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Broussonetia papyrifera, known as paper mulberry, is a potential feed stock for bioethanol production because of its cellulose-rich composition. Lignin in dilute acid pretreated Broussonetia papyrifera was fractionated to three different fractions, and their physiochemical properties were determined by FT-IR, GPC and NMR analyses. Different structural characteristics were observed from each lignin fraction. Cellobiohydrolases I (CBH) adsorption to each lignin was understood by the lignin properties. The results showed that aliphatic hydroxyl groups in lignin showed positive correlations with the maximum binding ability of CBH onto lignin samples. Also, the contents of phenolic compounds such as p-hydroxyphenyl benzoate (PB), syringyl (S) and guaiacyl (G) units in the lignin influenced their CBH binding., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Transcription of the cellulosomal cellulase/hemicellulase genes of Clostridium cellulovorans has been investigated by Northern blot, reverse transcriptase PCR (RT-PCR), primer extension, and S1 nuclease analysis. Northern hybridizations revealed that the cellulosomal cbpA gene cluster is transcribed as polycistronic mRNAs of 8 and 12 kb. The 8-kb mRNA coded for cbpA and exgS, and the 12-kb mRNA coded for cbpA, exgS, engH, and engK. The sizes of the mRNAs were about 3 kb for engE, 1.8 kb for manA, 2.7 kb for xynA, and 4 kb for pelA, indicating monocistronic transcription of these genes. Primer extension and S1 nuclease analysis of C. cellulovorans RNA showed that the transcriptional start sites of cbpA, engE, manA, and hbpA were located 233, 97, 64, and 61 bp upstream from the first nucleotide of each of the respective translation initiation codons. Alignment of the cbpA, engE, manA, and hbpA promoter regions provided evidence for highly conserved sequences that exhibited strong similarity to the sigma(A) consensus promoter sequences of gram-positive bacteria.

Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.

The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS. The C. cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.

A new cellulase gene, cel2, from the filamentous fungus Cochliobolus carbonum was cloned by using egl-1 of Trichoderma reesei as a heterologous probe. DNA blot analysis of cel2 showed that this gene is present as a single copy. The gene contains one 49-bp- intron. cel2 encodes a predicted protein (Cel2p) of 423 amino acids with a molecular mass of 45.8 kDa. The predicted pI is 4.96. It shows similarity to other endoglucanases from various fungi. From the comparison with other cellulase genes, cel2 belongs to family 7 of glucohydrolases. cel2 is located on a 2.5-Mb chromosome in C. carbonum and its expression is repressed by sucrose. A cel2 mutant of C. carbonum was created by transformation-mediated gene disruption. The pathogenicity of the mutant was indistinguishable from the wild type, indicating that cel2 by itself is not important for pathogenicity.

A large gene cluster for the Clostridium cellulovorans cellulosome has been cloned and sequenced upstream and downstream of the cbpA and exgS genes (C.-C. Liu and R. H. Doi, Gene 211:39-47, 1998). Gene walking revealed that the engL gene cluster (Y. Tamaru and R. H. Doi, J. Bacteriol. 182:244-247, 2000) was located downstream of the cbpA-exgS genes. Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain. The sequence of the clustered genes is cbpA-exgS-engH-engK-hbpA-engL-man A-engM-engN and is about 22 kb in length. The engN gene did not have a complete catalytic domain, indicating that engN is a truncated gene. This large gene cluster is flanked at the 5' end by a putative noncellulosomal operon consisting of nifV-orf1-sigX-regA and at the 3' end by noncellulosomal genes with homology to transposase (trp) and malate permease (mle). Since gene clusters for the cellulosome are also found in C. cellulolyticum and C. josui, they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may arise from a common ancestor with some evolutionary modifications.

The nucleotide sequence of P70, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been determined. The gene designated as exgS (Genbank Accession No. U34793) consists of 2112 bp and encodes a protein containing 703 amino acids with a molecular mass of 77.7 kDa. ExgS has a putative signal peptide sequence of 32 amino acids. The N-terminal region is separated from the C-terminal region by a short-Pro-Thr-Pro linker. The C-terminal region of ExgS contains a duplicated sequence (DS), each sequence consisting of 22 amino acids. exgS, located 67 bp downstream of cbpA in the chromosome, is immediately upstream of a gene encoding a family 9 type endoglucanase that we have designated as EngH. This gene cluster to date consists of regA-cbpA-exgS-engH. Recombinant ExgS (rExgS) containing no signal peptide was expressed in E. coli. The rExgS actively digested several forms of cellulose, including Avicel, Sigmacell101, crystalline cellulose, and xylan, but not carboxymethyl cellulose (CMC). Cellotetraose was the smallest oligosaccharide substrate for rExgS. The enzymatic studies indicated that ExgS was an exoglucanase and had some properties similar to that of CelS from C. thermocellum and CelF from C.cellulolyticum. An exoglucanase has now been found to be a component of the C. cellulovorans cellulosome as well as the previously reported endoglucanases.

Lignin can affect the enzymatic hydrolysis efficiency of lignocellulose. In this study, the lignin isolated from sugarcane bagasse (SCB) pretreated with p-toluenesulfonic acid (PL) was firstly aminated, and then the effects of PL and aminated PL (APL) on the bagasse enzymatic hydrolysis efficiency (EHE) were investigated. The results showed that the addition of PL and APL promoted the EHE, and EHE with APL (73.82 %) was higher than PL (51.39 %). To explore the reason, the data were further analyzed including cellulase adsorption capacity, enzyme activity, cellulase-lignin interaction, and molecular docking. It was found that APL adsorbed more cellulase (27.83 mg protein/g lignin) than PL (4.96 mg protein/g lignin), resulting from the greater interaction force and lower binding free energy between APL and cellulase. The addition of APL more remarkably enhanced the cellobiohydrolase and endoglucanase activities than PL due to more effectively inducing cellulase conformation optimization. [Display omitted] • Enzymatic hydrolysis of cellulosic substrate was enhanced from 45.83 % to 73.82 % by aminated lignin. • Aminated lignin adsorbed more cellulase molecules. • Activities of cellobiohydrolase (CBH) and endoglucosidase (EG) were promoted by aminated lignin. • Aminated lignin owned lower binding energy with cellulase. [ABSTRACT FROM AUTHOR]

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, β-1,4-xylanase, and β-glucosidase production. Cellulase and β-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. β-Glucosidase production in Δ creA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-β-1,4-glucanase, β-1,4-xylanase, and β-glucosidase 1 (bgl1). Interestingly, bgl1 expression in Δ creA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and β-glucosidase production in A. aculeatus. [ABSTRACT FROM AUTHOR]

