Crosstalk between Ras and inositol phosphate signaling revealed by lithium action on inositol monophosphatase in Schizophyllum commune.

Mushroom forming basidiomycete Schizophyllum commune has been used as a tractable model organism to study fungal sexual development. Ras signaling activation via G-protein-coupled receptors (GPCRs) has been postulated to play a significant role in the mating and development of S. commune. In this study, a crosstalk between Ras signaling and inositol phosphate signaling by inositol monophosphatase (IMPase) is revealed. Constitutively active Ras1 leads to the repression of IMPase transcription and lithium action on IMPase activity is compensated by the induction of IMPase at transcriptome level. Astonishingly, in S. commune lithium induces a considerable shift to inositol phosphate metabolism leading to a massive increase in the level of higher phosphorylated inositol species up to the inositol pyrophosphates. The lithium induced metabolic changes are not observable in a constitutively active Ras1 mutant. In addition to that, proteome profile helps us to elucidate an overview of lithium action to the broad aspect of fungal metabolism and cellular signaling. Taken together, these findings imply a crosstalk between Ras and inositol phosphate signaling.

Metal release and sequestration from black slate mediated by a laccase of Schizophyllum commune.

Schizophyllum commune is a filamentous basidiomycete which can degrade complex organic macromolecules like lignin by the secretion of a large repertoire of enzymes. One of these white rot enzymes, laccase, exhibits a broad substrate specificity and is able to oxidize a variety of substances including carbonaceous rocks. To investigate the role of laccase in bioweathering, laccase gene lcc2 was overexpressed, and the influence on weathering of black slate, originating from a former alum mine in Schmiedefeld, Germany, was examined. The metal release from the rock material was enhanced, associated with a partial metal accumulation into the mycelium. A sequestration of metals could be shown with fluorescent staining methods, and an accumulation of Zn, Cd, and Pb was visualized in different cell organelles. Additionally, we could show an increased metal resistance of the laccase overexpressing strain.

Comparison of Mono- and Dikaryotic Medicinal Mushrooms Lignocellulolytic Enzyme Activity.

Mono- and dikaryotic medicinal mushroom strains isolated from four wood-rotting basidiomycete fruiting bodies were comparatively evaluated for laccase, manganese peroxidase, cellulase, and xylanase activities in submerged cultivation in glucose or mandarin peel-containing media. Mandarin peels appeared to be the preferred growth substrate for laccase production by both mono- and dikaryotic Trametes multicolor 511 and T. versicolor 5 while glucose favored laccase activity secretion by Pleurotus ostreatus 2175. Lignocellulose-deconstructing enzyme profiles were highly variable between the studied monokaryotic and dikaryotic strains. A distinctive superiority of enzyme activity of the dikaryotic Trametes versicolor 5 and P. ostreatus 2175 over the same species monokaryotic isolates was revealed. By contrast, laccase, cellulase, and xylanase activities of the monokaryotic strain of T. multicolor 511 were rather higher than those in the dikaryotic culture. At the same time, hydrolases activity of Schizophyllum commune 632 was practically independent of the origin of the fungal culture. The results suggest that the monokaryotic isolates derived from the basidiomycetes fruiting bodies inherit parental properties but the capacity of individual monokaryotic cultures to produce lignocellulose-deconstructing enzymes can vary considerably.

Schizophyllum commune: An unexploited source for lignocellulose degrading enzymes.

Lignocellulose represents the most abundant source of carbon in the Earth. Thus, fraction technology of the biomass turns up as an emerging technology for the development of biorefineries. Saccharification and fermentation processes require the formulation of enzymatic cocktails or the development of microorganisms (naturally or genetically modified) with the appropriate toolbox to produce a cost-effective fermentation technology. Therefore, the search for microorganisms capable of developing effective cellulose hydrolysis represents one of the main challenges in this era. Schizophyllum commune is an edible agarical with a great capability to secrete a myriad of hydrolytic enzymes such as xylanases and endoglucanases that are expressed in a high range of substrates. In addition, a large number of protein-coding genes for glycoside hydrolases, oxidoreductases like laccases (Lacs; EC 1.10.3.2), as well as some sequences encoding for lytic polysaccharide monooxygenases (LPMOs) and expansins-like proteins demonstrate the potential of this fungus to be applied in different biotechnological process. In this review, we focus on the enzymatic toolbox of S. commune at the genetic, transcriptomic, and proteomic level, as well as the requirements to be employed for fermentable sugars production in biorefineries. At the end the trend of its use in patent registration is also reviewed.

Enhanced delignification of lignocellulosic substrates by Pichia GS115 expressed recombinant laccase.

