Ochratoxin A Biosynthesis Research Articles

Inhibitory effect of allyl and benzyl isothiocyanates on ochratoxin a producing fungi in grape and maize

The purpose of this study was to evaluate the inhibitory effect of allyl-isothiocyanate (AITC) and benzyl-isothiocyanate (BITC) on fungal growth and Ochratoxin A (OTA) production by Aspergillus ochraceus, A. carbonarius and A. niger. Here, we found that spore germination and fungal growth of the three fungi were significantly inhibited when the concentration of AITC and BITC was higher than 1.25 μg/mL. The inhibitory effect of AITC or BITC on A. carbonaceus and A. ochraceus was significantly stronger than that of A. niger. Scanning electron microscopy showed that the mycelia of all three fungi were changed by AITC and BITC. Compared with A. ochraceus and A. carbonarius, the damage to A. niger was lower. For OTA production, AITC and BITC could significantly down-regulated the expression of all five OTA biosynthesis genes in A. niger and A. carbonarius. In A. ochraceus, although several OTA biosynthesis genes were up-regulated, the key PKS gene was down-regulated by AITC and BITC. Twenty-five μg/mL of AITC or BITC could reduce the infection of the three fungi on grapes with inhibition rates of 28%–36% during 14 days and prolong the shelf life of grapes. In maize, the OTA production of the three fungi was significantly reduced by 25 μg/mL of AITC and BITC with the inhibition rates 68.04%–93.49% and 65.87%–75.45%, respectively. These results suggest that AITC and BITC can be used as natural fungicides to prevent A. niger, A. carbonarius and A. ochraceus from infecting grapes and maize and control OTA contamination.

Fungal G-Protein-Coupled Receptors: A Promising Mediator of the Impact of Extracellular Signals on Biosynthesis of Ochratoxin A.

G-protein-coupled receptors (GPCRs) are transmembrane receptors involved in transducing signals from the external environment inside the cell, which enables fungi to coordinate cell transport, metabolism, and growth to promote their survival, reproduction, and virulence. There are 14 classes of GPCRs in fungi involved in sensing various ligands. In this paper, the synthesis of mycotoxins that are GPCR-mediated is discussed with respect to ligands, environmental stimuli, and intra-/interspecific communication. Despite their apparent importance in fungal biology, very little is known about the role of ochratoxin A (OTA) biosynthesis by Aspergillus ochraceus and the ligands that are involved. Fortunately, increasing evidence shows that the GPCR that involves the AF/ST (sterigmatocystin) pathway in fungi belongs to the same genus. Therefore, we speculate that GPCRs play an important role in a variety of environmental signals and downstream pathways in OTA biosynthesis. The verification of this inference will result in a more controllable GPCR target for control of fungal contamination in the future.

Functional Role of Aspergillus carbonariusAcOTAbZIP Gene, a bZIP Transcription Factor within the OTA Gene Cluster.

Aspergillus carbonarius is the principal fungal species responsible for ochratoxin A (OTA) contamination of grapes and derived products in the main viticultural regions worldwide. In recent years, co-expressed genes representing a putative-OTA gene cluster were identified, and the deletion of a few of them allowed the partial elucidation of the biosynthetic pathway in the fungus. In the putative OTA-gene cluster is additionally present a bZIP transcription factor (AcOTAbZIP), and with this work, A. carbonarius ΔAcOTAbZIP strains were generated to study its functional role. According to phylogenetic analysis, the gene is conserved in the OTA-producing fungi. A Saccharomyces cerevisiae transcription factor binding motif (TFBM) homolog, associated with bZIP transcription factors was present in the A. carbonarius OTA-gene cluster no-coding regions. AcOTAbZIP deletion results in the loss of OTA and the intermediates OTB and OTβ. Additionally, in ΔAcOTAbZIP strains, a down-regulation of AcOTApks, AcOTAnrps, AcOTAp450, and AcOTAhal genes was observed compared to wild type (WT). These results provide evidence of the direct involvement of the AcOTAbZIP gene in the OTA biosynthetic pathway by regulating the involved genes. The loss of OTA biosynthesis ability does not affect fungal development as demonstrated by the comparison of ΔAcOTAbZIP strains and WT strains in terms of vegetative growth and asexual sporulation on three different media. Finally, no statistically significant differences in virulence were observed among ΔAcOTAbZIP strains and WT strains on artificially inoculated grape berries, demonstrating that OTA is not required by A. carbonarius for the pathogenicity process.

Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by Aspergillus carbonarius.

Aspergillus carbonarius is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by A. carbonarius, both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, laeA and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of A. carbonarius to produce and accumulate OTA in grapes.

Comparative Genomic Analysis of Ochratoxin A Biosynthetic Cluster in Producing Fungi: New Evidence of a Cyclase Gene Involvement.

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 Aspergillus and Penicillium 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 Aspergillus and 2 Penicillium 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.

Influence of an industrial dry-fermented sausage processing on ochratoxin A production by Penicillium nordicum

Dry-fermented sausages are prone to be colonised by Penicillium nordicum, which is one of the main ochratoxin A (OTA)-producing species. Its ability to produce this mycotoxin on dry-fermented sausages has been reported. However, the influence of the conditions of a traditional processing of a Spanish dry-fermented sausage and the intrinsic physicochemical parameters of this product such as water activity (aw) and pH on OTA production has not been studied yet. Thus, the aim of this study was to evaluate the influence of traditional processing (interaction of relative humidity (RH) x temperature x ripening days) on the evolution of pH and aw during maturation of dry-fermented sausage “salchichón” and its relationship with OTA synthesis by P. nordicum. The expression of otapks and otanps genes, both involved in the biosynthesis of the mycotoxin, was also assessed. For this, 27 raw sausages were inoculated with P. nordicum and ripened for 26 days in a drying chamber (3 days at 5 °C and 84% RH, 17 days at 12 °C and 84% RH, and 6 days at 12 °C and 80% RH). From results, although it seems that the pH slightly influenced on OTA biosynthesis, the aw had a great impact on this mycotoxin production. In fact, the two highest OTA concentrations found coincided with a dramatic rise of the aw value (0.92 aw) by day 18 of incubation when the RH of the drying chamber was still 84% and at the end of the incubation time when the aw decreased noticeably (0.87 aw). The expression of the otapks and otanps genes correlated with the OTA produced by P. nordicum. Results from this work confirm that the traditional processing of Spanish dry-fermented sausages favours itself OTA synthesis by P. nordicum. Our findings may help in informed decision-making in relation to RH/temperature of drying chambers and shortening of the ripening process. This may be then effectively incorporated into the hygienic production system in the framework of HACCP together with other measures including the use of Penicillium nalgiovense as protective culture or the monitoring of otapks gene expression, and aw during the processing of dry-fermented sausages. All these strategies together may put ochratoxigenic Penicillia at a disadvantage and minimise OTA contamination risks in dry-fermented sausages.

The Genomic Regions That Contain Ochratoxin A Biosynthetic Genes Widely Differ in Aspergillus Section Circumdati Species.

Aspergillus section Circumdati includes 27 species, some of which are considered ochratoxin A (OTA) producers. However, there is considerable controversy about their potential OTA synthesis ability. In this work, the complete genomes of 13 species of Aspergillus section Circumdati were analyzed in order to study the cluster of OTA biosynthetic genes and the region was compared to those previously reported in A. steynii and A. westerdijkiae. The results obtained reveal that the genomes of some species in this section, including A. affinis, A. cretensis, A. elegans, A. muricatus, A. pulvericola, A. roseoglobulosus, and A. subramanianii, contain a potentially functional OTA biosynthetic cluster. Therefore, they might be able to synthesize the toxin. On the contrary, A. melleus, A. ochraceus, A. ostianus, A. persii, A. sclerotiorum, A. sesamicola, and A. westlandensis contain a truncated version of the cluster that lacks many of the genes involved in OTA biosynthesis, which might be related to their inability to produce OTA. The gain/loss pattern is different in all species, which suggests that the genetic evolution of this region might be due to independent events.

Functional roles of LaeA, polyketide synthase, and glucose oxidase in the regulation of ochratoxin A biosynthesis and virulence in Aspergillus carbonarius.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks‐knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

Carbon Catabolite Repression Gene AoCreA Regulates Morphological Development and Ochratoxin A Biosynthesis Responding to Carbon Sources in Aspergillus ochraceus.

