Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/bit.20282

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.

Outlook for cellulase improvement: screening and selection strategies.

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).

Pretreatment technologies are aimed to increase enzyme accessibility to biomass and yields of fermentable sugars. In general, pretreatment methods fall into four different categories including physical, chemical, physico-chemical, and biological. This paper comprehensively reviews the lignocellulosic wastes to bioethanol process with a focus on pretreatment methods, their mechanisms, advantages and disadvantages as well as the combinations of different pretreatment technologies. Moreover, the new advances in plant “omics” and genetic engineering approaches to increase cellulose composition, reduce cellulose crystallinity, produce hydrolases and protein modules disrupting plant cell wall substrates, and modify lignin structure in plants have also been expansively presented.

Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment

The economic dependency on fossil fuels and the resulting effects on climate and environment have put tremendous focus on utilizing fermentable sugars from lignocellulose, the largest known renewable carbohydrate source. The fermentable sugars in lignocellulose are derived from cellulose and hemicelluloses but these are not readily accessible to enzymatic hydrolysis and require a pretreatment, which causes an extensive modifi cation of the lignocellulosic structure. A number of pretreatment technologies are under development and being tested in pilot scale. Hydrolysis of lignocellulose carbohydrates into fermentable sugars requires a number of different cellulases and hemicellulases. The hydrolysis of cellulose is a sequential breakdown of the linear glucose chains, whereas hemicellulases must be capable of hydrolysing branched chains containing different sugars and functional groups. The technology for pretreatment and hydrolysis has been developed to an extent that is close to a commercially viable level. It has become possible to process lignocellulose at high substrate levels and the enzyme performance has been improved. Also the cost of enzymes has been reduced. Still a number of technical and scientifi c issues within pretreatment and hydrolysis remain to be solved. However, signifi cant improvements in yield and cost reductions are expected, thus making large-scale fermentation of lignocellulosic substrates possible. © 2007 Society of Chemical Industry and John Wiley & Sons, Ltd

Enzymatic conversion of lignocellulose into fermentable sugars: challenges and

Plant cell wall proteins called expansins are thought to disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them. We describe here a novel gene with sequence similarity to plant expansins, isolated from the cellulolytic fungus Trichoderma reesei. The protein named swollenin has an N-terminal fungal type cellulose binding domain connected by a linker region to the expansin-like domain. The protein also contains regions similar to mammalian fibronectin type III repeats, found for the first time in a fungal protein. The swollenin gene is regulated in a largely similar manner as the T. reesei cellulase genes. The biological role of SWOI was studied by disrupting the swo1 gene from T. reesei. The disruption had no apparent effect on the growth rate on glucose or on different cellulosic carbon sources. Non-stringent Southern hybridization of Trichoderma genomic DNA with swo1 showed the presence of other swollenin-like genes, which could substitute for the loss of SWOI in the disruptant. The swollenin gene was expressed in yeast and Aspergillus niger var. awamori. Activity assays on cotton fibers and filter paper were performed with concentrated SWOI-containing yeast supernatant that disrupted the structure of the cotton fibers without detectable formation of reducing sugars. It also weakened filter paper as assayed by an extensometer. The SWOI protein was purified from A. niger var. awamori culture supernatant and used in an activity assay with Valonia cell walls. It disrupted the structure of the cell walls without producing detectable amounts of reducing sugars.

Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials

The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1 In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability The T reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions There was no phenotypic difference between the Δcre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T reesei

Genetic modification of carbon catabolite repression in Trichoderma reesei for improved protein production.

Genes encoding novel cellulases, and a gene encoding a protein that facilitates the action of such novel cellulases, the novel cellulases and a protein that facilitates the action of such cellulases, and enzyme preparations containing such proteins are described. The native hosts and the culture medium of said hosts containing said novel cellulases are also disclosed. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

Cellulases, the genes encoding them and uses thereof

The cloning and sequencing of the genes encoding phytase (phy) and pH 2.5-optimum acid phosphatase (aph) from Aspergillus niger var. awamori

A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures The carriers tested were the T reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide The recombinant Xyn11A produced had properties similar to those of the native xylanase It constituted 6 to 10% of the total proteins secreted by the transformants About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed Even in the best Xyn11A producers, the levels of the fusion mRNAs were only approximately 10% of the level of cel7A (cbh1) mRNA in the untransformed host strain

High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus Trichoderma reesei Requires a Carrier Polypeptide with an Intact Domain Structure

Marja Paloheimo

Papers

Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials

Genetic modification of carbon catabolite repression in Trichoderma reesei for improved protein production.

Cellulases, the genes encoding them and uses thereof

The cloning and sequencing of the genes encoding phytase (phy) and pH 2.5-optimum acid phosphatase (aph) from Aspergillus niger var. awamori

High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus Trichoderma reesei Requires a Carrier Polypeptide with an Intact Domain Structure