Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains

ABSTRACT: BACKGROUND: Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions. RESULTS: The protein composition of the secretome produced by industrial T. reesei (strain CL847) grown on a medium promoting the production of both cellulases and hemicellulases was explored using two-dimensional electrophoresis and MALDI-TOF or LC-MS/MS protein identification. A total of 22 protein species were identified. As expected, most of them are potentially involved in biomass degradation. The 2D map obtained was then used to compare the secretomes produced by CL847 and another efficient cellulolytic T. reesei strain, Rut-C30, the reference cellulase-overproducing strain using lactose as carbon source and inducer of cellulases. CONCLUSION: This study provides the most complete mapping of the proteins secreted by T. reesei to date. We report on the first use of proteomics to compare secretome composition between two cellulase-overproducing strains Rut-C30 and CL847 grown under similar conditions. Comparison of protein patterns in both strains highlighted many unexpected differences between cellulase cocktails. The results demonstrate that 2D electrophoresis is a promising tool for studying cellulase production profiles, whether for industrial characterization of an entire secretome or for a more fundamental study on cellulase expression at genome-wide scale

Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina

International audienceBackground: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood. Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and beta-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-ef-fective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans—a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxida-tive cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications

Pharmacovigilance des effets indésirables systémiques de la visudyne® au sein d'une cohorte de patients atteints de dégénérescence maculaire liée à l'âge

LYON1-BU Santé (693882101) / SudocSudocFranceF

Maîtrise de la production et de la mise en oeuvre d'enzymes ligninolytiques fongiques dans le domaine papetier

L'ELIMINATION DE LA LIGNINE DES COMPOSES LIGNOCELLULOSIQUES RESTE LE PROBLEME MAJEUR DE L'INDUSTRIE PAPETIERE. COUTEUSE EN ENERGIE ET FORTEMENT POLLUANTE, ELLE GENERE DES CHLOROLIGNINES FORTEMENT TOXIQUES POUR L'HOMME. CES CONSIDERATIONS ECONOMIQUES ET ECOLOGIQUES ONT INCITE LA PROFESSION A RECHERCHER DES INNOVATIONS TECHNOLOGIQUES. DANS CE CONTEXTE, LES PROCEDES BIOTECHNOLOGIQUES BASES SUR DES ENZYMES FONGIQUES LIGNINOLYTIQUES REPRESENTENT UNE ALTERNATIVE PARTICULIEREMENT SEDUISANTE. LES TRAVAUX REALISES DANS LE CADRE DE CETTE THESE CONCERNENT LA MAITRISE DE LA PRODUCTION ET DE LA MISE EN UVRE DANS LE DOMAINE PAPETIER DE DEUX TYPES D'ENZYMES LIGNINOLYTIQUES, LA LACCASE ET LA MANGANESE PEROXYDASE (MNP). DANS UN PREMIER TEMPS, NOUS AVONS SELECTIONNE PAR DES METHODES DE LA GENETIQUE FORMELLE DES SOUCHES MONOCARYOTIQUES DE PYCNOPORUS CINNABARINUS HYPERPRODUCTRICES DE LACCASES. DEUX SOUCHES ONT AINSI ETE SELECTIONNEES. PAR LA SUITE, DEUX ISOFORMES DE LA LACCASE ONT ETE ISOLEES ET CARACTERISEES CHEZ LA MEILLEURE DES SOUCHES SELECTIONNEE (P. CINNABARINUS SS3). NOS TRAVAUX SE SONT ENSUITE ORIENTES VERS LA MAITRISE DE LA PRODUCTION A L'ECHELLE PILOTE DE MNP PAR UNE SOUCHE HYPERPRODUCTRICE (PHANEROCHAETE CHRYSOSPORIUM I-1512). L'EXTRAPOLATION D'UNE TECHNIQUE DE CULTURE MISE AU POINT PAR NOTRE UNITE, ASSOCIANT UN SYSTEME IMMOBILISE AVEC UN REACTEUR DE TYPE AIR-LIFT A PERMIS D'OBTENIR DES TAUX DE PRODUCTION DE MNP SANS PRECEDENTS. ENFIN, DE NOUVEAUX PROCEDES METTANT EN UVRE IN VITRO LA LACCASE ET LA MNP SUR DEUX TYPES DE PATES INDUSTRIELLES (PEUPLIER ET PAILLE DE BLE) ONT ETE DEVELOPPES. LES POTENTIALITES DE CES ENZYMES ONT ETE CLAIREMENT DEMONTREES PERMETTANT : - DES GAINS D'ENERGIE LORS DE L'ETAPE DE RAFFINAGE, ENTRAINANT UNE REDUCTION DU COUT DE PRODUCTION ; - UNE DELIGNIFICATION SELECTIVE IMPORTANTE PERMETTANT UNE REDUCTION SIGNIFICATIVE DU REJET DE CHLOROLIGNINES DANS LES EFFLUENTS PAPETIERS.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

A PCR-based method to quantify fungal growth during pretreatment of lignocellulosic biomass

