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Trichoderma virens colonizes roots and develops a symbiotic relationship with plants where the fungal partner derives nutrients from plants and offers defence, in return. Tsp1, a small secreted cysteine-rich protein, was earlier found to be upregulated in co-cultivation of T. virens with maize roots. Tsp1 is well conserved in Ascomycota division of fungi, but none of its homologs have been studied yet. We have expressed and purified recombinant Tsp1, and resolved its structure to 1.25 A resolutions, from two crystal forms, using Se-SAD methods. The Tsp1 adopts a β barrel fold and forms dimer in structure as well as in solution form. DALI based structure analysis revealed the structure similarity with two known fungal effector proteins: Alt a1 and PevD1. Structure and evolutionary analysis suggested that Tsp1 belongs to a novel effector protein family. Tsp1 acted as an inducer of salicylic acid mediated susceptibility in plants, rendering maize plants more susceptible to a necrotrophic pathogen Cochliobolus heterostrophus, as observed using plant defence assay and RT-qPCR analysis.

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is widely used as a cellulase producer in the industry. Herein, we describe the rational engineering of the publicly available T. reesei QM9414 strain to achieve a remarkable high-level production of cellulase on glucose. Overexpression of the key cellulase regulator XYR1 by the copper-repressible promoter Ptcu1 was first implemented to achieve a full cellulase production in the context of catabolite repression (CCR) while eliminating the requirement of inducing sugars for enzyme production. The T. reesei bgl1 gene was further overexpressed to compensate for its low β-glucosidase activity on glucose. This overexpression resulted in a 102% increase in FPase activity compared with the CCR-released RUT-C30 strain cultured on Avicel. Moreover, the saccharification efficiency toward pretreated corncob residues by crude enzymes from the engineered strain on glucose increased by 85% compared with that treated by enzymes from RUT-C30 cultivated on Avicel. The engineered T. reesei strain thus shows great potential as a viable alternative to deliver commercial cellulases after further optimization for efficient saccharification of agricultural waste.

This review summarizes the findings on the use of the Penicillium verruculosum В1-537 (ΔniaD) recipient strain for the development of producers of enzymes—β-glucosidase, polysaccharide monooxygenase, and xylanases—that complement the basic cellulase complex and enhance its effectiveness. A special feature of the P. verruculosum strain is the secretion of a basic cellulase complex consisting of cellobiohydrolases and endoglucanases (EGs) that, in terms of their hydrolytic ability, surpass Hypocrea (Trichoderma) strains that are widely used in the laboratory and in industry. The capabilities of the P. verruculosum В1-537 (ΔniaD) expression system can be used to complement the basic cellulase complex with new enzymes, which are required to enhance its hydrolytic activity, and to develop new enzyme preparations tailored to the conversion of different types of renewable plant biomass (RPB). The use of an expression system based on P. verruculosum fungus makes it possible to enhance the ability of basic cellulase complex to convert different types of RPB.

Benzoic acid (BA) is potentially toxic to humans and recalcitrant in the environment. Although it may be degraded by fungi, potential degradation pathways has received limited study. In the present...

Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi–host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a β-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Å resolution and belonged to the orthorhombic space group P212121 (R meas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (V M) of the crystal is 2.32 Å3 Da−1, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.

The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-β-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced α-amylase with a molecular weight lower than that of native α-amylase without an effect on growth. To test whether the A. oryzae α-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged α-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure.

Trichoderma reeseiβ-glucosidase (Bgl1) is one of four enzymes demonstrated to act synergistically to degrade cellulose both in vitro and in vivo. Our work attempted to better understand the substrate specificity and potential biotechnological applications of Bgl1. T. reesei Bgl1H cleaves over 80% of the β-(1-4) and β-(1-3) linkages in β-glucan and 14% of the β-(1-4) linkages in amorphous cellulose, significantly more than any tested bacterial β-glucosidase. Bgl1H cleaves 50% of the β-(1-4) linkages in xyloglucan when supplemented with cellulase and α-xyloside. Approximately 20% conversion to glucose was obtained from insoluble β-(1,3)-linked curdlan using only Bgl1H; addition of a curdlanase resulted in conversion of approximately 70% of the curdlan to glucose. Bgl1H also produces xylose from xylooligosaccharides and debranched xylans. For both glucans and xylans, the relative rates of hydrolysis increase with increasing polysaccharide chain lengths. Bgl1H is able to partially degrade β-glucan in a variety of grain components; addition of endo-acting enzymes improved the enzyme’s performance on these grain components. The ability of this enzyme to produce monosaccharides from undigestible polysaccharides suggest it may have potential in improving utilization of carbohydrates in animal feed, fermentations, and other biotechnological applications.

A new strain of Trichoderma reesei (teleomorph Hypocrea jecorina) with high cellulase production was obtained by exposing the spores from T. reesei QM9414 to an ultraviolet light followed by selecting fast-growing colonies on plates containing CMC (1% w/v) as the carbon source. The mutant T. reesei RP698 reduced cultivation period to 5 days and increased tolerance to the end-products of enzymatic cellulose digestion. Under submerged fermentation conditions, FPase, CMCase, and Avicelase production increased up to 2-fold as compared to the original QM9414 strain. The highest levels of cellulase activity were obtained at 27 °C after 72 h with Avicel®, cellobiose, and sugarcane bagasse as carbon sources. The temperature and pH activity optima of the FPase, CMCase, and Avicelase were approximately 60 °C and 5.0, respectively. The cellulase activity was unaffected by the addition of 140 mM glucose in the enzyme assay. When T. reesei RP698 crude extract was supplemented by the addition of β-glucosidase from Scytalidium thermophilum, a 2.3-fold increase in glucose release was observed, confirming the low inhibition by the end-product of cellulose hydrolysis. These features indicate the utility of this mutant strain in the production of enzymatic cocktails for biomass degradation.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.