Methods of the pretreatment of reeds (grinding, hydrothermal treatment, treatment with acid or alkali solutions, organosolv, deep eutectic solvents) and their effect on the subsequent enzymatic hydrolysis by cellulase and hemicellulase complexes have been studied. Substrates with the highest reactivity were obtained by exposing the reeds to a weakly alkaline deep eutectic solvent (DES) and an alkali solution. The depth of enzymatic hydrolysis of these pretreated substrates was 63 and 59%, and the degree of conversion of polysaccharides (cellulose and hemicellulose) into monosaccharides taking into account the yield of the substrate at the preprocessing stage was 60 and 34%, respectively. After pretreatment of the reeds with acid DES, water steam, water, or a water/organic solution of sulfuric acid, the depth of enzymatic hydrolysis was 45, 25, 20, and 11% and the degree of polysaccharide conversion was 26, 18, 13, and 10%, respectively. The industrial enzyme preparation Agrocell Plus with a predominant content of cellobiohydrolases and endoglucanases was most effective in hydrolyzing the dust fraction of the reed, as was the reed pretreated with a solution of sulfuric acid or DES (acidic or alkaline). The industrial enzyme preparation Agroxil Plus, containing endoxylanase and cellobiohydrolases, was more effective in hydrolyzing the reed after hydrothermal pretreatment or alkali solution. The results of the hydrolysis of the reed pretreated with acidic or weakly alkaline DES under the action of individual (homogeneous) cellulases indicate that in both cases the key enzyme was cellobiohydrolase 1. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the cellulase production of four fungi: Aspergillus terreus NIH2624, Aspergillus clavatus NRRL1, Aspergillus versicolor CBS583.65 and Aspergillus phoenicis ATCC3157, under submerged cultivation conditions. When these fungi were cultured in shake flasks using Mandels and Weber's minimal medium with 1% sugarcane bagasse as a carbon source and 1.8 g/L of rice bran extract as a nitrogen source, A. terreus showed maximum cellulase production (filter paper activity (FPase) 3.35 U/mL; carboxymethyl cellulase activity (CMCase) 1.69 U/mL). Consequently, A. terreus was selected for the optimization study for cellulase production. Among the different tested carbon sources, A. terreus showed higher CMCase activity when it was cultivated on delignified sugarcane bagasse (1.64 U/mL) and higher FPase activity on sugarcane straw (7.95 U/mL). Regarding the nitrogen sources, the maximum FPase activity was observed when using rice bran (FPase, 8.90 U/mL) and soybean meal (FPase, 9.63 U/mL). The optimized fermentation medium (minimal medium with delignified sugarcane bagasse and rice bran as carbon and nitrogen sources, respectively) resulted in an enzymatic cocktail mainly composed of xylanases, with a maximum activity of 1701.85 U/mL for beechwood xylan, 77.12 U/mL for endoglucanase and 21.02 U/mL for cellobiohydrolase. Additionally, the enzymatic cocktail showed efficient activities for β-glucosidase, β-xylanase, arabinofuranosidase and lytic polysaccharide monoxygenases (LPMOs). This cellulase enzyme solution has the potential to efficiently hydrolyze lignocellulosic biomass, producing second-generation sugars in biorefineries. [ABSTRACT FROM AUTHOR]

The escalating prevalence of drug-resistant pathogens not only jeopardizes the effectiveness of existing treatments but also increases the complexity and severity of infectious diseases. Escherichia coli is one the most common pathogens across all healthcare-associated infections. Enzymatic treatment of bacterial biofilms, targeting extracellular polymeric substances (EPS), can be used for EPS degradation and consequent increase in susceptibility of pathogenic bacteria to antibiotics. Here, we characterized three recombinant cellulases from Thermothelomyces thermophilus: a cellobiohydrolase I (TthCel7A), an endoglucanase (TthCel7B), and a cellobiohydrolase II (TthCel6A) as tools for hydrolysis of E. coli and Gluconacetobacter hansenii biofilms. Using a design mixture approach, we optimized the composition of cellulases, enhancing their synergistic activity to degrade the biofilms and significantly reducing the enzymatic dosage. In line with the crystalline and ordered structure of bacterial cellulose, the mixture of exo-glucanases (0.5 TthCel7A:0.5 TthCel6A) is effective in the hydrolysis of G. hansenii biofilm. Meanwhile, a mixture of exo- and endo-glucanases is required for the eradication of E. coli 042 and clinical E. coli biofilms with significantly different proportions of the enzymes (0.56 TthCel7B:0.44 TthCel6A and 0.6 TthCel7A:0.4 TthCel7B, respectively). X-ray diffraction pattern and crystallinity index of E. coli cellulose are comparable to those of carboxymethyl cellulose (CMC) substrate. Our results illustrate the complexity of E. coli biofilms and show that successful hydrolysis is achieved by a specific combination of cellulases, with consistent recurrence of TthCel7B endoglucanase. [ABSTRACT FROM AUTHOR]

Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus , and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5–6.0 (CIB12) and 4.5–5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma. [ABSTRACT FROM AUTHOR]

Previously, we constructed engineered M. circinelloides strains that can not only utilize cellulose, but also increase the yield of γ-linolenic acid (GLA). In the present study, an in-depth analysis of lipid accumulation by engineered M. circinelloides strains using corn straw was to be explored. When a two-stage temperature control strategy was adopted with adding 1.5% cellulase and 15% inoculum, the engineered strains led to increases in the lipid yield (up to 1.56 g per 100 g dry medium) and GLA yield (up to 274 mg per 100 g dry medium) of 1.8- and 2.3-fold, respectively, compared with the control strain. This study proved the engineered M. circinelloides strains, especially for Mc-C2PD6, possess advantages in using corn straw to produce GLA. This work provided a reference for transformation from agricultural cellulosic waste to functional lipid in one step, which might play a positive role in promoting the sustainable development of biological industry. [ABSTRACT FROM AUTHOR]

Lignocellulose was directly used in itaconic acid production by a model filamentous fungus Neurospora crassa. The promoters of two clock control genes and cellobiohydrolase 1 gene were selected for heterologous genes expression by evaluating different types of promoters. The effect of overexpression of different cellulase was compared, and it was found that expression of cellobiohydrolase 2 from Trichoderma reesei increased the filter paper activity by 2 times, the cellobiohydrolase activity by 4.5 times, and that the itaconic acid titer was also significantly improved. A bidirectional cis-aconitic acid accumulation strategy was established by constructing the reverse glyoxylate shunt and expressing the transporter MTTA, which increased itaconic acid production to 637.2 mg/L. The simultaneous optimization of cellulase and metabolic pathway was more conducive to the improvement of cellulase activity than that of cellulase alone, so as to further increase itaconic acid production. Finally, through the combination of fermentation by optimized strains and medium optimization, the titers of itaconic acid using Avicel and corn stover as substrate were 1165.1 mg/L and 871.3 mg/L, respectively. The results prove the potential of the consolidated bioprocessing that directly converts lignocellulose to itaconic acid by a model cellulase synthesizing strain. [ABSTRACT FROM AUTHOR]

