Cell-free production of the bifunctional glycoside hydrolase GH78 from Xylaria polymorpha

The ability to catalyze diverse reactions with relevance for chemical and pharmaceutical research and industry has led to an increasing interest in fungal enzymes. There is still an enormous potential considering the sheer amount of new enzymes from the huge diversity of fungi. Most of these fungal enzymes have not been characterized yet due to the lack of high throughput synthesis and analysis methods. This bottleneck could be overcome by means of cell-free protein synthesis. In this study, cell-free protein synthesis based on eukaryotic cell lysates was utilized to produce a functional glycoside hydrolase (GH78) from the soft-rot fungus Xylaria polymorpha (Ascomycota). The enzyme was successfully synthesized under different reaction conditions. We characterized its enzymatic activities and immobilized the protein via FLAG-Tag interaction. Alteration of several conditions including reaction temperature, template design and lysate supplementation had an influence on the activity of cell-free synthesized GH78. Consequently this led to a production of purified GH78 with a specific activity of 15.4 U mg− 1. The results of this study may be foundational for future high throughput fungal enzyme screenings, including substrate spectra analysis and mutant screenings

Genome and secretome of Chondrostereum purpureum correspond to saprotrophic and phytopathogenic life styles

We thank R. Ullrich for his mycological expertise and K. Eismann for useful technical assistance.The basidiomycete Chondrostereum purpureum (Silverleaf fungus) is a saprotroph and plant pathogen commercially used for combatting forest “weed” trees in vegetation management. However, little is known about its lignocellulose-degrading capabilities and the enzymatic machinery that is responsible for the degradative potential, and it is not yet clear to which group of wood-rot fungi it actually belongs. Here, we sequenced and analyzed the draft genome of C. purpureum (41.2 Mbp) and performed a quantitative proteomic approach during growth in submerged and solid-state cultures based on soybean meal suspension or containing beech wood supplemented with phenol-rich olive mill residues, respectively. The fungus harbors characteristic lignocellulolytic hydrolases (GH6 and GH7) and oxidoreductases (e.g. laccase, heme peroxidases). High abundance of some of these genes (e.g. 45 laccases, nine GH7) can be explained by gene expansion, e.g. identified for the laccase orthogroup ORTHOMCL11 that exhibits a total of 18 lineage-specific duplications. Other expanded genes families encode for proteins more related to a pathogenic lifestyle (e.g. protease and cytochrome P450s). The fungus responds to the presence of complex growth substrates (lignocellulose, phenolic residues) by the secretion of most of these lignocellulolytic and lignin-modifying enzymes (e.g. alcohol and aryl alcohol oxidases, laccases, GH6, GH7). Based on the genetic and enzymatic constitution, we consider the ‘marasmioid’ fungus C. purpureum as a ‘phytopathogenic’ white-rot fungus (WRF) that possesses a complex extracellular enzyme machinery to accomplish efficient lignocellulose degradation during both saprotrophic and phytopathogenic life phases.The work was financially and scientifically supported by the European Union [integrated projects INDOX – KBBE-7-2013-613549; ENZOX2 – 720297], by the DFG project PeroxiDiv HO 1961/8-1 and the AiF project PeroxyMEER IGF 19636 BG/3. The work has been partly funded by the DFG Priority Program 1374 "Infrastructure-Biodiversity-Exploratories" with the projects HO 1961/6-1, KE 1742/2-1 and JE 724/7-4 (AOBJ: 635952) and the Spanish Ministry of Economy and Competitiveness [project AGL2012-32873]. RR thanks the JAE-Program of the Spanish National Research Council [CSIC] and EA thanks MINECO and FEDER Co-Funds [RyC-2013-12481]

Oxidoreductases on their way to industrial biotransformations

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations

Draft Genome Sequence of the Wood-Degrading Ascomycete Kretzschmaria deusta DSM 104547

