Élucidation de la voie de biosynthèse d’une mycotoxine, la patuline : caractérisation du cluster de gène et étude de la régulation

Penicillium expansum est un contaminant commun des pomaceae (pommes et poires) causant la pourriture bleue. Ce champignon est le principal responsable de la présence de patuline dans les pommes et ses produits dérivés. Actuellement, la voie de biosynthèse de la patuline n’est que partiellement élucidée et le cluster de gènes correspondant n’est décrit que chez Aspergillus clavatus, champignon tellurique incapable de se développer dans les pommes. La caractérisation moléculaire de la voie de biosynthèse de la patuline est la condition sine qua none à toute étude visant à comprendre la régulation de la biosynthèse de la patuline, mais également à toute action permettant de limiter sa synthèse. C’est pourquoi le premier objectif de cette thèse a été de caractériser le cluster de gènes spécifique de la voie de biosynthèse chez Penicillium expansum. Celui-ci est caractérisé par une taille de 40 kb et contient les 15 mêmes gènes qu’Aspergillus clavatus, les seules différences résidant dans l’organisation et l’orientation des gènes. La caractérisation de la seconde étape de la voie de biosynthèse de la patuline a été ensuite entreprise chez Aspergillus clavatus, organisme modèle. Le gène patG code pour l’acide 6-méthylsalicylique décarboxylase responsable de la conversion de l’acide 6-méthylsalicylique en m-crésol. Pour faire suite au premier objectif, la régulation de la voie de biosynthèse de la patuline a été étudiée. Pour cela, une souche mutante pour le facteur de régulation spécifique à la patuline patL a été généré puis la production de patuline ainsi que l’expression des gènes du cluster analysés. Les résultats de cette étude ont montré que le gène patL joue le rôle d’interrupteur au sein du cluster. L’absence de patL conduit à une extinction totale de l’expression des gènes du cluster et à une abscence de production de patuline par Penicillium expansum. Dans cette même étude, des tests de pathogénicité ont été entrepris sur des pommes de différentes variétés démontrant ainsi que la patuline peut être un facteur de virulence facilitant l’infection de certaines variétés de pommes telles que la Golden Delicious ou la Pink Lady. Enfin, l’influence de la lumière a été évaluée en analysant l’impact de différentes longueurs d’ondes sur la croissance et la production de patuline de Penicillium expansum. Que ce soit in-vitro ou in-vivo, la croissance et la production de patuline sont très affectés par les lumières blanche, bleue et rouge. Favoriser le stockage des pommes sous les lumières blanche, bleue ou rouge plutôt qu’à l’obscurité pourrait devenir un moyen de prévention contre la contamination par Penicillium expansum. En conclusion, cette thèse présente un aspect fondamental avec la caractérisation du cluster de gènes chez Penicillium expansum et la caractérisation de la seconde étape de la voie de biosynthèse de la patuline ; mais aussi un aspect appliqué avec l’utilisation des lumières de différentes couleurs comme méthode de prévention contre Penicillium expansum durant le stockage des pommes. ABSTRACT : Penicillium expansum is the common contaminant of apples and the causal agent of blue mold rot. This fungus is the main patulin producer in apple based products. Actually, the patulin biosynthesis is partially elucidates and the gene cluster has been elucidated in Aspergillus clavatus, a telluric fungi unable to grow on apples. The molecular characterization of the patulin biosynthetic pathway is the key step for a better understanding of the mechanisms leading to patulin production and will help to define strategies to reduce its presence in apple products. The first objective of this thesis was the characterization of the patulin gene cluster in Penicillium expansum. The latter includes the same 15 genes as in Aspergillus clavatus but in a different order and orientation. Then, the second step of this biosynthetic pathway has been characterized and the patG gene encode for the 6-methylsalicylic decarboxylase involved in the 6- methylsalicylic acid conversion into m-cresol. The second objective consists of the study of the patulin regulation. For that, a patL mutated strain was generated and the patulin production and the patulin gene cluster expression were assessed. The mutation of this gene results in a down-regulation of the rest of the genes in the cluster associated with a lack of patulin production. Pathogenicity tests on apples revealed that patulin could act as a virulence factor in some apple varieties, like Golden Delicious or Pink Lady. In the last part of this thesis, the influence of different wavelength lights on the growth and the patulin production by Penicillium expansum were assessed in vitro and in vivo. In both cases, growth and patulin production were significantly affected under white, blue and red lights. Consequently, the apple storage under these lights could be a good alternative to the storage in the dark. In conclusion, this thesis presents a fundamental aspect that consist in the characterization of the patulin gene cluster in Penicillium expansum and the characterization of the second step of this pathway. An applied aspect is also provided by the use of the different wavelength lights to prevent the Penicillium expansum contamination during apple storage