Carbon is one of the most important nutrients for the development and secondary metabolism in fungi. CreA is the major transcriptional factor mediating carbon catabolite repression, which is employed in the utilization of carbon sources. Aspergillus ochraceus contaminates various food and feed containing different carbon sources by producing ochratoxin A (OTA). However, little is known about the function of AoCreA in regulating the morphology and OTA production of A. ochraceus. To give an insight into the mechanism of the carbon sources regulating development of A. ochraceus and OTA production, we have identified AoCreA in A. ochraceus. The homologous recombination strategy was used to generate the AoCreA deletion mutant (ΔAoCreA). We have investigated the morphology and OTA production of the wild type (WT) and ΔAoCreA of A. ochraceus with media containing different carbon sources (glucose, fructose, maltose, D-xylose, D-mannose, acetate, D-galactose, D-mannitol and lactose). ΔAoCreA showed a significant growth and conidiation defect on all media as compared with WT. Glucose and maltose were the most inducing media for OTA production by A. ochraceus, followed by sucrose and the nutrient-rich Yeast Extract Sucrose (YES) and Potato Dextrose Agar (PDA). The deletion of AoCreA led to a drastic reduction of OTA production on all kinds of media except PDA, which was supported by the expression profile of OTA biosynthetic genes. Furthermore, infection studies of ΔAoCreA on oats and pears showed the involvement of AoCreA in the pathogenicity of A. ochraceus. Thus, these results suggest that AoCreA regulates morphological development and OTA biosynthesis in response to carbon sources in A. ochraceus.

Isolation of a Novel Kluyveromyces marxianus Strain QKM-4 and Evidence of Its Volatilome Production and Binding Potentialities in the Biocontrol of Toxigenic Fungi and Their Mycotoxins

To overcome the economic losses associated with fungi and their toxic metabolites, environmentally safe and efficient approaches are needed. To this end, biological control using yeasts and safe bacterial strains and their products are being explored to replace synthetic fungicides. In the present study, the biocontrol effect of a yeast strain of Kluyveromyces marxianus, QKM-4, against the growth and mycotoxin synthesis potential of key toxigenic fungi was evaluated. In vitro assays were performed to find the application of yeast volatile organic compounds (VOCs) against fungal contamination on important agricultural commodities. The removal of ochratoxin A (OTA) and deoxynivalenol (DON) by living and heat-inactivated yeast cells was also explored. VOCs produced by strain QKM-4 were able to significantly limit the fungal growth of 17 fungal species belonging to genera Aspergillus, Penicillium, and Fusarium. Yeast VOCs were able to reduce OTA biosynthesis potential of Penicillium verrucosum and Aspergillus carbonarius by 99.6 and 98.7%, respectively. In vivo application of QKM-4 VOCs against Fusarium oxysporum and A. carbonarius infection on tomatoes and grapes, respectively, determined a complete inhibition of fungal spore germination. GC/MS-based analysis of yeast VOCs identified long-chain alkanes, including nonadecane, eicosane, docosane, heptacosane, hexatriacontane, and tetracosane. In vitro testing of the mycotoxin-binding potential of the living and heat-inactivated QKM-4 cells showed a reduction of OTA and DON up to 58 and 49%, respectively, from artificially contaminated buffers. Our findings clearly demonstrate the strong antifungal potential of K. marxianus QKM-4 and propose this strain as a strong candidate for application in agriculture to safeguard food and feed products.

In vitro antifungal effects of spices on ochratoxin A production and related gene expression in Penicillium nordicum on a dry-cured fermented sausage medium

Penicillium nordicum, an ochratoxin A (OTA) producer, widely contaminates the surface of dry-cured fermented sausages. Although the meat industry uses antifungal synthetic compounds during ripening, consumers' preferences currently stand up for natural preservatives. The aim of this study was to evaluate the antifungal effect of spices commonly added to dry-cured fermented sausages during their processing on P. nordicum for establishing their value as an alternative strategy to synthetic compounds. The mould strain was grown on a dry-cured fermented sausage-based medium containing oregano, rosemary or thyme. Moreover, an antifungal commercial preparation containing potassium sorbate and natamycin was tested alone and in combination with the spices. The growth rate, OTA accumulation and relative expression of genes involved in OTA biosynthesis (otapks and otanps) and some stress pathways (Hog1 and Rho1) were evaluated. In the growth assessment, the oregano alone or in combination with the antifungal preparation significantly reduced the growth rate. Different patterns were observed at both sampling times for the OTA production and gene expression analyses, and even differences between treatments at the same incubation period were observed. Regarding OTA accumulation, significant reductions were found when adding oregano or rosemary in the presence and absence of the antifungal compounds. While a stimulation of the expression of most of the tested genes was obtained at the initial stages, a repression was generally found at the end of the incubation. This makes sense since the mycotoxin accumulation was usually higher at the initial stages than at the end of the incubation. Accordingly, such spices would allow satisfying the current consumers’ demand for natural preservatives minimising the hazard associated with the OTA presence in cured meats at the same time.