We report here the draft genome of Kretzschmaria (Ustulina) deusta, an ascomycetous fungus that colonizes and substantially degrades hardwood and can infest living broad-leaved trees. The genome was assembled into 858 contigs, with a total size of 46.5 Mb, and 11,074 protein-coding genes were predicted

Integrated biovalorization of wine and olive mill by-products to produce enzymes of industrial interest and soil amendments

An integral and affordable strategy for the simultaneous production of lignin-modifying and carbohydrate active enzymes and organic amendment, with the aid of a saprobe fungus was developed by using olive oil and wine extraction by-products. The polyporal fungus Trametes versicolor was cultivated in soy or barley media supplemented with dry olive mill residue (DOR) as well as with grape pomace and stalks (GPS) in solid state fermentation (SSF). This strategy led to a 4-fold increase in the activity of laccase, the principal enzyme produced by SFF, in DOR-soy media as compared to controls. T. versicolor managed to secrete lignin-modifying enzymes in GPS, although no stimulative effect was observed. GPS-barley media turned out to be the appropriate medium to elicit most of the carbohydrate active enzymes. The reuse of exhausted solid by-products as amendments after fermentation was also investigated. The water soluble compound polymerization profile of fermented residues was found to correlate with the effect of phytotoxic depletion. The incubation of DOR and GPS with T. versicolor not only reduced its phytotoxicity but also stimulated the plant growth. This study provides a basis for understanding the stimulation and repression of two groups of enzymes of industrial interest in the presence of different carbon and nitrogen sources from by-products, possible enzyme recovery and the final reuse as soil amendments

Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron

Some litter-decaying fungi secrete haem-thiolate peroxygenases that oxidize numerous organic compounds and therefore have a high potential for applications such as the detoxification of recalcitrant organic waste and chemical synthesis. Like P450 enzymes, they transfer oxygen functionalities to aromatic and aliphatic substrates. However, in contrast to this class of enzymes, they only require H2O2 for activity. Furthermore, they exhibit halogenation activity, as in the well characterized fungal chloroperoxidase, and display ether-cleavage activity. The major form of a highly glycosylated peroxygenase was produced from Agrocybe aegerita culture media, purified to apparent SDS homogeneity and crystallized under three different pH conditions. One crystal form containing two molecules per asymmetric unit was solved at 2.2 Å resolution by SAD using the anomalous signal of the haem iron. Subsequently, two other crystal forms with four molecules per asymmetric unit were determined at 2.3 and 2.6 Å resolution by molecular replacement

First Dye-Decolorizing Peroxidase from an Ascomycetous Fungus Secreted by Xylaria grammica

Background: Fungal DyP-type peroxidases have so far been described exclusively for basidiomycetes. Moreover, peroxidases from ascomycetes that oxidize Mn2+ ions are yet not known. Methods: We describe here the physicochemical, biocatalytic, and molecular characterization of a DyP-type peroxidase (DyP, EC 1.11.1.19) from an ascomycetous fungus. Results: The enzyme oxidizes classic peroxidase substrates such as 2,6-DMP but also veratryl alcohol and notably Mn2+ to Mn3+ ions, suggesting a physiological function of this DyP in lignin modification. The KM value (49 µM) indicates that Mn2+ ions bind with high affinity to the XgrDyP protein but their subsequent oxidation into reactive Mn3+ proceeds with moderate efficiency compared to MnPs and VPs. Mn2+ oxidation was most effective at an acidic pH (between 4.0 and 5.0) and a hypothetical surface exposed an Mn2+ binding site comprising three acidic amino acids (two aspartates and one glutamate) could be localized within the hypothetical XgrDyP structure. The oxidation of Mn2+ ions is seemingly supported by four aromatic amino acids that mediate an electron transfer from the surface to the heme center. Conclusions: Our findings shed new light on the possible involvement of DyP-type peroxidases in lignocellulose degradation, especially by fungi that lack prototypical ligninolytic class II peroxidases