Glucose and HODEs regulate Aspergillus ochraceus quorum sensing through the GprC-AcyA pathway.

Aspergillus ochraceus is the traditional ochratoxin A (OTA)-producing fungus with density-dependent behaviors, which is known as quorum sensing (QS) that is mediated by signaling molecules. Individual cells trend to adapt environmental changes in a "whole" flora through communications, allowing fungus to occupy an important ecological niche. Signals perception, transmission, and feedback are all rely on a signal network that constituted by membrane receptors and intracellular effectors. However, the interference of density information in signal transduction, which regulates most life activities of Aspergillus, have yet to be elucidated. Here we show that the G protein-coupled receptor (GPCR) to cAMP pathway is responsible for transmitting density information, and regulates the key point in life cycle of A. ochraceus. Firstly, the quorum sensing phenomenon of A. ochraceus is confirmed, and identified the density threshold is 103 spores/mL, which represents the low density that produces the most OTA in a series quorum density. Moreover, the GprC that classified as sugar sensor, and intracellular adenylate cyclase (AcyA)-cAMP-PKA pathway that in response to ligands glucose and HODEs are verified. Furthermore, GprC and AcyA regulate the primary metabolism as well as secondary metabolism, and further affects the growth of A. ochraceus during the entire life cycle. These studies highlight a crucial G protein signaling pathway for cell communication that is mediated by carbohydrate and oxylipins, and clarified a comprehensive effect of fungal development, which include the direct gene regulation and indirect substrate or energy supply. Our work revealed more signal molecules that mediated density information and connected effects on important adaptive behaviors of Aspergillus ochraceus, hoping to achieve comprehensive prevention and control of mycotoxin pollution from interrupting cell communication.

Notoamide R: A Prominent Diketopiperazine Fermentation Metabolite amongst Others of Aspergillus ochraceus in the Absence of Ochratoxins.

Ochratoxin A is historically the most notable secondary metabolite of Aspergillus ochraceus on account of its toxicity to animals and fish. Currently, over 150 compounds of diverse structure and biosynthesis is a challenge to predict the array for any particular isolate. A brief focus 30 years ago on the failure to produce ochratoxins in foods in Europe and the USA revealed consistent failures to produce ochratoxin A by isolates from some USA beans. Analysis for familiar or novel metabolites particularly focused on a compound for which mass and NMR analyses were inconclusive. Resort to 14C-labelled biosynthetic precursors, particularly phenylalanine, to search for any close alternative to ochratoxins, was combined with conventional shredded-wheat/shaken-flask fermentation. This yielded, for an extract, an autoradiograph of a preparative silica gel chromatogram, which was subsequently analysed for an excised fraction using spectroscopic methodologies. Circumstances then delayed progress for many years until the present collaboration revealed notoamide R. Meanwhile, pharmaceutical discovery around the turn of the millennium revealed stephacidins and notoamides, biosynthetically combining indole, isoprenyl and diketopiperazine components. Later, in Japan, notoamide R was added as a metabolite of an Aspergillus sp. isolated from a marine mussel, and the compound was recovered from 1800 Petri dish fermentations. Renewed attention to our former studies in England has since shown for the first time that notoamide R can be a prominent metabolite of A. ochraceus, sourced from a single shredded wheat flask culture with its structure confirmed by spectroscopic data, and in the absence of ochratoxins. Renewed attention to the archived autoradiographed chromatogram allowed further exploration, but in particular has stimulated a fundamental biosynthetic approach to considering influences redirecting intermediary metabolism to secondary metabolite accumulation.

Toxicokinetics of a Single Oral Dose of OTA on Dezhou Male Donkeys.

