using a fixed resin-bed we reduced the working volume by ~ 90% and removed the aqueous phase from the process. After a washing step, a highly concentrated illudin M fraction was obtained by isocratic elution with 80% methanol. The fraction was dried and extracted using a water/heptane mixture, enriching illudin M in the heptane phase. From heptane illudin M could be instantly crystalized by concentrating the solution, achieving a final purity > 95%. CONCLUSION: We have developed a robust, scalable and low-cost downstream process to obtain highly pure illudin M. By using solid phase extraction we reduced the production of solvent waste. Heptane from the final purification step could be recycled. The reduced amounts of solvents required, and the short purification time render this method a very economic and ecologic alternative to published processes.

Optimization of the production process for the anticancer lead compound illudin M: improving titers in shake-flasks.

BACKGROUND: The fungal sesquiterpenes Illudin M and S are important base molecules for the development of new anticancer agents due to their strong activity against some resistant tumor cell lines. Due to nonspecific toxicity of the natural compounds, improvement of the pharmacophore is required. A semisynthetic derivative of illudin S (Irofulven) entered phase II clinical trials for the treatment of castration-resistant metastatic prostate cancer. Several semisynthetic illudin M derivatives showed increased in vitro selectivity and improved therapeutic index against certain tumor cell lines, encouraging further investigation. This requires a sustainable supply of the natural compound, which is produced by Basidiomycota of the genus Omphalotus. We aimed to develop a robust biotechnological process to deliver illudin M in quantities sufficient to support medicinal chemistry studies and future preclinical and clinical development. In this study, we report the initial steps towards this goal. RESULTS: After establishing analytical workflows, different culture media and commercially available Omphalotus strains were screened for the production of illudin M.Omphalotus nidiformis cultivated in a medium containing corn steep solids reached ~ 38 mg L-1 setting the starting point for optimization. Improved seed preparation in combination with a simplified medium (glucose 13.5 g L-1; corn steep solids 7.0 g L- 1; Dox broth modified 35 mL), reduced cultivation time and enhanced titers significantly (~ 400 mg L-1). Based on a reproducible cultivation method, a feeding strategy was developed considering potential biosynthetic bottlenecks. Acetate and glucose were fed at 96 h (8.0 g L-1) and 120 h (6.0 g L-1) respectively, which resulted in final illudin M titer of ~ 940 mg L-1 after eight days. This is a 25 fold increase compared to the initial titer. CONCLUSION: After strict standardization of seed-preparation and cultivation parameters, a combination of experimental design, empirical trials and additional supply of limiting biosynthetic precursors, led to a highly reproducible process in shake flasks with high titers of illudin M. These findings are the base for further work towards a scalable biotechnological process for a stable illudin M supply.

Phoenicin Switch: Discovering the Trigger for Radical Phoenicin Production in Multiple Wild-Type Penicillium Species.

Society faces the challenge of storing energy from sustainable sources in inexpensive, nontoxic ways that do not deplete the limited resources of Earth. In this regard, quinone redox flow batteries have been proposed as ideal; however, industrially used quinones have traditionally been synthesized from fossil fuels. Therefore, we investigated the production of phoenicin (compound 1), a deep violet dibenzoquinone produced by certain Penicillium species, for its industrial potential. Strains grew as surface cultures on customized growth media with varying production parameters, and phoenicin production was assessed by ultrahigh-performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOF MS) analysis of the supernatant. Phoenicin production was reliant on the sucrose concentration, and by varying that, we produced 4.94 ± 0.56 g/L phoenicin on a Czapek yeast autolysate broth (CY)-based medium with Penicillium phoeniceum (CBS 249.32) as the production host, with 71.91% phoenicin purity in the resulting medium broth. Unexpectedly, metabolites corresponding to phoenicin polymers were tentatively identified in P. phoeniceum, of which the dimer (diphoenicin) was a major chromatographic peak. An MS-based metabolomics study was conducted on P. atrosanguineum using feature-based molecular networking and multivariate statistics, and it was found that few or no known secondary metabolites besides phoenicin were secreted into the growth medium. Finally, the effects of sucrose, sodium nitrate, and yeast extract (YE) in the growth medium were investigated in a 23 full factorial design. The results indicated an optimal sucrose concentration of 92.87 g/L on CY when NaNO3 and YE were fixed at 3 and 5 g/L, respectively. IMPORTANCE This work was undertaken to explore the production of fungal quinones in wild-type strains for use as electrolytes in redox flow batteries. As society converts energy production in a more sustainable direction, it becomes increasingly more important to store sustainable energy in smart ways. Conventional battery technologies imply the use of highly toxic, expensive, and rare metals; thus, quinone redox flow batteries have been proposed to be a desirable alternative. In this study, we explored the possibility of producing the fungal quinone phoenicin in Penicillium spp. by changing the growth parameters. The production of other secondary metabolites and known mycotoxins was also investigated in a metabolomics study. It was shown that phoenicin production was activated by optimizing the carbon concentration of the medium, resulting in high titers and purity of the single metabolite.

Subcellular localization of fungal specialized metabolites.

Fungal specialized metabolites play an important role in the environment and have impacted human health and survival significantly. These specialized metabolites are often the end product of a series of sequential and collaborating biosynthetic enzymes that reside within different subcellular compartments. A wide variety of methods have been developed to understand fungal specialized metabolite biosynthesis in terms of the chemical conversions and the biosynthetic enzymes required, however there are far fewer studies elucidating the compartmentalization of the same enzymes. This review illustrates the biosynthesis of specialized metabolites where the localization of all, or some, of the biosynthetic enzymes have been determined and describes the methods used to identify the sub-cellular localization.