The pH-Responsive Transcription Factor PacC Governs Pathogenicity and Ochratoxin A Biosynthesis in Aspergillus carbonarius.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. Aspergillus carbonarius is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in A. carbonarius pathogenicity using designed pacC gene knockout mutant. ΔAcpacC mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The ΔAcpacC mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of AcpacC resulted in a complete inhibition of OTA production at pH 7.0. Additionally, ΔAcpacC mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of pacC gene into ΔAcpacC mutant restored the WT phenotype. Our results demonstrate important roles of PacC of A. carbonarius in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

Transcriptome analysis of non-ochratoxigenic Aspergillus carbonarius strains and interactions between some black aspergilli species

Aspergillus carbonarius consistently produces large amounts of ochratoxin A (OTA), a mycotoxin with nephrotoxic effects on animals and humans. In the present study, we analyzed the transcriptional changes associated to OTA production in three atypical non-ochratoxigenic strains of A. carbonarius. In addition, in vitro interactions between ochratoxigenic strains of A. carbonarius and A. niger and non-ochratoxigenic strains of A. carbonarius and A. tubingensis were studied in order to evaluate their potential for controlling OTA production. RNA-seq analysis revealed that there are 696 differentially expressed genes identified in the three non-OTA-producing strains, including 280 up-regulated and 333 down-regulated genes. A functional and gene ontology enrichment analysis revealed that the processes related to metabolic and oxidation processes, associated with functions such as oxidoreductase and hydrolase activity were down regulated. All the genes related with OTA biosynthesis in A. carbonarius were the most down-regulated genes in non-ochratoxigenic strains. We also showed that these strains possess a deleterious mutation in the AcOTApks gene required for OTA biosynthesis. Moreover, one of these strains gave the best control of OTA production resulting in an OTA reduction of 98–100% in co-inoculation with an ochratoxigenic strain of A. niger and an OTA reduction of 79–89% with an ochratoxigenic strain of A. carbonarius. Results of this study provided novel insights into the knowledge of the OTA biosynthetic pathway in these non-ochratoxigenic wild strains, and showed the biocontrol potential of these strains.

Efficacy of the Combined Protective Cultures of Penicillium chrysogenum and Debaryomyces hansenii for the Control of Ochratoxin A Hazards in Dry-Cured Ham.

The ecological conditions during the ripening of dry-cured ham favour the development of moulds on its surface, being frequently the presence of Penicillium nordicum, a producer of ochratoxin A (OTA). Biocontrol using moulds and yeasts usually found in dry-cured ham is a promising strategy to minimize this hazard. The aim of this work is to evaluate the effect of previously selected Debaryomyces hansenii and Penicillium chrysogenum strains on growth, OTA production, and relative expression of genes involved in the OTA biosynthesis by P. nordicum. P. nordicum was inoculated against the protective cultures individually and combined on dry-cured ham for 21 days at 20 °C. None of the treatments reduced the growth of P. nordicum, but all of them decreased OTA concentration. The lower production of OTA could be related to significant repression of the relative expression of otapksPN and otanpsPN genes of P. nordicum. The efficacy of the combined protective cultures was tested in 24 dry-cured hams in industrial ripening (an 8 month-long production). OTA was detected in nine of the 12 dry-cured hams in the batch inoculated only with P. nordicum. However, in the batch inoculated with both P. nordicum and the combined protective culture, a considerable reduction of OTA contamination was observed. In conclusion, although the efficacy of individual use P. chrysogenum is great, the combination with D. hansenii enhances its antifungal activity and could be proposed as a mixed protective culture to control the hazard of the presence of OTA in dry-cured ham.

Interacting climate change factors (CO2 and temperature cycles) effects on growth, secondary metabolite gene expression and phenotypic ochratoxin A production by Aspergillus carbonarius strains on a grape-based matrix