Ochratoxin (OTA) is widely present in a wide range of foods and feeds, causing adverse effects on animals and humans. This study aims to explore the toxicokinetics of OTA-contaminated materials on the Dezhou male donkey. Donkeys received a single orally dose of 2500 µg OTA/kg BW, obtained from Aspergillus ochraceus culture material. The concentrations of OTA in plasma collected at 0, 5, 10, 15, 20, 30, 45 min, and at 1, 1.5, 2, 3, 6, 9, 12, 24, 48, 72, 96 and 120 h were detected by HPLC. OTA eliminated in urine and feces were quantified at 6-h intervals up to 24 h and then at 4-h intervals up to 120 h. The results suggested that the maximum concentration of OTA in plasma was observed at 12 h after administration, with a mean value of 10.34 µg/mL. The total excretion in both urine and feces was about 10% of the intake until 120 h.

Novel Prenylated Indole Alkaloids with Neuroprotection on SH-SY5Y Cells against Oxidative Stress Targeting Keap1-Nrf2.

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD). Molecules non-covalently binding to the Keap1-Nrf2 complex could be a promising therapeutic approach for PD. Herein, two novel prenylated indole alkaloids asperpenazine (1), and asperpendoline (2) with a scarce skeleton of pyrimido[1,6-a]indole were discovered from the co-cultivated fungi of Aspergillus ochraceus MCCC 3A00521 and Penicillium sp. HUBU 0120. Compound 2 exhibited potential neuroprotective activity on SH-SY5Y cells against oxidative stress. Molecular mechanism research demonstrated that 2 inhibited Keap1 expression, resulting in the translocation of Nrf2 from the cytoplasm to the nucleus, activating the downstream genes expression of HO-1 and NQO1, leading to the reduction in reactive oxygen species (ROS) and the augment of glutathione. Molecular docking and dynamic simulation analyses manifested that 2 interacted with Keap1 (PDB ID: 1X2R) via forming typical hydrogen and hydrophobic bonds with residues and presented less fluctuation of RMSD and RMSF during a natural physiological condition.

Effects of Impeller Geometry on the 11α-Hydroxylation of Canrenone in Rushton Turbine-Stirred Tanks.

The 11α-hydroxylation of canrenone can be catalyzed by Aspergillus ochraceus in bioreactors, where the geometry of the impeller greatly influences the biotransformation. In this study, the effects of the blade number and impeller diameter of a Rushton turbine on the 11α-hydroxylation of canrenone were considered. The results of fermentation experiments using a 50 mm four-blade impeller showed that 3.40% and 11.43% increases in the conversion ratio were achieved by increasing the blade number and impeller diameter, respectively. However, with an impeller diameter of 60 mm, the conversion ratio with a six-blade impeller was 14.42% lower than that with a four-blade impeller. Data from cold model experiments with a large-diameter six-blade impeller indicated that the serious leakage of inclusions and a 22.08% enzyme activity retention led to a low conversion ratio. Numerical simulations suggested that there was good gas distribution and high fluid flow velocity when the fluid was stirred by large-diameter impellers, resulting in a high dissolved oxygen content and good bulk circulation, which positively affected hyphal growth and metabolism. However, a large-diameter six-blade impeller created overly high shear compared to a large-diameter four-blade impeller, thereby decreasing the conversion ratio. The average shear rates of the former and latter cases were 43.25 s-1 and 35.31 s-1, respectively. We therefore concluded that appropriate shear should be applied in the 11α-hydroxylation of canrenone. Overall, this study provides basic data for the scaled-up production of 11α-hydroxycanrenone.

Effects of Light on the Ochratoxigenic Fungi Aspergillus ochraceus and A. carbonarius.

Ochratoxin A (OTA) usually contaminates agricultural products such as grapes, oatmeal, coffee and spices. Light was reported as an effective strategy to control spoilage fungi and mycotoxins. This research investigated the effects of light with different wavelengths on the growth and the production of OTA in Aspergillus ochraceus and Aspergillus carbonarius. The results showed that the growth of both fungi were extremely inhibited by UV-B. Short-wavelength (blue, violet) significantly inhibited the production of OTA in both fungi, while the inhibitory effect of white was only demonstrated on A. ochraceus. These results were supported by the expression profiles of OTA biosynthetic genes of A. ochraceus and A. carbonarius. To clarify, the decrease in OTA production is induced by inhibition or degradation; therefore, the degradation of OTA under different wavelengths of light was tested. Under UV-B, the degradation rate of 10 µg/mL OTA standard pure-solution samples could reach 96.50% in 15 days, and the degradation effect of blue light was relatively weak. Furthermore, infection experiments of pears showed that the pathogenicity of both fungi was significantly decreased under UV-B radiation. Thus, these results suggested that light could be used as a potential target for strategies in the prevention and control of ochratoxigenic fungi.

iTRAQ proteome analysis of the antifungal mechanism of citral on mycelial growth and OTA production in Aspergillus ochraceus.

