Exploring the use of hydrophilic ionic liquids for the lysis of fungal structures.

Hybrid in silico and ultrasound-assisted enzymatic extraction as a functionalizing strategy for cassava starch: Structural, physicochemical and morphological insights.

The food enzyme containing cellulase (1,4-(1,3;1,4)-β-d-glucan-4-glucanohydrolase; EC 3.2.1.4) and endo-1,3(4)-β-glucanase (3-(1,3,1,4)-β-d-glucan 3(4)-glucanohydrolase; EC 3.2.1.6) is produced with the non-genetically modified Trichoderma reesei strain 480KY by Kerry Ingredients & Flavours Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in eight food manufacturing processes. Since residual amounts of food enzyme-total organic solids (TOS) are removed in two processes, dietary exposure was calculated for the remaining six food manufacturing processes. It was estimated to be up to 2.681 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1014 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 378. A search for the homology of the two amino acid sequences to known allergens was made and a match with one respiratory allergen was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The food enzyme endo-1,4-β-xylanase (4-β-d-xylan xylanohydrolase; EC 3.2.1.8) is produced with the genetically modified Trichoderma reesei strain DP-Nzd66 by Genencor International B.V. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in the processing of cereals and other grains for the production of baked products. The dietary exposure was estimated to be up to 0.609 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1214 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1993. A search for the homology of the amino acid sequence of the endo-1,4-β-xylanase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The food enzyme cellulase (4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase; EC 3.2.1.4) is produced with the genetically modified Trichoderma reesei strain AR-852 by AB Enzymes GmbH. The safety of this food enzyme was evaluated previously, and it did not give rise to safety concerns when used in five food manufacturing processes. Subsequently, the applicant has requested to extend its use to include one additional process and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of six food manufacturing processes. As the food enzyme-total organic solids (TOS) are removed in one process, the dietary exposure to the food enzyme-TOS was estimated for the remaining five food manufacturing processes. It was estimated to be up to 0.494 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level (NOAEL) previously reported (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 2024. Based on the revised margin of exposure and the previous evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

The chemical diversity of antifungal sorbicillinoids produced by the marine-derived fungus Trichoderma reesei HK1-12 was investigated by using bioassay-guided separation combined with molecular networking technology. A novel sorbicillinoid monomer, trichocillin A (1), along with four known analogues (4-5), was isolated from the fungal cultures. Furthermore, 12 monomeric, dimeric, trimeric, or hybrid sorbicillinoids (6-17) were tentatively identified from the molecular networks, six of which (7, 8, 11-13, and 15) represent previously unreported structures. Vertinolide (2) exhibited antifungal activity against Rhizoctonia solani with an inhibition rate of 90.9% at 50 μg/mL in vitro and comparable in vivo curative effects at 400 μg/mL (66.5%) to those of the spore suspension of HK1-12 (69.8%) and the positive control tebuconazole·trifloxystrobin (71.9%). This study indicated that sorbicillinoids are key antibiosis agents responsible for T. reesei HK1-12 as a potential biocontrol agent of R. solani.

An application was submitted to the Food Standards Agency and Food Standards Scotland in April 2023 by AB Enzymes Finland Oy (“the applicant”) for the extension of use of an additive consisting of 6-phytase produced by Trichoderma reesei (CBS 126897) (Quantum ® Blue) under the category ‘zootechnical additive’, functional group ‘digestibility enhancer’, to suckling and weaned piglets and other porcine species and ornamental birds so that the authorisation covers all avian species and all porcine species. The European Food Safety Authority (EFSA) concluded that the final product does not give rise to any safety concerns with regards to the genetic modification of the production strain, as neither the viable cells of the production strain nor its DNA were detected (2013a, 2024). No antibiotic resistance genes from the genetic modification in the production strain were identified. Under the intended conditions of use, Quantum ® Blue is considered safe for the target species. The use of the additive in animal nutrition is considered safe for consumers and for the environment. All forms of the additive should be considered potential respiratory sensitisers, and any inhalation exposure presents a risk. Nevertheless, the Panel noted that the solid formulations are dust-free, which makes inhalation exposure unlikely. Due to the absence of new data, no conclusions could be drawn regarding the potential of the various formulations to cause skin or eye irritation, or to act as skin sensitisers. Quantum ® Blue is efficacious in poultry, pigs for fattening and sows at 250 FTU/kg and in weaned and suckling piglets at 500 FTU/kg, as well as in all laying avian species at 150 FTU/kg. Regarding the extension of use, the additive has the potential to be efficacious in ornamental birds and in porcine species for fattening/reared for reproduction and reproductive animals at 250 FTU/kg, and in suckling and weaned piglets of minor porcine species at 500 FTU/kg complete feed. FSA/FSS has reviewed the applicant’s authorisation application, supporting documentation, and other regulators’ risk assessments, most notably the EFSA risk assessment opinion, and considers sufficient evidence has been demonstrated to conclude without the need for further questions or risk assessment.

