Trichoderma Reesei Research Articles

Enhanced production of select phytocannabinoids in medical Cannabis cultivars using microbial consortia.

The root microbiome of medical cannabis plants has been largely unexplored due to past legal restrictions in many countries. Microbes that live on and within the tissue of Cannabis sativa L. similar to other plants, provide advantages such as stimulating plant growth, helping it absorb minerals, providing protection against pathogen attacks, and influencing the production of secondary metabolites. To gain insight into the microbial communities of C. sativa cultivars with different tetrahydrocannabinol (THC) and cannabidiol (CBD) profiles, a greenhouse trial was carried out with and without inoculants added to the growth substrate. Illumina MiSeq metabarcoding was used to analyze the root and rhizosphere microbiomes of the five cultivars. Plant biomass production showed higher levels in three of five cultivars inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis and microbial suspension. The blossom dry weight of the cultivar THE was greater when inoculated with R. irregularis and microbial suspension than with no inoculation. Increasing plant biomass and blossom dry weight are two important parameters for producing cannabis for medical applications. In mature Cannabis, 12 phytocannabinoid compounds varied among cultivars and were affected by inoculants. Significant differences (p ≤ 0.01) in concentrations of cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabigerol (CBG), cannabidiol (CBD), and cannabigerolic acid (CBGA) were observed in all Cannabis cultivars when amended with F, K1, and K2 inoculants. We found microbes that were shared among cultivars. For example, Terrimicrobium sp., Actinoplanes sp., and Trichoderma reesei were shared by the cultivars ECC-EUS-THE, CCL-ECC, and EUS-THE, respectively. Actinoplanes sp. is a known species that produces phosphatase enzymes, while Trichoderma reesei is a fungal train that produces cellulase and contributes to organic matter mineralization. However, the role of Terrimicrobium sp. as an anaerobic bacterium remains unknown. This study demonstrated that the use of inoculants had an impact on the production of phytocannabinoids in five Cannabis cultivars. These inoculants could have useful applications for optimizing cannabis cultivation practices and increasing the production of phytocannabinoids.

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi.

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications.

Response surface methodology for the mixed fungal fermentation of Codonopsis pilosula straw using Trichoderma reesei and Coprinus comatus.

The objective of this study was to investigate the cellulose degradation rate (CDR) and lignin degradation rate (LDR) of Codonopsis pilosula straw (CPS) and the optimal fermentation parameters for mixed fungal fermentation. Single-factor tests were used to study the effects of the fungal ratio (Trichoderma reesei: Coprinus comatus), fungal inoculum, corn flour content, and fermentation time on the degradation rate of cellulose and lignin. Based on the results of this experiment, the optimal fermentation factors were identified, and the effects of various factors and their interactions on the degradation rates of cellulose and lignin were further evaluated using the response surface method. The quadratic polynomial mathematical model of degradation rates of the cellulose and lignin in CPS by mixed fungus fermentation was established using Design Expert software v8.0.6. Under the optimal parameters for fungal fermentation of CPS straw (fungal ratio 4:6, fungal inoculum 8%, corn flour content 10%, fermentation time of 15 d), the CDR and LDR reached 13.65% and 10.73%, respectively. Collectively, the mixed fungal fermentation of CPS resulted in decreased lignin and cellulose content, better retention of nutrients, and enhanced fermentation quality. The results of this study indicate that fermentation using Trichoderma reesei and Coprinus comatus is a productive method for straw degradation, providing a theoretical basis for the development of CPS as feed.