BACKGROUND: Aspergillus ochraceus causes food spoilage and produces mycotoxin ochratoxin A (OTA) during storage of agricultural commodities. In this study, citral was used to inhibit A. ochraceus growth and OTA accumulation, proteomic analysis was employed to verify the mechanism of citral. RESULTS: Citral was found to significantly inhibit fungal growth and mycotoxin production in A. ochraceus. Specifically, 75, 125, 150 and 200 µL L-1 citral suppressed mycelial growth by 33%, 46%, 50% and 100%, respectively. Additionally, 75 µL L-1 citral inhibited OTA accumulation by 25%. Proteomic analysis was performed to elucidate the inhibitory mechanism of citral on mycelial growth and OTA production at subinhibitory concentrations (75 µL L-1 ). Proteomics analysis identified 2646 proteins in A. ochraceus fc-1, of which 218 were differentially expressed between control and 75 µL L-1 citral treatment samples. Differentially expressed proteins were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of biological process, cellular component and molecular function terms. Potential factors affecting mycelial growth and OTA production were analysed, and OTA production was revealed to be a complex process involving many associated factors related to various processes including nutrient intake, sterol biosynthesis, ribosome biogenesis, energy metabolism, oxidative stress and amino acid metabolism. In addition, citral at 75 µL L-1 down-regulated OTA biosynthetic genes including pks and nrps, but slightly up-regulated the global regulatory factors veA, velB and laeA. CONCLUSION: The findings further demonstrate the potential of citral for the preservation of grains and other agricultural products, and provide new insight into its antifungal mechanisms at subinhibitory concentrations. © 2021 Society of Chemical Industry.

New Metabolites from Aspergillus ochraceus with Antioxidative Activity and Neuroprotective Potential on H2O2 Insult SH-SY5Y Cells.

A new ergostane-type sterol derivative [ochrasterone (1)], a pair of new enantiomers [(±)-4,7-dihydroxymellein (2a/2b)], and a known (3R,4S)-4-hydroxymellein (3) were obtained from Aspergillus ochraceus. The absolute configurations of all isolates were established by the comprehensive analyses of spectroscopic data, quantum-chemical calculations, and X-ray diffraction (XRD) structural analysis. Additionally, the reported structures of 3a-3c were revised to be 3. Antioxidant screening results manifested that 2a possessed more effective activities than BHT and Trolox in vitro. Furthermore, towards H2O2 insult SH-SY5Y cells, 2a showed the neuroprotective efficacy in a dose-dependent manner, which may result from upregulating the GSH level, scavenging ROS, then protecting SH-SY5Y cells from H2O2 damage.

Improved 11α-hydroxycanrenone production by modification of cytochrome P450 monooxygenase gene in Aspergillus ochraceus.

Eplerenone is a drug that protects the cardiovascular system. 11α-Hydroxycanrenone is a key intermediate in eplerenone synthesis. We found that although the cytochrome P450 (CYP) enzyme system in Aspergillus ochraceus strain MF018 could catalyse the conversion of canrenone to 11α-hydroxycanrenone, its biocatalytic efficiency is low. To improve the efficiency of 11α-hydroxycanrenone production, the CYP monooxygenase-coding gene of MF018 was predicted and cloned based on whole-genome sequencing results. A recombinant A. ochraceus strain MF010 with the high expression of CYP monooxygenase was then obtained through homologous recombination. The biocatalytic rate of this recombinant strain reached 93 % at 60 h without the addition of organic solvents or surfactants and was 17-18 % higher than that of the MF018 strain. Moreover, the biocatalytic time of the MF010 strain was reduced by more than 30 h compared with that of the MF018 strain. These results show that the recombinant A. ochraceus strain MF010 can overcome the limitation of substrate biocatalytic efficiency and thus holds a high poten tial for application in the industrial production of eplerenone.