A comparative study on crystal violet and congo red dyes removal from wastewater using Trichoderma reesei biomass: Equilibrium, kinetic, thermodynamic studies, and error analysis

Supplementing XYR1-mutated Trichoderma reesei strain cultivation with (SO2-ethanol-water) softwood pulp improves cellulase production

Banana, contributing to food security and the economy in Côte d’Ivoire, is subjected to multiple biotic and abiotic stresses. The intensive use of chemical inputs promotes resistance in pathogenic fungi while contributing to environmental pollution. The phyllospheric microbiome of banana, whose structure varies according to geographical conditions, represents a potentially exploitable resource for the development of biological control strategies. This study aimed to evaluate the antagonistic potential of phyllospheric fungi associated with banana. Fifty‑four leaf samples (27 apparently healthy and 27 symptomatic) were collected from nine plantations distributed across three localities: Zokoguhé, Bribouo and Gonaté. The analytical approach consisted of isolating fungi present in the leaf samples and assessing their antifungal activities through solid‑medium confrontation assays between antagonists and pathogens. The fungi isolated from banana leaves belonged to the genera Trichoderma, Aspergillus, Fusarium, Cladosporium, Mucor, Exophiala, Rhizomucor, Citalidium, Pestalotiopsis, Aureobasidium, Scytalidium and Alternaria. Among them, Trichoderma reesei, with a highest prevalence of 83.33%, appears to be a core member of the fungal community associated with banana leaves. It showed the strongest antagonistic activity, inhibiting Fusarium oxysporum growth by 54.56%. These findings demonstrate that the banana phyllosphere harbors fungal communities of interest, capable of exerting biocontrol activity.

Spontaneous strain degeneration, defined as the loss of an essential biological function during prolonged usage, is frequently observed in microorganisms and poses a significant challenge to the biotechnology industry. In Trichoderma reesei, a filamentous fungus widely used for large-scale cellulase production, spontaneous loss of cellulase productivity has been reported. However, studies on this phenomenon have focused solely on industrial strains derived from the Rut-C30 lineage. This study analyzes strain degeneration in a different industrial lineage of T. reesei, RL-P37, and its hypercellulase-producing descendant, GEN-3A. We found that RL-P37 and GEN-3A are also affected by the degeneration phenomenon, with the highly productive GEN-3A showing greater susceptibility. The degenerated phenotype was characterized by reduced cellulase productivity, altered growth behavior, and distinct morphological changes. In particular, cellulase hyperproduction was associated with bulbous, highly branched hyphae, while these morphological traits were lost in degenerated isolates. Our study establishes a framework for characterizing strain degeneration in T. reesei, highlights the trade-off between productivity and stability, and identifies distinctive morphological signatures linked to cellulase hyperproduction and degeneration, which may serve as early phenotypic indicators for industrial strain monitoring.

Elimination of calnexin overcomes the secretion bottleneck of Trichoderma reesei β-glucosidase Cel3B which boosts corncob saccharification.

Tunable structure and reinforcement of polyvinyl alcohol (PVA) hydrogels using fungal chitin particles.

The fungus Trichoderma reesei is widely employed for industrial enzyme production. However, the mechanisms coordinating enzyme production with amino acid biosynthesis and metabolism remain incompletely understood. Here, we characterized a novel C2H2 zinc finger transcription factor, TrTRC-1, in T. reesei. The deletion of TrTRC-1 enhanced cellulase, xylanase, and extracellular protein production by 9.8-32.4% compared to that in T. reesei Rut C30, while simultaneously stimulating mycelial growth and conidiation. Transcriptomic analysis revealed that the deletion of TrTRC-1 significantly altered the expression of genes associated with primary metabolic pathways, amino acid biosynthesis/metabolism, and carbohydrate-active enzymes. In a 5 L bioreactor, T. reesei ΔTrTRC-1 achieved an FPase activity of 17.35 IU/mL and exhibited robust saccharification efficiency on lignocellulosic biomass. These findings demonstrate that TrTRC-1 serves as a multifunctional regulator in T. reesei, advancing our understanding of the regulatory network that balances enzyme production with cellular metabolism and providing a strategic basis for constructing microbial chassis strains with industrial application prospects.

