Broad-specificity GH131 ß-glucanases are a hallmark of fungi and oomycetes that colonize plants.

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with ß-1,4, ß-1,3 and mixed ß-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.

The ectomycorrhizal basidiomycete Laccaria bicolor releases a secreted ß-1,4 endoglucanase that plays a key role in symbiosis development.

In ectomycorrhiza, root ingress and colonization of the apoplast by colonizing hyphae is thought to rely mainly on the mechanical force that results from hyphal tip growth, but this could be enhanced by secretion of cell-wall-degrading enzymes, which have not yet been identified. The sole cellulose-binding module (CBM1) encoded in the genome of the ectomycorrhizal Laccaria bicolor is linked to a glycoside hydrolase family 5 (GH5) endoglucanase, LbGH5-CBM1. Here, we characterize LbGH5-CBM1 gene expression and the biochemical properties of its protein product. We also immunolocalized LbGH5-CBM1 by immunofluorescence confocal microscopy in poplar ectomycorrhiza. We show that LbGH5-CBM1 expression is substantially induced in ectomycorrhiza, and RNAi mutants with a decreased LbGH5-CBM1 expression have a lower ability to form ectomycorrhiza, suggesting a key role in symbiosis. Recombinant LbGH5-CBM1 displays its highest activity towards cellulose and galactomannans, but no activity toward L. bicolor cell walls. In situ localization of LbGH5-CBM1 in ectomycorrhiza reveals that the endoglucanase accumulates at the periphery of hyphae forming the Hartig net and the mantle. Our data suggest that the symbiosis-induced endoglucanase LbGH5-CBM1 is an enzymatic effector involved in cell wall remodeling during formation of the Hartig net and is an important determinant for successful symbiotic colonization.

Metabolic Engineering of Fusarium oxysporum to Improve Its Ethanol-Producing Capability.

Fusarium oxysporum is one of the few filamentous fungi capable of fermenting ethanol directly from plant cell wall biomass. It has the enzymatic toolbox necessary to break down biomass to its monosaccharides and, under anaerobic and microaerobic conditions, ferments them to ethanol. Although these traits could enable its use in consolidated processes and thus bypass some of the bottlenecks encountered in ethanol production from lignocellulosic material when Saccharomyces cerevisiae is used-namely its inability to degrade lignocellulose and to consume pentoses-two major disadvantages of F. oxysporum compared to the yeast-its low growth rate and low ethanol productivity-hinder the further development of this process. We had previously identified phosphoglucomutase and transaldolase, two major enzymes of glucose catabolism and the pentose phosphate pathway, as possible bottlenecks in the metabolism of the fungus and we had reported the effect of their constitutive production on the growth characteristics of the fungus. In this study, we investigated the effect of their constitutive production on ethanol productivity under anaerobic conditions. We report an increase in ethanol yield and a concomitant decrease in acetic acid production. Metabolomics analysis revealed that the genetic modifications applied did not simply accelerate the metabolic rate of the microorganism; they also affected the relative concentrations of the various metabolites suggesting an increased channeling toward the chorismate pathway, an activation of the γ-aminobutyric acid shunt, and an excess in NADPH regeneration.

A GH115 α-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan.

BACKGROUND: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. RESULTS: We report the characterisation of a recombinant α-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me)GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an α-l-arabinofuranosidase (AbfA), and a ß-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. CONCLUSIONS: Our GH115 α-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.

Challenges in ethanol production with Fusarium oxysporum through consolidated bioprocessing.

Fusarium oxysporum has been reported as being able to both produce the enzymes necessary to degrade lignocellulosic biomass to sugars and also ferment the monosaccharides to ethanol under anaerobic or microaerobic conditions. However, in order to become an economically feasible alternative to other ethanol-producing microorganisms, a better understanding of its physiology, metabolic pathways, and bottlenecks is required, together with an improvement in its efficiency and robustness. In this report, we describe the challenges for the future and give additional justification for our recent publication.

Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates.

The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel® and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS (13)C-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates.

Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism. RESULTS: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source. CONCLUSIONS: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics.

Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris.

Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut(+) strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed.

Assessment of the biomass hydrolysis potential in bacterial isolates from a volcanic environment: biosynthesis of the corresponding activities.

The biomass degrading enzymatic potential of 101 thermophilic bacterial strains isolated from a volcanic environment (Santorini, Aegean Sea, Greece) was assessed. 80 % of the strains showed xylanolytic activity in Congo Red plates, while only eight could simultaneously hydrolyze cellulose. Fifteen isolates were selected on the basis of their increased enzyme production, the majority of which was identified as Geobacilli through 16S rDNA analysis. In addition, the enzymatic profile was evaluated in liquid cultures using various carbon sources, a procedure that revealed lack of correlation on xylanase levels between the two cultivation modes and the inability of solid CMC cultures to fully unravel the cellulose degrading potential of the isolates. Strain SP24, showing more than 99 % 16S DNA similarity with Geobacillus sp. was further studied for its unique ability to simultaneously exhibit cellulase, xylanase, ß-glucosidase and ß-xylosidase activities. The first two enzymes were produced mainly extracellularly, while the ß-glycosidic activities were primarily detected in the cytosol. Maximum enzyme production by this strain was attained using a combination of wheat bran and xylan in the growth medium. Bioreactor cultures showed that aeration was necessary for both enhanced growth and enzyme production. Aeration had a strong positive effect on cellulase production while it negatively affected expression of ß-glucosidase. Xylanase and ß-xylosidase production was practically unaffected by aeration levels.

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum.

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 µM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 µmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 µM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.

Homologous overexpression of xylanase in Fusarium oxysporum increases ethanol productivity during consolidated bioprocessing (CBP) of lignocellulosics.

In an effort to increase ethanol productivity during the consolidated bioprocessing (CBP) of lignocellulosics by Fusarium oxysporum, we attempted the constitutive homologous overexpression of one of the key process enzymes, namely an endo-xylanase. The endo-ß-1,4-xylanase 2 gene was incorporated into the F. oxysporum genome under the regulation of the gpdA promoter of Aspergillus nidulans. The transformation was effected through Agrobacterium tumefaciens and resulted in 12 transformants, two of which were selected for further study due to their high extracellular xylanase activities under normally repressing conditions (glucose as sole carbon source). During natural induction conditions (growth on xylan) though, the extracellular enzyme levels of the transformants were only marginally higher (5-10%) compared to the wild type despite the significantly stronger xylanase 2 mRNA signals. SDS-PAGE verified enzyme assay results that there was no intracellular xylanase 2 accumulation in the transformants, suggesting the potential regulation in a post transcriptional or translational level. The fermentative performance of the transformants was evaluated and compared to that of the wild type in simple CBP systems using either corn cob or wheat bran as sole carbon sources. Both transformants produced approximately 60% more ethanol compared to the wild type on corn cob, while for wheat bran this picture was repeated for only one of them. This result is attributed to the high extracellular xylanase activities in the transformants' fermentation broths that were maintained 2-2.5-fold higher compared to the wild type.