Biochar with enhanced performance prepared from bio-regulated lignocellulose for efficient removal of organic pollutants from wastewater

This work presented a novelty method for preparing lignocellulosic materials with reinforced performances, that is, the lignocellulose of corn straw was bio-regulated into cellulose, hemicellulose, and lignin via three types of microorganisms (Aspergillus niger, Myrothecium verrucaria, Trichoderma reesei) with cross treatment. Three different types of biochar were then prepared by the carbonization and combined alkali activation processes. The results showed that the great quantity of micro/mesopores endowed biochars ultra-high specific surface areas up to 2881, 2986, and 2878 m2 g−1. Biochars exhibited ultra-high maximum adsorption capacities for crystal violet (CV) and tetracycline hydrochloride (TC), which was higher than that of most adsorbents including the corn straw-based carbon and multi-walled carbon nanotube. After five cycles, the samples maintained a removal rate of over 70% for CV and over 60% for TC, proving their good potential for recycling. This study showed that the bio-regulation of lignocellulose was not only an effective method to develop materials with enhanced adsorption performances, but also provided a feasible idea for the comprehensive utilization of lignocellulose.

RGS4 impacts carbohydrate and siderophore metabolism in Trichoderma reesei

BackgroundAdaptation to complex, rapidly changing environments is crucial for evolutionary success of fungi. The heterotrimeric G-protein pathway belongs to the most important signaling cascades applied for this task. In Trichoderma reesei, enzyme production, growth and secondary metabolism are among the physiological traits influenced by the G-protein pathway in a light dependent manner.ResultsHere, we investigated the function of the SNX/H-type regulator of G-protein signaling (RGS) protein RGS4 of T. reesei. We show that RGS4 is involved in regulation of cellulase production, growth, asexual development and oxidative stress response in darkness as well as in osmotic stress response in the presence of sodium chloride, particularly in light. Transcriptome analysis revealed regulation of several ribosomal genes, six genes mutated in RutC30 as well as several genes encoding transcription factors and transporters. Importantly, RGS4 positively regulates the siderophore cluster responsible for fusarinine C biosynthesis in light. The respective deletion mutant shows altered growth on nutrient sources related to siderophore production such as ornithine or proline in a BIOLOG phenotype microarray assay. Additionally, growth on storage carbohydrates as well as several intermediates of the D-galactose and D-arabinose catabolic pathway is decreased, predominantly in light.ConclusionsWe conclude that RGS4 mainly operates in light and targets plant cell wall degradation, siderophore production and storage compound metabolism in T. reesei.

Dissolved oxygen on xylanase production by Trichoderma reesei using Reutalis trisperma press cake as an additional substrate

Dissolved oxygen (DO) level in media plays a big role in the succession of the fermentation process by a microorganism. In this project, Trichoderma reesei was cultivated in two types of media, media A and B, both in 250 mL shake flask and 1.5 L fermenter at varying DO level. Media A and B contained the same carbon source and mineral salts, the difference between both media was the presence of press cake in Media B as urea and ammonium sulfate replacement, whereas the nitrogen sources in media A consists of urea and ammonium sulfate. The batch fermentation process was conducted in a 7.8 C/N ratio with an initial pH of 6. The purified enzyme was shown to convert xylan to xylose. Media B that contained Reutealis trisperma press cake gives a higher xylanase activity than media A without press cake, for both shake flask and fermenter cultivations. A xylanase activity of 101.07±1.65 U/mL and 39.30±0.64 U/mL was produced in a shake flask under 140 rpm for 96 hours of fermentation with ratio of 0.6 media B/flask volume and 0.6 media A/flask volume, respectively. A xylanase activity of 205.90±0.84 U/mL and 88.87±1.45 U/mL was produced in 72 hours at 0.8 L/min of aeration rate in a 1.5 L fermenter with media A and media B, respectively. Thus, this study identified the Reutealis trisperma cake can be used as a beneficial additional substrate and nitrogen sources. The increase in DO level, which indicated by increasing aeration rate, can generate a higher xylanase activity for both media in shake flask and fermenter.

