Ingénierie de la voie des pentoses phosphate chez la levure Saccharomyces cerevisiae (applications en œnologie)

Il existe un intérêt croissant pour le développement de levures S. cerevisiae œnologiques à rendement abaissé de conversion des sucres en alcool. Nous proposons ici une approche originale basée sur la réorientation du flux carboné vers la voie des pentoses phosphate (VPP). Dans un premier temps, nous avons montré que le flux à travers la VPP est limité par le niveau de réoxydation du NADPH et par la capacité de la voie elle même. Nous avons ensuite mis en évidence le rôle crucial du facteur de transcription Stb5 dans le maintien d'un flux basal à travers la VPP. La surexpression de STB5, couplée à l'introduction d'un système de réoxydation du NADPH, est une stratégie intéressante pour amplifier le flux à travers la VPP. En parallèle, une stratégie d'évolution dirigée basée sur l'adaptation des souches sur gluconate, un hexose mal assimilé et incorporé au niveau de la VPP, a été développée. Des souches évoluées présentant une meilleure assimilation du gluconate ont été obtenues après 70, 180 et 240 générations. En fermentation, ces souches produisent la même quantité d'éthanol que la souche parentale mais présentent des phénotypes complètement nouveaux, en particulier des performances fermentaires accrues, de faibles besoins en azote, une production d'acétate réduite et une forte production de composés aromatiques. L'analyse 13C-flux et transcriptomique d'une souche évoluée ECA5 révèle une amplification de la VPP d'un facteur 1.5 par rapport à la souche parentale EC1118, en lien avec la surexpression de GND1 et TKL1. L'expression de nombreux gènes du métabolisme azoté et de la voie Ehrlich, de l'homéostasie des protons et de la glycolyse est augmentée chez ECA5, alors que les gènes de stress et de la respiration sont globalement réprimés, de façon cohérente avec les phénotypes observés. Outre le développement de nouvelles souches d'intérêt œnologique, ce travail apporte un éclairage nouveau sur le fonctionnement de la VPP et sur ses liens avec le métabolisme central et secondaireThere is an ever-growing interest in the development of S. cerevisiae wine yeast strains with reduced ethanol yield. We proposed a novel approach based on rerouting the carbon flux towards the pentose phosphate (PP) pathway. First, we showed that the flux through the PP pathway is limited both by the absence of a mechanism for reoxidation of NADPH and by the intrinsic capacity of the pathway. We also showed that the transcription factor Stb5 plays a key role in maintaining a basal flux through the PP pathway to meet the requirements for NADPH and biosynthetic precursors. Over-expression of STB5 is a potentially useful strategy for increasing the flux through the PP pathway, provided that an alternative system of reoxidation of NADPH is expressed. In parallel, we investigated an evolutionary engineering strategy based on long-term batch culture on gluconate, a substrate poorly assimilated by S. cerevisiae cells and metabolized by the PP pathway. We selected strains that had evolved a greater gluconate consumption capacity after 70, 180 and 240 generations. During wine fermentation, these evolved strains produced similar amounts of ethanol as the parental strain but displayed completely novel phenotypes, including higher fermentation rates, lower nitrogen requirements, lower levels of acetate production, and enhanced production of aroma compounds. 13C flux analysis and transcripomic analysis of one of these strains, ECA5, showed a greater flux through the PP pathway consistent with the observed increased expression of GND1 and TKL1. The expression of genes associated with nitrogen metabolism, the Ehrlich pathway, proton homeostasis and glycolysis was stronger than in the parental strain, whereas genes involved in stress response and respiration were down-regulated, in agreement with the phenotypes of ECA5. In addition to providing strains with considerable potential for wine making, this work sheds new light on the operation of PP pathway and its links with central and secondary metabolismMONTPELLIER-SupAgro La Gaillarde (341722306) / SudocSudocFranceF

Assessment of micropollutants toxicity by using a modified Saccharomyces cerevisiae model

International audienc

The Saccharomyces cerevisiae zinc factor protein Stb5p is required as a basal regulator of the pentose phosphate pathway

Correspondance auteur: Dequin S. E-mail: [email protected] audienceIn Saccharomyces cerevisiae, the oxidative stress-activated zinc cluster protein Stb5p activates genes involved in NADPH production and most genes of the pentose phosphate (PP) pathway. To gain insight into the role of Stb5p, we studied the behaviour of stb5 deletion mutants during aerobic and anaerobic growth on glucose. stb5 mutants were auxotrophic for methionine and pyrimidine nucleotides. The methionine auxotrophy phenotype was air dependent, suggesting an impaired aerobic NADPH status. Consistent with this, the acetate level was reduced and the alpha-ketoglutarate level was increased in the stb5 mutant. stb5 cells also required pyrimidine nucleotides for aerobic and anaerobic growth, consistent with a reduction in 5-phosphoribosyl-1-pyrophosphate production caused by a reduced flux through the PP pathway. Strains overexpressing STB5 could not grow on glucose. This growth defect was restored by overproduction of an NADPH-butanediol dehydrogenase, which reoxidizes the excess NADPH in the oxidative PP pathway. These findings suggest a major role for the transcription factor Stb5p in maintaining a basal flux through the PP pathway to meet the NADPH requirements for aerobic growth, and to provide the nucleotide precursors. Our data also demonstrate the potential use of a system based on overproduction of this transcription factor to increase flux through the PP pathwa

Développement d'une méthode automatique de suivi de la croissance de Borrelia burgdorferi