Background: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. Results: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in β -glucosidase activity compared to batch processes using the same type and amount of carbon sources. Conclusion: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology. [ABSTRACT FROM AUTHOR]

Forest productivity usually declines with the increase in the age of stand, which may be associated with the disturbance in the balance of nitrogen (N) and phosphorus (P) nutrients. However, the effects of stand age on plantation nutrient balance and productivity in plantation is still unclear. Therefore, an experiment was conducted in this study to investigate the influence of the increase in the age of stand (6‐, 10‐, 15‐, 25‐, 30‐, and 34‐year‐old stands) on plant growth and the dynamics of N and P in Castanopsis hystrix plantations of Southern China. The increase in the age of stand reduced plantation productivity and leaf N:P. Moreover, productivity was found to be positively correlated with leaf N:P, reflecting that the decrease in productivity was related to changes in N and P contents in leaves. Reduction in leaf N:P was associated with declining N content and stable P content in leaves. The decrease in leaf N content may be due to the declining NO3−–N and microbial biomass N contents in soil, although activities of N‐acetylglucosaminidase, cellobiohydrolase, and polyphenol oxidase decreased with the increase in stand age. The stability of leaf P content may be associated with the increase in soil total P and microbial biomass P, boosting P supply to plants. In contrast, decreased soil phosphatase activity with the increase in the age of stand likely suggests that no additional P was needed through P mineralization. The findings indicate that increasing stand age would improve N consumption but alleviate relative P limitations. Thus, more attention needs to be paid to N fertilizer management (e.g., 10–15‐year stages) to sustain C. hystrix productivity with stand age. [ABSTRACT FROM AUTHOR]

Biochar (BC) was used in Trichoderma sp. culture to produce high-activity cellulase on a laboratory scale. The biochar was added into the flask before being applied to the fermenter to identify the enhancement effect and to determine the best amount of addition and the most suitable incubation period. Cellulase production was performed with a working volume of 4 L, and enzymatic hydrolysis was conducted to evaluate the saccharification ability of the enzyme. During incubation, the activities of three enzymes (Endoglucanase (EG), β-glucosidase (BGL), and cellobiohydrolase (CBH)) were measured for three days, and the cellulase activity was determined using a filter paper unit (FPU). In flask scale, EG, BGL, and CBH activities were increased by 1.4, 2.1, and 1.8 folds, respectively, and the incubation period was shortened by adding BC. In the fermenter scale, EG, BGL, and CBH activities were noticeably enhanced by 12.1, 5.8, and 7.2 folds, respectively, and FPU was 42.1 (9.8 folds). Additionally, the conversion rates of cellulose and steam exploded softwood and hardwood were 109.4%, 75.4%, and 87.3%, which were similar to a commercial enzyme (Cellic CTec II). This study demonstrated that biochar could be used to produce high-activity cellulase in a shorter period and suggests a novel method for effective cellulase production. [ABSTRACT FROM AUTHOR]

The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes (CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. sclerotiorum degrades cellulose remain elusive. Here, we unveil a novel secretory cellobiohydrolase, SsdchA, characterized by a signal peptide and a Glyco_hydro_7 (GH7) domain. SsdchA exhibits a robust expression of during early infection stages. Interestingly, colony morphology and growth rates remain unaffected across the wild-type, SsdchA deletion strains and SsdchA overexpression strains on potato dextrose agar (PDA) medium. Nevertheless, the pathogenicity and cellobiohydrolase activity decreased in the SsdchA deletion strains, but enhanced in the SsdchA overexpression strains. Moreover, the heterologous expression of SsdchA in Arabidopsis thaliana leads to reduced cellulose content and heightened susceptibility to S. sclerotiorum. Collectively, our data underscore the pivotal role of the novel cellobiohydrolase SsdchA in the pathogenicity of S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Cellobiohydrolase (CBH), belonging to glycoside hydrolase family 6 (GH6), plays an essential role in cellulose saccharification, but its low thermotolerance presents a challenge in improving the reaction efficiency. Based on a report that chimeric CBH II (GH6) engineered to remove non-disulfide-bonded free Cys shows increased thermotolerance, we previously mutated the two free Cys residues to Ser in GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCelöA) and obtained a thermotolerant double mutant, C240S/C393S (Yamaguchi et al., J. Appl. Glycosci. 2020; 67: 79~86). Here, characterization of the double mutant revealed that its activity towards both amorphous and crystalline cellulose was higher than that of the wild-type enzyme at elevated temperature, suggesting that the catalytic domain is the major contributor to the increased thermotolerance. To investigate the role of each free Cys residue, we prepared both single mutants, C240S and C393S, of the catalytic domain of PcCelôA and examined their residual activity at high temperature and the temperature-dependent changes of folding by means of circular dichroism measurements and thermal shift assay. The results indicate that the C393S mutation is the main contributor to both the increased thermotolerance of C240S/C393S and the increased activity of the catalytic domain at high temperature. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 6 cellobiohydrolase (GH6 CBH) is a group of cellulases capable of hydrolyzing crystalline cellulose. However, the synergistic reaction of GH6 CBH with other cellulases is hindered by its relatively low thermotolerance. We previously obtained a thermotolerant double mutant, C240S/C393S, of GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCelôA) by replacing the two free cysteine (Cys) residues, C240 and C393, with serine (Yamaguchi et al., J Appl Glycosci. 2020; 67;79-86). In the accompanying paper (Part I; Yamaguchi et al., J Appl Glycosci. 2024; 71: 55-62), we measured the temperature dependence of the activity and folding of C240S/C393S and its single mutants, C240S and C393S, and found that replacement of C393 was the major contributor to the increased thermotolerance of C240S/C393S. Here, in order to investigate the mechanism involved, we crystallized the wild-type and the mutant enzymes and compared their X-ray crystal structures. The overall structures of the wild-type and the three mutant enzymes were similar. However, C240S/C393S had the lowest relative B-factor at both the N-terminal loop (residues 172-177) and the C-terminal loop (residues 390-425). This result suggests that reduced structural fluctuation of the substrate-enclosing loops, possibly due to stronger hydrogen bonding involving C393, could account for the increased thermotolerance of C240S/C393S. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 7 (GH7) cellulases are key enzymes responsible for carbon cycling on earth through their role in cellulose degradation and constitute highly important industrial enzymes as well. Although these enzymes are found in a wide variety of evolutionarily distant organisms across eukaryotes, they exhibit remarkably conserved features within two groups: exo‐acting cellobiohydrolases and endoglucanases. However, recently reports have emerged of a separate clade of GH7 endoglucanases from protist symbionts of termites that are 60–80 amino acids shorter. In this work, we describe the first crystal structure of a short GH7 endoglucanase, RsSymEG1, from a symbiont of the lower termite Reticulitermes speratus. A more open flat surface and shorter loops around the non‐reducing end of the cellulose‐binding cleft indicate enhanced access to cellulose chains on the surface of cellulose microfibrils. Additionally, when comparing activities on polysaccharides to a typical fungal GH7 endoglucanase (Trichoderma longibrachiatum Cel7B), RsSymEG1 showed significantly faster initial hydrolytic activity. We also examine the prevalence and diversity of GH7 enzymes that the symbionts provide to the termite host, compare overall structures and substrate binding between cellobiohydrolase and long and short endoglucanase, and highlight the presence of similar short GH7s in other organisms. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass holds promise as a renewable feedstock for various applications, but its efficient conversion requires cost-effective degradation strategies. The main objective of this study was to investigate the effect of the growth conditions of Cellulomonas phragmiteti in the production of (hemi)cellulosic supernatants. To meet this aim, different lignocellulosic residues were used as carbon sources for growth using defined mineral or nutritive culture media. Cell-free culture supernatants with xylanolytic activity were produced in all the conditions evaluated, but the highest xylanase activity (15.3 U/mL) was achieved in Luria–Bertani (LB) medium containing 1% waste paper. Under these conditions, almost negligible β-glucosidase, cellobiohydrolase, β-xylosidase, and α-arabinofuranosidase activity was detected. The xylanolytic supernatant showed tolerance to salt and displayed maximal catalytic efficiency at pH 6 and 45 °C, along with good activity in the ranges of 45–55 °C and pH 5–8. As it showed good stability at 45 °C, the supernatant was employed for the hydrolysis of birchwood xylan (50 g/L) under optimal conditions, releasing 10.7 g/L xylose in 72 h. Thus, C. phragmiteti was found to produce a xylanolytic enzymatic supernatant efficiently by utilizing the cheap and abundant lignocellulosic residue of waste paper, and the produced supernatant has promising attributes for industrial applications. [ABSTRACT FROM AUTHOR]

