Cyclodepsipeptide Biosynthesis in Hypocreales Fungi and Sequence Divergence of The Non-Ribosomal Peptide Synthase Genes.
ABSTRACT: Fungi from the Hypocreales order synthesize a range of toxic non-ribosomal cyclic peptides with antimicrobial, insecticidal and cytotoxic activities. Entomopathogenic Beauveria, Isaria and Cordyceps as well as phytopathogenic Fusarium spp. are known producers of beauvericins (BEAs), beauvenniatins (BEAEs) or enniatins (ENNs). The compounds are synthesized by beauvericin/enniatin synthase (BEAS/ESYN1), which shows significant sequence divergence among Hypocreales members. We investigated ENN, BEA and BEAE production among entomopathogenic (Beauveria, Cordyceps, Isaria) and phytopathogenic (Fusarium) fungi; BEA and ENNs were quantified using an LC-MS/MS method. Phylogenetic analysis of partial sequences of putative BEAS/ESYN1 amplicons was also made. Nineteen fungal strains were identified based on sequence analysis of amplified ITS and tef-1? regions. BEA was produced by all investigated fungi, with F. proliferatum and F. concentricum being the most efficient producers. ENNs were synthesized mostly by F. acuminatum, F. avenaceum and C. confragosa. The phylogeny reconstruction suggests that ancestral BEA biosynthesis independently diverged into biosynthesis of other compounds. The divergent positioning of three Fusarium isolates raises the possibility of parallel acquisition of cyclic depsipeptide synthases in ancient complexes within Fusarium genus. Different fungi have independently evolved NRPS genes involved in depsipeptide biosynthesis, with functional adaptation towards biosynthesis of overlapping yet diversified metabolite profiles.
Project description:Members of the fungal genus Fusarium can produce numerous secondary metabolites, including the nonribosomal mycotoxins beauvericin (BEA) and enniatins (ENNs). Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) that contains both peptide synthetase and S-adenosyl-l-methionine-dependent N-methyltransferase activities. Several Fusarium species can produce ENNs, BEA or both, but the mechanism(s) enabling these differential metabolic profiles is unknown. In this study, we analyzed the primary structure of ESYN1 by sequencing esyn1 transcripts from different Fusarium species. We measured ENNs and BEA production by ultra-performance liquid chromatography coupled with photodiode array and Acquity QDa mass detector (UPLC-PDA-QDa) analyses. We predicted protein structures, compared the predictions by multivariate analysis methods and found a striking correlation between BEA/ENN-producing profiles and ESYN1 three-dimensional structures. Structural differences in the ? strand's Asn789-Ala793 and His797-Asp802 portions of the amino acid adenylation domain can be used to distinguish BEA/ENN-producing Fusarium isolates from those that produce only ENN.
Project description:Beauvericin (BEA) and enniatins (ENNs) are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1? gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.
Project description:Beauvericin (BEA) is a cyclodepsipeptide mycotoxin, showing insecticidal, antibiotic and antimicrobial activities, as well as inducing apoptosis of cancer cell lines. BEA can be produced by multiple fungal species, including saprotrophs, plant, insect and human pathogens, particularly belonging to <i>Fusarium</i>, <i>Beauveria</i> and <i>Isaria</i> genera. The ability of <i>Trichoderma</i> species to produce BEA was until now uncertain. Biosynthesis of BEA is governed by a non-ribosomal peptide synthase (NRPS), known as beauvericin synthase (BEAS), which appears to present considerable divergence among different fungal species. In the present study we compared the production of beauvericin among <i>Fusarium</i> and <i>Trichoderma</i> strains using UPLC methods. <i>BEAS</i> fragments were sequenced and analyzed to examine the level of the gene's divergence between these two genera and confirm the presence of active <i>BEAS</i> copy in <i>Trichoderma</i>. Seventeen strains of twelve species were studied and phylogenetic analysis showed distinctive grouping of <i>Fusarium</i> and <i>Trichoderma</i> strains. The highest producers of beauvericin were <i>F. proliferatum</i> and <i>F. nygamai</i>. <i>Trichoderma</i> strains of three species (<i>T. atroviride</i>, <i>T. viride</i>, <i>T. koningiopsis</i>) were minor BEA producers. The study showed beauvericin production by <i>Fusarium</i> and <i>Trichoderma</i> species and high variance of the non-ribosomal peptide synthase gene among fungal species from the <i>Hypocreales</i> order.