In situ assay of the reducing sugars in hydrophilic natural deep eutectic solvents by a modified DNS method

The DNS assay has been already a universally method for the in situ determination of reducing sugars (RS) in aqueous phase solvents. Today, “greener biorefineries” require in situ measurement of reducing sugars in non-aqueous solvents such as natural deep eutectic solvents (NADESs). Therefore, that DNS assay cannot be performed successfully in such eutectic phase solvent becomes a puzzle that must be solved. In this work, an improved DNS method, which we call the DNS'' method, was established and implemented successfully in hydrophilic eutectic phase solvent systems (hiNADES). The tickets to the success of DNS'' method are the inserted two essential steps, water-adding disintegration and pH alkalization. Then we tested two excellent RS solvents (ChCl_Gly and ChCl_LA) from six synthetic ChCl-based hiNADESs. The optimal disintegration conditions for assaying RS in ChCl_LA/NaOH (ChCl_Gly/H2O) were volume ratio 3: 1 (3: 1), molar concentration 2.0 mol/L (0 mol/L). Thereafter, we drew the standard curves of seven RSs in ChCl_Gly and ChCl_LA, which are glucose, xylose, fructose, mannose, fucose, rhamnose, and maltose. We determined that HMF interferes with DNS assay in hiNADES. The principles and standard procedures of DNS'' method were also investigated. Finally, the enzymatic saccharification of microcrystalline cellulose (MCC) by two commercial Trichoderma reesei cellulases (Celluclast® 1.5 L and Cellic® CTec2) in neat hiNADES was tracked using the DNS'' method.

Safety evaluation of the food enzyme endo-polygalacturonase from the genetically modified Trichoderma reesei strain RF6197.

The food enzyme endo-polygalacturonase ((1-4)-α-d-galacturonan glycanohydrolase; EC 3.2.1.15) is produced with the genetically modified Trichoderma reesei strain RF6197 by AB Enzymes GmbH. 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 five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, production of wine and wine vinegar, coffee demucilation and production of plant extracts as flavouring preparations. Since residual amounts of the total organic solids (TOS) are removed during the coffee demucilation and the production of flavouring extracts, dietary exposure was calculated only for the remaining three food processes. It was estimated to be up to 0.156 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 1,000 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 6,410. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and matches were found with a number of pollen allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen 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.

Safety evaluation of the food enzyme pectinesterase from the genetically modified Trichoderma reesei strain RF6201.

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Trichoderma reesei strain RF6201 by AB Enzymes GmbH. 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 five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, production of wine and wine vinegar, coffee demucilation and production of plant extracts as flavouring preparations. Since residual amounts of the total organic solids (TOS) are removed during the coffee demucilation and the production of flavouring extracts, dietary exposure was calculated only for the remaining three food processes. It was estimated to be up to 0.532 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 1,000 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 1,880. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found with pollen allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure, particularly in individuals sensitised to pollen allergens, 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.

The Potential of microalgae for cellulose degradation and utilization for biofuel application

As society is becoming more reliant on fuels, a more sustainable form of energy must be investigated. Another presiding issue is the output of cellulose left over from other plants that are used for biofuels, such as corn. However, the microalgae, Chlamydomonas Reinhardtii, a unicellular organism, is an ideal source of energy, as there is evidence that it contains the genes that are responsible for the encoding of Cellulases, which allow for the degradation of cellulose, such as endoglucanases. Chlamydomonas Reinhardtii typically lives in both soil and water environments, a photosynthetic organism that utilizes light as an energy source. The uncommon trait for microalgae to express cellulase allows for external sources of carbon to be utilized by the microorganism, which could affect the biological output of macromolecules common in biofuels such as lipids and carbohydrates. The study aims to compare not only the cellulase expression levels of Chlamydomonas Reinhardtii, but also see how the lipid output of the microalgae compares to other microorganisms used in the biofuel industry such as Chlorella Vulgaris, another phototrophic microalgae, which is used for direct fuel. Additionally, Trichoderma Reesei will also be compared, which is another microorganism that is used for biofuel production. However, the industry utilizes Trichoderma Reesei’s ability to produce cellulase, rather than just taking directly from the microorganism. The conclusions unfortunately did not show any cellulase expression, and biofuel output favored algae

