Biosynthesis Of Ochratoxin Research Articles

Influence of light on ochratoxin biosynthesis by Penicillium

Light has a profound influence on ochratoxin biosynthesis by Penicillia. When incubated under constant daylight of a certain intensity, ochratoxin A biosynthesis is decreased by about 20-30% compared to incubation under constant darkness. Under day/night oscillation, the ochratoxin A polyketide synthase gene, a key gene of the ochratoxin A biosynthesis pathway, is rhythmically expressed, and moreover, the amount of ochratoxin also oscillates between the amounts produced either during constant darkness or during constant light. This indicates a partial degradation of ochratoxin A (20-30%) under light conditions until a certain lower limit is reached. This behavior is dependent on the light intensity. At 1,600 Lux, only weak effects could be observed; however, at 2,800 Lux, the effects became significant. After growth under constant light conditions, Penicillium produced ochratoxin B at amounts which are 5 times higher than after growth in constant dark or in alternating light/dark conditions. Growth experiments in the dark on medium with increasing amounts of ochratoxin A revealed that externally applied ochratoxin is moderately toxic. However, if the same growth experiments are carried out under light conditions, the growth inhibiting activity of ochratoxin A is greatly increased, indicating that light amplifies the toxic activity of ochratoxin. Because of the oscillation of the concentration of ochratoxin A during night and day incubation, Penicillium seems to have developed an adaptive mechanism to reduce the amount of ochratoxin A during daylight below a toxic level.

Identification of a splicing coactivator gene that affects the production of ochratoxin a in Aspergillus carbonarius

Ochratoxin A is a mycotoxin produced by some fungi species. Among them, Aspergillus carbonarius is considered a powerful producer. Genes involved in the ochratoxin A biosynthesis pathway have been identified in some producer species. However, there are few studies that purpose to identify these genes in A. carbonarius. The use of insertion mutants to identify genes associated with certain properties has been increased in the literature. In this work, the region of T-DNA integration was investigated in one A. carbonarius ochratoxin-defective mutant previously obtained by Agrobacterium tumefaciens-mediated transformation, in order to find an association between interrupted gene and the biosynthesis of ochratoxin A. The integration occurred in a gene that possibly encodes a splicing coactivator protein. The analysis of the relative expression of the splicing coativator gene from A. carbonarius wild type strain in four different media showed high correlation between the transcript levels and the ochratoxin A production.

Lipoperoxidation affects ochratoxin A biosynthesis in Aspergillus ochraceus and its interaction with wheat seeds

In Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus, lipoperoxidative signalling is crucial for the regulation of mycotoxin biosynthesis, conidiogenesis, and sclerotia formation. Resveratrol, which is a lipoxygenase (LOX) and cyclooxygenase inhibitor, downmodulates the biosynthesis of ochratoxin A (OTA) in Aspergillus ochraceus. In the genome of A. ochraceus, a lox-like sequence (AoloxA; National Center for Biotechnology Information (NCBI) accession number: DQ087531) for a lipoxygenase-like enzyme has been found, which presents high homology (100 identities, 100 positives %, score 555) with a lox gene of Aspergillus fumigatus (NCBI accession number: XM741370). To study how inhibition of oxylipins formation may affect the A. ochraceus metabolism, we have used a DeltaAoloxA strain. This mutant displays a different colony morphology, a delayed conidia formation, and a high sclerotia production. When compared to the wild type, the DeltaAoloxA strain showed a lower basal activity of LOX and diminished levels of 13-hydroperoxylinoleic acid (HPODE) and other oxylipins derived from linoleic acid. The limited oxylipins formation corresponded to a remarkable inhibition of OTA biosynthesis in the DeltaAoloxA strain. Also, wheat seeds (Triticum durum cv Ciccio) inoculated with the DeltaAoloxA mutant did not accumulate 9-HPODE, which is a crucial element in the host defence system. Similarly, the expression of the pathogenesis-related protein 1 (PR1) gene in wheat seeds was not enhanced. The results obtained contribute to the current knowledge on the role of lipid peroxidation governed by the AoloxA gene in the morphogenesis, OTA biosynthesis, and in host-pathogen interaction between wheat seeds and A. ochraceus.