Cellulose, a sustainable source of fermentable glucose and nanomaterials, exists as highly-ordered, solvent-inaccessible fibrils held by intra- and intermolecular hydrogen bonds. Cellulose hydrolysis rates and extents are limited by the availability and accessibility of productive cellulase binding sites at the water–cellulose interface. There is a need to understand how spatial heterogeneity of celluloses impacts hydrolysis kinetics. We report a real-time, in situ infrared spectromicroscopy study of enzymatic cellulose hydrolysis in buffer with micrometer-scale spatial mapping. Algal cellulose depletion by a purified cellobiohydrolase Cel7A was tracked as time-resolved decreases in the absorption peak intensities of the glycosidic bond (1161 cm−1), C2–O2 (1112 cm−1), C3–O3 (1059 cm−1), and C6–O6 rotamers (1034 cm−1, 1013 cm−1, and 997 cm−1). Depletion kinetics varied spatially, with peak intensities decreasing to zero in some areas and plateauing in others. Hydration impacted cellulose ordering where C6–O6 and C3–O3 peaks were sharp, narrow, and centered at higher frequencies when hydrated, but broader, less well-defined and centered at lower frequencies when dried. Temporal trends of the Lateral Order Index (LOI), Total Crystallinity Index (TCI), and Hydrogen Bonding Index (HBI) indicated that hydrolysis by Cel7A preferentially removed less extensively hydrogen bonded cellulose, without significantly affecting the overall crystallinity of highly ordered cellulose. [ABSTRACT FROM AUTHOR]

The Penicillium verruculosum filamentous fungus is a highly active producer of cellulolytic complex enzymes, cellobiohydrolases, endoglucanases and β-glucosidases. Using the CRISPR/Cas9 genome editing system, previously adapted to P. verruculosum, a strain with a knockout of the xlnR gene encoding XlnR, one of the main transcription factors of filamentous fungi, has been obtained. The transcription level of cellulolytic genes was determined by quantitative PCR for the P. verruculosum B1-221-151 strain and the new P. verruculosum ΔxlnR strain. The XlnR protein was shown to activate transcription of the cbh1, egl2, and bgl1 genes encoding cellobiohydrolase 1, endoglucanase 2, and β-glucosidase, respectively, in the presence of xylose and xylooligosaccharides in the growth medium. It was found that other factors are also involved in the activation of transcription of these genes by cellobiose, cellotriose, sophorose, and gentiobiose, which has a complex effect on the biosynthesis of the cellulolytic complex of enzymes produced by the P. verruculosum fungus. [ABSTRACT FROM AUTHOR]

The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (P cbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (T cbh1) to construct the new expression cassette lacA:: Δ cbh1 , which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi. One-Sentence Summary The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei. [ABSTRACT FROM AUTHOR]

Trichoderma reesei was induced to produce cellulase by a combination of glucose and β-disaccharide; however, lower levels of auxiliary proteins for degrading lignocellulosic biomass were detected by iTRAQ analysis compared with cellulose as an inducer, especially cellulose induced protein 1 (CIP1). In this study, A pdc1 promoter-driven overexpression of the endogenous Trcip1 gene was observed in T. reesei Rut C30, and the Trcip1 transcription levels of the two transformants, T. reesei OE-cip1-1 and OE-cip1-2, demonstrated 31.2- and 164.6-fold increases, respectively, but there was no significant change in cellobiohydrolase, endoglucanase and filter paper activity at 48 h. The crude enzyme was then used to hydrolyze corn stover. For T. reesei OE-cip1-1 and OE-cip1-2, the hydrolysis efficiency increased by 25.0 and 28.6% with a solid loading of 5% at 2 h, respectively. Simultaneously, 85.5 and 85.2 g/L glucose were released using a cellulase cocktail at high solid loading (20%), and these glucose release rates were significantly greater than that of T. reesei Rut C30 cellulase (77.4 g/L) at 120 h. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction (XRD) showed that the enhanced hydrolysis efficiency was primarily triggered by the decrease in the crystallinity of lignocellulose, and the fiber structure had varying degrees of loosening and disintegration. [ABSTRACT FROM AUTHOR]

