Since its inception, the carbohydrate-active enzymes database (CAZy; http://www.cazy.org
 ) has described the families of enzymes that cleave or build complex carbohydrates, namely the glycoside hydrolases (GH), the polysaccharide lyases (PL), the carbohydrate esterases (CE), the glycosyltransferases (GT) and their appended non-catalytic carbohydrate-binding modules (CBM). The recent discovery that members of families CBM33 and family GH61 are in fact lytic polysaccharide monooxygenases (LPMO), demands a reclassification of these families into a suitable category. Because lignin is invariably found together with polysaccharides in the plant cell wall and because lignin fragments are likely to act in concert with (LPMO), we have decided to join the families of lignin degradation enzymes to the LPMO families and launch a new CAZy class that we name “Auxiliary Activities” in order to accommodate a range of enzyme mechanisms and substrates related to lignocellulose conversion. Comparative analyses of these auxiliary activities in 41 fungal genomes reveal a pertinent division of several fungal groups and subgroups combining their phylogenetic origin and their nutritional mode (white vs. brown rot). The new class introduced in the CAZy database extends the traditional CAZy families, and provides a better coverage of the full extent of the lignocellulose breakdown machinery.

/pdf/expansion-of-the-enzymatic-repertoire-of-the-cazy-database-23c38ut1lr.pdf

Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes

Plants represent a vast, renewable resource and are well suited to provide sustainably for humankind’s transportation fuel needs. To produce infrastructure-compatible fuels from biomass, two challenges remain: overcoming plant cell wall recalcitrance to extract sugar and phenolic intermediates, and reduction of oxygenated intermediates to fuel molecules. To compete with fossil-based fuels, two primary routes to deconstruct cell walls are under development, namely biochemical and thermochemical conversion. Here, we focus on overcoming recalcitrance with biochemical conversion, which uses low-severity thermochemical pretreatment followed by enzymatic hydrolysis to produce soluble sugars. Many challenges remain, including understanding how pretreatments affect the physicochemical nature of heterogeneous cell walls; determination of how enzymes deconstruct the cell wall effectively with the aim of designing superior catalysts; and resolution of issues associated with the co-optimization of pretreatment, enzymatic hydrolysis, and fermentation. Here, we highlight some of the scientific challenges and open questions with a particular focus on problems across multiple length scales.

Deconstruction of lignocellulosic biomass to fuels and chemicals

The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies. Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414. Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.

https://link.springer.com/content/pdf/10.1186%2F1754-6834-5-1.pdf

A versatile toolkit for high throughput functional genomics with Trichoderma reesei

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family

Trichoderma is a genus of common filamentous fungi that display a remarkable range of lifestyles and interactions with other fungi, animals and plants. Because of their ability to antagonize plant-pathogenic fungi and to stimulate plant growth and defence responses, some Trichoderma strains are used for biological control of plant diseases. In this Review, we discuss recent advances in molecular ecology and genomics which indicate that the interactions of Trichoderma spp. with animals and plants may have evolved as a result of saprotrophy on fungal biomass (mycotrophy) and various forms of parasitism on other fungi (mycoparasitism), combined with broad environmental opportunism.

/pdf/trichoderma-the-genomics-of-opportunistic-success-35ia0o8871.pdf

Trichoderma : the genomics of opportunistic success

Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.

/pdf/genome-sequencing-and-analysis-of-the-biomass-degrading-3cuwfw4we8.pdf

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).

Corrigendum: Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina )

/pdf/corrigendum-genome-sequencing-and-analysis-of-the-biomass-7snfo2epqm.pdf

Melissa Jackson

Papers

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).

Corrigendum: Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)