Project description:Beauvericin is a depsipeptide mycotoxin. The production of several beauvericin analogues has previously been shown among various genera among Hypocreales fungi. This includes so-called beauvenniatins, in which one or more N-methyl-phenylalanine residues is exchanged with other amino acids. In addition, a range of "unnatural" beauvericins has been prepared by a precursor addition to growth medium. Our aim was to get insight into the natural production of beauvericin analogues among different Hypocreales fungi, such as Fusarium and Isaria spp. In addition to beauvericin, we tentatively identified six earlier described analogues in the extracts; these were beauvericin A and/or its structural isomer beauvericin F, beauvericin C, beauvericin J, beauvericin D, and beauvenniatin A. Other analogues contained at least one additional oxygen atom. We show that the additional oxygen atom(s) were due to the presence of one to three N-methyl-tyrosine moieties in the depsipeptide molecules by using different liquid chromatography?mass spectrometry-based approaches. In addition, we also tentatively identified a beauvenniatin that contained N-methyl-leucine, which we named beauvenniatin L. This compound has not been reported before. Our data show that N-methyl-tyrosine containing beauvericins may be among the major naturally produced analogues in certain fungal strains.
Project description:Most of the fungi from the <i>Fusarium</i> genus are pathogenic to cereals, vegetables, and fruits and the products of their secondary metabolism mycotoxins may accumulate in foods and feeds. Non-ribosomal cyclodepsipeptides are one of the main mycotoxin groups and include beauvericins (BEAs), enniatins (ENNs), and beauvenniatins (BEAEs). When ingested, even small amounts of these metabolites significantly affect human and animal health. On the other hand, in view of their antimicrobial activities and cytotoxicity, they may be used as components in drug discovery and processing and are considered as suitable candidates for anti-cancer drugs. Therefore, it is crucial to expand the existing knowledge about cyclodepsipeptides and to search for new analogues of these compounds. The present manuscript aimed to highlight the extensive variability of cyclodepsipeptides by describing chemistry, biosynthesis, and occurrence of BEAs, ENNs, and BEAEs in foods and feeds. Moreover, the co-occurrence of <i>Fusarium</i> species was compared to the amounts of toxins in crops, vegetables, and fruits from different regions of the world.
Project description:In 2017-2018, extensive symptoms of sudden decline and fruit rot were observed on date palms in southern Tunisia. Samples of diseased plants were randomly collected in six localities. Based on morphological identification, <i>Fusarium</i> was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative <i>Fusarium</i> strains at species level and investigate their phylogenetic relationships. The main species detected was <i>Fusarium</i> <i>proliferatum</i>, and at a much lesser extent, <i>Fusarium</i> <i>brachygibbosum</i>, <i>Fusarium</i> <i>caatingaense</i>, <i>Fusarium</i> <i>clavum</i>, <i>Fusarium</i> <i>incarnatum</i><i>,</i> and <i>Fusarium</i> <i>solani</i>. Pathogenicity on the <i>Deglet</i> <i>Nour</i> variety plantlets and the capability to produce mycotoxins were also assessed. All <i>Fusarium</i> species were pathogenic complying Koch's postulates. <i>Fusarium</i> <i>proliferatum</i> strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All <i>F.</i> <i>brachygibbosum</i> strains produced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. <i>Fusarium</i> <i>caatingaense</i>, <i>F.</i> <i>clavum</i>, <i>F.</i> <i>incarnatum</i> produced only BEA. <i>Fusarium</i> <i>solani</i> strains produced MON, BEA, and ENNs. This work reports for the first time a comprehensive multidisciplinary study of <i>Fusarium</i> species on date palms, concerning both phytopathological and food safety issues.
Project description:Pineapple (Ananas comosus var. comosus) is an important perennial crop in tropical and subtropical areas. It may be infected by various Fusarium species, contaminating the plant material with mycotoxins. The aim of this study was to evaluate Fusarium species variability among the genotypes isolated from pineapple fruits displaying fungal infection symptoms and to evaluate their mycotoxigenic abilities. Forty-four isolates of ten Fusarium species were obtained from pineapple fruit samples: F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incarnatum, F. oxysporum, F. polyphialidicum, F. proliferatum, F. temperatum and F. verticillioides. Fumonisins B1-B3, beauvericin (BEA) and moniliformin (MON) contents were quantified by high-performance liquid chromatography (HPLC) in pineapple fruit tissue. Fumonisins are likely the most dangerous metabolites present in fruit samples (the maximum FB1 content was 250 μg g(-1) in pineapple skin and 20 μg ml(-1) in juice fraction). In both fractions, BEA and MON were of minor significance. FUM1 and FUM8 genes were identified in F. fujikuroi, F. proliferatum, F. temperatum and F. verticillioides. Cyclic peptide synthase gene (esyn1 homologue) from the BEA biosynthetic pathway was identified in 40 isolates of eight species. Based on the gene-specific polymerase chain reaction (PCR) assays, none of the isolates tested were found to be able to produce trichothecenes or zearalenone.
