Lignocellulosic biomass, the most abundant natural resource on earth, can be used for cellulosic ethanol production but requires a pretreatment to improve enzyme access to the polymeric sugars while obtaining value from the other components. γ-Valerolactone (GVL) is a promising candidate for biomass pretreatment since it is renewable and bio-based. In the present work, the effect of a pretreatment based on GVL on the enzymatic saccharification of white birch was evaluated at a laboratory scale and the importance of the washing procedure for the subsequent saccharification was demonstrated. Both the saccharification yield and the production of cellulosic ethanol were higher using a noncommercial enzyme crude from Talaromyces amestolkiae than with the commercial cocktail Cellic CTec2 from Novozymes. Furthermore, the production of extracellular cellulases by T. amestolkiae has been optimized in 2 L bioreactors, with improvements ranging from 40% to 75%. Finally, it was corroborated by isoelectric focus that optimization of cellulase secretion by T. amestolkiae did not affect the pattern production of the main β-glucosidases and endoglucanases secreted by this fungus.

Cellulose is a major renewable resource for a wide variety of sustainable industrial products. However, for its utilization, finding new efficient enzymes for plant cell wall depolymerization is crucial. In addition to microbial sources, cellulases also exist in plants, however, are less studied. Fleshy fruit ripening includes enzymatic cell wall hydrolysis, leading to tissue softening. Therefore, bilberry (Vaccinium myrtillus L.), which produces small fruits that undergo extensive and rapid softening, was selected to explore cellulases of plant origin. We identified 20 glycoside hydrolase family 9 (GH9) cellulases from a recently sequenced bilberry genome, including four of which showed fruit ripening-specific expression and could be associated with fruit softening based on phylogenetic, transcriptomic and gene expression analyses. These four cellulases were secreted enzymes: two B-types and two C-types with a carbohydrate binding module 49. For functional characterization, these four cellulases were expressed in Pichia pastoris. All recombinant enzymes demonstrated glucanase activity toward cellulose and hemicellulose substrates. Particularly, VmGH9C1 demonstrated high activity and ability to degrade cellulose, xyloglucan, and glucomannan. In addition, all the enzymes retained activity under wide pH (6-10) and temperature ranges (optimum 70 °C), revealing the potential applications of plant GH9 cellulases in the industrial bioprocessing of lignocellulose.

Trichoderma reesei, the main filamentous fungus used for industrial cellulase production, was long considered to be asexual. The recent discovery of the mating type locus in the natural isolate QM6a and the possibility to cross this sterile female strain with a fertile natural female strain opened up a new avenue for strain optimization. We crossed the hyperproducer RutC30 with a compatible female ascospore-derived isolate of the wild-type strain CBS999.97 and analyzed about 300 offspring. A continuous distribution of secreted protein levels was observed in the progeny, confirming the involvement of several mutated loci in the hyperproductive phenotype. A bias toward MAT1-2 strains was identified for higher producers, but not directly linked to the Mating-type locus itself. Transgressive phenotypes were observed in terms of both productivity and secretome quality, with offspring that outperform their parents for three enzymatic activities. Genomic sequences of the 10 best producers highlighted the genetic diversity generated and the involvement of parental alleles in hyperproduction and fertility.
OBJECTIVE: The filamentous fungus Trichoderma reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars. The filamentous fungus T. reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars, which can in turn be fermented to produce second-generation biofuels and bioproducts. Production performance improvement, which is essential to reduce production cost, relies on classical mutagenesis and genetic engineering techniques. Although sexual reproduction is a powerful tool for improving domesticated species, it is often difficult to apply to industrial fungi since most of them are considered asexual. In this study, we demonstrated that outbreeding is an efficient strategy to optimize T. reesei. Crossing between a natural isolate and a mutagenized strain generated a biodiverse progeny with some offspring displaying transgressive phenotype for cellulase activities.

Cellulases have been widely used in many fields such as animal feed, textile, food, lignocellulose bioconversion, etc. Efficient and low-cost production of cellulases is very important for its industrial application, especially in bioconversion of lignocellulosic biomass. Filamentous fungi are currently widely used in industrial cellulase production due to their ability to secrete large amounts of active free cellulases extracellularly. This review comprehensively summarized the research progress on cellulases from filamentous fungi in recent years, including filamentous fungi used for cellulase production and its modification strategies, enzyme compositions, characterization methods and application of fungal cellulase systems, and the production of fungal cellulase includes production processes, factors affecting cellulase production such as inducers, fermentation medium, process parameters and their control strategies. Also, the future perspectives and research topics in fungal cellulase production are presented in the end of the review. The review helps to deepen the understanding of the current status of fungal cellulases, thereby promoting the production technology progress and industrial application of filamentous fungal cellulase.