Abstract Fungi cause diverse, serious socio-economic problems, including biodeterioration of valuable products and materials that spawns a biocides industry worth ~$11 billion globally. To help combat environmental fungi that commonly colonise material products, this study tested the hypothesis that combination of an approved fungicide with diverse agents approved by the FDA (Food and Drug Administration) could reveal potent combinatorial activities with promise for fungicidal applications. The strategy to use approved compounds lowers potential development risks for any effective combinations. A high-throughput assay of 1280 FDA-approved compounds was conducted to find those that potentiate the effect of iodopropynyl-butyl-carbamate (IPBC) on the growth of Trichoderma virens; IPBC is one of the two most widely used Biocidal Products Regulations–approved fungicides. From this library, 34 compounds in combination with IPBC strongly inhibited fungal growth. Low-cost compounds that gave the most effective growth inhibition were tested against other environmental fungi that are standard biomarkers for resistance of synthetic materials to fungal colonisation. Trifluoperazine (TFZ) in combination with IPBC enhanced growth inhibition of three of the five test fungi. The antifungal hexetidine (HEX) potentiated IPBC action against two of the test organisms. Testable hypotheses on the mechanisms of these combinatorial actions are discussed. Neither IPBC + TFZ nor IPBC + HEX exhibited a combinatorial effect against mammalian cells. These combinations retained strong fungal growth inhibition properties after incorporation to a polymer matrix (alginate) with potential for fungicide delivery. The study reveals the potential of such approved compounds for novel combinatorial applications in the control of fungal environmental opportunists. Key points • Search with an approved fungicide to find new fungicidal synergies in drug libraries. • New combinations inhibit growth of key environmental fungi on different matrices. • The approach enables a more rapid response to demand for new biocides.

The glycoside hydrolase family 3 β-glucosidase Cel3A from Neurospora crassa (NcCel3A) is highly specific for cellobiose. The crystal structure of NcCel3A has been solved to 2.25 Å resolution. The structure is a dimer and exhibits a high degree of N-glycosylation., The glycoside hydrolase family 3 (GH3) β-glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β-Glucosidases hydrolyse the β-1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an R cryst and R free of 0.18 and 0.22, respectively. NcCel3A is an extensively N-glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β-glucosidase from Rasamsonia emersonii. NcCel3A is a three-domain protein with a number of extended loops that deepen the active-site cleft of the enzyme. These structures characterize this subfamily of GH3 β-glucosidases and account for the high cellobiose specificity of this subfamily.

To expand the range of soluble carbon sources for our enzyme production system, we investigated the properties of sucrose utilization and its effect on cellulase production by Trichoderma reesei M2-1. We performed batch cultivation of T. reesei M2-1 on sucrose and related sugars along with cellobiose, which was used as a cellulase inducer. The results clearly revealed that the hydrolysis products of sucrose, i.e. glucose and fructose, but not sucrose, can be used as a carbon source for enzyme production. In a 10-day continuous feeding experiment using invertase-treated sucrose/cellobiose, the fungal strain produced cellulases with a filter paper-degrading activity of 20.3 U/mL and production efficiency of 254 U/g-carbon sources. These values were comparable with those of glucose/cellobiose feeding (21.2 U/mL and 265 U/g-carbon sources, respectively). Furthermore, the comparison of the specific activities clearly indicated that the compositions of both produced enzymes were similar. Therefore, enzymatically hydrolyzed sucrose can be utilized as an alternative carbon source to glucose in our enzyme production system with T. reesei M2-1.

Trichoderma virens produces viridin/viridiol, heptelidic (koningic) acid, several volatile sesquiterpenes and gliotoxin (Q strains) or gliovirin (P strains). We earlier reported that deletion of the terpene cyclase vir4 and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH, designated as vGPD) associated with the "vir" cluster abrogated the biosynthesis of several volatile sesquiterpene metabolites. Here we show that, the deletion of this GAPDH also impairs the biosynthesis of heptelidic acid (a non-volatile sesquiterpene), viridin (steroid) and gliovirin (non-ribosomal peptide), indicating regulation of non-volatile metabolite biosynthesis by this GAPDH that is associated with a secondary metabolism gene cluster. To gain further insights into the details of this novel form of regulation, we identified the terpene cyclase gene responsible for heptelidic acid biosynthesis (hereafter designated as has1) and prove that the expression of this gene is regulated by vGPD. Interestingly, deletion of has1 impaired biosynthesis of heptelidic acid (HA), viridin and gliovirin, but not of volatile sesquiterpenes. Deletion of the vir cluster associated terpene cyclase gene (vir4), located next to the vGPD gene, did not impair biosynthesis of HA, viridin or gliovirin. We thus unveil a novel circuitry of regulation of secondary metabolism where an HA-tolerant GAPDH isoform (vGPD) regulates HA biosynthesis through the transcriptional regulation of the HA-synthase gene (which is not part of the "vir" cluster). Interestingly, impairment of HA biosynthesis leads to the down-regulation of biosynthesis of other non-volatile secondary metabolites, but not of volatile secondary metabolites. We thus provide evidence that the "vir" cluster associated, HA-tolerant GAPDH in T. virens participates in the biosynthesis of volatile sesquiterpenes as a biosynthetic enzyme, and regulates the production of non-volatile metabolites via regulation of HA biosynthesis. The orthologue of the "vir" cluster in Aspergillus oryzae was earlier reported to synthesize HA by another group. Our study thus proves that the same gene cluster can code for unrelated metabolites in different species.

Malaria remains a worldwide threat, afflicting over 200 million people each year. The emergence of drug resistance against existing therapeutics threatens to destabilize global efforts aimed at controlling Plasmodium spp. parasites, which is expected to leave vast portions of humanity unprotected against the disease. To address this need, systematic testing of a fungal natural product extract library assembled through the University of Oklahoma Citizen Science Soil Collection Program has generated an initial set of bioactive extracts that exhibit potent antiplasmodial activity (EC(50) < 0.30 μg/mL) and low levels of toxicity against human cells (less than 50% reduction in HepG2 growth at 25 μg/mL). Analysis of the two top-performing extracts from Trichoderma sp. and Hypocrea sp. isolates revealed both contained chemically diverse assemblages of putative peptaibol-like compounds that were responsible for their antiplasmodial actions. Purification and structure determination efforts yielded 30 new peptaibols and lipopeptaibols (1–14 and 28–43), along with 22 known metabolites (15–27 and 44–52). While several compounds displayed promising activity profiles, one of the new metabolites, harzianin NPDG I (14), stood out from the others due to its noteworthy potency (EC(50) = 0.10 μM against multi-drug-resistant P. falciparum line Dd2) and absence of gross toxicity toward HepG2 at the highest concentrations tested (HepG2 EC(50) > 25 μM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for translating peptaibols into a bioactive scaffold worthy of further development.

In Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1S387V, Tr_Cre1S388V, Tr_Cre1T389V, and Tr_Cre1T390V, respectively. Transcription of cel7a in Tr_ Cre1S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-β-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1S388V were significantly increased by up to 2- to 3-fold compared with that of other transformants and the parent strain. The results suggested that S388 is critical site of phosphorylation for triggering CCR at the terminus of Cre1. To our knowledge, this is the first report demonstrating an improvement of cellulase production in T. reesei under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in T. reesei under CCR., Graphical Abstract Schematic diagram on transcriptional regulation of cellulase expression in mutant and parent strains. We developed a precision engineering strategy by mimicking dephosphorylation at a key residue S388 of Cre1 to improve the production of cellulase in fungal species under CCR, highlighting that this technology can accelerate the industrial process of lignocellulosic biorefinery.

Eleven fungal endophytes were isolated from the plant parts of Z. nimmonii (J. Graham) Dalzell, an endemic species of the Western Ghats, India, a biodiversity hotspot area. The endophytic isolates were characterized by the sequencing of the Internal Transcribed Spacer (ITS) regions and designated as strains by depositing ITS sequences in the Gen Bank sequence database. All the strains were cultured in Potato Dextrose broth (PDB, 500 mL) contained in Erlenmeyer flasks to obtain the secondary metabolites. The culture filtrate was extracted with ethyl-acetate (EA) three times and concentrated by flash evaporation to obtain EA crude dry extract. The strains were evaluated for the antibacterial potentials against six pathogenic bacterial strains viz., Bacillus subtilis (MTCC 121), Staphylococcus aureus (MTCC 7443) Pseudomonas aeruginosa (MTCC 7093), Escherichia coli (MTCC 729), Enterobacter aerogenes (MTCC 111) and Klebsiella pneumoniae (MTCC 661). Nine endophytic fungal extracts except Alternaria consortiale and Hypocrea lixi showed inhibitory activities against at least two of the six test bacterial strains. Bipolaris specifera (KM114290) exhibited the highest inhibition zones ranging from 15.1 ± 0.3 to 26.7 ± 1.1 mm (diameter), against all six test bacteria in the agar disk diffusion assay, and with Minimum Inhibitory Concentrations (MIC’s) of 0.04–0.14 mg/mL, followed by Aspergillus terreus. B. specifera extract was therefore selected and characterized for the identification of antibacterial compounds by chromatographic techniques. Seven antibacterial compounds viz., (1) Bicyclo[3.2.0]heptan-2-one, 6-hydroxy-5-methyl-6-vinyl; (2) Adipic acid divinyl ester; (3) 1,4-Naphthoquinone, 6-acetyl-2,5-dihydroxy; (4) Decanedioic acid, 3,7-dimethyl ester; (5) (Z)-4-Hexenoic acid 2-acetyl-2-methyl-ethyl ester and (6) Butanoic acid 2-acetyl-3-methyl-methyl ester and (7) Caffeic acid, were identified through liquid and gas chromatography. These compounds are mainly volatile esters of fatty acids, phenolics and adipic acid found rare in nature. This study envisages the possible drug discovery using endophytes from traditional and endemic medicinal species.

Mines and caves are unusual ecosystems containing unique fungi and are greatly understudied compared to other environments. The Soudan Mine in Tower, MN, an iron ore mine that closed in 1963 after operating for 80 years, was sampled to explore fungal diversity and to investigate taxa that tolerate heavy metals for potential bioprocessing technologies or as sources of bioactive molecules for drug discovery and possible biocontrol for white-nose syndrome (WNS) of bats. The mine is 714 m deep, has 18 levels and contains large quantities of wooden timbers, in contrast to many other oligotrophic subterranean environments. Fungi were cultured from samples and the ITS region was sequenced for identification and phylogenetic analysis. Results show Ascomycota are the dominant fungi followed by Basidiomycota and Mucoromycota. Out of 164 identified taxa, 108 belong to the Ascomycota and 26 and 31 to Basidiomycota and Mucoromycota, respectively. There are also 46 taxa that do not match (

The Rhus verniciflua Stokes (RVS) extract is used as a traditional herbal medicine in Southeast Asian countries such as Korea and China. In the present study, one phenolic acid and six flavonoids were isolated from an 80% ethanol RVS extract to examine their antimicrobial activities. These compounds were identified as 3′,4′,7-trihydroxyflavone (1), methyl gallate (2), gallic acid (3), fusti (4), fisetin (5), butin (6), and sulfuretin (7) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. The antimicrobial activities of compounds 5 and 6 (at a dose of 16 μg/mL each) were superior to that of the control, cycloheximide (at a dose of 25 μg/mL), against Hypocrea nigricans; additionally, the activities of compounds 1 and 2 (at a dose of 8 μg/mL each) were superior to the control against Penicillium oxalicum. Also, chemical compounds 1 and 5 (at a dose of 16 μg/mL each) had higher activities than the control (25 μg/mL) against Trichoderma virens. Chemical compound 1 (at a dose of 8 μg/mL) had a similar activity to that of the control against Bacillus subtilis. The obtained results suggest that the RVS extract could be a promising food and nutraceutical source because of the antimicrobial properties of its phenolic compounds.