Aspergillus westerdijkiae polyketide synthase gene “ aoks1” is involved in the biosynthesis of ochratoxin A

Ochratoxin A (OTA) is a potential nephrotoxic, teratogenic, immunogenic, hepatotoxic and carcinogenic mycotoxin, produced by Aspergillus westerdijkiae NRRL 3174. Herein we describe the characterization of a putative OTA-polyketide synthase gene “ aoks1”, cloned by using gene walking approach. The predicted amino acid sequence of the 2 kb clone display 34–60% similarities to different polyketide synthase genes including lovastatine biosynthesis gene “ lovb” in A. terreus, compactin biosynthesis gene “ mlcA” in Penicillium citrinum and OTA biosynthesis gene “ otapksPN” in P. nordicum. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aoks1 expression was found to be associated with OTA biosynthesis. Further a mutant, in which the aoks1 gene was inactivated by Escherichia coli hygromycin B phosphotransferase gene, lost the capacity to produce OTA, but still producing mellein. To our knowledge this report describes for the first time characterization of a gene involved in OTA biosynthesis, with the information about mellein which was proposed in the literature to be an intermediate OTA. This study also suggests that aoks1 may be the second polyketide synthase gene required for OTA biosynthesis in A. westerdijkiae NRRL 3174.

Molecular and chemical monitoring of growth and ochratoxin a biosynthesis ofP. verrucosum in wheat stored at different moisture conditions

The growth and ochratoxin A producing activity at the phenotypical and molecular level ofP. verrucosum in wheat stored at different relative moisture conditions have been followed. Growth has been measured by determination of colony forming units (cfu). The expression of the ochratoxin A polyketide synthase (otapksPV) has been followed over time by reverse transcriptase Real Time PCR, whereas the biosynthesis of ochratoxin A by the fungus has been followed by HPLC. The remoisted and inoculated wheat was kept in perforated plastic bags and stored in tower silos. The bags were adjusted to 24%, 19% and 14% (m/m) relative moisture and stored for 6 month (September 06 - February 06) at ambient temperatures. A high increase of the cfu number up to a level of about lx10(8) cfu/g could be observed in the 24% sample. Interestingly no growth could be detected in the 14% and 19% samples. This result was supported by the quantitative Real Time PCR data. In congruence with this growth behaviour ochratoxin A could be detected from the first to the last time point in the 24% sample. Interestingly the ochratoxin A amounts measured over time did not show increasing or constant values, but varied from higher to lower values. After storage of three month (November 2006) the ochratoxin A concentration in the wheat was highest. The expression of theotapksPV gene mirrors this behaviour. The expression of this gene also fluctuates around a certain value, showing high activity after three month of storage.

Suppression of ochratoxin biosynthesis by naturally occurring alkaloids

The effects of four alkaloids on the biosynthesis of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin were examined on four OTA-producing aspergilli: Aspergillus auricomus, A. sclerotiorum and two isolates of A. alliaceus. Piperine and piperlongumine, natural alkaloids of Piper longum, significantly inhibited OTA production at 0.001% (w/v) for all aspergilli examined. Piperine and piperlongumine affected the polyketide synthesis step of OTA production and inhibited production of citrinin. Curcumin, a constituent of tumeric, completely inhibited mycelial growth of A. alliaceus isolate 791 at 0.1% (w/v) and decreased OTA production by ∼70% at 0.01% (w/v). Sesamin, a constituent of sesame oil, inhibited OTA and OTB production by 60 and 45%, respectively, at 0.1% (w/v), showing its effect was on chloroperoxidase and polyketide synthase activity. The potential advantage of these natural products to reduce ochratoxin contamination of agricultural commodities is discussed.

