Yitzhak Hadar

Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus

Abstract. The genus Pleurotus comprises a group of edible ligninolytic mushrooms with medicinal properties and important biotechnological and environmental applications. The cultivation of Pleurotus spp is an economically important food industry worldwide which has expanded in the past few years. P. ostreatus is the third most important cultivated mushroom for food purposes. Nutritionally, it has unique flavor and aromatic properties; and it is considered to be rich in protein, fiber, carbohydrates, vitamins and minerals. Pleurotus spp are promising as medicinal mushrooms, exhibiting hematological, antiviral, antitumor, antibiotic, antibacterial, hypocholesterolic and immunomodulation activities. The bioactive molecules isolated from the different fungi are polysaccharides. One of the most important aspects of Pleurotus spp is related to the use of their ligninolytic system for a variety of applications, such as the bioconversion of agricultural wastes into valuable products for animal feed and other food products and the use of their ligninolytic enzymes for the biodegradation of organopollutants, xenobiotics and industrial contaminants. In this Mini-Review, we describe the properties of Pleurotus spp in relation to their biotechnological applications and potential.

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Siderophores of Pseudomonas putida as an iron source for dicot and monocot plants

Iron uptake from ferrated (59Fe) pseudobactin (PSB), a Pseudomonas putida siderophore, by various plant species was studied in nutrient solution culture under short term (10 h) and long term (3 weeks) conditions. In the short term experiments, 59Fe uptake rate from 59FePSB by dicots (peanuts, cotton and sunflower) was relatively low when compared with 59Fe uptake rate from 59FeEDDHA. Iron uptake rate from 59FePSB was pH and concentration dependent, as was the Fe uptake rate from 59FeEDDHA. The rate was about 10 times lower than that of Fe uptake from the synthetic chelate. Results were similar for long term experiments.

The Role of Ligand Exchange in the Uptake of Iron from Microbial Siderophores by Gramineous Plants

The siderophore rhizoferrin, produced by the fungus Rhizopus arrhizus, was previously found to be as an efficient Fe source as Fe-ethylenediamine-di(o-hydroxphenylacetic acid) to strategy I plants. The role of this microbial siderophore in Fe uptake by strategy II plants is the focus of this research. Fe-rhizoferrin was found to be an efficient Fe source for barley (Hordeum vulgare L.) and corn (Zea mays L.). The mechanisms by which these Gramineae utilize Fe from Fe-rhizoferrin and from other chelators were studied. Fe uptake from 59Fe-rhizoferrin, 59Fe-ferrioxamine B, 59Fe-ethylenediaminetetraacetic acid, and 59Fe-2[prime]-deoxymugineic acid by barley plants grown in nutrient solution at pH 6.0 was examined during periods of high (morning) and low (evening) phytosiderophore release. Uptake and translocation rates from Fe chelates paralleled the diurnal rhythm of phytosiderophore release. In corn, however, similar uptake and translocation rates were observed both in the morning and in the evening. A constant rate of the phytosiderophores release during 14 h of light was found in the corn cv Alice. The results presented support the hypothesis that Fe from Fe-rhizoferrin is taken up by strategy II plants via an indirect mechanism that involves ligand exchange between the ferrated microbial siderophore and phytosiderophores, which are then taken up by the plant. This hypothesis was verified by in vitro ligand-exchange experiments.

Oxidative biodegradation of phosphorothiolates by fungal laccase

Organophosphorus (OP) insecticides and nerve agents that contain P‐S bond are relatively more resistant to enzymatic hydrolysis. Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonate) (ABTS) displayed complete and rapid oxidative degradation of the nerve agents VX and Russian VX (RVX) and the insecticide analog diisopropyl‐Amiton with specific activity: k sp=2200, 667 and 1833 nmol min−1 mg−1, respectively (pH 7.4, 37°C). A molar ratio of 1:20 for OP/ABTS and 0.05 M phosphate at pH 7.4 provided the highest degradation rate of VX and RVX. The thermostable laccase purified from the fungus Chaetomium thermophilium (Ct) in the presence of ABTS caused a 52‐fold slower degradation of VX with k sp=42 nmol min−1 mg−1. The enzymatic biodegradation products were identified by 31P‐NMR and GC/MS analysis.