Cellulase Production in Bamboo Solid Media using Trichoderma reesei RUT-C30 and its Temperature Dependence

Harnessing agro-industrial by-products for food protein production: Ex-ante process design for precision fermentation using Trichoderma reesei

ABSTRACT Agricultural waste management is critical for reducing environmental pollution and enhancing soil health, particularly the treatment of organic waste containing heavy metals. This study investigates the distinct effects of mesophilic and thermophilic microbial inoculation on composting. Using rice straw and swine manure as feedstocks, composting piles were inoculated with mesophilic (Bacillus subtilis and Trichoderma reesei) or thermophilic (Geobacillus stearothermophilus and Aspergillus fumigatus) microorganisms, alongside a control group with no inoculation. The results revealed that thermophilic inoculation significantly enhanced organic degradation and humification, leading to more efficient stabilization of heavy metals such as Pb, Zn, and Cr, especially during the thermophilic and mature phases. Microbial community analysis showed that thermophilic inoculation created a more connected microbial network and boosted the microbial functionality. Spearman correlation analysis indicated that the enhanced key metabolic pathways of IT, particularly those involved in organic matter degradation and heavy metal detoxification, were associated with reduced metal bioavailability during thermophilic phase. These findings highlighting the potential of thermophilic inoculants for sustainable waste management and environmental protection.

The food enzyme phospholipase A2 (phosphatidylcholine 2‐acylhydrolase; EC 3.1.1.4) is produced with the genetically modified Trichoderma reesei strain RF8793 by AB Enzymes GmbH. In a previous opinion, the total organic solids (TOS) values could not be calculated, which led to the inability to establish the representativeness of the batch used for toxicological examination and to calculate the dietary exposure. In this assessment, EFSA completes the safety evaluation of this food enzyme by taking into account the chemical compositional data from new food enzyme batches. The food enzyme is intended to be used in three food manufacturing processes. Dietary exposure to the food enzyme–TOS was estimated to be up to 0.008 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 125,000. Based on the new data and the previous evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The food enzyme chymosin (EC 3.4.23.4) is produced with the genetically modified Trichoderma reesei strain DP‐Nyj88 by Genencor International BV. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in the processing of dairy products for the production of cheese. Dietary exposure was estimated to be up to 0.013 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 76,923. A search for the homology of the amino acid sequence of the chymosin to known allergens was made, and matches with five respiratory allergens and one injected allergen were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

BackgroundThe degradation of agricultural wastes is crucial for sustainable economic and environmental development, necessitating efficient cellulolytic enzymes to enable high-value bioconversion. The filamentous fungus Trichoderma reesei is a widely used cellulase producer for deconstructing agricultural wastes in biomass conversion. However, its enzyme system remains suboptimal and requires further refinement to achieve economical bioconversion of agricultural wastes.ResultsHerein, a hyper-cellulolytic T. reesei mutant strain CU7-4 derived from the industrial strain RUT-C30 was obtained by UV mutagenesis. When degrading the different pretreated corncob residues, CU7-4 exhibited a 20% improvement in saccharification efficiency compared to the parental strain RUT-C30. Furthermore, comparative proteomics was employed to decipher the variation between the secretomes of CU7-4 and RUT-C30. It was found that the discrepancy of the protein proportion between the secretomes may enable the changed saccharification efficiency towards the pretreated corncob residues. Then, three small secreted proteins (SSP1, EPL1, CUT1) and two β-glucosidases (Cel3H, Cel3F) were identified through the significant differences analysis in protein abundance between CU7-4 and RUT-C30, combined with responding to the essential transcriptional regulator Xyr1. Further investigation of these five proteins was conducted. Deletion of SSP1 and EPL1 was certified to facilitate degrading corncob residues and corn stover. Overexpression of Cel3F improved the activities of cellobiohydrolase and β-glucosidase, and the in vitro addition of Cel3F significantly promoted the saccharification efficiency of RUT-C30 toward corncob residues.ConclusionsThis study not only expands the protein functions for deciphering the mechanism of lignocellulose degradation, but also provides valuable protein targets for engineering the robust and powerful lignocellulolytic enzyme system, thereby facilitating the efficient degradation of agricultural wastes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-025-02695-6.