The endoglucanase belonging to glycoside hydrolase family 61 are little studied. In present study, a β-endoglucanase of ~37 kDa induced on autoclaved mycelium of Fusarium oxysporum was cloned and characterized. The molecular characterization of β-endoglucanase encoding gene revealed presence of a single intron and an open reading frame of 1044-bp which encoded a protein of 347 amino acid residues. The phylogenetic analysis of Eglu revealed its similarity to endo-β-glucanases of other Trichoderma spp. The catalytic site of β-endoglucanase contained Asp, Asn, His and Tyr residues. The cDNA encoding β-glucanase was cloned into E. coli and Pichia pastoris using pQUA-30 and pPIC9K vector system, respectively. The comparison of structure revealed that most similar structure to Eglu is Hypocrea jecorina template 5o2w.1.A of glycoside hydrolase family 61.The biochemical characterization of β-endoglucanase purified from T. saturnisporum isolate and the recombinant protein expressed in E. coli and P. pastoris was active under acidic conditions with a pH optima of 5 and temperature optima of 60 °C. The purified and expressed enzyme preparation was able to inhibit growth of F.oxysporum at 1 × 105 spores/mL which clearly revealed its significance in plant pathogen suppression.

To develop a versatile Trichoderma reesei (teleomorph Hypocrea jecorina) expression system for the high-purity production of heterologous proteins.The versatile T. reesei expression system is based on xyn1 and xyn2 promoters, A824V transition in XYRI, and a bicomponent carbon source strategy. Red fluorescent protein gene rfp and alkaline endoglucanase EGV gene egv3 from Humicola insolens were used as reporter genes to test our versatile expression system CONCLUSIONS: The versatile T. reesei expression system can be applied to produce heterologous proteins with high purity and high yield.

The inverting glycoside hydrolase Trichoderma reesei (Hypocrea jecorina) Cel6A is a promising candidate for protein engineering for more economical production of biofuels. Until recently, its catalytic mechanism had been uncertain: The best candidate residue to serve as a catalytic base, Asp-175, is farther from the glycosidic cleavage site than in other glycoside hydrolase enzymes. Recent unbiased transition path sampling simulations revealed the hydrolytic mechanism for this more distant base, employing a water wire; however, it is not clear why the enzyme employs a more distant catalytic base, a highly conserved feature among homologs across different kingdoms. In this work, we describe molecular dynamics simulations designed to uncover how a base with a longer side chain, as in a D175E mutant, affects procession and active site alignment in the Michaelis complex. We show that the hydrogen bond network is tuned to the shorter aspartate side chain, and that a longer glutamate side chain inhibits procession as well as being less likely to adopt a catalytically productive conformation. Furthermore, we draw comparisons between the active site in Trichoderma reesei Cel6A and another inverting, processive cellulase to deduce the contribution of the water wire to the overall enzyme function, revealing that the more distant catalytic base enhances product release. Our results can inform efforts in the study and design of enzymes by demonstrating how counterintuitive sacrifices in chemical reactivity can have worthwhile benefits for other steps in the catalytic cycle.

This study is aimed to improving extracellular lipase production by Trichoderma strains using oil palm empty fruit bunch (OPEFB) medium on solid state fermentation with the assistance of statistical optimization. Two selected Trichoderma strains (Trichoderma sp.1 and Hypocrea neorufa.1) with high lipase producing potential were employed in a stepwise optimization. Initially, the influence of nutritional (carbon, nitrogen sources, and inducers) and environmental (moisture and temperature) factors on lipase production was evaluated using One-Factor-At-A-time (OFAT) method. Next, three most influential factors (Glucose, Urea and Olive oil) resulting from OFAT were optimized for lipase production by Trichoderma sp.1 using the central composite design under response surface methodology. Experiments conducted under optimum conditions (10.38% (w/w) glucose, 0.86% (w/w) urea, and 7.38% (v/w) olive oil) were further improved the lipase production from 0.195 to 0.390 Ug−1, which is about 2 folds greater than un-optimized condition. This study demonstrated that OPEFB can be utilized as inexpensive substrate for improving the lipase secretion by Trichoderma sp.1.

For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose-degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncated HjLPMO9A revealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site.

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 A resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

The relationship between unfolding and inactivation of Hypocrea orientalis β-glucosidase has been investigated for the first time. The secretion of β-glucosidase from H. orientalis is induced by raw cassava residues. The enzyme was 75 kD without glycosylation. Guanidine hydrochloride (GuHCl) could reversibly inactivate the enzyme with an estimated IC50 value of 0.4 M. The inactivation kinetics model by GuHCl has been established and the microscopic inactivation rate constants are determined. The values of forward inactivation rate constants of free enzyme are found to be larger than that of substrate–enzyme complex suggesting the enzyme could be protected by substrate during denaturation. Conformational change of the enzyme during denaturation is observed as the intrinsic fluorescence emission peaks appeared red-shift (334–354 nm) with intensity decreased following increase of GuHCl concentrations. Inactivation extent is found to be greater than conformation change of the whole enzyme, indicating that the active site of H. orientalis β-glucosidase might be a more flexible region than the whole enzyme.

A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7-22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19-29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K m and V max values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.

This study evaluated the effectiveness of solid-state fermentation with Arxula adeninivorans or Hypocrea jecorina to improve hygienic quality and digestibility of banana peels by mono-gastric animals. Green peels of cooking bananas ( Musa AAA-group) were solid-state fermented in ziploc plastic bags for 14 d either non-inoculated or inoculated with A. adeninivorans or H. jecorina. Colonies of aerobic bacteria, lactic acid bacteria, Enterobacteriaceae , yeasts and moulds were enumerated. In-vitro digestibility (total tract and pre-caecal) of dry matter (DM), organic matter (OM) and crude protein (CP) was also determined. Solid-state fermentation significantly improved (P in-vitro total tract digestibility (9.9% for DM and 10.1% for OM), and in-vitro pre-caecal digestibility (25.0% for DM, 30.9% for OM and 74.5% for CP); however, no significant effect (P>0.05) due to inoculation was observed. Conversely, inoculation (with A. adeninivorans or H. jecorina ) significantly improved (P Enterobacteriaceae and mould counts than the non-inoculated treatment. However, H. jecorina triggered a significantly superior (P in-vitro pre-caecal digestibility than A. adeninivorans .

