Filamentous fungi are able to synthesise a remarkable range of secondary metabolites, which play various key roles in the interaction between fungi and the rest of the biosphere, determining their ...ecological fitness. Many of them can have a beneficial activity to be exploited, as well as negative impact on human and animal health, as in the case of mycotoxins contaminating large quantities of food, feed, and agricultural products worldwide and posing serious health and economic risks. The elucidation of the molecular aspects of mycotoxin biosynthesis has been greatly sped up over the past decade due to the advent of next-generation sequencing technologies, which greatly reduced the cost of genome sequencing and related omic analyses. Here, we briefly highlight the recent progress in the use and integration of omic approaches for the study of mycotoxins biosynthesis. Particular attention has been paid to genomics and transcriptomic approaches for the identification and characterisation of biosynthetic gene clusters of mycotoxins and the understanding of the regulatory pathways activated in response to physiological and environmental factors leading to their production. The latest innovations in genome-editing technology have also provided a more powerful tool for the complete explanation of regulatory and biosynthesis pathways. Finally, we address the crucial issue of the interpretation of the combined omics data on the biology of the mycotoxigenic fungi. They are rapidly expanding and require the development of resources for more efficient integration, as well as the completeness and the availability of intertwined data for the research community.
The widespread use of Next-Generation Sequencing has opened a new era in the study of biological systems by significantly increasing the catalog of fungal genomes sequences and identifying gene ...clusters for known secondary metabolites as well as novel cryptic ones. However, most of these clusters still need to be examined in detail to completely understand the pathway steps and the regulation of the biosynthesis of metabolites. Genome sequencing approach led to the identification of the biosynthetic genes cluster of ochratoxin A (OTA) in a number of producing fungal species. Ochratoxin A is a potent pentaketide nephrotoxin produced by
and
species and found as widely contaminant in food, beverages and feed. The increasing availability of several new genome sequences of OTA producer species in JGI Mycocosm and/or GenBank databanks led us to analyze and update the gene cluster structure in 19
and 2
OTA producing species, resulting in a well conserved organization of OTA core genes among the species. Furthermore, our comparative genome analyses evidenced the presence of an additional gene, previously undescribed, located between the polyketide and non-ribosomal synthase genes in the cluster of all the species analyzed. The presence of a SnoaL cyclase domain in the sequence of this gene supports its putative role in the polyketide cyclization reaction during the initial steps of the OTA biosynthesis pathway. The phylogenetic analysis showed a clustering of OTA SnoaL domains in accordance with the phylogeny of OTA producing species at species and section levels. The characterization of this new OTA gene, its putative role and its expression evidence in three important representative producing species, are reported here for the first time.
The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on ...food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin.
The strawberry tree (
) is a medicinal plant and an important source of biocompounds, potentially useful for pharmaceutical and chemical applications to prevent or treat several human diseases. The ...strawberry tree fruits have usually been used to produce traditional products such as jams and jellies and to obtain fermented alcoholic drinks, representing the most valuable derivative products. Other fermented products are potentially interesting for their nutritional value; however, the fermentation process needs to be controlled and standardized to obtain high-quality products/ingredients. In this work, we investigated two different fermentative procedures, using strawberry tree whole fruit and fruit paste as matrices inoculated with a selected starter strain of
LI 180-7. The physical, chemical, microbiological and nutritional properties of fermented products were evaluated, as well as their antioxidant activity. The new obtained fermented products are enriched in organic acids (acetic acid varied from 39.58 and 57.21 mg/g DW and lactic acid from 85.33 to 114.1 mg/g DW) and have better nutritional traits showing a higher amount of total polyphenols (phenolic acids, flavonoids and anthocyanins) that ranged from 1852 mg GAE/100 g DW to 2682 mg GAE/100 g DW. Also, the amount of isoprenoid increased ranging from 155.5 μg/g DW to 164.61 μg/g DW. In this regard, the most promising strategy seemed to be the fermentation of the fruit paste preparation; while the extract of fermented whole fruits showed the most powerful antioxidant activity. Finally, a preliminary attempt to produce a food prototype enriched in fermented strawberry tree fruits suggested the whole fruit fermented sample as the most promising from a preliminary sensory analysis.
Polyketide synthase (PKSs) and nonribosomal peptide synthetase (NRPSs) are large multimodular enzymes involved in biosynthesis of polyketide and peptide toxins produced by fungi. Furthermore, hybrid ...enzymes, in which a reducing PKS region is fused to a single NRPS module, are also responsible of the synthesis of peptide-polyketide metabolites in fungi. The genes encoding for PKSs and NRPSs have been exposed to complex evolutionary mechanisms, which have determined the great number and diversity of metabolites. In this study, we considered the most important polyketide and peptide mycotoxins and, for the first time, a phylogenetic analysis of both PKSs and NRPSs involved in their biosynthesis was assessed using two domains for each enzyme: β-ketosynthase (KS) and acyl-transferase (AT) for PKSs; adenylation (A) and condensation (C) for NRPSs. The analysis of both KS and AT domains confirmed the differentiation of the three classes of highly, partially and non-reducing PKSs. Hybrid PKS-NRPSs involved in mycotoxins biosynthesis grouped together in the phylogenetic trees of all the domains analyzed. For most mycotoxins, the corresponding biosynthetic enzymes from distinct fungal species grouped together, except for PKS and NRPS involved in ochratoxin A biosynthesis, for which an unlike process of evolution could be hypothesized in different species.