Utilization of energy-rich crop residues by ruminants is restricted by the presence of lignin, which is recalcitrant to digestion. Application of lignin degrading enzymes on the lignocellulosic biomass exposes the cellulose for easy digestion by ruminants. Laccases have been found to be considerably effective in improving the digestibility by way of delignification. However, laccase yields from natural hosts are not sufficient for industrial scale applications, which restricts their use. A viable option would be to express the laccase gene in compatible hosts to achieve higher production yields. A codon-optimized synthetic variant of Schizophyllum commune laccase gene was cloned into a pPIC9K vector and expressed in P. pastoris GS115 (his4) under the control of an alcohol oxidase promoter. Colonies were screened for G418 resistance and the methanol utilization phenotype was established. The transformant yielded a laccase activity of 344 U·mL-1 after 5 days of growth at 30°C (0.019 g·mL-1 wet cell weight). The laccase protein produced by the recombinant Pichia clone was detected as two bands with apparent molecular weights of 55 kDa and 70 kDa on SDS-PAGE. Activity staining on native PAGE confirmed the presence of bioactive laccase. Treatment of five common crop residues with recombinant laccase recorded a lignin loss ranging between 1.64% in sorghum stover, to 4.83% in finger millet, with an enhancement in digestibility ranging between 8.71% in maize straw to 24.61% in finger millet straw. Treatment with recombinant laccase was effective in enhancing the digestibility of lignocellulosic biomass for ruminant feeding through delignification. To date, a number of hosts have been adventured to produce laccase in large quantities, but, to our knowledge, there are no reports of the expression of laccase protein from Schizophyllum commune in Pichia pastoris, and also on the treatment of crop residues using recombinant laccase for ruminant feeding.

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and ß-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external ß-xylosidase.

Stone-Eating Fungi: Mechanisms in Bioweathering and the Potential Role of Laccases in Black Slate Degradation With the Basidiomycete Schizophyllum commune.

Many enzymes, such as laccases, are involved in the saprotrophic lifestyle of fungi and the effects of those may be linked to enhanced bioweathering on stone surfaces. To test this hypothesis, we studied the decomposition of kerogen-enriched lithologies, especially with black slate containing up to 20% of Corg. Indeed, a formation of ditches with attached hyphal material could be observed. To address enzymes involved, proteomics was performed and one group of enzymes, the multicopper oxidase family members of laccases, was specifically investigated. A role in bioweathering of rocks containing high contents of organic carbon in the form of kerogen could be shown using the basidiomycete Schizophyllum commune, a white rot fungus that has been used as a model organism to study the role of filamentous basidiomycete fungi in bioweathering of black slate.

Proteomic characterization and schizophyllan production by Schizophyllum commune ISTL04 cultured on Leucaena leucocephala wood under submerged fermentation.

In this study Schizophyllum commune ISTL04 was grown on Leucaena leucocephala wood (LLW) for secretome analysis and schizophyllan production. There is no report on extracellular protein profile and schizophyllan production on woody biomass by this fungus under submerged fermentation conditions. Leucaena leucocephala, a promising bioenergy crop having high holocellulose content was used as substrate without pretreatment. The maximum sugar, extracellular protein and exopolysaccharide (EPS) production during fermentation was found to be 8.53±0.07mgmL-1, 391±7.51mgL-1 and 4.2±0.1gL-1 or 0.21gg-1LLW on day 18 respectively. The secretome profile was dominated by glycoside hydrolases followed by carbohydrate esterase and other oxidative enzymes. EPS was further characterized by FTIR and GC-MS for functional group, monomer composition and linkage analysis and was identified as schizophyllan. The result indicated that LLW can be utilized as a low cost substrate for enzyme cocktail and schizophyllan production.

Transcriptional analysis of genes encoding ß-glucosidase of Schizophyllum commune KUC9397 under optimal conditions.

The present study was conducted to determine the gene responsible for beta-glucosidase (BGL) production and to generate a full-length complementary DNA (cDNA) of one of the putative BGL genes, which showed a significant expression level when Schizophyllum commune KUC9397 was grown in optimized medium. The relative expression levels of seven genes encoding BGL of S. commune KUC9397 were determined with real-time quantitative reverse transcription PCR in cellulose-containing optimized medium (OM) compared to glucose-containing basal medium (BM). The most abundant transcript was bgl3a in OM. The transcript number of the bgl3a increased more than 57.60-fold when S. commune KUC9397 was grown on cellulose-containing OM compared to that on glucose-containing BM. The bgl3a was identified, and a deduced amino acid sequence of bgl3a shared homology (97%) with GH3 BGL of S. commune H4-8. This is the first report showing the transcription levels of genes encoding BGL and identification of full-length cDNA of glycoside hydrolase 3 (GH3) BGL from S. commune. Furthermore, this study is one of the steps for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

One-Step Partially Purified Lipases (ScLipA and ScLipB) from Schizophyllum commune UTARA1 Obtained via Solid State Fermentation and Their Applications.