Crop straws provide enormous lignocellulose resources transformable for sustainable biofuels and valuable bioproducts. However, lignocellulose recalcitrance basically restricts essential biomass enzymatic saccharification at large scale. In this study, the mushroom-derived cellobiohydrolase (LeGH7) was introduced into Trichoderma reesei (Rut-C30) to generate two desirable strains, namely GH7-5 and GH7-6. Compared to the Rut-C30 strain, both engineered strains exhibited significantly enhanced enzymatic activities, with β-glucosidases, endocellulases, cellobiohydrolases, and xylanase activities increasing by 113 %, 140 %, 241 %, and 196 %, respectively. By performing steam explosion and mild alkali pretreatments with mature straws of five bioenergy crops, diverse lignocellulose substrates were effectively digested by the crude enzymes secreted from the engineered strains, leading to the high-yield hexoses released for bioethanol production. Notably, the LeGH7 enzyme purified from engineered strain enabled to act as multiple cellulases and xylanase at higher activities, interpreting how synergistic enhancement of enzymatic saccharification was achieved for distinct lignocellulose substrates in major bioenergy crops. Therefore, this study has identified a novel enzyme that is active for simultaneous hydrolyses of cellulose and xylan, providing an applicable strategy for high biomass enzymatic saccharification and bioethanol conversion in bioenergy crops.

The Victoria Falls rainforest is a protected site whose forest floors harbor a host of cellulolytic microorganisms involved in biomass degradation. This study collected decaying logs and soil from the rainforest for bioprospecting cellulases from their metagenomes. Metagenomic DNA was isolated from the compound sample. Degenerate cellulase primers were used to amplify cellulase genes in the metagenome. The resulting amplicons cloned into Z-competent Escherichia coli DH5α were analyzed by functional screening for the production of cellulase extracellularly. Functional screening of the clones resulted in one clone (Clone-i) testing positive for extracellular cellulase production. Submerged fermentation of Clone-i was carried out for cellulase production. The cellulases were characterized to determine their activity\'s optimum pH and temperature. The diversity of the cellulases produced by Clone-i was determined. Clone-i\'s optimum enzyme activity was observed after 72 hours of incubation at 50°C and pH 5. Clone-i produced 80% more exoglucanases as compared to endoglucanases. The cellulolytic Clone-i\' isolate shows Victoria Falls rainforest\'s potential as an enzyme bioprospecting site, reflecting that metagenomics is a valuable tool in microbial ecology.

Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7 days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.

Colletotrichum lindemuthianum is a phytopathogenic fungus that causes anthracnose in common beans (Phaseolus vulgaris) and presents a great diversity of pathotypes with different levels of virulence against bean varieties worldwide. The purpose of this study was to establish whether pathotypic diversity is associated with differences in the mycelial growth and secretion of plant-cell-wall-degrading enzymes (PCWDEs). We evaluated growth, hemicellulase and cellulase activity, and PCWDE secretion in four pathotypes of C. lindemuthianum in cultures with glucose, bean hypocotyls and green beans of P. vulgaris, and water hyacinth (Eichhornia crassipes). The results showed differences in the mycelial growth, hemicellulolytic activity, and PCWDE secretion among the pathotypes. Glucose was not the preferred carbon source for the best mycelial growth in all pathotypes, each of which showed a unique PCWDE secretion profile, indicating different levels of carbon catabolite regulation (CCR). The pathotypes showed a high differential hemicellulolytic capacity to degrade host and water hyacinth tissues, suggesting CCR by pentoses and that there are differences in the absorption and metabolism of different monosaccharides and/or disaccharides. We propose that different levels of CCR could optimize growth in different host tissues and could allow for consortium behavior in interactions with bean crops.

Unravelling the multifaceted and bidirectional interactions between microbiota and host physiology represents a major scientific challenge. Here, we utilise the nematode model, Pristionchus pacificus, coupled to a laboratory-simulated decay process of its insect host, to mimic natural microbiota succession and investigate associated tripartite interactions. Metagenomics reveal that during initial decay stages, the population of vitamin B-producing bacteria diminishes, potentially due to a preferential selection by nematodes. As decay progresses to nutrient-depleted stages, bacteria with smaller genomes producing less nutrients become more prevalent. Lipid utilisation and dauer formation, representing key nematode survival strategies, are influenced by microbiota changes. Additionally, horizontally acquired cellulases extend the nematodes\' reproductive phase due to more efficient foraging. Lastly, the expressions of Pristionchus species-specific genes are more responsive to natural microbiota compared to conserved genes, suggesting their importance in the organisms\' adaptation to its ecological niche. In summary, we show the importance of microbial successions and their reciprocal interaction with nematodes for insect decay in semi-artificial ecosystems.

Biomass-degrading enzymes produced by microorganisms have a great potential in the processing of agricultural wastes. In order to produce suitable biomass-degrading enzymes for releasing sugars and aroma compounds from tobacco scraps, the feasibility of directly using the scraps as a carbon source for enzyme production was investigated in this study. By comparative studies of ten fungal strains isolated from tobacco leaves, Aspergillus brunneoviolaceus Ab-10 was found to produce an efficient enzyme mixture for the saccharification of tobacco scraps. Proteomic analysis identified a set of plant biomass-degrading enzymes in the enzyme mixture, including amylases, hemicellulases, cellulases and pectinases. At a substrate concentration of 100 g/L and enzyme dosage of 4 mg/g, glucose of 17.6 g/L was produced from tobacco scraps using the crude enzyme produced by A. brunneoviolaceus Ab-10. In addition, the contents of 23 volatile molecules, including the aroma compounds 4-ketoisophorone and benzyl alcohol, were significantly increased after the enzymatic treatment. The results provide a strategy for valorization of tobacco waste by integrating the production of biomass-degrading enzymes into the tobacco scrap processing system.