Background Amylases are used in various industrial processes and a key requirement for the efficiency of these processes is the use of enzymes with high catalytic activity at ambient temperature. Unfortunately, most amylases isolated from bacteria and filamentous fungi have optimal activity above 45 °C and low pH. For example, the most commonly used industrial glucoamylases, a type of amylase that degrades starch to glucose, are produced by Aspergillus strains displaying optimal activities at 45–60 °C. Thus, isolating new amylases with optimal activity at ambient temperature is essential for improving industrial processes. In this report, a glucoamylase secreted by the cold-adapted yeast Tetracladium sp. was isolated and biochemically characterized. Results The effects of physicochemical parameters on enzyme activity were analyzed, and pH and temperature were found to be key factors modulating the glucoamylase activity. The optimal conditions for enzyme activity were 30 °C and pH 6.0, and the K m and k cat using soluble starch as substrate were 4.5 g/L and 45 min−1, respectively. Possible amylase or glucoamylase encoding genes were identified, and their transcript levels using glucose or soluble starch as the sole carbon source were analyzed. Transcription levels were highest in medium supplemented with soluble starch for the potential glucoamylase encoding gene. Comparison of the structural model of the identified Tetracladium sp. glucoamylase with the solved structure of the Hypocrea jecorina glucoamylase revealed unique structural features that may explain the thermal lability of the glucoamylase from Tetracladium sp. Conclusion The glucoamylase secreted by Tetracladium sp. is a novel cold-adapted enzyme and its properties should render this enzyme suitable for use in industrial processes that require cold-active amylases, such as biofuel production. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0693-x) contains supplementary material, which is available to authorized users.

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 103 -104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc.

The facile synthesis of protein–inorganic hybrid nanoflowers was evaluated for the efficient immobilization of recombinant his-tagged enzymes, which have a broad range of potential applications. In this study, we report the preparation of a metal–protein hybrid nanoflower system for efficient immobilization of the recombinant enzymes L-arabinitol 4-dehydrogenase from Hypocrea jecorina (HjLAD) and NADH oxidase from Streptococcus pyogenes (SpNox). Compared with free enzymes, synthesized hybrid nanoflowers exhibited enhanced enzymatic activities of 246 and 144% for HjLAD and SpNox, respectively. We have demonstrated that immobilized enzymes retained high catalytic activity and improved the tolerance towards pH and temperature changes. Synthesized nanoflowers also retained high storage stability and reusability. In addition, the immobilized enzymes exhibited significantly enhanced L-xylulose production under co-factor regeneration conditions than the free enzyme combination. These results demonstrate that variations in the concentration of metals and synthesis conditions of nanoflowers can be extended to efficiently immobilize recombinant his-tagged enzymes.

A new nitrogen-containing compound, trichothioneic acid, was discovered from the metabolites of fungal strain FKI-7573 using a mass spectrometry screening method guided by odd number of molecular weights, which indicates compounds that contain an odd number of nitrogen atoms. Strain FKI-7573 was isolated from soil collected in Obihiro, Hokkaido, Japan, and identified as Trichoderma virens by a sequence analysis of the internal transcribed spacer region, including 5.8S ribosomal RNA. The structure of trichothioneic acid was determined by mass spectrometry, nuclear magnetic resonance spectroscopy, electronic circular dichroism spectra, and chemical degradation analyses. These analyses revealed that trichothioneic acid consists of heptelidic acid and l-ergothioneine, and contains three nitrogen atoms. Trichothioneic acid exhibited hydroxyl radical-scavenging and singlet oxygen-quenching activities.

Water-soluble kraft lignin-based polyoxyethylene ether (KL-PEG), synthesized from the black liquor of kraft pulping and PEG, was used to improve the enzymatic hydrolysis efficiency of dilute acid pretreated (DA-pretreated) Eucalyptus hardwood and cellulase stability. The physicochemical properties of KL-PEG polymer such as solubility, surface tension, charge and aggregation behavior in the solution were first studied. KL-PEG could enhance the enzymatic hydrolysis of Avicel and DA-pretreated Eucalyptus from 63.6% and 58.3% to 78.5% and 93.8%, respectively. The enzymatic activity of cellulase after the enzymatic hydrolysis of Avicel and DA-pretreated Eucalyptus for 72 h remained approximately 84% and 44% in the presence of KL-PEG polymer. KL-PEG could improve the stability and longevity of the cellulase, facilitate the recovery and save the amount of cellulase. The efficient utilization of the pulping black liquor lignin was of great significance to alleviate the pressure brought by the shortage of petrochemical resources, and build an energy-saving and low-carbon society.

β-Glucosidases enhance enzymatic biomass conversion by relieving cellobiose inhibition of endoglucanases and cellobiohydrolases. However, the susceptibility of these enzymes to inhibition and transglycosylation at high glucose or cellobiose concentrations severely limits their activity and, consequently, the overall efficiency of enzyme mixtures. We determined the impact of these two processes on the hydrolytic activity of the industrially relevant family 3 β-glucosidases from Hypocrea jecorina, HjCel3A and HjCel3B, and investigated the underlying molecular mechanisms through kinetic studies, binding free energy calculations, and molecular dynamics (MD) simulations. HjCel3B had a 7-fold higher specificity for cellobiose than HjCel3A but greater tendency for glucose inhibition. Energy decomposition analysis indicated that cellobiose has relatively weak electrostatic interactions with binding site residues, allowing it to be easily displaced by glucose and free to inhibit other hydrolytic enzymes. HjCel3A is, thus, preferable as an industrial β-glucosidase despite its lower activity caused by transglycosylation. This competing pathway to hydrolysis arises from binding of glucose or cellobiose at the product site after formation of the glycosyl-enzyme intermediate. MD simulations revealed that binding is facilitated by hydrophobic interactions with Trp-37, Phe-260, and Tyr-443. Targeting these aromatic residues for mutation to reduce substrate affinity at the product site would therefore potentially mitigate transglycosidic activity. Engineering improved variants of HjCel3A and other structurally similar β-glucosidases would have a significant economic effect on enzymatic biomass conversion in terms of yield and production cost as the process can be consequently conducted at higher substrate loadings.