Gene expression as an indication for ochratoxin A biosynthesis inPenicillium nordicum

The expression level of theotapksPN gene ofPenicillium nordicum was determined by Reverse Transcription Real Time PCR in relation to several growth parameters known to have an influence on ochratoxin A biosynthesis, like pH, medium composition and temperature. Generally there was a good correlation between the expression level of this key gene and ochratoxin A biosynthesis. The results, which were obtained by Real Time PCR for a single gene, could generally be confirmed by microarray analysis with a whole set of genes related to ochratoxin A biosynthesis. Growth conditions which favour the expression of the ochratoxin A biosynthetic genes also resulted in the biosynthesis of ochratoxin A by the fungal culture, indicating that the influence of abiotic parameters is mediated via the regulation of transcription of the ochratoxin A biosynthetic genes. The influence of several parameters, like pH, salt concentration and temperature on the expression of the ochratoxin A biosynthetic genes has been analysed. Optimum conditions for the activation of the ochratoxin A biosynthesis genes were: pH 6.0-8.0, 25 °C and a sodium chloride concentration of 20 g/l. This was paralleled by the capacity ofP. nordicum to produce ochratoxin A under the same conditions. After combining the most inhibitory conditions (15 °C, sodium chloride 10 g/l, pH 5.0) nearly a complete deactivation of the ochratoxin A biosynthetic genes could be observed resulting in a complete abolishment of ochratoxin A biosynthesis. In contrast, a high expression and a high production of ochratoxin A could be observed after growth ofP. nordicum under more optimal conditions (25 °C, 20 g/1 sodium chloride, pH 8.0).

Molecular monitoring of environmental conditions influencing the induction of ochratoxin A biosynthesis genes in Penicillium nordicum

A real-time polymerase chain reaction (PCR) system specific for the ochratoxin A polyketide synthase gene (otapksPN) of Penicillium nordicum has been used to analyze environmental conditions, influencing the induction of that key gene of the ochratoxin A biosynthetic pathway. Generally, the induction of that gene coincides very well with the biosynthesis of ochratoxin A, demonstrating that its induction can be used as a molecular signal to monitor ochratoxin A production. It could be shown, that the expression of the otapksPN gene is greatly dependent on the media used. In YES medium expression is highest, followed by minimal medium which support ochratoxin A production and minimal medium which suppresses ochratoxin A production. The amount of ochratoxin A produced shows the same tendency. The amount produced is highest on YES medium and decreases successively to the two minimal media. The system was also used to determine the influence of environmental parameters like temperature, pH and NaCl concentration on the expression of the otapksPN gene and on ochratoxin A production in parallel. It could be shown that under acidic conditions, below pH 5.0, the expression of the otapksPN gene as well as the ochratoxin A concentration were reduced. In case of salt concentration again both measures coincide, having both highest values at increasing NaCl concentrations. In case of the temperature, however, expression of the otapksPN gene was uncoupled to ochratoxin A production. The expression was high at all temperatures tested, however, clear differences in the biosynthesis of ochratoxin A by P. nordicum could be observed at the different temperatures, showing highest production at 25 degrees C. The importance of these data are discussed with reference to the natural habitat of P. nordicum.

Biosynthesis of ochratoxins by Aspergillus ochraceus

Shaken liquid fermentation of an isolate of Aspergillus ochraceus showed growth-associated production of ochratoxins A and B, followed by production of a related polyketide diaporthin. Later, between 150 and 250 h, mellein accumulated transitorily. In contrast, shaken solid substrate (shredded wheat) fermentation over 14 days produced mainly ochratoxins A and B (ratio ca. 5:1) in very high yield (up to 10 mg/g). In these systems experiments with 14C-labelled precursors and putative intermediates revealed temporal separation of early and late stages of the ochratoxin biosynthetic pathway, but did not support an intermediary role for mellein. The pentaketide intermediate ochratoxin β was biotransformed very efficiently into both ochratoxins A and B, 14 and 19%, respectively. The already chlorinated ochratoxin α was only biotransformed significantly (4.85%) into ochratoxin A, indicating that chlorination is mainly a penultimate biosynthetic step in the biosynthesis of ochratoxin A. This was supported by poor (1.5%) conversion of radiolabelled ochratoxin B into ochratoxin A. Experiments implied that some ochratoxin B may arise by dechlorination of ochratoxin A.

Isolation of Variants of Petromyces albertensis with Altered Ochratoxin Production

Conidia of a non-stromatal isolate of the ochratoxin producing Petromyces albertensis species were irradiated in order to obtain variants altered in ochratoxin production. Variants producing various amounts of ochratoxins, including an ochratoxin non-producing variant were readily isolated. These variants are planned to be applied to reveal details of ochratoxin biosynthesis within this teleomorphic species.