Little is known on the impact that climate change (CC) may have on Aspergillus carbonarius and Ochratoxin A (OTA) contamination of grapes, especially in the Mediterranean region where in CC scenarios temperature are expected to increase by +2–5 °C and CO2 from 400 to 800/1200 ppm. This study examined the effect of (i) current and increased temperature in the alternating 11.5 h dark/12.5 h light cycle (15–28 °C vs 18–34 °C), representative of the North Apulia area, South Italy and (ii) existing and predicted CO2 concentrations (400 vs 1000 ppm), on growth, expression of biosynthetic genes (AcOTApks, AcOTAnrps, AcOTAhal, AcOTAp450, AcOTAbZIP) and regulatory genes of Velvet complex (laeA/veA/velB, “velvet complex”) involved in OTA biosynthesis and OTA phenotypic production by three strains of A. carbonarius. The experiments made on a grape-based matrix showed that elevated CO2 resulted in a general stimulation of growth and OTA production. These results were also supported by the up-regulation of both structural and regulatory genes involved in the OTA biosynthesis. Our work has shown for the first time that elevated CO2 concentration in the Mediterranean region may result in an increased risk of OTA contamination in the wine production chain.

Capsaicin, an inhibitor of Ochratoxin A production by Aspergillus section Nigri strains in grapes (Vitis vinifera L.)

ABSTRACTFood decay by spoilage fungi leads to significant economic losses and hazards to consumers’ health due to the potential of mycotoxin occurrence. Ochratoxin A (OTA) is a mycotoxin known as nephrotoxic and carcinogenic to humans. Natural capsaicin was evaluated for its effectiveness against the growth of five Aspergillus section Nigri strains and accumulation of OTA in inoculated black grapes. Results showed that capsaicin was effective in inhibiting fungal growth and OTA production by new four Aspergillus section Nigri strains (ATHUM 6997, 6998, 6999, 7000) and by Aspergillus carbonarius as well. Moreover, capsaicin addition exhibited maximum inhibition of OTA produced by ATHUM 6997, 6998, 6999, and 7000 in black grapes at 28.9%, 8.6%, 68.4%, and 78.1%, respectively. Inhibition percentage of OTA production by A. carbonarius in grapes treated with capsaicin was estimated at 61.5%. These results suggest that capsaicin influences the OTA biosynthesis pathway of all Aspergillus section Nigri strains and therefore could be used as an effective natural preservative against OTA contamination of vineyards. Risk assessment revealed that when grapes are treated with capsaicin, consumers are less exposed to OTA.

Role of Some Food-Grade Synthesized Flavonoids on the Control of Ochratoxin A in Aspergillus carbonarius.

Ochratoxin A (OTA) is a mycotoxin with a serious impact on human health. In Mediterranean countries, the black Aspergilli group, in particular Aspergillus carbonarius, causes the highest OTA contamination. Here we describe the synthesis of three polyphenolic flavonoids: 5-hydroxy-6,7-dimethoxy-flavone (MOS), 5,6-dihydroxy-7-methoxy-flavone (NEG), and 5,6 dihydroxy-flavone (DHF), as well as their effect on the prevention of OTA biosynthesis and lipoxygenase (LOX) activity in A. carbonarius cultured in a conducive liquid medium. The best control effect on OTA biosynthesis was achieved using NEG and DHF. In fungal cultures treated with these compounds at 5, 25, and 50 μg/mL, OTA biosynthesis significantly decreased throughout the 8-day experiment. NEG and DHF appear to have an inhibiting effect also on the activity of LOX, whereas MOS, which did not significantly inhibit OTA production, had no effect on LOX activity. The presence of free hydroxyls in catecholic position in the molecule appears to be a determining factor for significantly inhibiting OTA biosynthesis. However, the presence of a methoxy group in C-7 in NEG could slightly lower the molecule’s reactivity increasing OTA inhibition by this molecule at 5 μg/mL. Polyphenolic flavonoids present in edible plants may be easily synthesized and used to control OTA biosynthesis.

VeA Gene Acts as a Positive Regulator of Conidia Production, Ochratoxin A Biosynthesis, and Oxidative Stress Tolerance in Aspergillus niger.

The veA gene is a key regulator governing morphogenetic development and secondary metabolism in many fungi. Here, we characterized and disrupted a veA orthologue in an ochratoxigenic Aspergillus niger strain. Morphological development, ochratoxin A (OTA) biosynthesis, and oxidative stress tolerance in the wild-type and veA disruption strains were further analyzed. Accordingly, the link between the veA gene and development of specific gene brlA, OTA biosynthesis key gene pks, and oxidative-stress-tolerance-related gene cat was explored. Results demonstrated that the veA gene acts as a positive regulator of conidia production, OTA biosynthesis, and oxidative stress tolerance in A. niger, regardless of light conditions. Darkness promoted conidial production and OTA biosynthesis in the A. niger wild-type strain. Our results contribute to a better understanding of the veA regulatory mechanism and suggest the veA gene as a potential target for developing control strategies for A. niger infection and OTA biosynthesis.