The genus Trichoderma is ubiquitous in the environment, particularly in soils. Trichoderma species could be readily isolated from soil by all available conventional methods, largely because they grow rapidly and also because of their abundant conidiation. Based on the phylogenetic study, several researchers reported that Trichoderma and Hypocrea form a single holomorph genus, within which two major clades can be distinguished. The species of Trichoderma possess diverse biotechnological applications such as they act as biofungicide for controlling various plant diseases, as biofertilizers for plant growth promotion. Trichoderma secrete diverse volatile compounds including alcohols, aldehydes and ketones, ethylene, hydrogen cyanide, and monoterpenes, as well as nonvolatile compounds including peptaibols and diketopiperazine-like gliotoxin and gliovirin which are known to exhibit antibiotic activity. The interaction of Trichoderma with the host plant results in parasitism/predation; production of antibiotic is combined with mycoparasitism (penetration and infection), production of cell wall-degrading enzymes or lytic enzymes, competition for nutrients or for space, and establishment of induced resistance in the plant.

Background The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) displays increased cellulase expression while growing on inducers such as lactose or cellulose. However, the mechanism of cellulase induction in T. reesei is not yet completely characterized. Here, a protein annotated as β-glucosidase (BGL3I) was found to be involved in cellulase induction in T. reesei. The effects of BGL3I on cellulase production have not yet been fully understood. Results Deletion of the bgl3i gene had no influence on the growth of T. reesei, but significantly increased its cellulase activities. Deletion of bgl3i also resulted in decreased extracellular galactosidase activity, but significantly increased transcription of lactose permeases, which might be involved in lactose transport. Furthermore, deletion of bgl3i enhanced the transcription levels of intracellular β-glucosidases cel1a, cel1b and the regulator xyr1, which are all essential for lactose induction in T. reesei. BGL3I was found to have a relatively high ability to hydrolyze sophorose, which is proposed to be the strongest natural inducer of cellulase synthesis in T. reesei. Conclusions BGL3I may take part in the complex regulating system of cellulase induction. The deletion of bgl3i offers a new strategy to improve T. reesei strain performance.

Trichoderma virens genome harbors two isoforms of GAPDH, one (gGPD) involved in glycolysis and the other one (vGPD) in secondary metabolism. vGPD is expressed as part of the “vir” cluster responsible for the biosynthesis of volatile sesquiterpenes. The secondary metabolism-associated GAPDH is tolerant to the anti-cancer metabolite heptelidic acid (HA), produced by T. virens. Characterizing the HA-tolerant form of GAPDH, thus has implications in cancer therapy. In order to get insight into the mechanism of HA-tolerance of vGPD, we have purified recombinant form of this protein. The protein displays biochemical and biophysical characteristics analogous to the gGPD isoform. It exists as a tetramer with Tm of about 56.5 °C, and displays phosphorylation enzyme activity with Km and Kcat of 0.38 mM and 2.55 sec−1, respectively. The protein weakly binds to the sequence upstream of the vir4 gene that codes for the core enzyme (a terpene cyclase) of the “vir” cluster. The EMSA analysis indicates that vGPD may not act as a transcription factor driving the “vir” cluster, at least not by directly binding to the promoter region. We also succeeded in obtaining small crystals of this protein. We have constructed structural models of vGPD and gGPD of T. virens. In silico constrained docking analysis reveals weaker binding of heptelidic acid in vGPD, compared to gGPD protein.

Eight cadinane derivatives, trichocadinins H − N (1–7) and methylhydroheptelidate (8), and two carotane derivatives, 14-O-methyltrichocarotin G (9) and 14-O-methyl CAF-603 (10), including eight new ones (1–6, 9, and 10), were isolated from the culture of Trichoderma virens RR-dl-6-8 obtained from the organohalogen-enriched marine red alga Rhodomela confervoides. Their structures and relative configurations were established by analysis of NMR and mass spectroscopic data, and the absolute configurations were assigned on the basis of ECD curves, highlighted by the ECD diversity of carboxylic acid derivatives. Among the isolates, 1 with a halogen atom and 8, a new naturally occurring compound, are 2,3-seco-cadinane sesquiterpenes, and the epimeric 2 and 3 feature a 2-nor-cadinane skeleton. A commercially-sourced compound with the same planar structure as that of 7 has been reported in a patent, but its configuration was not given. Compounds 1–10 exhibited growth inhibition of some marine phytoplankton species.

Preconditioning of lignocellulosic biomass unfortunately leads to the formation of degradation byproducts that severely inhibit the subsequent enzymatic hydrolysis. This study attempts to prioritize these degradation compounds such that a basis for their mitigation can be developed. Rice straw prehydrolyzates, produced by hot water pretreatment at 220 °C and 52 min, were fractionated using centrifugal partition chromatography (CPC) into phenolics, furans, organic acids, monomeric and oligomeric sugars. Purified inhibitors were tested against substrate conversion efficiencies of exo-cellulase enzyme from Hypocrea jecorina. Results showed that rice straw phenolics at 1 g/L reduced the specific hydrolysis rate by 92% compared to that of control. It was followed by acetic acid, which reduced enzyme efficiency by 87% at 1 g/L. The CPC purified xylo-oligosaccharides only inhibited the initial substrate hydrolysis rate of exo-cellulase, that which recovered over time and was comparable to that of the control aft...

A bottleneck in enzymatic starch hydrolysis, like in biofuel industry, is relatively slow degradation of branched structures compared to linear ones. This research aimed to evaluate glucoamylases for their activity towards branched gluco-oligosaccharides. The activity of seven modified glucoamylases and two homologs was compared to that of a reference glucoamylase obtained from a commercial enzyme cocktail 'Distillase® SSF'. All enzymes were evaluated for their activity towards panose (glc(α1-6)glc(α1-4)glc), pullulan and a purified branched gluco-oligosaccharide with a degree of polymerisation of 5 (bDP5) identified as glc(α1-4)[glc(α1-4)glc(α1-6)]glc(α1-4)glc. The enzymes degraded bDP5 differently, which was mainly due to variation in their capability to cleave α-(1→6)-linked or the α-(1→4)-linked glucosyl residue at the non-reducing end of the branched glucosyl residue. By comparing the enzyme activity towards bDP5 with those towards panose and pullulan, it was suggested that the activity towards bDP5 could be estimated only when the activity towards both commercial substrates was evaluated.