Carbon-13 nuclear magnetic resonance assignments and biosynthesis of ochratoxin A

The carbon-13 chemical shifts have been assigned to all the carbons in the isocoumarin portion of ochartoxin A. Incorporation of carbon-13 enriched acetate was used to confirm the biosynthesis of ochratoxin A.

Mycotoxins – Their Biosynthesis in Fungi: Ochratoxins – Metabolites of Combined Pathways

Little work has been done on the biosynthesis of ochratoxins. Nevertheless. a scheme based on known chemical and enzymatic reactions and incorporating known facts about ochratoxin biosynthesis is proposed as a working model. The scheme calls for formation of an unbranched pentaketide from acetyl and malonyl CoA, its cyclization and aromatization to an is ocoumarin, methylation and oxidation to the carboxy derivative which is chlorinated by chloroperoxidase before acyl activation for reaction with an ester of phenylalanine which arises via the shikimic acid pathway. The amide, ochratoxin A, is considered the final product of this biosynthetic pathway.

Metabolism of Glutamic Acid in Aspergillus ochraceus During the Biosynthesis of Ochratoxin A

The uptake and utilization of glutamic acid in the biosynthesis of ochratoxin A by Aspergillus ochraceus were studied. Uniformly labeled L-[ 14 C]glutamic acid was incorporated into both the phenylalanine and isocoumarin moieties of ochratoxin A. Penicillic acid was also labeled. During the early stages of development, the amino acid was used mainly for the synthesis of ribonucleic acid and protein. A portion of glutamic acid was oxidized and was recovered as metabolic 14 CO 2 . The initial uptake velocity of glutamic acid decreased with age and was pH and temperature dependent. No relationship was found between the initial uptake velocities and ochratoxin A biosynthesis.

Metabolism of Glutamic Acid in Aspergillus ochraceus During the Biosynthesis of Ochratoxin A

The uptake and utilization of glutamic acid in the biosynthesis of ochratoxin A by Aspergillus ochraceus were studied. Uniformly labeled L[14C]glutamic acid was incorporated into both the phenylalanine and isocoumarin moieties of ochratoxin A. Penicillic acid was also labeled. During the early stages of development, the amino acid was used mainly for the synthesis of ribonucleic acid and protein. A portion of glutamic acid was oxidized and was recovered as metabolic 14CO-2. The initial uptake velocity of glutamic acid decreased with age and was pH and temperature dependent. No relationship was found between the initial uptake velocities and ochratoxin A biosynthesis.

Application of 13 C-NMR to the biosynthetic investigations. I. Biosynthesis of ochratoxin A.

Incorporation of L-phenylalanine-U-14C, Na-acetate-2-14C into ochratoxin A was demonstrated in Aspergillus ochraceus, suggesting that the biosynthesis of ochratoxin A involves so called acetate-malonate and shikimic acid pathways. By the aid of 13C-NMR spectrometric method, the participation of Na-formate-13C for the formation of carboxyl group was confirmed.

The biosynthesis of the ochratoxins, metabolites of aspergillus ochraceus

The biosynthesis of ochratoxin A (Ia) was studied in Aspergillus ochraceus Wilh. by experiments in which dl-phenylalanine [U- 14C], sodium acetate [1- 14C] and methionine [ 14CH 3] were supplied to a resting culture of the fungus. The carboxyl carbon of the amide group is shown to be derived from the C 1-pool.

Biosynthesis of ochratoxin A.

Biosynthesis of ochratoxin A by Aspergillus ochraceus Wilh. was investigated by radiolabeling experiments in which phenylalanine-1-(14)C and sodium acetate-2-(14)C were supplied to the fungus in sucrose-yeast extract medium. Results showed that phenylalanine was incorporated unaltered into the phenylalanine moiety of ochratoxin A, whereas the isocoumarin moiety of ochratoxin A was mostly derived via acetate condensation.

Biosynthesis of Ochratoxin A1

Biosynthesis of ochratoxin A by Aspergillus ochraceus Wilh. was investigated by radiolabeling experiments in which phenylalanine-1-(14)C and sodium acetate-2-(14)C were supplied to the fungus in sucrose-yeast extract medium. Results showed that phenylalanine was incorporated unaltered into the phenylalanine moiety of ochratoxin A, whereas the isocoumarin moiety of ochratoxin A was mostly derived via acetate condensation.