Knockout of the transcriptional repressor Trctf1 is known to enhance the yield of cellulose-induced cellulase synthesis in Trichoderma reesei. However, different inducers possess distinct induction mechanisms, and the effect of Trctf1 on cellulase synthesis with soluble inducers remains unknown. To evaluate the effect of the Trctf1 gene on cellulase synthesis and develop a high-yielding cellulase strain, we established a CRISPR–Cas9 genome editing system in T. reesei Rut C30 using codon-optimized Cas9 protein and in vitro transcribed RNA. This study demonstrated that T. reesei ΔTrctf1 with the Trctf1 gene knocked out showed no statistically significant differences in cellulase, cellobiohydrolase, endoglucanase, and β−glucosidase production when induced with MGD (the mixture of glucose and sophorose). However, when induced with lactose, the activities of these enzymes increased by 20.2%, 12.4%, and 12.9%, respectively, with no statistically significant differences in β−glucosidase activity. The hydrolysis efficiency on corn stover of cellulases produced by T. reesei ΔTrctf1 under different inducers was not significantly different from that of wild-type cellulases, indicating that Trctf1 gene deletion has little effect on the cellulase cocktail. These findings contribute to a better understanding of the molecular mechanisms underlying the regulation of T. reesei cellulase synthesis by different soluble inducers, as well as the construction of high-yield cellulase gene−engineered strains. [ABSTRACT FROM AUTHOR]

Lignin structure is known to have significant effects on the enzymatic hydrolysis efficiency (EHE) of lignocellulose. In this study, the lignin produced from corn stalk pretreated with p-toluenesulfonic acid (PCS) was used to prepare carboxylated and quaternized lignin (CQCL) and epoxidized lignin (ECL), and the effect of the two modified lignin forms on the EHE of PCS was investigated. The results showed that EHE after adding CQCL (83.7%) was higher than adding ECL (60.8%). To explore the reasons, the unproductive adsorption, cellulase-lignin interaction, and molecular dynamics experiments were conducted. The results showed that the smaller hydrophobic interaction and electrostatic attraction between CQCL and cellulase caused less unproductive adsorption between CQCL and cellulase than ECL. Additionally, CQCL and ECL changed differently the conformations of key amino acid residues of cellobiohydrolase I and endoglucanase II, which was also responsible for the higher EHE after adding CQCL. [ABSTRACT FROM AUTHOR]

Proteins 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 structureswere 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. [ABSTRACT FROM AUTHOR]

Background: The 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. Results: We 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. Conclusions: In 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. [ABSTRACT FROM AUTHOR]

Chitosan is a non-cytotoxic polysaccharide that, upon hydrolysis, releases oligomers of different sizes that may have antioxidant, antimicrobial activity and the inhibition of cancer cell growth, among other applications. It is, therefore, a hydrolysis process with great biotechnological relevance. Thus, this study aims to use a crude enzyme concentrate (CEC) produced by a filamentous fungus to obtain oligomers with different molecular weights. The microorganism was cultivated in a liquid medium (modified Czapeck—with carboxymethylcellulose as enzyme inducer). The enzymes present in the CEC were identified by LC-MS/MS, with an emphasis on cellobiohydrolase (E.C 3.2.1.91). The fungus of the Aspergillus genus was identified by amplifying the ITS1-5.8S-ITS2 rDNA region and metaproteomic analysis, where the excreted enzymes were identified with sequence coverage greater than 84% to A. nidulans. Chitosan hydrolysis assays compared the CEC with the commercial enzyme (Celluclast 1.5 L®). The ability to reduce the initial molecular mass of chitosan by 47.80, 75.24, and 93.26% after 2.0, 5.0, and 24 h of reaction, respectively, was observed. FTIR analyses revealed lower absorbance of chitosan oligomers' spectral signals, and their crystallinity was reduced after 3 h of hydrolysis. Based on these results, we can conclude that the crude enzyme concentrate showed a significant technological potential for obtaining chitosan oligomers of different sizes. [ABSTRACT FROM AUTHOR]

Research on the gut microbiota, which involves a large and complex microbial community, is an important part of infectious disease control. In China, few studies have been reported on the diversity of the gut microbiota of wild marmots. To obtain full details of the gut microbiota, including bacteria, fungi, viruses and archaea, in wild marmots, we have sequenced metagenomes from five sample-sites feces on the Hulun Buir Grassland in Inner Mongolia, China. We have created a comprehensive database of bacterial, fungal, viral, and archaeal genomes and aligned metagenomic sequences (determined based on marmot fecal samples) against the database. We delineated the detailed and distinct gut microbiota structures of marmots. A total of 5,891 bacteria, 233 viruses, 236 fungi, and 217 archaea were found. The dominant bacterial phyla were Firmicutes, Proteobacteria, Bacteroidetes, and Actinomycetes. The viral families were Myoviridae, Siphoviridae, Phycodnaviridae, Herpesviridae and Podoviridae. The dominant fungi phyla were Ascomycota, Basidiomycota, and Blastocladiomycota. The dominant archaea were Biobacteria, Omoarchaea, Nanoarchaea, and Microbacteria. Furthermore, the gut microbiota was affected by host species and environment, and environment was the most important factor. There were 36,989 glycoside hydrolase genes in the microbiota, with 365 genes homologous to genes encoding β-glucosidase, cellulase, and cellulose β-1,4- cellobiosidase. Additionally, antibiotic resistance genes such as macB, bcrA, and msbA were abundant. To sum up, the gut microbiota of marmot had population diversity and functional diversity, which provides a basis for further research on the regulatory effects of the gut microbiota on the host. In addition, metagenomics revealed that the gut microbiota of marmots can degrade cellulose and hemicellulose. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass is a source of carbohydrates that can be used in the production of biogas. The aim of this study was to obtain biogas from biomass waste (leaves, stems and seed bagasse) of Ricinus communis, applying pretreatments such as temperature and humidity. We examined the effect of these pretreatments on the biomass, two enzymatic pretreatments (cellulase and cellobiohydrolase), two chemicals (NaOH and HCl) and two controls (dried castor straw and seed bagasse) on the methane content. The experiment was performed in two anaerobic digestion (AD) assays at a controlled temperature (37 °C) and at room temperature, with a hydraulic retention time (HRT) of 55 days. The results showed that the residues of the seed bagasse produced the highest biogas yields both at room temperature and at the controlled temperature since this material at 37 °C produced 460.63 mL gVS−1 under cellulase pretreatment; at room temperature, the highest level of production was found for the control (263.41 mL gVS−1). The lowest yields at the controlled temperature and room temperature were obtained from residues of Ricinus communis treated with cellobiohydrolase and the seed bagasse treated with alkaline (15.15 mL gVS−1 and 78.51 mL gVS−1, respectively). Meanwhile, the greatest amount of methane was produced by seed bagasse treated with cellobiohydrolase at a controlled temperature (92.2% CH4) and the lowest content of CH4 (15.5%) was obtained at a controlled temperature from castor straw under the control treatment. [ABSTRACT FROM AUTHOR]