Natural products are a crucial source of antimicrobial agents, but reliance on low-resolution bioactivity-guided approaches has led to diminishing interest in discovery programmes. Here, we demonstrate that two in-house automated informatic platforms can be used to target classes of biologically active natural products, specifically, peptaibols. We demonstrate that mass spectrometry-based informatic approaches can be used to detect natural products with high sensitivity, identifying desired agents present in complex microbial extracts. Using our specialised software packages, we could elaborate specific branches of chemical space, uncovering new variants of trichopolyn and demonstrating a way forward in mining natural products as a valuable source of potential pharmaceutical agents.

Cel3A from the thermophilic fungus R. emersonii has proven to be more efficient in the hydrolysis of β-glycosidic linkages than Cel3A from H. jecorina., The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase from H. jecorina (HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

An efficient biocatalytic cell-free system containing L-arabinitol dehydrogenase (LAD) for L-arabinitol oxidation and NADH oxidase (Nox) for cofactor regeneration was successfully constructed and used for L-rare sugar production. The recombinant LAD (HjLAD) from Hypocrea jecorina suffered from NADH inhibition. Applying Nox to the cell-free HjLAD system drove the thermodynamically unfavorable equilibrium from L-arabinitol to L-xylulose, along with the regeneration of the oxidized cofactor NAD+. Response surface methodology was used to optimize the conditions for L-xylulose production. The optimal conditions for biocatalytic production of L-xylulose were found to be 4.9 U mL−1 HjLAD, 8.2 mM NAD+ at pH 8.0, and 30.9 °C; a high conversion rate of 78.4% was achieved under these conditions. We demonstrate a cell-free enzyme coupling system enabling efficient cofactor recycling and providing high yields of L-xylulose. The results indicate that this coupling system provides a new biocatalytic method for L-rare sugar production.

Thermal inactivation kinetics of Hypocrea orientalis β-glucosidase and effect of glucose on thermostability of the enzyme have been determined in this paper. Kinetic studies showed that the thermal inactivation was irreversible and first-order reaction. The microscopic rate constants for inactivation of free enzyme and substrate-enzyme complex were both determined, which suggested that substrates can protect β-glucosidase against thermal deactivation effectively. On the other hand, glucose was found to protect β-glucosidase from heat inactivation to remain almost whole activity below 70°C at 20mM concentration, whereas the apparent inactivation rate of BG decreased to be 0.3×10(-3)s(-1) in the presence of 5mM glucose, smaller than that of sugar-free enzyme (1.91×10(-3)s(-1)). The intrinsic fluorescence spectra results showed that glucose also had stabilizing effect on the conformation of BG against thermal denaturation. Docking simulation depicted the interaction mode between glucose and active residues of the enzyme to produce stabilizing effect.

Highlights • Cladosporium halotolerans removes 96 % of Mn2+ in two weeks by chelating mechanisms. • Hypocrea jecorina removes Mn2+ by oxidizing mechanisms with 56 % balance. • Laccases could be involved in Mn removal mechanisms played by C. halotolerans., Manganese (Mn) is toxic at higher concentrations requiring its removal before returning the wastewater to the environment. This article reported the Mn removal of two fungi strains isolated from mine wastewater. ITS rRNA region sequencing identified the fungi strains as Cladosporium halotolerans and Hypocrea jecorina. Mn2+ removal assays were performed in Sabouraud broth with 50 mg L−1 Mn2+ supplemented and bioleaching assays using MnO2 instead of MnSO4 at the same conditions. C. halotolerans removed 96 % of 50 mg L−1 Mn2+ at two weeks without MnO2 bioleaching with 649.9 mg of biomass and H. jecorina removed about 50 % of Mn2+ in 21 days from initial 50 mg of Mn2+ L-1 with 316.8 mg of biomass. Extracellular laccases were present in C. halotolerans agar regardless of the Mn addition. Mn adsorbed was detected on C. halotolerans hyphae. Mn oxidation was positive to H. jecorina by reaction of its medium with Leucoberbelin blue.

Ras-GTPases are nucleotide hydrolases involved in key cellular processes. In fungi, Ras-GTPases regulate conidiation, development, virulence, and interactions with other fungi or plants. Trichoderma spp. are filamentous saprophytic fungi, widely distributed along all latitudes, characterized by their rapid growth and metabolic diversity. Many species of this genus interact with other fungi, animals or plants. Furthermore, these fungi are used as biocontrol agents due to their ability to antagonize phytopathogenic fungi and oomycetes, through competence, antibiosis, and parasitism. However, the genetic and molecular regulation of these processes is scarcely described in these fungi. In this work, we investigated the role of the gene tbrg-1 product (GenBank accession number XP_013956100; JGI ID: Tv_70852) of T. virens during its interaction with other fungi and plants. Sequence analyses predicted that TBRG-1 bears the characteristic domains of Ras-GTPases; however, its size (1011 aa) is 3- to 4-times bigger compared with classical GTPases. Interestingly, phylogenetic analyses grouped the TBRG-1 protein with hypothetical proteins of similar sizes, sharing conserved regions; whereas other known Ras-GTPases were perfectly grouped with their respective families. These facts led us to classify TBRG-1 into a new family of Ras-GTPases, the Big Ras-GTPases (BRG). Therefore, the gene was named tbrg-1 (TrichodermaBigRas-GTPase-1). Quantification of conidia and scanning electron microscopy showed that the mutants-lacking tbrg-1 produced less conidia, as well as a delayed conidiophore development compared to the wild-type (wt). Moreover, a deregulation of conidiation-related genes (con-10, con-13, and stuA) was observed in tbrg-1-lacking strains, which indicates that TBRG-1 is necessary for proper conidiophore and conidia development. Furthermore, the lack of tbrg-1 affected positively the antagonistic capability of T. virens against the phytopathogens Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum, which was consistent with the expression patterns of mycoparasitism-related genes, sp1 and cht1, that code for a protease and for a chitinase, respectively. Furthermore, the antibiosis effect of mycelium-free culture filtrates of Δtbrg-1 against R. solani was considerably enhanced. The expression of secondary metabolism-related genes, particularly gliP, showed an upregulation in Δtbrg-1, which paralleled an increase in gliotoxin production as compared to the wt. These results indicate that TBRG-1 plays a negative role in secondary metabolism and antagonism. Unexpectedly, the biocontrol activity of Δtbrg-1 was ineffective to protect the tomato seeds and seedlings against R. solani. On the contrary, Δtbrg-1 behaved like a plant pathogen, indicating that TBRG-1 is probably implicated in the recognition process for establishing a beneficial relationship with plants.