Filamentous fungi have shown great potential in the pretreatment of lignocellulosic biomass and their use in bio-processes based on Solid State Fermentation (SSF) opens promising perspectives for plant biomass valorization. Obviously, quantification of the fungal biomass throughout the fermentation is a crucial parameter for SSF evaluation and control, both at the laboratory and industrial scale. Here we provide a qPCR-based method as a reliable alternative to conventional methods to estimate mycelial growth during plant biomass treatment. For the three strains analyzed, the lowest detection limit ranged from 58 to 272μg mycelium dry weight per gram biomass (dry weight). We show that the qPCR-based method allows fungal growth monitoring during fermentation and provides relevant information for selection of the most appropriate fungal strains in specific SSF/reactor condition

Subcellular partitioning of metals in Aporrectodea caliginosa along a gradient of metal exposure in 31 field-contaminated soils

Subcellular fractionation of metals in organisms was proposed as a better way to characterize metal bioaccumulation. Here we report the impact of a laboratory exposure to a wide range of field-metal contaminated soils on the subcellular partitioning of metals in the earthworm Aporrectodea caliginosa. Soils moderately contaminated were chosen to create a gradient of soil metal availability; covering ranges of both soil metal contents and of several soil parameters. Following exposure, Cd, Pb and Zn concentrations were determined both in total earthworm body and in three subcellular compartments: cytosolic, granular and debris fractions. Three distinct proxies of soil metal availability were investigated: CaCl2-extractable content dissolved content predicted by a semi-mechanistic model and free ion concentration predicted by a geochemical speciation model. Subcellular partitionings of Cd and Pb were modified along the gradient of metal exposure, while stable Zn partitioning reflected regulation processes. Cd subcellular distribution responded more strongly to increasing soil Cd concentration than the total internal content, when Pb subcellular distribution and total internal content were similarly affected. Free ion concentrations were better descriptors of Cd and Pb subcellular distribution than CaCl2 extractable and dissolved metal concentrations. However, free ion concentrations and soil total metal contents were equivalent descriptors of the subcellular partitioning of Cd and Pb because they were highly correlated. Considering lowly contaminated soils, our results raise the question of the added value of three proxies of metal availability compared to soil total metal content in the assessment of metal bioavailability to earthworm

Fungal pretreatment of wheat straw for anaerobic digestion

International audienceRecently, there has been a considerable interest for the production of renewable energy from lignocellulosic biomass which presents several advantages. It is, however, a technological challenge because of the difficulty to hydrolyze lignin contained in this biomass. This limitation can be overcome by pretreatments. Among them, low-cost fungal pretreatments are attractive. This study focuses on the selection of a fungal strain, in order to increase methane production from wheat straw. After a screening on 176 strains, thirteen were preselected and used to pretreat straw. BMP of those straws were measured. Compared to the control straw and considering pretreated VS, up to 43% more methane were obtained. Taking into account the dry weight loss observed during the pretreatment in non-optimized conditions, it was found up to 21% more methane compared to the control straw

Efficient biomass pretreatment using the White-rot Fungus Polyporus Brumalis.

Implementation of cheap and eco-friendly biomass pretreatment processes is necessary to develop sustainable biorefineries. In nature, white-rot basidiomycetes are able to degrade lignin efficiently and selectively and are thus of great interest in such bioprocesses. In this study, five basidiomycetes strains were evaluated for their ability to pretreat wheat straw under solid state fermentation. Fungal pretreatments were carried out in glass-column reactors under operating conditions approaching industrial practices. The pretreatment efficiency was evaluated through the quantification of dry weight losses and subsequent hydrolysis of the carbohydrate fraction by enzymatic cocktails. The highest lignin to cellulose losses ratio was obtained using a strain of Polyporus brumalis which exhibited high ligninolytic capabilities. This selectivity along with the low dry weight loss makes the pretreatment profitable by enhancing cellulose and hemicellulose conversion yields. Therefore P. brumalis can be viewed as a promising strain to pretreat lignocellulosic biomass for biorefinery applications

A structural equation model of soil metal bioavailability to earthworms: confronting causal theory and observations using a laboratory exposure to field-contaminated soils

Structural equation models (SEM) are increasingly used in ecology as multivariate analysis that can represent theoretical variables and address complex sets of hypotheses. Here we demonstrate the interest of SEM in ecotoxicology, more precisely to test the three-step concept of metal bioavailability to earthworms. The SEM modeled the three-step causal chain between environmental availability, environmental bioavailability and toxicological bioavailability. In the model, each step is an unmeasured (latent) variable reflected by several observed variables. In an exposure experiment designed specifically to test this SEM for Cd, Pb and Zn, Aporrectodea caliginosa was exposed to 31 agricultural field-contaminated soils. Chemical and biological measurements used included CaC12-extractable metal concentrations in soils, free ion concentration in soil solution as predicted by a geochemical model, dissolved metal concentration as predicted by a semi-mechanistic model, internal metal concentrations in total earthworms and in subcellular fractions, and several biomarkers. The observations verified the causal definition of Cd and Pb bioavailability in the SEM, but not for Zn. Several indicators consistently reflected the hypothetical causal definition and could thus be pertinent measurements of Cd and Pb bioavailability to earthworm in field-contaminated soils. SEM highlights that the metals present in the soil solution and easily extractable are not the main source of available metals for earthworms. This study further highlights SEM as a powerful tool that can handle natural ecosystem complexity, thus participating to the paradigm change in ecotoxicology from a bottom-up to a top-down approach