Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions

International audienceBackground: On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions. Results: The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold β-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high β-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars. Conclusions: The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail

The length and depth of real algebraic groups

Let $G$ be a connected real algebraic group. An unrefinable chain of $G$ is a chain of subgroups $G=G_0>G_1>...>G_t=1$ where each $G_i$ is a maximal connected real subgroup of $G_{i-1}$. The maximal (respectively, minimal) length of such an unrefinable chain is called the length (respectively, depth) of $G$. We give a precise formula for the length of $G$, which generalises results of Burness, Liebeck and Shalev on complex algebraic groups and also on compact Lie groups. If $G$ is simple then we bound the depth of $G$ above and below, and in many cases we compute the exact value. In particular, the depth of any simple $G$ is at most $9$

Modélisation et optimisation de la production de cellulases par Trichoderma reesei pour les bioraffineries lignocellulosiques

Dans le contexte énergétique et climatique mondial, le coût élevé des enzymes Cellulolytiques (cellulases) freine le développement des bioraffineries lignocellulosiques, pour produire des biocarburants et composés chimiques à partir d'une matière première végétale renouvelable. L'objectif de ce travail est de caractériser et de modéliser le métabolisme du micro-organisme Trichoderma reesei, afin d'optimiser le protocole industriel de production de cellulases. Cette étude a été réalisée sur des milieux modèles représentatifs de ceux attendus à l'échelle industrielle. Tout d'abord, la stoechiométrie des réactions de croissance et de production a été établie, puis une étude cinétique a été menée pour mesurer précisément le comportement du micro-organisme à forte induction de la production de cellulases. Le modèle résultant a été utilisé pour optimiser le protocole industriel de production. Ensuite, l'intégration de cette étape dans une bioraffinerie lignocellulosique a été étudiée, avec l'effet sur le métabolisme i) des mélanges de sucres disponibles, ii) des composés inhibiteurs issus de la dégradation de la lignocellulose, et iii) du changement d'échelle. Ces travaux ont fait progresser de façon substantielle les connaissances du métabolisme de T. reesei en ce qui concerne la production de cellulases, et les modèles développés sont des outils d'aide rationnelle à la définition d'un procédé de production de cellulases intégré dans une bioraffinerie lignocellulosique.In the global energetic and climatic context, the high cost of the cellulolytic enzymes (cellulases) postpones the development of lignocellulosic biorefineries, dedicated to produce biofuels and chemical compounds from renewable vegetable feedstocks. The aim of this work was to measure and model the metabolism of the micro-organism Trichoderma reesei, in order to optimize the industrial protocol for the production of cellulase. This study was carried out using synthetic media representative of industrial ones. First, the stoichiometries of growth and protein production reactions were determined. Then, a kinetic study was conducted to precisely measure the specific rates of T. reesei at high induction of cellulase production. The resulting model was used to optimize the industrial production protocol. Finally the integration of this step in a lignocellulosic biorefinery was studied by determining the impacts on the metabolism of i) available sugar mixtures, ii) inhibitory compounds from lignocellulosic biomass degradation, and iii) scale-up. These results significantly contributed to improve the knowledge of T. reesei metabolism on cellulase production. The developed models are rational tools for the optimization of a cellulase production protocol suited to lignocellulosic biorefineries

Modélisation et optimisation de la production de cellulases par Trichoderma reesei pour les bioraffineries lignocellulosiques

Dans le contexte énergétique et climatique mondial, le coût élevé des enzymes Cellulolytiques (cellulases) freine le développement des bioraffineries lignocellulosiques, pour produire des biocarburants et composés chimiques à partir d'une matière première végétale renouvelable. L'objectif de ce travail est de caractériser et de modéliser le métabolisme du micro-organisme Trichoderma reesei, afin d'optimiser le protocole industriel de production de cellulases. Cette étude a été réalisée sur des milieux modèles représentatifs de ceux attendus à l'échelle industrielle. Tout d'abord, la stoechiométrie des réactions de croissance et de production a été établie, puis une étude cinétique a été menée pour mesurer précisément le comportement du micro-organisme à forte induction de la production de cellulases. Le modèle résultant a été utilisé pour optimiser le protocole industriel de production. Ensuite, l'intégration de cette étape dans une bioraffinerie lignocellulosique a été étudiée, avec l'effet sur le métabolisme i) des mélanges de sucres disponibles, ii) des composés inhibiteurs issus de la dégradation de la lignocellulose, et iii) du changement d'échelle. Ces travaux ont fait progresser de façon substantielle les connaissances du métabolisme de T. reesei en ce qui concerne la production de cellulases, et les modèles développés sont des outils d'aide rationnelle à la définition d'un procédé de production de cellulases intégré dans une bioraffinerie lignocellulosique.In the global energetic and climatic context, the high cost of the cellulolytic enzymes (cellulases) postpones the development of lignocellulosic biorefineries, dedicated to produce biofuels and chemical compounds from renewable vegetable feedstocks. The aim of this work was to measure and model the metabolism of the micro-organism Trichoderma reesei, in order to optimize the industrial protocol for the production of cellulase. This study was carried out using synthetic media representative of industrial ones. First, the stoichiometries of growth and protein production reactions were determined. Then, a kinetic study was conducted to precisely measure the specific rates of T. reesei at high induction of cellulase production. The resulting model was used to optimize the industrial production protocol. Finally the integration of this step in a lignocellulosic biorefinery was studied by determining the impacts on the metabolism of i) available sugar mixtures, ii) inhibitory compounds from lignocellulosic biomass degradation, and iii) scale-up. These results significantly contributed to improve the knowledge of T. reesei metabolism on cellulase production. The developed models are rational tools for the optimization of a cellulase production protocol suited to lignocellulosic biorefineries.CLERMONT FD-Bib.électronique (631139902) / SudocSudocFranceF