Biocontrol agents and natural compounds against mycotoxinogenic fungi

Mycotoxins are toxic fungal secondary metabolites that contaminate food and feed. Mycotoxin contamination occurs as soon as environmental conditions are favorable for fungal growth and mycotoxin production, in the fields, during storage of raw materials and during industrial processes

Streptomyces roseolus, A Promising Biocontrol Agent Against Aspergillus flavus, the Main Aflatoxin B1 Producer

Crop contamination by aflatoxin B1 is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute to the substitution of phytopharmaceutical products are thus needed. For this, a promising method resides in the use of biocontrol agents. Several actinobacteria strains have demonstrated to effectively reduce the aflatoxin B1 concentration. Nevertheless, the molecular mechanism of action by which these biological agents reduce the mycotoxin concentration has not been determined. The aim of the present study was to test the potential use of Streptomyces roseolus as a biocontrol agent against aflatoxin B1 contamination. Co-cultures with Aspergillus flavus were conducted, and the molecular fungal response was investigated through analyzing the q-PCR expression of 65 genes encoding relevant fungal functions. Moreover, kojic and cyclopiazonic acid concentrations, as well as morphological fungal changes were also analyzed. The results demonstrated that reduced concentrations of aflatoxin B1 and kojic acid were respectively correlated with the down-regulation of the aflatoxin B1 gene cluster and kojR gene expression. Moreover, a fungal hypersporulated phenotype and a general over-expression of genes involved in fungal development were observed in the co-culture conditio

Phenyllactic acid produced by Geotrichum candidum reduces Fusarium sporotrichioides and F. langsethiae rowth and T-2 Toxin Concentration

Fusarium sporotrichioides and F. langsethiae are present in barley crops. Their toxic metabolites, mainly T-2 toxin, affect the quality and safety of raw material and final products such as beer. Therefore, it is crucial to reduce Fusarium spp. proliferation and T-2 toxin contamination during the brewing process. The addition of Geotrichum candidum has been previously demonstrated to reduce the proliferation of Fusarium spp. and the production of toxic metabolites, but the mechanism of action is still not known. Thus, this study focuses on the elucidation of the interaction mechanism between G. candidum and Fusarium spp. in order to improve this bioprocess. First, over a period of 168 h, the co-culture kinetics showed an almost 90% reduction in T-2 toxin concentration, starting at 24 h. Second, sequential cultures lead to a reduction in Fusarium growth and T-2 toxin concentration. Simultaneously, it was demonstrated that G. candidum produces phenyllactic acid (PLA) at the early stages of growth, which could potentially be responsible for the reduction in Fusarium growth and T-2 toxin concentration. To prove the PLA effect, F. sporotrichioides and F. langsethiae were cultivated in PLA supplemented medium. The expected results were achieved with 0.3 g/L of PLA. These promising results contribute to a better understanding of the bioprocess, allowing its optimization at an up-scaled industrial level

Antimicrobial activities of novel bipyridine compounds produced by a new strain of Saccharothrix isolated from Saharan soil