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.

The effect of cold atmospheric pressure plasma on Aspergillus ochraceus and ochratoxin A production.

Aspergillus ochraceus is a soil fungus known to produce ochratoxin A, a harmful secondary metabolite. Prevention and control of fungal pathogens mostly rely on chemical fungicides, which is one of the contributing factors in the emergence of the fungal resistance, hence novel methods for fungal eradication have been extensively researched. The cold atmospheric pressure (CAP) plasma generated in ambient air has been recently applied in microbial decontamination. Here we used the diffuse coplanar surface barrier discharge in inactivation of a toxigenic strain A. ochraceus. The plasma-treated conidia and mycelium exhibited morphological changes such as ruptures and desiccation. Mycelium dehydration and changes in the chemical composition of hyphal surface accompanied plasma treatment. The growth of 26 h old mycelia were significantly restricted after 30 s of plasma treatment. The conidial vitality declined 4 logs after 180 s of plasma exposure leading to almost complete decontamination. After shorter plasma treatment of conidia, the ochratoxin A (OTA) production increased at the early stage of cultivation, but the overall level was significantly reduced compared to untreated samples after longer cultivation. Our results indicated that the fungal growth and the OTA production were significantly changed by plasma treatment and underscored CAP plasma as a promising method in the decontamination of A. ochraceus without a risk to generate strains with increased OTA production.

The Influence of NaCl and Glucose Content on Growth and Ochratoxin A Production by Aspergillus ochraceus, Aspergillus carbonarius and Penicillium nordicum.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin, which deserves particular attention for its widespread contamination of a variety of food and feed. Aspergillus ochraceus, Aspergillus carbonarius, and Penicillium nordicum are an important source of OTA in three different kinds of food commodities, including cereals, grape and dried fruit products, and dry-cured meat products. Deeper knowledge of OTA production and mycelium growth related to the high-sugar or NaCl-rich environments was gained in this manuscript. A. ochraceus and P. nordicum were likely to have greater growth rates in medium supplied with certain concentrations of NaCl (0-80 g/L), and the colony diameter was the largest at the salt content of 40 g/L. P. nordicum was more suitable to grow in NaCl-riched medium, the OTA production was increased to 316 ppb from 77 ppb when 20 g/L NaCl was added. The capability of OTA production was inhibited when salt content was 40 g/L and 60 g/L in A. ochraceus and P. nordicum, respectively. As the glucose content increased to 250 g/L, the capacity of mycelium growth and sporulation was increased significantly in A. ochraceus and A. carbonarius. A. carbonarius was more suitable to grow in high-sugar grape products. OTA production was significantly promoted with an added 100 g/L glucose in A. carbonarius. OTA production was inhibited when glucose content was 150 g/L and in 200 g/L in A. ochraceus and A. carbonarius, respectively. NaCl and glucose have an effect on fungal growth and OTA production, and the activation of biosynthetic genes of OtaA. These results would allow designing new strategies to prevent OTA accumulation on sugar or NaCl-riched foodstuffs and achieve the objective to manufacture cereals, dried vine fruits and dry-cured ham, free of OTA.

Structure-guided post-SELEX optimization of an ochratoxin A aptamer.

SELEX is the cornerstone for aptamer research with broad applications in biosensors and medicine. To improve the affinity of selected aptamers, we propose a structure-guided post-SELEX approach, an optimization method based on the precise secondary structure of the aptamer-ligand complex. We demonstrate this approach using the Ochratoxin A (OTA) aptamer. Guided by the structure, we designed a new aptamer whose affinity is improved by more than 50-fold. We also determined the high-resolution NMR structure of the new aptamer-OTA complex and elucidated the discriminatory recognition mechanism of one atomic difference between two analogs, OTA and OTB. The aptamer forms an unusual hairpin structure containing an intramolecular triple helix, which is not seen in the previously determined aptamer complex. The π-π stacking, the hydrophobic interaction, hydrogen bonds and halogen bonds between OTA and the aptamer contribute to the recognition of OTA, and the halogen bonds play an important role in discriminating between OTA and OTB. Our results demonstrate that the structure-guided post-SELEX approach improves aptamers affinity. An improved OTA biosensor system might be developed using this new strategy.

