Biology and biotechnology of Trichoderma

Fungi of the genus Trichoderma are soilborne, green-spored ascomycetes that can be found all over the world. They have been studied with respect to various characteristics and applications and are known as successful colonizers of their habitats, efficiently fighting their competitors. Once established, they launch their potent degradative machinery for decomposition of the often heterogeneous substrate at hand. Therefore, distribution and phylogeny, defense mechanisms, beneficial as well as deleterious interaction with hosts, enzyme production and secretion, sexual development, and response to environmental conditions such as nutrients and light have been studied in great detail with many species of this genus, thus rendering Trichoderma one of the best studied fungi with the genome of three species currently available. Efficient biocontrol strains of the genus are being developed as promising biological fungicides, and their weaponry for this function also includes secondary metabolites with potential applications as novel antibiotics. The cellulases produced by Trichoderma reesei, the biotechnological workhorse of the genus, are important industrial products, especially with respect to production of second generation biofuels from cellulosic waste. Genetic engineering not only led to significant improvements in industrial processes but also to intriguing insights into the biology of these fungi and is now complemented by the availability of a sexual cycle in T. reesei/Hypocrea jecorina, which significantly facilitates both industrial and basic research. This review aims to give a broad overview on the qualities and versatility of the best studied Trichoderma species and to highlight intriguing findings as well as promising applications

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

The yeast Geotrichum candidum encodes functional lytic polysaccharide monooxygenases

Background: Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that have revolutionized our understanding of lignocellulose degradation. Fungal LPMOs of the AA9 family target cellulose and hemicelluloses. AA9 LPMO-coding genes have been identified across a wide range of fungal saprotrophs (Ascomycotina, Basidiomycotina, etc.), but so far they have not been found in more basal lineages. Recent genome analysis of the yeast Geotrichum candidum (Saccharomycotina) revealed the presence of several LPMO genes, which belong to the AA9 family. Results: In this study, three AA9 LPMOs from G. candidum were successfully produced and biochemically characterized. The use of native signal peptides was well suited to ensure correct processing and high recombinant production of GcLPMO9A, GcLPMO9B, and GcLPMO9C in Pichia pastoris. We show that GcLPMO9A and GcLPMO9B were both active on cellulose and xyloglucan, releasing a mixture of soluble C1- and C4-oxidized oligosaccharides from cellulose. All three enzymes disrupted cellulose fibers and significantly improved the saccharification of pretreated lignocellulosic biomass upon addition to a commercial cellulase cocktail. Conclusions: The unique enzymatic arsenal of G. candidum compared to other yeasts could be beneficial for plant cell wall decomposition in a saprophytic or pathogenic context. From a biotechnological point of view, G. candidum LPMOs are promising candidates to further enhance enzyme cocktails used in biorefineries such as consolidated bioprocessing

Influence of the carbohydrate-binding module on the activity of a fungal aa9 lytic polysaccharide monooxygenase on cellulosic substrates

Background: Cellulose-active lytic polysaccharide monooxygenases (LPMOs) secreted by filamentous fungi play a key role in the degradation of recalcitrant lignocellulosic biomass. They can occur as multidomain proteins fused to a carbohydrate-binding module (CBM). From a biotech perspective, LPMOs are promising innovative tools for producing nanocelluloses and biofuels, but their direct action on cellulosic substrates is not fully understood. Results: In this study, we probed the role of the CBM from family 1 (CBM1) appended to the LPMO9H from Podospora anserina (PaLPMO9H) using model cellulosic substrates. Deletion of the CBM1 weakened the binding to cellulose nanofibrils, amorphous and crystalline cellulose. Although the release of soluble sugars from cellulose was drastically reduced under standard conditions, the truncated LPMO retained some activity on soluble oligosaccharides. The cellulolytic action of the truncated LPMO was demonstrated using synergy experiments with a cellobiohydrolase (CBH). The truncated LPMO was still able to improve the efficiency of the CBH on cellulose nanofibrils in the same range as the full-length LPMO. Increasing the substrate concentration enhanced the performance of PaLPMO9H without CBM in terms of product release. Interestingly, removing the CBM also altered the regioselectivity of PaLPMO9H, significantly increasing cleavage at the C1 position. Analysis of the insoluble fraction of cellulosic substrates evaluated by optical and atomic force microscopy confirmed that the CBM1 module was not strictly required to promote disruption of the cellulose network. Conclusions: Absence of the CBM1 does not preclude the activity of the LPMO on cellulose but its presence has an important role in driving the enzyme to the substrate and releasing more soluble sugars (both oxidized and nonoxidized), thus facilitating the detection of LPMO activity at low substrate concentration. These results provide insights into the mechanism of action of fungal LPMOs on cellulose to produce nanocelluloses and biofuels

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production