The actinobacterium strain ABH26 closely related to Saccharothrix xinjiangensis, isolated from an Algerian Saharan soil sample, exhibited highly antagonist activity against Gram-positive bacteria, yeasts and filamentous fungi. Its ability to produce antimicrobial compounds was investigated using several solid culture media. The highest antimicrobial activity was obtained on Bennett medium. The antibiotics secreted by strain ABH26 on Bennett medium were extracted by methanol and purified by reverse-phase HPLC using a C18 column. The chemical structures of the compounds were determined after spectroscopic (1H NMR, 13C NMR, 1H-1H COSY and 1H-13C HMBC spectra), and spectrometric (mass spectrum) analyses. Two new cyanogriside antibiotics named cyanogriside I (1) and cyanogriside J (2), were characterized along with three known caerulomycins, caerulomycin A (3), caerulomycin F (4) and caerulomycinonitrile (5). This is the first report of cyanogrisides and caerulomycins production by a member of the Saccharothrix genus. The minimum inhibitory concentrations (MIC) of these antibiotics were determined against pathogenic microorganisms

Impact of veA on the development, aggressiveness, dissemination and secondary metabolism of Penicillium expansum

Penicillium expansum, the causal agent of blue mould disease, produces the mycotoxins patulin and citrinin amongst other secondary metabolites. Secondary metabolism is associated with fungal development, which responds to numerous biotic and abiotic external triggers. The global transcription factor VeA plays a key role in the coordination of secondary metabolism and differentiation processes in many fungal species. The specific role of VeA in P. expansum remains unknown. A null mutant PeΔveA strain and a complemented PeΔveA:veA strain were generated in P. expansum and their pathogenicity on apples was studied. Like the wild‐type and the complemented strains, the null mutant PeΔveA strain was still able to sporulate and to colonize apples, but at a lower rate. However, it could not form coremia either in vitro or in vivo, thus limiting its dissemination from natural substrates. The impact of veA on the expression of genes encoding proteins involved in the production of patulin, citrinin and other secondary metabolites was evaluated. The disruption of veA drastically reduced the production of patulin and citrinin on synthetic media, associated with a marked down‐regulation of all genes involved in the biosynthesis of the two mycotoxins. Moreover, the null mutant PeΔveA strain was unable to produce patulin on apples. The analysis of gene expression revealed a global impact on secondary metabolism, as 15 of 35 backbone genes showed differential regulation on two different media. These findings support the hypothesis that VeA contributes to the pathogenicity of P. expansum and modulates its secondary metabolism

Elucidation of a mycotoxin biosynthesis pathway, the patulin : gene cluster characterization and study of its regulation