Iron Nutrition and Interactions in Plants

In most soils, FellI oxides (group name) are the common source of Fe for plant nutrition. Since this Fe has to be supplied via solution, the solubility and the dissolution rate of the Fe oxides are essential for the Fe supply. Hydrolysis constants and solubility products (Ksp) describing the effect of pH on FeIII ion concentration in solution are available for the well-known Fe oxides occurring in soils such as goethite, hematite, ferrihydrite. Ksp values are usually extremely low «Fe3+)· (OH)3 = 10-37 _1044 ). However, for each mineral type, Ksp may increase by several orders of magnitude with decreasing crystal size and it decreases with increasing Al substitution assuming ideal solid solution between the pure end-members. Based on such calculations a poorly crystalline goethite with a crystal size of 5 nm may well reach the solubility of ferrihydrite. The variations in Ksp are of relevance for soils because crystal size and Al substitution of soil Fe oxides vary considerably and can now be determined relatively easily. The concentration of Fe2+ in soil solutions is often much higher than that of Fe(llI) ions. Therefore, redox potential strongly influences the activity of Fell. At a given pH and Eh , the activity of Fell is higher the higher Ksp of the FeIII oxide and thus also varies with the type of Fe oxide present. Besides the solubility, it is the dissolution rate which governs the supply of soluble Fe to the plant roots. Dissolution of Fe oxides takes place either by protonation, complexation or, most important, by reduction. Numerous dissolution rate studies with various FellI oxides were conducted in strong mineral acids (protonation) and they have shown that besides the Fe oxide species, crystal size and/or crystal order and substitution are important determinative factors. For example, in soils, small amounts of a more highly soluble metaor instable Fe oxide such as ferrihydrite with a large specific surface (several hundred m2g -1) may be essential for the Fe supply to the plant root. Its higher dissolution rate can also be used to quantify its amount in soils. Ferrihydrite can be an important component in soils with high amounts of organic matter and/or active redox dynamics, whereas highly aerated and strongly weathered soils are usually very low in ferrihydrite. On the other hand, dissolution rates of goethites decrease as their Al substitution increases. Much less information exists on the rate of reductive and chelative dissolution of Fe oxides which generally simulate soil conditions better than dissolution by protonation. Here again, type of oxide, crystal size and substitution are important factors. Organic anions such as oxalate, which are adsorbed at the surface, may weaken the Fe3+ -0 bonds and thereby increase reductive dissolution. As often observed in weathering, the dissolution features of the crystals appear to follow zones of weakness in the crystal.

Cellulase-Xylanase Synergy in Designer Cellulosomes for Enhanced Degradation of a Complex Cellulosic Substrate