A Consensus Ochratoxin A Biosynthetic Pathway: Insights from the Genome Sequence of Aspergillus ochraceus and a Comparative Genomic Analysis.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by Aspergillus and Penicillium species that widely contaminates food and feed. We sequenced and assembled the complete ∼37-Mb genome of Aspergillusochraceus fc-1, a well-known producer of OTA. Key genes of the OTA biosynthetic pathway were identified by comparative genomic analyses with five other sequenced OTA-producing fungi: A. carbonarius, A. niger, A. steynii, A. westerdijkiae, and Penicillium nordicum OTA production was completely inhibited in the deletion mutants (ΔotaA, ΔotaB, ΔotaC, ΔotaD, and ΔotaR1), and OTA biosynthesis was restored by feeding a postblock substrate to the corresponding mutant. The OTA biosynthetic pathway was unblocked in the ΔotaD mutant by the addition of heterologously expressed halogenase. OTA biosynthesis begins with a polyketide synthase (PKS), OtaA, utilizing acetyl coenzyme A (acetyl-CoA) and malonyl-CoA to synthesize 7-methylmellein, which is oxidized to OTβ by cytochrome P450 monooxygenase (OtaC). OTβ and l-β-phenylalanine are combined by a nonribosomal peptide synthetase (NRPS), OtaB, to form an amide bond to synthesize OTB. Finally, OTB is chlorinated by a halogenase (OtaD) to OTA. The otaABCD genes were expressed at low levels in the ΔotaR1 mutant. A second regulator, otaR2, which is adjacent to the biosynthetic gene, could modulate only the expression of otaA, otaB, and otaD Thus, we have identified a consensus OTA biosynthetic pathway that can be used to prevent and control OTA synthesis and will help us understand the variation and production of the intermediate components in the biosynthetic pathway.IMPORTANCE Ochratoxin A (OTA) is a significant mycotoxin that contaminates cereal products, coffee, grapes, wine, cheese, and meat. OTA is nephrotoxic, carcinogenic, teratogenic, and immunotoxic. OTA contamination is a serious threat to food safety, endangers human health, and can cause huge economic losses. At present, >20 species of the genera Aspergillus and Penicillium are known to produce OTA. Here we demonstrate that a consensus OTA biosynthetic pathway exists in all OTA-producing fungi and is encoded by a gene cluster containing four highly conserved biosynthetic genes and a bZIP transcription factor.

Effect of Cinnamaldehyde on Morphological Alterations of Aspergillus ochraceus and Expression of Key Genes Involved in Ochratoxin A Biosynthesis.

Ochratoxin A (OTA) is a potent nephrotoxic, hepatotoxic, and teratogenic compound which is a significant mycotoxin contaminates cereals during storage. Aspergillus ochraceus is the most common producer of OTA in cereals and cereal-derived products. Cinnamaldehyde is a natural substance derived from plant cinnamon playing an important role in the reduction of OTA contamination. In this study, the antifungal and antitoxigenic effect of cinnamaldehyde was investigated with its mechanisms of inhibition of fungal growth at the morphological and ultrastructural levels, and inhibition of OTA biosynthesis at the transcriptional level. Significant A. ochraceus growth was inhibited at 0.4–1.6 mmol/L with fumigation. A. ochraceus exposed to 0.4 mmol/L of cinnamaldehyde indicated irreversible harmful morphological and ultrastructural modifications such as the folding of the cell, the loss of integrity of the cell wall, the disruption of plasma membrane, the destruction of the mitochondria, and the absence of intracellular organelles. These alterations may be attributed to its inhibition of enzymatic reactions that regulate cell wall synthesis, thus disturbing the morphogenesis and growth of A. ochraceus. In the presence of cinnamaldehyde, the tested biosynthetic and regulatory genes like pks, nrps, veA, laeA and velB were highly downregulated. Moreover, the downregulation effect of cinnamaldehyde increased proportionally with the concentrations. These results suggest that the decrease of OTA production by cinnamaldehyde is attributed to the downregulation of the transcriptional levels of OTA biosynthetic and regulatory genes besides the inhibition of fungal growth. The study reveals the mechanisms of the antifungal and antitoxigenic activities of cinnamaldehyde against A. ochraceus, and further emphasizes that cinnamaldehyde could be a safe and effective natural agents against OTA contamination during cereals storage.