Edible jellyfish are mainly consumed and marketed in Southeastern Countries, generally produced by a multi-phase drying process, using mixtures of salt and alum. Recently, jellyfish have become very ...attractive also for Western food markets. They are novel food in Europe and no recognized handling/processing steps have been set up yet. Moreover, no specific food safety and quality parameters are available. In this study, we identified a set of safety and quality parameters for jellyfish, based on standards and process hygiene criteria used in Europe for other products. These assays were tested on three different jellyfish preparations that can be used as raw materials for subsequent food processing. All jellyfish samples revealed the absence of pathogens (
spp. and
),
and
spp., even if a limited presence of
was observed. No biogenic amine histamine was detected and negligible levels of total volatile basic nitrogen (TVB-N) were revealed. Total bacterium, yeast and mold counts were negligible or undetectable by conventional accredited methods, and conversely the results were higher when optimized saline conditions were used. This study, for the first time, established a set of quality and safety parameters necessary for first-operations and subsequent processing of jellyfish as novel food. Highlights: Jellyfish can represent a novel food in Europe. Identification of safety and quality parameters for jellyfish food products. Saline conditions are essential for improving safety and quality assessment of jellyfish as food.
The inhibitory action of 20 antagonistic
isolates against the aflatoxigenic isolate
ITEM 9 (Af-9) and their efficacy in reducing aflatoxin formation in vitro were examined. Production of metabolites ...with inhibitory effect by the
isolates was also investigated. Antagonistic effect against Af-9 was assessed by inhibition of radial growth of the colonies and by fungal interactions in dual confrontation tests. A total of 8 out of 20 isolates resulted in a significant growth inhibition of 3-day-old cultures of Af-9, ranging from 13% to 65%. A total of 14 isolates reduced significantly the aflatoxin B
(AfB
) content of 15-day-old Af-9 cultures; 4 were ineffective, and 2 increased AfB
. Reduction of AfB
content was up to 84.9% and 71.1% in 7- and 15-day-old cultures, respectively. Since the inhibition of Af-9 growth by metabolites of
was not necessarily associated with inhibition of AfB
production and vice versa, we investigated the mechanism of reduction of AfB
content at the molecular level by examining two strains: one (T60) that reduced both growth and mycotoxin content; and the other (T44) that reduced mycotoxin content but not Af-9 growth. The expression analyses for the two regulatory genes
and
, and the structural genes
,
,
and
of the aflatoxin biosynthesis cluster indicated that neither strain was able to downregulate the aflatoxin synthesis, leading to the conclusion that the AfB
content reduction by these
strains was based on other mechanisms, such as enzyme degradation or complexation. Although further studies are envisaged to identify the metabolites involved in the biocontrol of
and prevention of aflatoxin accumulation, as well as for assessment of the efficacy under controlled and field conditions,
spp. qualify as promising agents and possible alternative options to other biocontrol agents already in use.
Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and ...novel compounds. A comparative analysis of the OTA-biosynthetic cluster in
, and
has revealed a high synteny in OTA cluster organization in five structural genes (
,
,
, and
). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene,
, located in the OTA cluster between the
and
genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in
. In the present study, we demonstrated an upregulation of the cyclase gene in
under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in
.
The genus Aspergillus is among the most abundant and widely distributed organism on earth, and at the moment comprises 339 known species. It is one of the most important economically fungal genus and ...the biotechnological use of Aspergillus species is related to production of soy sauce, of different hydrolytic enzymes (amylases, lipases) and organic acid (citric acid, gluconic acid), as well as biologically active metabolites such as lovastatin. Although they are not considered to be major cause of plant diseases, Aspergillus species are responsible for several disorders in various plants and plant products, especially as opportunistic storage moulds. The notable consequence of their presence is contamination of foods and feeds by mycotoxins, among which the most important are aflatoxins, ochratoxin A, and, at a less extent, fumonisins. Aflatoxins B
, B
, G
, G
are the most toxic and carcinogenic mycotoxins, due to their extreme hepatocarcinogenicity; ochratoxin A is a potent nephrotoxin, it is also carcinogenic, teratogenic, and immunotoxic in rats and possibly in humans; fumonisins are hepatotoxic and nephrotoxic with potential carcinogenic effects on rat and mice. In this chapter we summarize the main aspects of morphology, ecology, epidemiology, and toxigenicity of Aspergillus foodborne pathogens which belong to sections Flavi, Circumdati, and Nigri, occurring in several agricultural products and responsible of aflatoxin, ochratoxin A, and fumonisins contamination of food and feed.
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