Cellulolytic enzymes for bioconversion of lignocellulose to fermentable sugar provide an economically viable solution for numerous biofuels production, but currently problematic due to the high cost of commercial cellulase. It has been assessed the secretome of full cellulase and hemicellulase, as well as auxiliary enzyme activity, were assessed in 17 fungal strains. The breakdown of pretreated poplar with various crude enzymes was investigated, and the synergistic effects of crude enzymes and commercial cellulase were evaluated. Correlation coefficients between different enzyme activities and glucose hydrolysis from pretreated poplar were also discussed. It was found cellobiohydrolase and xylanase activity were positively correlated with glucose release, but β-glucosidase activity was indeterminate and lysis polysaccharide monooxygenase activity had a poor correlation. The highest cellulose conversion rates from the pretreated poplar utilized commercial cellulase LLC02 combined with Trichoderma reesei ATCC 24449, Aspergillus niger SM24 and Aspergillus niger SM751 were 94.8 %, 91.6 %, and 103.6 %, respectively. These findings demonstrate the fact that coupling an appropriate amount of secretome cellulolytic enzyme cocktail with cellulase boosts woody biomass hydrolysis, implying that crude enzyme cocktails from selected fungal strains have a high potential for minimizing commercial cellulase costs for industrial applications. [ABSTRACT FROM AUTHOR]

Rice straw, containing harmful bacteria and being of low value, created a significant issue that limits its use. This study aimed to improve hygienic quality of fermented rice straw and create value-added products (α-tocopherol or organic acids) using Bacillus tequilensis QH1 (QH), waste spinach effluent (S), and their combination (QHS) for short- (7 day) and long-term (42 day) fermentation, without treatment as a control (CK). Long-term fermentation and QH reduced relative abundance of pathogenic and saprophytic Staphylococcus saparophytics from 53.9% to 5.24% and 12.6% and Enterobacter sp. from 4.34% to 1.75% and 0.43%. B. tequilensis QH1 secreted endoglucanase (42.20 U/g), β-glucosidase (27.27 U/g), cellobiohydrolase (44.52 U/g), and laccase (17.2 U/g) and facilitated degradation of hemicellulose and lignin. The QHS increased organic acids production by 63.7% on day 7 over the CK. Yields of α-tocopherol were higher in QH and QHS, at 1.39 and 1.46 times more than raw material (7.24 mg/kg DM), due to up-regulating biosynthesis of phenylalanine, tyrosine, and tryptophan. The S decreased α-tocopherol yield, and QH motivated B. cellulasensis emergence and together degraded lignin to synthesize α-tocopherol. In conclusion, utilizing combination of B. tequilensis QH1 and waste spinach effluent to produce hygienic and value-added rice straw was feasible. [Display omitted] • Rice straw had poor hygiene due to containing pathogenic and saprophytic bacteria. • Pathogenic and saprophytic bacteria was reduced by QH at long-term fermentation. • B. cellulasensis and B. tequilensis QH1 degraded lignin to yield α-tocopherol. • Productions of organic acid and α-tocopherol were increased by QHS. [ABSTRACT FROM AUTHOR]

Filamentous fungi grow through elongation of their apical region by exocytosis and secrete enzymes that can be of commercial or industrial importance. Their hyphae exhibit extensive branching, making it difficult to control hyphal growth for observation and analysis. Therefore, although hyphal morphology and productivity are closely related, the relationship between the two has not yet been clarified. Conventional morphology and productivity studies have only compared the results of macro imaging of fungal pellets cultured in bulk with the averaged products in the culture medium. Filamentous fungi are multicellular and their expression differs between different hyphae. To truly understand the relationship between morphology and productivity, it is necessary to compare the morphology and productivity of individual hyphae. To achieve this, we developed a microfluidic system that confines hyphae to individual channels for observation and investigated the relationship between their growth, morphology, and enzyme productivity. Furthermore, using Trichoderma reesei, a potent cellulaseproducing fungus, as a model, we developed a cellulase detection assay with 4-MUC substrate to detect hyphal growth and enzyme secretion in a microfluidic device in real time. Using a strain that expresses cellobiohydrolase I (CBH I) fused with AcGFP1, we compared fluorescence from the detection assay with GFP fluorescence intensity, which showed a strong correlation between the two. These results indicate that extracellular enzymes can be easily detected in the microfluidic device in real time because the production of cellulase is synchronized in T. reesei. This microfluidic system enables real-time visualization of the dynamics of hypha and enzymes during carbon source exchange and the quantitative dynamics of gene expression. This technology can be applied to many biosystems from bioenergy production to human health. [ABSTRACT FROM AUTHOR]

In order to figure out the induction mechanisms of glycoside hydrolase genes in Aspergillus aculeatus, we screened approximately 9,000 transfer DNA (T-DNA)-inserted mutants for positive regulators involved in the induction. Since the mutants possess the orotidine 5′-monophosphate decarboxylase gene as a reporter gene to monitor the cellulose-responsive expression of the cellobiohydrolase I gene (cbhI), candidate strains were isolated by counterselection against 5-fluoroorotic acid (5-FOA). One 5-FOA-resistant mutant harboring the T-DNA at the uge5 locus showed reduced cellulose utilization and cbhI expression. A. aculeatus Uge5 is homologous to Aspergillus fumigatus uge5 (Afu5g10780; E-value, 0.0; identities, 93%), which catalyzes the conversion of uridine diphosphate (UDP)-glucose to UDP-galactopyranose. The uge5 deletion mutant in A. aculeatus (Δuge5) showed reduced conidium formation on minimal media supplemented with galactose, locust bean gum (LBG), and guar gum as a carbon source. β-1,4-Endoglucanase and β-1,4-mannanase production in submerged culture containing LBG was reduced to 10% and 6% of the control strain at day 5, respectively, but no difference was observed in cultures containing wheat bran. The expression of major cellulolytic and mannolytic genes in the presence of mannobiose in Δuge5 was reduced to less than 15% of the control strain, while cellobiose-responsive expression was only modestly reduced at early inducing time points. Since all test genes were controlled by a transcription factor ManR, these data demonstrate that Uge5 is involved in inducer-dependent selective expression of genes controlled via ManR. Key points: • UDP-glucose 4-epimerase (Uge5) regulates expression of glycosyl hydrolase genes. • ManR regulates both cellobiose- and mannobiose-responsive expression. • Uge5 plays a key role in mannobiose-responsive expression. [ABSTRACT FROM AUTHOR]