Lipases with unique characteristics are of value in industrial applications, especially those targeting cost-effectiveness and less downstream processes. The aims of this research were to: (i) optimize the fermentation parameters via solid state fermentation (SSF); and (ii) study the performance in hydrolysis and esterification processes of the one-step partially purified Schizophyllum commune UTARA1 lipases. Lipase was produced by cultivating S. commune UTARA1 on sugarcane bagasse (SB) with used cooking oil (UCO) via SSF and its production was optimized using Design-Expert® 7.0.0. Fractions 30% (ScLipA) and 70% (ScLipB) which contained high lipase activity were obtained by stepwise (NH4)2SO4 precipitation. Crude fish oil, coconut oil and butter were used to investigate the lipase hydrolysis capabilities by a free glycerol assay. Results showed that ScLipA has affinities for long, medium and short chain triglycerides, as all the oils investigated were degraded, whereas ScLipB has affinities for long chain triglycerides as it only degrades crude fish oil. During esterification, ScLipA was able to synthesize trilaurin and triacetin. Conversely, ScLipB was specific towards the formation of 2-mono-olein and triacetin. From the results obtained, it was determined that ScLipA and ScLipB are sn-2 regioselective lipases. Hence, the one-step partial purification strategy proved to be feasible for partial purification of S. commune UTARA1 lipases that has potential use in industrial applications.

Microbial Glucuronoyl Esterases: 10 Years after Discovery.

A carbohydrate esterase called glucuronoyl esterase (GE) was discovered 10 years ago in a cellulolytic system of the wood-rotting fungus Schizophyllum commune Genes coding for GEs were subsequently found in a number of microbial genomes, and a new family of carbohydrate esterases (CE15) has been established. The multidomain structures of GEs, together with their catalytic properties on artificial substrates and positive effect on enzymatic saccharification of plant biomass, led to the view that the esterases evolved for hydrolysis of the ester linkages between 4-O-methyl-d-glucuronic acid of plant glucuronoxylans and lignin alcohols, one of the crosslinks in the plant cell walls. This idea of the function of GEs is further supported by the effects of cloning of fungal GEs in plants and by very recently reported evidence for changes in the size of isolated lignin-carbohydrate complexes due to uronic acid de-esterification. These facts make GEs interesting candidates for biotechnological applications in plant biomass processing and genetic modification of plants. This article is a brief summary of current knowledge of these relatively recent and unexplored esterases.

Feruloyl esterases from Schizophyllum commune to treat food industry side-streams.

Agro-industrial side-streams are abundant and renewable resources of hydroxycinnamic acids with potential applications as antioxidants and preservatives in the food, health, cosmetic, and pharmaceutical industries. Feruloyl esterases (FAEs) from Schizophyllum commune were functionally expressed in Pichia pastoris with extracellular activities of 6000UL(-1). The recombinant enzymes, ScFaeD1 and ScFaeD2, released ferulic acid from destarched wheat bran and sugar beet pectin. Overnight incubation of coffee pulp released caffeic (>60%), ferulic (>80%) and p-coumaric acid (100%) indicating applicability for the valorization of food processing wastes and enhanced biomass degradation. Based on substrate specificity profiling and the release of diferulates from destarched wheat bran, the recombinant FAEs were characterized as type D FAEs. ScFaeD1 and ScFaeD2 preferably hydrolyzed feruloylated saccharides with ferulic acid esterified to the O-5 position of arabinose residues and showed an unprecedented ability to hydrolyze benzoic acid esters.

Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

BACKGROUND: The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. RESULTS: MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

Direct ethanol production from cellulosic materials by consolidated biological processing using the wood rot fungus Schizophyllum commune.

In the present study, ethanol production from polysaccharides or wood chips was conducted in a single reactor under anaerobic conditions using the white rot fungus Schizophyllum commune NBRC 4928, which produces enzymes that degrade lignin, cellulose and hemicellulose. The ethanol yields produced from glucose and xylose were 80.5%, and 52.5%, respectively. The absolute yields of ethanol per microcrystalline cellulose (MCC), xylan and arabinogalactan were 0.26g/g-MCC, 0.0419g/g-xylan and 0.0508g/g-arabinogalactan, respectively. By comparing the actual ethanol yields from polysaccharides with monosaccharide fermentation, it was shown that the rate of saccharification was slower than that in fermentation. S. commune NBRC 4928 is concluded to be suitable for CBP because it can produce ethanol from various types of sugar. From the autoclaved cedar chip, only little ethanol was produced by S. commune NBRC 4928 alone but ethanol production was enhanced by combined use of ethanol fermenting and lignin degrading fungi.