Enhancing feeding value of corn distiller’s grains with solubles via fungal co-cultured solid-state fermentation for monogastric animal nutrition

Corn distiller’s dried grains with solubles (cDDGS) as a major co-product from dry-grind corn-ethanol processes have imbalanced key amino acids (AA), high indigestible fiber and phytate, and risk of mycotoxin contamination, which limit its inclusion rate in monogastric animal diets. This study used cellulolytic fungus Trichoderma reesei (TR), two zygomycete fungi Mucor indicus (MI) and Rizopus oryzae (RO) and their co-cultures to ferment corn wet distiller’s grains with solubles (WDGS) with the supply of soybean hulls and urea via solid-state fermentation at 28 °C for up to 9 days. Compared to control (non-inoculation) and monoculture, TR and RO co-culture (TR+RO) after 6 and 9 days showed more balanced branch-chain AA (BCAA) of valine (Val), leucine (Leu), and isoleucine (Ile), higher phytate reduction (56–61%), lower concentration of deoxynivalenol (DON), zearalenone (ZEN) and total aflatoxins (AFT). However, the treatments with MI (MI, RO+MI, TR+MI) showed increase of DON while reduction of ZEN compared to treatment without MI. Due to fast growth of RO and MI, the co-culture of TR+MI and TR+RO after 3 days of fermentation improved total AA by 32 % and 28 %, respectively, higher than monoculture of TR (18 % after 3 days). Compared with co-culture, monoculture of TR produced feed with higher in vitro dry matter digestibility (IVDMD) with 22–43 % improvement from control, and higher in vitro digestibility of total AA (IVDAA) with 35–50 % improvement from control, the similar level was also achieved by co-culture of TR+RO after 6 days of fermentation. Therefore, co-culture of TR+RO and monoculture of TR in fungal bioprocessing could provide a feasible solution to improve the overall feeding value of the cDDGS.

Chiral recognition mechanism of cellobiohydrolase Cel7A for ligands based on the β‐blocker propranolol: The effect of explicit water molecules on binding and selectivities

AbstractProteins are useful chiral selectors. In order to understand the recognition mechanism and chiral discrimination, the binding of the (R)‐ and (S)‐enantiomers of a series of designed amino alcohol inhibitors based on propranolol to cellobiohydrolase Cel7A (Trichoderma reesei) has been studied more closely. X‐ray crystal structures were determined of the protein complex with the (R)‐ and (S)‐enantiomers of the strongest binding propranolol analogue. The combination of the structural data, thermodynamic data from capillary electrophoresis and microcalorimetry experiments and computational modelling give a clearer insight into the origin of the enantioselectivity and its opposite thermodynamic signature. The new crystal structures were used in computational molecular flexible dockings of the propranolol analogues using the program Glide. The results indicated that several water molecules in the active site were essential for the docking of the (R)‐enantiomers but not for the (S)‐enantiomers. The results are discussed in relation to the enantiomeric discrimination of the enzyme. Both dissociation constants (Kd values) and thermodynamical data are included to show the effects of the structural modifications in the ligand on enthalpy and entropy in relation to the enantioselectivity.

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source

BackgroundThe filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei.ResultsWe chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain.ConclusionsIn general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.

Enhanced biovalorization of palm biomass wastes as biodiesel feedstocks through integrated solid-state and submerged fermentations by fungal co-cultures

Palm empty fruit bunches (EFB) were valorized into fungal lipids by oleaginous fungus Aspergillus tubingensis TSIP9 under solid-state fermentation (SSF) and submerged fermentation (SmF). An integrated SSF-SmF process increased lipid production from 116.2 ± 0.1 mg/g-EFB under SSF and 60.1 ± 0.2 under SmF up to 124.9 ± 0.5 mg/g-EFB, possibly due to the combined benefits of dispersed mycelia forming during SSF and better mass transfer during SmF. As A. tubingensis lacks sufficient β-glucosidase, it was co-cultured with high β-glucosidase-producing Trichoderma reesei QM 9414. The co-cultures improved overall lipid yields likely due to synergistic interaction of the two fungi. After inoculum size was optimized and the co-cultures were performed in bioreactors, the lipid yield was increased up to 205.1 ± 1.1 mg/g-EFB. The fatty acid composition of fungal lipids indicated their potential use as biodiesel feedstocks. The fungal fermentation of EFB also provided cellulose pulp residues. These strategies could be practical options for low-cost biovalorization of biomass wastes.