Project description:A total of 470 edible vegetable oil samples including peanut, soybean, rapeseed, sesame seed, corn, blend, and others collected from eight provinces of China were analyzed for the concentrations of beauvericin (BEA), enniatin A (ENA), A1 (ENA1), B (ENB), and B1 (ENB1) by ultraperformance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). Concentrations of BEA, ENB, and ENB1 (average = 5.59 μg/kg, 5.16 μg/kg, and 4.61 μg/kg) in all positive samples were higher than those for ENA and ENA1 (average = 0.85 μg/kg and 1.88 μg/kg). Frequencies of BEA and ENNs in all analyzed samples were all higher than 50% with the exception of ENA1 (36.6%, 172/470). Levels of BEA and ENNs in all analyzed samples varied based on their sample types and geographical distributions (Kruskal–Wallis test, p < 0.05). The soybean and peanut oil samples were found to be more easily contaminated by BEA and ENNs than other oil samples. Concentrations of BEA and ENNs in samples obtained from Heilongjiang, Shandong and Guizhou were higher than those found in samples from other provinces. Besides, frequencies of mycotoxin co-contaminations were high and their co-contamination types also varied by oil types. BEA-ENA-ENA1-ENB-ENB1 was the most commonly found toxin combination type, almost in one third of the analyzed samples (30%, 141/470). Overall, these results indicate that co-occurrence of BEA and ENNs in analyzed Chinese edible vegetable oil samples is highly common, and it is vital to monitor them, both simultaneously and on a widespread level.
Project description:Emerging mycotoxins such as moniliformin (MON), enniatins (ENs), beauvericin (BEA), and fusaproliferin (FUS) may contaminate maize and negatively influence the yield and quality of grain. The aim of this study was to determine the content of emerging Fusarium mycotoxins in Serbian maize from the 2016, 2017, and 2018 harvests. A total of 190 samples from commercial maize production operations in Serbia were analyzed for the presence of MON, ENs, BEA, and FUS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained results were interpreted together with weather data from each year. MON, BEA, and FUS were major contaminants, while other emerging mycotoxins were not detected or were found in fewer samples (<20%). Overall contamination was highest in 2016 when MON and BEA were found in 50-80% of samples. In 2017 and 2018, high levels of MON, FUS, and BEA were detected in regions with high precipitation and warm weather during the silking phase of maize (July and the beginning of August), when the plants are most susceptible to Fusarium infections. Since environmental conditions in Serbia are favorable for the occurrence of mycotoxigenic fungi, monitoring Fusarium toxins is essential for the production of safe food and feed.
Project description:Recent surveys report the occurrence of <i>Aspergillus</i> and <i>Penicillium</i> metabolites (aflatoxins (AFLs), ochratoxin A (OTA), cyclopiazonic and mycophenolic acids (MPA), sterigmatocystin (STC), citrinin), <i>Fusarium</i> (trichothecenes, zearalenone (ZEA), fumonisins (FBs), enniatins (ENNs)) and <i>Alternaria</i> (alternariol (AOH), its methyl ether (AME), tentoxin (TE), and tenuazonic acid (TNZ)) toxins in dry <i>Camellia sinensis</i> and herbal tea samples. Since tea is consumed in the form of infusion, correct risk assessment needs evaluation of mycotoxins' transfer rates. We have studied the transfer of AFLs, OTA, STC, deoxynivalenol (DON), ZEA, FBs, T-2, and HT-2 toxins, AOH, AME, TE, ENN A and B, beauvericin (BEA), and MPA from the spiked green tea matrix into an infusion under variation of preparation time and water characteristics (total dissolved solids (TDS) and pH). Analytes were detected by HPLC-MS/MS. The main factors affecting transfer rate proved to be mycotoxins' polarity, pH of the resulting infusion (for OTA, FB2, and MPA) and matrix-infusion contact period. The concentration of mycotoxins increased by 20-50% within the first ten minutes of infusing, after that kinetic curve changed slowly. The concentration of DON and FB2 increased by about 10%, for ZEA, MPA, and STC it stayed constant, while for T-2, TE, AOH, and AFLs G1 and G2 it went down. Maximum transfer correlated well with analytes polarity. Maximum transfer of ENNs, BEA, STC, ZEA, and AOH into infusion was below 25%; AFLs-25-45%; DON, TE, and T-2 toxins 60-90%, FB1-80-100%. The concentration of OTA, MPA, and FB2 in the infusion depended on its pH. At pH about four, 20%, 40%, and 60% of these toxins transferred into an infusion, at pH about seven, their concentrations doubled. Water TDS did not affect transfer significantly.