Glucuronoyl esterases are active on the polymeric substrate methyl esterified glucuronoxylan.

Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic deesterification was monitored by (1)H NMR spectroscopy and evaluated on the basis of the decrease of the signal of the ester methyl group and increase of the signal of methanol. The results show for the first time the action of enzymes on polymeric substrate, which imitates more closely the natural substrate in plant cell walls than the low molecular mass artificial substrates used up to present.

Active fungal GH115 α-glucuronidase produced in Arabidopsis thaliana affects only the UX1-reactive glucuronate decorations on native glucuronoxylans.

BACKGROUND: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of α-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 α-glucuronidases cleave the internal α-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 α-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2. RESULTS: The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta. CONCLUSIONS: Active GH115 α-glucuronidase has been produced for the first time in plants. The cell wall-targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.

Immobilized lipase from Schizophyllum commune ISTL04 for the production of fatty acids methyl esters from cyanobacterial oil.

Novel lipase from model mushroom Schizophyllum commune strain ISTL04 produced by solid state fermentation of Leucaena leucocephala seeds, was immobilized onto Celite for enzymatic FAMEs production from cyanobacterial endolith Leptolyngbya ISTCY101. The isolate showed vigorous growth and produced remarkable lipase activity of 146.5 U g(-1) dry solid substrate, without any external lipase inducer. Single-factor experiments were carried out to study the effects of various reaction parameters on the FAMEs yield. The best conditions for enzymatic transesterification as revealed by the results were: 1:3 oil to methanol molar ratio, added at 3h intervals, 12% water content, 1581.5 U g(-1) immobilized lipase, temperature 45 °C, and time 24h. Under these conditions, the maximum FAMEs yield reached 94%. The immobilized lipase was able to produce >90% of the relative FAMEs yield after four repeated transesterification cycles. This immobilized lipase exhibited potential for application in biodiesel industry.

Enzymatic degradation of lignin-carbohydrate complexes (LCCs): model studies using a fungal glucuronoyl esterase from Cerrena unicolor.

Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and É£-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis-Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding É£-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass.

Optimization of manganese peroxidase production from Schizophyllum sp. F17 in solid-state fermentation of agro-industrial residues.

Manganese peroxidase (MnP), a crucial enzyme in lignin degradation, has wide potential applications in environmental protection. However, large-scale industrial application of this enzyme is limited due to several factors primarily related to cost and availability. Special attention has been paid to the production of MnP from inexpensive sources, such as lignocellulosic residues, using solid-state fermentation (SSF) systems. In the present study, a suitable SSF medium for the production of MnP by Schizophyllum sp. F17 from agro-industrial residues has been optimized. The mixed solid medium, comprising pine sawdust, rice straw, and soybean powder at a ratio of 0.52:0.15:0.33, conferred a maximum enzyme activity of 11.18 U/g on the sixth day of SSF. The results show that the use of wastes such as pine sawdust and rice straw makes the enzyme production more economical as well as helps solve environmental problems.

Glucoamylase is a major allergen of Schizophyllum commune.

BACKGROUND: Schizophyllum commune is one of the causative agents of basidiomycosis including disorders such as allergic bronchopulmonary mycosis, allergic fungal sinusitis, and mucoid impaction of bronchi, the incidence of those of which has been increasing. These mycoses are difficult to diagnose because only a limited number of diagnostic tools are currently available. The biggest problem is that no specific antigens of S. commune have been identified to enable serodiagnosis of the disease. OBJECTIVE: In this study, we attempted to identify a major antigen of S. commune to establish a reliable serodiagnostic method. METHODS: We used mass spectrometry to identify an antigen that reacted with the serum of a patient with allergic bronchopulmonary mycosis caused by S. commune. The protein was expressed in Escherichia coli, highly purified, and the patient sera IgG and IgE titres against the protein were determined by enzyme-linked immunosorbent assay. RESULTS: The protein identified as a major antigen of S. commune was named Sch c 1; it was a homolog of glucoamylase. The IgG and IgE titres against Sch c 1 in patient sera were significantly higher than those in healthy volunteer sera (P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: Sch c 1 is recognized by the host immune system of patients as an antigen/allergen. The purified glucoamylase Sch c 1 is a promising candidate antigen for the serodiagnosis of S. commune-induced mycosis.