Detoxification strategy of wheat straw hemicellulosic hydrolysate for cultivating Trichoderma reesei: a contribution towards the wheat straw biorefinery

The urgency for reducing the dependence on fossil-based materials is increasing the interest in the utilization of renewable feedstocks. Lignocellulosic residual biomass can be used as feedstock to produce chemicals and energy without generating food security problems. Wheat straw (WS) has a clear potential for developing sustainable processes in a circular bioeconomy context. However, the development of processes requires a strategy for utilizing the hemicellulosic, cellulosic, and lignin fractions. This work covers the utilization of the hemicellulosic fraction as the first stage of a wheat straw biorefinery. The aim was to evaluate the hydrolysis of WS by using liquid hot water (LHW) treatment, the detoxification of the produced wheat straw hydrolysate (WSH), and the cultivation of Trichoderma reesei using it as the only carbon source as proof of detoxification. LHW treatment was performed at 160 °C and 90 min and yielded a WSH rich in monomeric and oligomeric saccharides (~ 14 g/L) and containing degradation products in low concentration (furfural, HMF, and acetic acid). As part of the development of the extraction and detoxification strategy, we determined the specific inhibition thresholds for T. reesei for the mentioned degradation products. Detoxification was carried out by evaporation by modifying the % of volume evaporated and the pH of the solution. Approximately 55.9% of acetic acid and 100% of furfural were removed from the WSH. The fungal biomass obtained in the medium containing WSH was equivalent to 98% of the biomass obtained in the control medium.

Use of a Cellulase from Trichoderma reesei as an Adjuvant for Enterococcus faecalis Biofilm Disruption in Combination with Antibiotics as an Alternative Treatment in Secondary Endodontic Infection.

Apical periodontitis is an inflammation leading to the injury and destruction of periradicular tissues. It is a sequence of events that starts from root canal infection, endodontic treatment, caries, or other dental interventions. Enterococcus faecalis is a ubiquitous oral pathogen that is challenging to eradicate because of biofilm formation during tooth infection. This study evaluated a hydrolase (CEL) from the fungus Trichoderma reesei combined with amoxicillin/clavulanic acid as a treatment against a clinical E. faecalis strain. Electron microscopy was used to visualize the structure modification of the extracellular polymeric substances. Biofilms were developed on human dental apices using standardized bioreactors to evaluate the antibiofilm activity of the treatment. Calcein and ethidium homodimer assays were used to evaluate the cytotoxic activity in human fibroblasts. In contrast, the human-derived monocytic cell line (THP-1) was used to evaluate the immunological response of CEL. In addition, the secretion of the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine IL-10 were measured by ELISA. The results demonstrated that CEL did not induce the secretion of IL-6 and TNF-α when compared with lipopolysaccharide used as a positive control. Furthermore, the treatment combining CEL with amoxicillin/clavulanic acid showed excellent antibiofilm activity, with a 91.4% reduction in CFU on apical biofilms and a 97.6% reduction in the microcolonies. The results of this study could be used to develop a treatment to help eradicate persistent E. faecalis in apical periodontitis.