Production of xylanase enzyme by fungi strains has gained interest in the recent years due to its high productivity, high catalytic power, as well as its potential applications in different areas such as feed, food, textile, and biofuel industries. The conventional methodologies, to produce enzymes, involve complex apparatus and chemical solvents and are associated with high costs and low-yield recovery. To obtain the high-yield recovery of the enzymes, modern enzyme extraction methods are taken into account. Aqueous two-phase systems (ATPS) are an alternative separative methodology for the purification and recovery of the enzymes and other biomolecules. The advantages of ATPS are easy scale-up and extraction, volume reduction, and rapid separation. The objective of this study was to produce Trichoderma harzianum xylanase by solid-state fermentation (SSF) using corn cobs as a support/substrate and employing ATPS for its partial recovery. In this study, the results showed the ability of a microorganism to grow on the corn cobs and to produce the xylanase enzyme. Xylanolytic activity reached 7.85 U/g of corn cobs. The enzyme was efficiently concentrated by ATPS. In addition, a high purification factor (10-fold) and considerable enzyme recovery (%ER) (84%) percentage were obtained. Fil: Ricardo GOMEZ GARCIA. Autonomous University Of Coahuila, Saltillo, Coahuila,; México Fil: Miguel MEDINA-Morales. Autonomous University Of Coahuila, Saltillo, Coahuila,; México Fil: Raúl RODRIGUEZ. Autonomous University Of Coahuila, Saltillo, Coahuila,; México Fil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina Fil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina Fil: Cristobal AGUILAR. Autonomous University Of Coahuila, Saltillo, Coahuila,; México

At the catalytic site for the hydrolysis of cellulose the enzyme cellobiohydrolase Cel7A binds the enantiomers of the adrenergic beta-blocker propranolol with different selectivity. Methyl-to-hydroxymethyl group modifications of propranolol, which result in higher affinity and improved selectivity, were herein studied by 1 H,1 H and 1 H,13 C scalar spin-spin coupling constants as well as utilizing the nuclear Overhauser effect (NOE) in conjunction with molecular dynamics simulations of the ligands per se, which showed the presence of all-antiperiplanar conformations, except for the one containing a vicinal oxygen-oxygen arrangement governed by the gauche effect. For the ligand-protein complexes investigated by NMR spectroscopy using, inter alia, transferred NOESY and saturation-transfer difference (STD) NMR experiments the S-isomers were shown to bind with a higher affinity and a conformation similar to that preferred in solution, in contrast to the R-isomer. The fact that the S-form of the propranolol enantiomer is pre-arranged for binding to the protein is also observed for a crystal structure of dihydroxy-(S)-propranolol and Cel7A presented herein. Whereas the binding of propranolol is entropy driven, the complexation with the dihydroxy analogue is anticipated to be favored also by an enthalpic term, such as for its enantiomer, that is, dihydroxy-(R)-propranolol, because hydrogen-bond donation replaces the corresponding bonding from hydroxyl groups in glucosyl residues of the natural substrate. In addition to a favorable entropy component, albeit lesser in magnitude, this represents an effect of enthalpy-to-entropy compensation in ligand-protein interactions.

Trichoderma reesei (syn. Hypocrea jecorina) is the model organism for industrial production of plant cell wall degradating enzymes. The integration of light and nutrient signals for adaptation of enzyme production in T. reesei emerged as an important regulatory mechanism to be tackled for strain improvement. Gene regulation specific for cellulase inducing conditions is different in light and darkness with substantial regulation by photoreceptors. Genes regulated by light are clustered in the genome, with several of the clusters overlapping with CAZyme clusters. Major cellulase transcription factor genes and at least 75% of glycoside hydrolase encoding genes show the potential of light dependent regulation. Accordingly, light dependent protein complex formation occurs within the promoters of cellulases and their regulators. Additionally growth on diverse carbon sources is different between light and darkness and dependent on the presence of photoreceptors in several cases. Thereby, also light intensity plays a regulatory role, with cellulase levels dropping at higher light intensities dependent in the strain background. The heterotrimeric G-protein pathway is the most important nutrient signaling pathway in the connection with light response and triggers posttranscriptional regulation of cellulase expression. All G-protein alpha subunits impact cellulase regulation in a light dependent manner. The downstream cAMP pathway is involved in light dependent regulation as well. Connections between the regulatory pathways are mainly established via the photoreceptor ENV1. The effect of photoreceptors on plant cell wall degradation also occurs in the model filamentous fungus Neurospora crassa. In the currently proposed model, T. reesei senses the presence of plant biomass in its environment by detection of building blocks of cellulose and hemicellulose. Interpretation of the respective signals is subsequently adjusted to the requirements in light and darkness (or on the surface versus within the substrate) by an interconnection of nutrient signaling with light response. This review provides an overview on the importance of light, photoreceptors and related signaling pathways for formation of plant cell wall degrading enzymes in T. reesei. Additionally, the relevance of light dependent gene regulation for industrial fermentations with Trichoderma as well as strategies for exploitation of the observed effects are discussed.

Trichoderma reesei is the anamorph form of Hypocrea jecorina (H. jecorina) and belongs to a soft rot ascomycetal fungus that is used in commercial applications, such as the production of enzymes; this fungus is an efficient cell factory for protein production, a property that is exploited by the enzyme industry. The most important property of H. jecorina for commercial applications is that it can secrete a variety of cellulases involved in lignocellulose hydrolysis, a property that has made H. jecorina the most widely used cellulase in the world, producing filamentous fungi for research and other applications. In this chapter, the functions of transcription factors that regulate cellulase and hemicellulase will be introduced, and the expression of cellulases and hemicellulases regulated by chromatin remodeling will also be discussed. Furthermore, the transcriptional regulation of cellulase and hemicellulase in H. jecorina by external signals will be discussed.