Penicillium expansum est un contaminant commun des pomaceae (pommes et poires) causant la pourriture bleue. Ce champignon est le principal responsable de la présence de patuline dans les pommes et ses produits dérivés. Actuellement, la voie de biosynthèse de la patuline n’est que partiellement élucidée et le cluster de gènes correspondant n’est décrit que chez Aspergillus clavatus, champignon tellurique incapable de se développer dans les pommes. La caractérisation moléculaire de la voie de biosynthèse de la patuline est la condition sine qua none à toute étude visant à comprendre la régulation de la biosynthèse de la patuline, mais également à toute action permettant de limiter sa synthèse. C’est pourquoi le premier objectif de cette thèse a été de caractériser le cluster de gènes spécifique de la voie de biosynthèse chez Penicillium expansum. Celui-ci est caractérisé par une taille de 40 kb et contient les 15 mêmes gènes qu’Aspergillus clavatus, les seules différences résidant dans l’organisation et l’orientation des gènes. La caractérisation de la seconde étape de la voie de biosynthèse de la patuline a été ensuite entreprise chez Aspergillus clavatus, organisme modèle. Le gène patG code pour l’acide 6-méthylsalicylique décarboxylase responsable de la conversion de l’acide 6-méthylsalicylique en m-crésol. Pour faire suite au premier objectif, la régulation de la voie de biosynthèse de la patuline a été étudiée. Pour cela, une souche mutante pour le facteur de régulation spécifique à la patuline patL a été généré puis la production de patuline ainsi que l’expression des gènes du cluster analysés. Les résultats de cette étude ont montré que le gène patL joue le rôle d’interrupteur au sein du cluster. L’absence de patL conduit à une extinction totale de l’expression des gènes du cluster et à une abscence de production de patuline par Penicillium expansum. Dans cette même étude, des tests de pathogénicité ont été entrepris sur des pommes de différentes variétés démontrant ainsi que la patuline peut être un facteur de virulence facilitant l’infection de certaines variétés de pommes telles que la Golden Delicious ou la Pink Lady. Enfin, l’influence de la lumière a été évaluée en analysant l’impact de différentes longueurs d’ondes sur la croissance et la production de patuline de Penicillium expansum. Que ce soit in-vitro ou in-vivo, la croissance et la production de patuline sont très affectés par les lumières blanche, bleue et rouge. Favoriser le stockage des pommes sous les lumières blanche, bleue ou rouge plutôt qu’à l’obscurité pourrait devenir un moyen de prévention contre la contamination par Penicillium expansum. En conclusion, cette thèse présente un aspect fondamental avec la caractérisation du cluster de gènes chez Penicillium expansum et la caractérisation de la seconde étape de la voie de biosynthèse de la patuline ; mais aussi un aspect appliqué avec l’utilisation des lumières de différentes couleurs comme méthode de prévention contre Penicillium expansum durant le stockage des pommes.Penicillium expansum is the common contaminant of apples and the causal agent of blue mold rot. This fungus is the main patulin producer in apple based products. Actually, the patulin biosynthesis is partially elucidates and the gene cluster has been elucidated in Aspergillus clavatus, a telluric fungi unable to grow on apples. The molecular characterization of the patulin biosynthetic pathway is the key step for a better understanding of the mechanisms leading to patulin production and will help to define strategies to reduce its presence in apple products. The first objective of this thesis was the characterization of the patulin gene cluster in Penicillium expansum. The latter includes the same 15 genes as in Aspergillus clavatus but in a different order and orientation. Then, the second step of this biosynthetic pathway has been characterized and the patG gene encode for the 6-methylsalicylic decarboxylase involved in the 6- methylsalicylic acid conversion into m-cresol. The second objective consists of the study of the patulin regulation. For that, a patL mutated strain was generated and the patulin production and the patulin gene cluster expression were assessed. The mutation of this gene results in a down-regulation of the rest of the genes in the cluster associated with a lack of patulin production. Pathogenicity tests on apples revealed that patulin could act as a virulence factor in some apple varieties, like Golden Delicious or Pink Lady. In the last part of this thesis, the influence of different wavelength lights on the growth and the patulin production by Penicillium expansum were assessed in vitro and in vivo. In both cases, growth and patulin production were significantly affected under white, blue and red lights. Consequently, the apple storage under these lights could be a good alternative to the storage in the dark. In conclusion, this thesis presents a fundamental aspect that consist in the characterization of the patulin gene cluster in Penicillium expansum and the characterization of the second step of this pathway. An applied aspect is also provided by the use of the different wavelength lights to prevent the Penicillium expansum contamination during apple storage

Impact of the Inoculation Method of <i>Geotrichum candidum</i>, Used as Biocontrol Agent, on T-2 Toxin Produced by <i>Fusarium sporotrichioides</i> and <i>F. langsethiae</i> during the Malting Process