A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing <it>Trichoderma reesei</it> strains

<p>Abstract</p> <p>Background</p> <p>During bioprocess development, secondary screening is a key step at the boundary between laboratory and industrial conditions. To ensure an effective high-throughput screening, miniaturized laboratory conditions must mimic industrial conditions, especially for oxygen transfer, feeding capacity and pH stabilization.</p> <p>Results</p> <p>A feeding strategy has been applied to develop a simple screening procedure, in which a stoichiometric study is combined with a standard miniaturization procedure. Actually, the knowledge of all nutriments and base or acid requirements leads to a great simplification of pH stabilization issue of miniaturized fed-batch cultures. Applied to cellulase production by <it>Trichoderma reesei</it>, this strategy resulted in a stoichiometric mixed feed of carbon and nitrogen sources. While keeping the pH between shake flask and stirred bioreactor comparable, the developed shake flask protocol reproduced the strain behaviour under stirred bioreactor conditions. Compared to a an already existing miniaturized shake flasks protocol, the cellulase concentration was increased 5-fold, reaching about 10 g L^-1. Applied to the secondary screening of several clones, the newly developed protocol succeeded in selecting a clone with a high industrial potential.</p> <p>Conclusions</p> <p>The understanding of a bioprocess stoichiometry contributed to define a simpler and more effective miniaturization. The suggested strategy can potentially be applied to other fed-batch processes, for the screening of either strain collections or experimental conditions.</p

RNA-seq analysis of glucose fermentation by the natural Isopropanol producer Clostridium beijerinckii DSM6423

Purpose: C. beijerinckii DSM 6423 is the most cited natural IpOH producer. Improving the natural production of this strain through a targeted approach required a full sequencing and characterization of its genome, together with transcriptomic analyses of its own regulations.The goals of this study are then to evaluate the transcriptional profile (RNA-Seq) of C. beijerinckii DSM6423, a natural isopropanol producer, during a fermentation of glucose in controlled bioreactors. Methods: A RNA-Seq approach was chosen in order to have a timelapse study of DSM 6423 throughout the fermentation process. Three independent duplicate fermentations of DSM 6423 were carried out in bioreactors on three different weeks, showing good reproducibility. On each cultivation, five biomass samples were collected for RNA-Seq analyses.and DNA was eliminated after DNAse I treatment (AM1906, Invitrogen). The 15 resulting RNA samples were sequenced and analyzed using the previously reconstructed genome of DSM 6423. Results: Using a data analysis workflow (TAMARA) developed by the Genoscope platform of Evry, we were able to highlight the transcriptional regulation along the fermentation by calculating the transcription profiles of each gene, using the 3h sample as reference. Clustering was performed using CAST algorithm revealed 8 clusters containing 953 genes and corresponding to genes up-regulated at 6, 8, 11 or 24 hours and gene down-regulated at 6, 8, 11 or 24 hours. Conclusion: Such analyses were carried out in this study and provide useful data to better understand the genetic background and the physiological specificities of C. beijerinckii DSM6423 isopropanol producer. Notably, this work is the first omic study of a natural IBE producer. The data gathered needs time for proper exploitation, but a better understanding of the metabolic pathways and various genes involved opens the door for future targeted approaches

Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations

Abstract Background Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. Results This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l $$^{-1}$$ - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) $$^{-1}$$ - 1 , the lowest rate of 0.3 g (l h) $$^{-1}$$ - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in \upbeta β -glucosidase activity compared to batch processes using the same type and amount of carbon sources. Conclusion We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology

Characterization of three bacterial glycoside hydrolase family 9 endoglucanases with different modular architectures isolated from a compost metagenome

International audienceBackground: Environmental bacteria express a wide diversity of glycoside hydrolases (GH). Screening and characterization of GH from metagenomic sources provides an insight into biomass degradation strategies of non-cultivated prokaryotes. Methods: In the present report, we screened a compost metagenome for lignocellulolytic activities and identified six genes encoding enzymes belonging to family GH9 (GH9a-f). Three of these enzymes (GH9b, GH9d and GH9e) were successfully expressed and characterized. Results: A phylogenetic analysis of the catalytic domain of pro-and eukaryotic GH9 enzymes suggested the existence of two major subgroups. Bacterial GH9s displayed a wide variety of modular architectures and those harboring an N-terminal Ig-like domain, such as GH9b and GH9d, segregated from the remainder. We purified and characterized GH9 endoglucanases from both subgroups and examined their stabilities, substrate specificities and product profiles. GH9e exhibited an original hydrolysis pattern, liberating an elevated proportion of oligosaccharides longer than cellobiose. All of the enzymes exhibited processive behavior and a synergistic action on crystalline cellulose. Synergy was also evidenced between GH9d and a GH48 enzyme identified from the same metagenome. Conclusions: The characterized GH9 enzymes displayed different modular architectures and distinct substrate and product profiles. The presence of a cellulose binding domain was shown to be necessary for binding and digestion of insoluble cellulosic substrates, but not for processivity. General significance: The identification of six GH9 enzymes from a compost metagenome and the functional variety of three characterized members highlight the importance of this enzyme family in bacterial biomass deconstruction