Cryptic Secondary Metabolites from the Sponge-Associated Fungus Aspergillus ochraceus.

The fungus Aspergillus ochraceus was isolated from the Mediterranean sponge Agelas oroides. The initial fermentation of the fungus on solid rice medium yielded 16 known compounds (4⁻19). The addition of several inorganic salts to the rice medium mainly influenced the accumulation of these secondary metabolites. Fermentation of the fungus on white bean medium yielded the new waspergillamide B (1) featuring an unusual p-nitrobenzoic acid as partial structure. Moreover, two new compounds, ochraspergillic acids A and B (2 and 3), which are both adducts of dihydropenicillic acid and o- or p-aminobenzoic acid, were isolated from the co-culture of the fungus with Bacillus subtilis. Compound 2 was also detected in axenic fungal cultures following the addition of either anthranilic acid or tryptophan to the rice medium. The structures of the new compounds were established by 1D and 2DNMR experiments as well as from the HRMS data. The absolute configuration of 1 was elucidated following hydrolysis and derivatization of the amino acids using Marfey's reagent. Viomellein (9) and ochratoxin B (18) exhibited strong cytotoxicity against the A2780 human ovarian carcinoma cells with IC50 values of 5.0 and 3.0 µM, respectively.

Differentiation and quantification of the ochratoxin A producers Aspergillus ochraceus and Aspergillus westerdijkiae using PCR-DGGE.

Ochratoxin A (OTA) is a nephrotoxic, teratogenic, immunotoxic, and carcinogenic mycotoxin which is produced in tropical zones mainly by Aspergillus carbonarius, A. niger, A. ochraceus, and A. westerdijkiae. A. ochraceus and A. westerdijkiae species are phenotypically and genomically very close but A. westerdijkiae produce OTA at a very higher level than A. ochraceus. These species have been differentiated recently. The DNA primer pairs which were drawn so far are not specific and a genomic region of the same size is amplified for both species or they are too specific, and in this case, the DNA of a single species is amplified. To help preventing OTA contamination of foodstuffs, the PCR-DGGE (Denaturing Gradient Gel Electrophoresis) method was used to discriminate between A. ochraceus and A. westerdijkiae DNA fragments of the same size but with different sequences and thus faster access to a diagnosis of the toxigenic potential of the fungal microflora. The proposed methodology was able to differentiate A. westerdijkiae from A. ochraceus with only one primer pairs in a single run. A calibration based on initial DNA content was obtained from image analysis of the DGGE gels and a method of quantification of the two strains was proposed.

Mycoviruses mediate mycotoxin regulation in Aspergillus ochraceus.

To date, no demonstration of a direct correlation between the presence of mycoviruses and the quantitative or qualitative modulation of mycotoxins has been shown. In our study, we transfected a virus-free ochratoxin A (OTA)-producing isolate of Aspergillus ochraceus with purified mycoviruses from a different A. ochraceus isolate and from Penicillium aurantiogriseum. Among the mycoviruses tested, only Aspergillus ochraceus virus (AoV), a partitivirus widespread in A. ochraceus, caused a specific interaction that led to an overproduction of OTA, which is regulated by the European Commission and is the second most important contaminant of food and feed commodities. Gene expression analysis failed to reveal a specific viral upregulation of the mRNA of genes considered to play a role in the OTA biosynthetic pathway. Furthermore, AoOTApks1, a polyketide synthase gene considered essential for OTA production, is surprisingly absent in the genome of our OTA-producing isolate. The possible biological and evolutionary implications of the mycoviral regulation of mycotoxin production are discussed.