In malt production, steeping and germination steps offer favorable environmental conditions for fungal proliferation when barley is already contaminated by Fusarium species, T-2 toxin producers. However, the use of G. candidum as a biocontrol agent can prevent this proliferation. Indeed, in previous work, a correlation between phenyllactic acid (PLA) production by G. candidum and the reduction in Fusarium sporotrichioides and F. langsethiae growth and T-2 toxin concentration was demonstrated. In the present study, to improve the efficiency of G. candidum, the effects of the inoculum concentration and the inoculation method of G. candidum on PLA and T-2 toxin concentrations were evaluated. First, co-culture experiments with Fusarium species and G. candidum were conducted in a liquid synthetic medium. The results showed that inoculation of G. candidum in the freeze-dried form at 0.4 g/L allowed the production of PLA from the second day of incubation associated with a reduction in T-2 toxin concentration of 82% and 69% produced by F. sporotrichioides and F. langsethiae, respectively. Moreover, the activated form of G. candidum at 0.4 g/L enhanced PLA concentration leading to better T-2 toxin reduction. Second, experiments were conducted on artificially infected barley kernels with both Fusarium species under conditions mimicking the malting step. As for co-culture experiments, the use of the activated form of G. candidum was established as the best condition for T-2 toxin concentration reduction for a 3 day malting period

Impact of the Inoculation Method of Geotrichum candidum, Used as Biocontrol Agent, on T-2 Toxin Produced by Fusarium sporotrichioides and F. langsethiae during the Malting Process

In malt production, steeping and germination steps offer favorable environmental conditions for fungal proliferation when barley is already contaminated by Fusarium species, T-2 toxin producers. However, the use of G. candidum as a biocontrol agent can prevent this proliferation. Indeed, in previous work, a correlation between phenyllactic acid (PLA) production by G. candidum and the reduction in Fusarium sporotrichioides and F. langsethiae growth and T-2 toxin concentration was demonstrated. In the present study, to improve the efficiency of G. candidum, the effects of the inoculum concentration and the inoculation method of G. candidum on PLA and T-2 toxin concentrations were evaluated. First, co-culture experiments with Fusarium species and G. candidum were conducted in a liquid synthetic medium. The results showed that inoculation of G. candidum in the freeze-dried form at 0.4 g/L allowed the production of PLA from the second day of incubation associated with a reduction in T-2 toxin concentration of 82% and 69% produced by F. sporotrichioides and F. langsethiae, respectively. Moreover, the activated form of G. candidum at 0.4 g/L enhanced PLA concentration leading to better T-2 toxin reduction. Second, experiments were conducted on artificially infected barley kernels with both Fusarium species under conditions mimicking the malting step. As for co-culture experiments, the use of the activated form of G. candidum was established as the best condition for T-2 toxin concentration reduction for a 3 day malting period

Effect of Streptomyces roseolus Cell-Free Supernatants on the Fungal Development, Transcriptome, and Aflatoxin B1 Production of Aspergillus flavus

International audienceCrop contamination by aflatoxin B1 (AFB1), an Aspergillus-flavus-produced toxin, is frequently observed in tropical and subtropical regions. This phenomenon is emerging in Europe, most likely as a result of climate change. Alternative methods, such as biocontrol agents (BCAs), are currently being developed to reduce the use of chemicals in the prevention of mycotoxin contamination. Actinobacteria are known to produce many bioactive compounds, and some of them can reduce in vitro AFB1 concentration. In this context, the present study aims to analyze the effect of a cell-free supernatant (CFS) from Streptomyces roseolus culture on the development of A. flavus, as well as on its transcriptome profile using microarray assay and its impact on AFB1 concentration. Results demonstrated that in vitro, the S. roseolus CFS reduced the dry weight and conidiation of A. flavus from 77% and 43%, respectively, and was therefore associated with a reduction in AFB1 concentration reduction to levels under the limit of quantification. The transcriptomic data analysis revealed that 5198 genes were differentially expressed in response to the CFS exposure and among them 5169 were downregulated including most of the genes involved in biosynthetic gene clusters. The aflatoxins’ gene cluster was the most downregulated. Other gene clusters, such as the aspergillic acid, aspirochlorine, and ustiloxin B gene clusters, were also downregulated and associated with a variation in their concentration, confirmed by LC-HRMS