There is a high level of conservation of tryptophans within the active site architecture of the cellulase family, whereas the function of the four tryptophans in the catalytic domain of Cel7A is unclear. By mutating four tryptophan residues in the catalytic domain of Cel7A from Penicillium piceum (PpCel7A), the binding affinity between PpCel7A and p‐nitrophenol‐d‐cellobioside (pNPC) was reduced as determined by Michaelis–Menten constants, molecular dynamics simulations, and fluorescence spectroscopy. Furthermore, PpCel7A variants showed a reduced level of cellobiohydrolase (CBH) activity against cellulose analogs or natural cellulose. Therefore, it could be concluded four tryptophan residues in Cel7A played a critical role in substrate binding. Mutagenesis results indicated that the W390 stacking interactions at the −2 site played an essential role in facilitating substrate distortion to the −1 site. As soon as the function was altered, the mutation would inevitably affect the catalytic activity against the natural substrate. Interestingly, no clear relationship was found between the CBH activity of PpCel7A variants against pNPC and Avicel. p‐Nitrophenol contains many electrophilic groups that may result in overestimation of the binding constant between tryptophan residues and pNPC in comparison with the natural substrate. Consequently, screening improved cellulase using cellulose analogs would divert attention from the target direction for lignocellulose biorefinery. Clarifying mechanism of catalytic diversity on the natural cellulose or cellulose analogs may give better insight into cellulase screening and selecting strategy. [ABSTRACT FROM AUTHOR]

Trichoderma reesei is a powerful fungal cell factory for the production of cellulolytic enzymes due to its outstanding protein secretion capacity. Endoplasmic reticulum-associated degradation (ERAD) plays an integral role in protein secretion that responds to secretion pressure and removes misfolded proteins. However, the role of ERAD in fungal growth and endogenous protein secretion, particularly cellulase secretion, remains poorly understood in T. reesei. Here, we investigated the ability of T. reesei to grow under different stresses and to secrete cellulases by disrupting three major genes (hrd1, hrd3 and der1) involved in the critical parts of the ERAD pathway. Under the ER stress induced by high concentrations of DTT, knockout of hrd1, hrd3 and der1 resulted in severely impaired growth, and the mutants Δhrd1 and Δhrd3 exhibited high sensitivity to the cell wall-disturbing agents, CFW and CR. In addition, the absence of either hrd3 or der1 led to the decreased heat tolerance of this fungus. These mutants showed significant differences in the secretion of cellulases compared to the parental strain QM9414. During fermentation, the secretion of endoglucanase in the mutants was essentially consistent with that of the parental strain, while cellobiohydrolase and β-glucosidase were declined. It was further discovered that the transcription levels of the endoglucanase-encoding genes (eg1 and eg2) and the cellobiohydrolase-encoding gene (cbh1) were not remarkedly changed. However, the β-glucosidase-encoding gene (bgl1) was significantly downregulated in the ERAD-deficient mutants, which was presumably due to the activation of a proposed feedback mechanism, repression under secretion stress (RESS). Taken together, our results indicate that a defective ERAD pathway negatively affects fungal growth and cellulase secretion, which provides a novel insight into the cellulase secretion mechanism in T. reesei. [ABSTRACT FROM AUTHOR]

Vaccination is critical for population protection from pathogenic infections. However, its efficiency is frequently compromised by a failure of antigen retention and presentation. Herein, we designed a dextran-binding protein DexBP, which is composed of the carbohydrate-binding domains of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A, together with the sequence of the fluorescent protein mCherry. DexBP was further prepared by engineered Escherichia coli cells and grafted to magnetic nanoparticles. The magnetic nanoparticles were integrated with a dextran/poly(vinyl alcohol) framework and a reactive oxygen species-responsive linker, obtaining magnetic polymeric microgels for carrying pathogen antigen. Similar to amoeba aggregation, the microgels self-assembled to form aggregates and further induced dendritic cell aggregation. This step-by-step assembly retained antigens at lymph nodes, promoted antigen presentation, stimulated humoral immunity, and protected the mice from life-threatening systemic infections. This study developed a magnetic microgel-assembling platform for dynamically regulating immune response during protection of the body from dangerous infections. [ABSTRACT FROM AUTHOR]

In this study, exposure experiments were conducted to assess the effects of polystyrene nanoparticles (PS) and amine-modified polystyrene nanoparticles (APS) at environmental concentrations (1, 10, and 100 µg L− 1) on two fungal species (Geotrichum candidum and Aspergillus niger), isolated from leaf litter in streams, concerning their growth and metabolic activity. Results showed that PS at 1 and 10 µg L− 1 have hormesis effects on G. candidum growth. Compared with G. candidum, A. niger had higher sensitivity to nanoplastic exposure. Besides, the peroxidase and cellobiohydrolase activities of A. niger were significantly inhibited by nanoplastics (except 1 µg L− 1 PS), which would weaken its metabolic activity in carbon cycling. These results provided a new thought on how the growth and functions of aquatic fungi cope with the stress induced by nanoplastics. Overall, the study provided evidence for the different responses of aquatic fungi to nanoplastics in streams. [ABSTRACT FROM AUTHOR]

Significant engineering of Saccharomyces cerevisiae is required to enable consolidated bioprocessing (CBP) of lignocellulose to ethanol. Genome modification in S. cerevisiae has been successful partly due to its efficient homology-directed DNA repair machinery, and CRISPR technology has made multi-gene editing even more accessible. Here, we tested the integration of cellulase encoding genes to various sites on the yeast genome to inform the best strategy for creating cellulolytic strains for CBP. We targeted endoglucanase (EG) or cellobiohydrolase (CBH) encoding genes to discreet chromosomal sites for single-copy integration or to the repeated delta sites for multi-copy integration. CBH1 activity was significantly higher when the gene was targeted to the delta sequences compared to single gene integration loci. EG production was comparable, though lower when the gene was targeted to a chromosome 10 site. We subsequently used the information to construct a strain containing three cellulase encoding genes. While individual cellulase activities could be assayed and cellulose conversion demonstrated, it was shown that targeting specific genes to specific loci had dramatic effects on strain efficiency. Since marker-containing plasmids could be cured from these strains, additional genetic changes can subsequently be made to optimize strains for CBP conversion of lignocellulose. [ABSTRACT FROM AUTHOR]

Cellulose which is extremely produced by plants, can be used for biofuel production but this function needs chemical or enzymatic digestion. Cellulose hydrolysis of plant wastes for ethanol production requires a mixture of three enzyme groups, including endoglucanases, exoglucanases, and beta-glucosidases. The cellobiohydrolase enzyme (Cel6B) from Thermobifidia fusca has been used for cellulase activity extensively. This research aimed to express recombinant Cel6B enzyme in Pichia pastoris. For this purpose, cel6B gene in control of AOX1 promoter (methanol inducible) was introduced into Pichia pastoris. Amplification of cel6B gene was performed by PCR technique and then introduced into the Phil-S1 yeast vector. The recombinant construct contained the cel6B gene sequence and PHO1 signal sequence as secretion signal was transferred into Pichia pastoris GS115 strain. The transformed yeast cells expressed the recombinant Cel6B to yield 2.104 U (μmol/min)/ml of culture medium. Purified recombinant enzyme showed the best activity at 60 °C and pH 4.5 and this was agreed with optimum conditions for recombinant Cel6B enzymes which were produced in other systems. The purity of the enzyme was examined by SDS-PAGE technique, and a single band with a molecular weight about 59.6 kDa was observed. As cel6B gene sequence was not optimized for expression in the Pichia pastoris yeast, this could be one of the reasons for low level activity of recombinant Cel6B enzyme. This thermostable enzyme can be used for cellulolytic digestion of biomaterials in biofuel production research and other uses. [ABSTRACT FROM AUTHOR]