National audienceBorrelia (ou Borreliella) burgdorferi (Bb) est l’agent infectieux responsable de la maladie de Lyme. Même si de nombreuses études ont été conduites sur cet organisme, beaucoup de question reste sans réponse concernant sa physiologie et sa pathogénicité. En effet, la culture de Bb présente de nombreuse difficultés. Par exemple, la méthode standard de suivi de croissance de Bb actuelle est un dénombrement manuel au microscope utilisant une chambre de comptage de Petroff Hausser, ce qui demande du temps et présente une certaine variabilité. Dans cette étude, nous proposons une méthode alternative, plus rapide et automatisée, représentant une amélioration qui pourra permettre d’accélérer les études dans ce champs de recherche

The aesthetics, between history and pedagogy. Bibliography

International audienceThau lagoon (extended on 7500 hectares) is located in the south of France and is the main shellfish-farming center in the Mediterranean Sea. Around this lagoon, numerous activities such as wastewater treatment plants, harbor and vine cultivation represent a potential source of pollution. Although subject to microbiological and chemical monitoring (heavy metals), there is currently no global study realized on the contamination of air and water. This work proposes an integrated approach based on a non-target analysis by SBSE/HSSE coupled with GC-MS and a target analysis in order to identify chemical pollutants (heavy metals, 34 pharmaceuticals, 12 pesticides, 2 industrial products and 6 toxins). This monitoring was carried out during one year on 6 sampling points and on two matrices: air (non-target analysis) and water (non-target and target analysis). Concerning the targeted analysis, very low concentrations ranging from the pg/L to the ng/L were found in water. Among pollutants, most frequently found are: atrazine, its metabolites, azithromycin, clarithromycin, atenolol and sotalol at low concentrations ranging from 2.44 pg/L to 0.6 ng/L. In contrast, in non-targeted analysis, 116 different molecules were found including 77 and 79 in the air and water respectively. Only 28 molecules were common to both matrices. To access the exposure risk of workers and residents of the lagoon, we have in second approach decided to select some molecules from the non-target analysis and to quantify them. The selection was based on the toxicity of the molecules (Toxtree software) and their frequency of detection. Only the molecules found in more than 20% of sampling were conserved for the analysis. Examples of potentially dangerous susbtances included in this list are: - triethyl phosphate (organophosphorus flame-retardant and intermediate in the manufacture for pesticides), - diisobutyl phthalate (endocrine disruptor). This substance is considered of very high concern (SVHC) and its use is strictly regulated (Annex XVII of REACH1) - 2-furanmethanol. This molecule is included in the list of hazardous substances harmonised for classification and labelling in the EU (Annex VI to CLP regulation)2 and is suspected to be carcinogenic. The next step of the study will be to made analysis on the shellfishes grown in the lagoon. All of these elements will allow to trace pollutants in the Thau lagoon and to determine the potential sources of exposition and potential bioaccumulation

Inter-laboratory exercise on antibiotic drugs analysis in aqueous samples.

International audienceAn inter-laboratory exercise was organized under the PHARMAS EU project, by the Advanced School of Public Health (EHESP), in order to evaluate the performances of analytical methods for the measurement of antibiotics in waters (surface and tap). This is the first time such an exercise on antibiotics has been organized in Europe, using different kinds of analytical methods and devices. In this exercise thirteen laboratories from five countries (Canada, France, Italy, the Netherlands and Portugal) participated, and a total number of 78 samples were distributed. During the exercise, 2 testing samples (3 bottles of each) prepared from tap water and river water, respectively, spiked with antibiotics, were sent to participants and analyzed over a period of one month. A final number of 77 (98.7%) testing samples were considered. Depending on substances studied by each participant, 305 values in duplicate were collected, with the results for each sample being expressed as the target concentration. A statistical study was initiated using 611 results. The mean value, standard deviation, coefficient of variation, standard uncertainty of the mean, median, the minimum and maximum values of each series as well as the 95% confidence interval were obtained from each participant laboratory. In this exercise, 36 results (6% of accounted values) were outliers according to the distribution over the median (box plot). The outlier results were excluded. In order to establish the stability of testing samples in the course of the exercise, differences between variances obtained for every type of sample at different intervals were evaluated. The results showed no representative variations and it can be considered that all samples were stable during the exercise. The goals of this inter-laboratory study were to assess results variability when analysis is conducted by different laboratories, to evaluate the influence of different matrix samples, and to determine the rate at which participating laboratories successfully completed the tests initiated

Villes connectées : détection du risque biologique

International audienceLa sécurité dans les villes est un enjeu majeur dans les pays européens. Le projet IMPETUS vise à développer une plateforme numérique pour améliorer la gestion des évènements.Parmi les outils disponibles, il apparait des manques pour la sécurisation. Un outil pour détecter une attaque biologique est proposé. Il fonctionne en continu et analyse de manière régulière la concentration en bactéries dans l’air. Il permet d’alerter lorsqu’une concentration mesurée présente une valeur supérieure à un seuil. Suite à cette alerte, une phase d’identification de l’organisme sera entreprise. Les campagnes d’essais réalisées dans 3 villes ont permis de montrer une différence entre le contenu microbien de l’air dans des salles vides, des salles avec des activités classiques et des salles avec une simulation d’attaque biologique

Villes connectées : détection du risque biologique

International audienceLa sécurité dans les villes est un enjeu majeur dans les pays européens. Le projet IMPETUS vise à développer une plateforme numérique pour améliorer la gestion des évènements.Parmi les outils disponibles, il apparait des manques pour la sécurisation. Un outil pour détecter une attaque biologique est proposé. Il fonctionne en continu et analyse de manière régulière la concentration en bactéries dans l’air. Il permet d’alerter lorsqu’une concentration mesurée présente une valeur supérieure à un seuil. Suite à cette alerte, une phase d’identification de l’organisme sera entreprise. Les campagnes d’essais réalisées dans 3 villes ont permis de montrer une différence entre le contenu microbien de l’air dans des salles vides, des salles avec des activités classiques et des salles avec une simulation d’attaque biologique