ABSTRACT Designer cellulosomes are precision-engineered multienzyme complexes in which the molecular architecture and enzyme content are exquisitely controlled. This system was used to examine enzyme cooperation for improved synergy among Thermobifida fusca glycoside hydrolases. Two T. fusca cellulases, Cel48A exoglucanase and Cel5A endoglucanase, and two T. fusca xylanases, endoxylanases Xyn10B and Xyn11A, were selected as enzymatic components of a mixed cellulase/xylanase-containing designer cellulosome. The resultant mixed multienzyme complex was fabricated on a single scaffoldin subunit bearing all four enzymes. Conversion of T. fusca enzymes to the cellulosomal mode followed by their subsequent incorporation into a tetravalent cellulosome led to assemblies with enhanced activity (~2.4-fold) on wheat straw as a complex cellulosic substrate. The enhanced synergy was caused by the proximity of the enzymes on the complex compared to the free-enzyme systems. The hydrolytic properties of the tetravalent designer cellulosome were compared with the combined action of two separate divalent cellulase- and xylanase-containing cellulosomes. Significantly, the tetravalent designer cellulosome system exhibited an ~2-fold enhancement in enzymatic activity compared to the activity of the mixture of two distinct divalent scaffoldin-borne enzymes. These results provide additional evidence that close proximity between cellulases and xylanases is key to the observed concerted degradation of the complex cellulosic substrate in which the integrated enzymes complement each other by promoting access to the relevant polysaccharide components of the substrate. The data demonstrate that cooperation among xylanases and cellulases can be augmented by their integration into a single designer cellulosome. IMPORTANCE Global efforts towards alternative energy programs are highlighted by processes for converting plant-derived carbohydrates to biofuels. The major barrier in such processes is the inherent recalcitrance to enzymatic degradation of cellulose combined with related associated polysaccharides. The multienzyme cellulosome complexes, produced by anaerobic bacteria, are considered to be the most efficient systems for degradation of plant cell wall biomass. In the present work, we have employed a synthetic biology approach by producing artificial designer cellulosomes of predefined enzyme composition and architecture. The engineered tetravalent cellulosome complexes contain two different types of cellulases and two distinct xylanases. Using this approach, enhanced synergistic activity was observed on wheat straw, a natural recalcitrant substrate. The present work strives to gain insight into the combined action of cellulosomal enzyme components towards the development of advanced systems for improved degradation of cellulosic material. Global efforts towards alternative energy programs are highlighted by processes for converting plant-derived carbohydrates to biofuels. The major barrier in such processes is the inherent recalcitrance to enzymatic degradation of cellulose combined with related associated polysaccharides. The multienzyme cellulosome complexes, produced by anaerobic bacteria, are considered to be the most efficient systems for degradation of plant cell wall biomass. In the present work, we have employed a synthetic biology approach by producing artificial designer cellulosomes of predefined enzyme composition and architecture. The engineered tetravalent cellulosome complexes contain two different types of cellulases and two distinct xylanases. Using this approach, enhanced synergistic activity was observed on wheat straw, a natural recalcitrant substrate. The present work strives to gain insight into the combined action of cellulosomal enzyme components towards the development of advanced systems for improved degradation of cellulosic material.

Niche and host-associated functional signatures of the root surface microbiome

Plant microbiomes are critical to host adaptation and impact plant productivity and health. Root-associated microbiomes vary by soil and host genotype, but the contribution of these factors to community structure and metabolic potential has not been fully addressed. Here we characterize root microbial communities of two disparate agricultural crops grown in the same natural soil in a controlled and replicated experimental system. Metagenomic (genetic potential) analysis identifies a core set of functional genes associated with root colonization in both plant hosts, and metatranscriptomic (functional expression) analysis revealed that most genes enriched in the root zones are expressed. Root colonization requires multiple functional capabilities, and these capabilities are enriched at the community level. Differences between the root-associated microbial communities from different plants are observed at the genus or species level, and are related to root-zone environmental factors.

Suppression of Rhizoctonia solani and Sclerotium rolfsii diseases in container media containing composted separated cattle manure and composted grape marc

Abstract Composts may serve as a substitute for peat as the organic component in container media. Media containing composted grape marc (CGM) or composted separated cattle manure (CSM) were suppressive to diseases caused by Rhizoctonia solani and Sclerotium rolfsii. Radish damping-off severity, as well as disease build-up, were reduced in both CSM or CGM compared with peat. R. solani root rot in pothos was significantly controlled in CGM and CGM-peat mixtures. Both composts were suppressive to disease caused by S. rolfsii in beans and chickpeas. The mechanism of suppression is probably due to the presence of antagonistic microorganisms in the composts, as gamma irradiation of the composts eliminated the suppressive effect. The use of CGM and CSM in container media will provide a high-quality peat substitute suppressive to soil-borne pathogens.

Cartapip™: a biopulping product for control of pitch and resin acid problems in pulp mills

This paper addresses a biological treatment of wood chips using specific isolants of the ascomycete, Ophiostoma piliferum, which results in a reduced extractive content, specifically pitch also known as resin, of the wood chips. Natural isolants of O. piliferum cause blue stain in the sapwood of conifers. Colorless isolants of O. piliferum, isolated in the laboratory, marketed as Cartapip™ have been shown not to cause discoloration of wood. Hyphae preferentially colonize ray parenchyma cells and resin canals. Studies have shown that not only does overall pitch content decrease with treatment of wood chips by the O. piliferum strains tested, but also many free resin acids and fatty acids are significantly reduced. Additionally, treatment of wood chips with the colorless strains of O. piliferum results in biocontrol since other microorganisms including staining organisms have significantly reduced growth on the wood chips. Therefore bleach chemicals can be saved in the processing of mechanical pulp to give higher brightness paper.