Cellobiohydrolase GH7 (Cel7A) is a crucial component of an efficient cellulase system. High cellulase activity was identified in the secretome of a locally identified fungal strain of Aspergillus fumigatus (NITDGPKA3). To investigate the potentiality of Cel7A as a biocatalyst, we have cloned the gene from isolated strain of A. fumigatus. The enzyme (AfCel7A) was expressed in Pichia pastoris and purified. Biochemical properties of this enzyme have been explored and compared with those of Trichoderma reesei Cel7A (TrCel7A), the primary source of industrial cellulase. Higher catalytic activity (Kcat/KM) in AfCel7A than that in TrCel7A suggested a comparative structural analysis with Cel7A of other fungal origins (GcaCel7A, RemCel7A, ThaCel7A, and HirCel7A), including TrCel7A. Variations mainly were observed in the loop region. The molecular dynamics simulation with cellononaose ligand clearly stated that AfCel7A is more compact and stable than TrCel7A. Binding energy analysis indicated that the binding of AfCel7A with cellulose is more potent than that of TrCel7A. Residues Asn49, Asn103, Asp107, Gln175, Glu212, Asp259, Glu217 are typical for binding cellulose in both AfCel7A and TrCel7A. Overall the biochemical and structural characterisation suggests that AfCel7A is a potential biocatalyst for industrial use. [ABSTRACT FROM AUTHOR]

The fluorimetry method for assaying cellobiohydrolase activity (CBHA), originally developed for pure cultures of microorganims, has been modified for studying samples of the digestive tract content of wild small mammals. Several fixation methods for the samples have been studied. The optimal fixation agent is 86% glycerol. A mathematical model of the dependence of the measured CBHA values on the concentration of chyme in samples has been developed. The model confirmed the effect of the chyme concentration on the analyzed results in the range of 5–15 g/L. The optimal concentration of chyme is equal to 10 g/L; i.e., a sample should contain 0.2 g of chyme in 1 mL of 86% glycerol, so the sample collection and fixation could be easily performed in the field. [ABSTRACT FROM AUTHOR]

It has been demonstrated that LPMO reactions can be driven by light, using the photosynthetic pigment chlorophyllin to achieve efficient oxidative degradation of cellulose. However, the effect of chlorophyllin on cellulases remains unclear. This study discovered that chlorophyllin does not affect the hydrolytic activity of cellulases under dark conditions. However, under light exposure, chlorophyllin-derived reactive oxygen species (ROS) exhibit a strong inhibitory effect on cellulases. These ROS primarily inhibit the hydrolytic action of endoglucanase II (Cel5A) and cellobiohydrolase II (Cel6A), while the action of cellobiohydrolase I and β-glucosidase remains unaffected. Scavenger studies revealed that singlet oxygen (1O₂) is the key inhibitory ROS responsible for the inhibition of Cel5A and Cel6A. The removal of 1O₂ by sodium azide effectively mitigates this inhibition, increasing the conversion yield of cellulose to glucose by 25.9 % when using the light-driven LPMO system in conjunction with cellulases. This study provides new insights into the role of chlorophyllin-derived 1O₂ in hindering hydrolytic action of cellulases and demonstrates the successful mitigation of this inhibition by sodium azide, thereby enhancing the cooperative degradation of cellulose to glucose by the light-driven LPMO system and cellulases. • The effect of chlorophyllin on the hydrolytic action of cellulases was studied. • Chlorophyllin-derived ROS inhibited the activities of Cel5A and Cel6A. • 1O₂ was identified as the primary inhibitory ROS for Cel5A and Cel6A. • The inhibition was effectively mitigated by the removal of 1O₂ using sodium azide. [ABSTRACT FROM AUTHOR]

White-rot fungi produce a wide spectrum of lignocellulose-degradation enzymes, which can be used in bioenergy, bioremediation, and other industrial applications. This study identified a cellobiohydrolase II (Cel6A, GH6 cellobiohydrolase, EC 3.2.1.91) with high hydrolytic activity toward crystalline cellulose from a white-rot fungus Lentinus sp. WR2. Both native (n Ls Cel6A) and recombinant (r Ls Cel6A) enzymes expressed in Pichia pastoris were purified and characterized. Three N- glycosylation sites at Asn102, Asn145, and Asn392 containing high-mannose glycans, were confirmed by mass spectrometry. To elucidate the functional role of N- linked glycans, three deglycosylated mutants of r Ls Cel6A, i.e., N102A, N145A, and N392A, were created and characterized for their biochemical and kinetic properties. While no discernible changes in the secondary structure of the three mutants were determined by circular dichroism spectrometry, deterioration of thermostability was revealed in N392A but not in N102A and N145A. Structure modeling and molecular dynamics analyses revealed that the N -linked glycan on Asn392 may restrict the flexibility of the C-terminal loop in Ls Cel6A, affecting the protein integrity and appropriate dynamics for the enzymatic function. In summary, this study identified a novel Ls Cel6A enzyme with high catalytic activity against insoluble forms of cellulose and demonstrated the role of N- linked glycosylation in the thermostability of the enzyme. [ABSTRACT FROM AUTHOR]

Cellobiohydrolase, a workhorse enzyme for cellulose degradation, has the potential for application in biofuels, biochemicals, biomaterials, etc. However, highly efficient cellobiohydrolase is desirable to improve the economic feasibility of industrial applications. In this work, two variants Tr Cel7AT97A and Tr Cel7AK166A were designed to increase the flexibility of the entrance of the catalytic tunnel. We performed the molecular dynamics simulations using NAMD for 100 ns at 300 K in the NPT (isothermal–isobaric) ensemble and then we found that the region flexibility at the catalytic tunnel entrances of Tr Cel7AT97A and Tr Cel7AK166A was improved, compared to that of Tr Cel7A, based on the calculation of the average root mean square fluctuation. The specific activities of Tr Cel7AT97A and Tr Cel7AK166A were increased by 37 % and 68 %, respectively, compared with that of Tr Cel7A at 45 °C for 12 h. An effective approach named revive amino acid flexibility for enzyme activity based on amino acid flexibility was proposed for protein engineering of cellobiohydrolase Tr Cel7A. [Display omitted] • Tr Cel7AK166A exhibited outstanding cellulose-degrading activity at 45 °C. • Flexibility of catalytic tunnel entrance of Tr Cel7A was improved by engineering. • Cellulose-degrading activity of Tr Cel7A is affected by the amino acid flexibility. [ABSTRACT FROM AUTHOR]