Improving cold-adaptability of mesophilic cellulase complex with a novel mushroom cellobiohydrolase for efficient low-temperature ensiling

Low ambient temperature poses a challenge for rice straw-silage processing in cold climate regions, as cold limits enzyme and microbial activity in silages. Here, a novel cold-active cellobiohydrolase (VvCBHI-I) was isolated from Volvariella volvacea, which exhibited outstanding cellobiohydrolase activity at 10–30 °C. The crude cellulase complex in the VvCBHI-I-expressing transformant T1 retained 50% relative activity at 10 °C, while the wildtype Trichoderma reesei showed <5% of the activity. VvCBHI-I greatly improved the saccharification efficiency of the cellulase complex with pretreated rice straw as substrate at 10 °C. In rice straw silage, pH (<4.5) and lactic acid content (>4.6%) remained stable after 15-day ensiling with the cellulase complex from T1 and Lactobacillus plantarum. Moreover, the proportions of cellulose and hemicellulose decreased to 29.84% ± 0.15% and 21.25% ± 0.26% of the dried material. This demonstrates the crucial potential of mushroom-derived cold-active cellobiohydrolases in successful ensiling in cold regions.

Alleviating vacuolar transport improves cellulase production in Trichoderma reesei

Increasing cellulase production in cellulolytic fungus Trichoderma reesei is of interest for biofuels and biorefineries. Previous studies indicated that secreted protein was occasionally accumulated in vacuoles; this phenomenon has also been reported in T. reesei. Therefore, alleviating vacuolar transport seems to be a promising strategy for improving cellulase production in T. reesei. Herein, we found that knockout of vps10, vps13, and vps21, among 11 vacuolar protein sorting factors, improved cellulase production in T. reesei. The filter paper activity in Δvps10, Δvps13, and Δvps21 increased by 1.28-, 2.45-, and 2.11-fold than that of the parent strain. Moreover, the β-glucosidase activity in Δvps13 and Δvps21 increased by 3.22- and 3.56-fold after 6days of fermentation. Furthermore, we also found that the vacuolar trafficking towards vacuoles was partially impaired in three knockout mutants, and disruption of vps13 alleviated the autophagy process. These results indicated that alleviated transport and degradation towards vacuole in Δvps10, Δvps13, and Δvps21 might improve cellulase production. Of note, the expression of cellulase genes in Δvps13 and Δvps21 was dramatically increased in the late induction phase compared to the parent. These results suggested that Vps13 and Vps21 might influence the cellulase production at transcription level. And further transcriptome analysis indicated that increased cellulase gene expression might be attributed to the differential expression of sugar transporters. Our study unravels the effect of alleviating vacuolar transport through knockout vps10, vps13, and vps21 for efficient cellulase secretion, providing new clues for higher cellulase production in T. reesei. KEY POINTS: • Disruption of vps10, vps13 or vps21 improves cellulase production • Vacuolar transport is impaired in three vps KO mutants • Deletion of vps13 or vps21 increases the transcript of cellulase genes in late stage.

BsEXLX of engineered Trichoderma reesei strain as dual-active expansin to boost cellulases secretion for synergistic enhancement of biomass enzymatic saccharification in corn and Miscanthus straws

In this study, bacterial BsEXLE1 gene was overexpressed into T. reesei (Rut-C30) to generate a desirable engineered TrEXLX10 strain. While incubated with alkali-pretreated Miscanthus straw as carbon source, the TrEXLX10 secreted the β-glucosidases, cellobiohydrolases and xylanses with activities raised by 34%, 82% and 159% compared to the Rut-C30. Supplying EXLX10-secreted crude enzymes and commercial mixed-cellulases for two-step lignocellulose hydrolyses of corn and Miscanthus straws after mild alkali pretreatments, this work measured consistently higher hexoses yields released by the EXLX10-secreted enzymes for synergistic enhancements of biomass saccharification in all parallel experiments examined. Meanwhile, this study detected that the expansin, purified from EXLX10-secreted solution, was of exceptionally high binding activities with wall polymers, and further determined its independent enhancement for cellulose hydrolysis. Therefore, this study raised a mechanism model to highlight EXLX/expansin dual-activation roles for both secretion of stable biomass-degradation enzymes at high activity and biomass enzymatic saccharification in bioenergy crops.