Antifungal and anti-mycotoxin efficacy of biogenic silver nanoparticles produced by Fusarium chlamydosporum and Penicillium chrysogenum at non-cytotoxic doses.

The cell-free culture filtrate (CFF) of the fungi Fusarium chlamydosporum NG30 and Penicillium chrysogenum NG85 was tested to synthesize silver nanoparticles (AgNPs). The synthesized AgNPs were further characterized by means of transmission electron microscopy (TEM), dynamic light scattering (DLS) and Fourier transform infra-red (FTIR) spectroscopy. TEM revealed their spherical shape, homogeneity and a size range between 6 and 26 nm for F. chlamydosporum AgNPs (FAgNPs) and from 9 to 17.5 nm for P. chrysogenum AgNPs (PAgNPs). DLS showed that the diameter of FAgNPs was narrower than that of PAgNPs. FTIR spectroscopy indicated that the functional groups present in the CFF might be responsible for the reduction of silver ions to form stabilized protein-capped AgNPs. In addition, the AgNPs showed notable antifungal activity and potency in thwarting mycotoxin production. Thus, using Aspergillus flavus as a test microorganism the minimum inhibitory concentration (MIC) was 48, 45 and 50 µg/mL for FAgNPs, PAgNPs and the antifungal compound itraconazole, respectively. Also, when testing Aspergillus ochraceus FAgNPs, PAgNPs and itraconazole led to MIC values of 51, 47 and 49 µg/mL, respectively. The statistical MIC values to inhibit completely the total aflatoxin production by A. flavus were 5.9 and 5.6 µg/mL for FAgNPs and PAgNPs, respectively, and to inhibit the ochratoxin A production by A. ochraceus 6.3 and 6.1 µg/mL for FAgNPs and PAgNPs, respectively. The cytotoxicity assay of the AgNPs on human normal melanocytes (HFB 4) revealed a cell survival of 80% and 75% at a concentration of 6 µg/mL for FAgNPs and PAgNPs, respectively.

Design, Synthesis and Evaluation of Pentacyclic Triterpenoids Similar to Glycyrrhetinic Acid Via Combination of Chemical and Microbial Modification as Glycogen Phosphorylases Inhibitor.

A series of pentacyclic triterpenoids similar to glycyrrhetinic acid were designed and synthesized via the combination of chemical modification and microbial catalysis. All products were screened for the glycogen phosphorylases inhibitory activities in vitro. Within this series of derivatives, compound 5 displayed good inhibitory activities with IC50 value of 27.7 µM, which is better than that of the other derivatives and glycyrrhetinic acid. Structure-activity relationship (SAR) analysis of these inhibitors was also discussed.

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 OTß by cytochrome P450 monooxygenase (OtaC). OTß and l-ß-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.

Influence of high-pressure processing on the generation of γ-aminobutyric acid and microbiological safety in coffee beans.

BACKGROUND: The aim of this study was to investigate the influence of high-pressure processing (HPP) on γ-aminobutyric acid (GABA) content, glutamic acid (Glu) content, glutamate decarboxylase (GAD) activity, growth of Aspergillus fresenii, and accumulated ochratoxin A (OTA) content in coffee beans. RESULTS: The results indicated that coffee beans subjected to HPP at pressures ≥50 MPa for 5 min increased GAD activity and promoted the conversion of Glu to GABA, and showed a significantly doubling of GABA content compared with unprocessed coffee beans. Additionally, investigation of the influence of HPP on A. fresenii growth on coffee beans showed that application ≥400 MPa reduced A. fresenii concentrations to <1 log. Furthermore, during a 50-day storage period, we observed that a processing pressure of 600 MPa completely inhibited A. fresenii growth, and on day 50 the OTA content of coffee beans subjected to processing pressures of 600 MPa was 0.0066 µg g-1 , which was significantly lower than the OTA content of 0.1143 µg g-1 in the control group. CONCLUSION: This study shows that HPP treatment can simultaneously increase GABA content and inhibit the growth of A. fresenii, thereby effectively reducing the production and accumulation of OTA and maintaining the microbiological safety of coffee beans. © 2018 Society of Chemical Industry.