Search

1 p., Los Agaricomycetes han desarrollado complejas maquinarias enzimáticas que les permiten descomponer los diferentes polímeros vegetales, incluida la lignina. Entre ellos, los Agaricales saprótrofos se caracterizan por su diversidad de hábitats y estilos de vida. El análisis de 52 genomas de Agaricomycetes aquí realizado revela que los Agaricales poseen una gran diversidad de enzimas hidrolíticas y oxidativas para la descomposición de la lignocelulosa. En base a las familias de genes con mayor velocidad evolutiva (dominios de unión a celulosa, glicosil hidrolasa GH43, monooxigenasas líticas de polisacáridos, peroxidasas ligninolíticas, enzimas de la superfamilia de glucosa-metanol-colina oxidasas/deshidrogenasas, lacasas y peroxigenasas), reconstruimos los estilos de vida de los ancestros que dieron lugar a los actuales Agaricomycetes degradadores de lignocelulosa. Los cambios en el conjunto de herramientas enzimáticas de los Agaricales ancestrales se correlacionaron con la evolución de su capacidad para crecer no solo sobre madera, sino también sobre hojarasca de bosques y madera en descomposición, siendo los descomponedores de la hojarasca de praderas el grupo ecofisiológico más reciente. En este contexto, las anteriores familias de enzimas se analizaron en relación con la diversidad de estilos de vida. Las peroxidasas aparecen como un componente central del set enzimático de los Agaricomycetes saprotrófos, consistente con su papel esencial en la degradación de la lignina y sus altas tasas evolutivas. Esto incluye no solo expansiones/pérdidas de genes de peroxidasas, sino también la presencia generalizada en Agaricales de nuevos tipos de peroxidasas que no se encuentran en Polyporales degradadores de madera, y en otros órdenes de Agaricomycetes., Projectos/contratos BIO2017-86559-R, BIO2015-73697-JIN, AGL2014-55971-R, NSF-grant-1457721, CEFOX-031B0831B, PIE-201620E081, ANR-11-LABX-0002-01, US-DOE-DE-AC02-05CH11231

1 p., Pleurotus eryngii es un hongo de prados y pastizales de interés biotecnológico por su capacidad para transformar el material lignocelulósico no leñoso. En este estudio combinamos análisis transcriptómicos, exoproteómicos y metabolómicos con objeto de ofrecer una explicación sobre los aspectos enzimáticos relacionados con la degradación de la paja de trigo. Durante la fase temprana de crecimiento encontramos un conjunto de enzimas extracelulares inducidas y constitutivas formado por glicosil hidrolasas, polisacárido liasas y carbohidrato esterasas activas sobre polisacáridos,lacasas activas sobre lignina, y una cantidad sorprendente de aril-alcohol oxidasas (AAOs). A tiempos largos identificamos una mayor diversidad y abundancia de enzimas, representada por oxidorreductasas implicadas en la despolimerización de celulosa y lignina, muchas de ellas inducidas desde la fase temprana de crecimiento. Estas enzimas oxidativas incluyeron monooxigenasas líticas de polisacáridos (LPMOs), celobiosa deshidrogenasa implicada en la activación de las LPMOs, y peroxidasas ligninolíticas (principalmente manganeso peroxidasas), junto a una gran abundancia de AAOs productoras de H2O2. Algunas de las enzimas más relevantes activas sobre polisacáridos aparecieron unidas a módulos de unión a celulosa. Esto se relacionó con el hábitat de P. eryngii.También elucidamos aspectos del catabolismo intracelular de compuestos aromáticos, un tema poco investigado en los basidiomicetos degradadores de lignina. Este enfoque multiómico revela que, aunque la descomposición de la paja de trigo no se traduce en grandes cambios (de acuerdo con análisis de 2D-NMR, entre otros), se produce la activación de enzimas hidrolíticas y oxidativas de gran interés biotecnológico en procesos dirigidos al aprovechamiento de la biomasa vegetal., Proyectos GENOBIOREF (BIO2017-86559-R), MICINN (cofinanciado con fondos FEDER); PIE-202120E019 y PIE-201620E081, CSIC; y contratos DE-AC02-05CH11231 y DE-AC36-08GO28308, U.S. DOE

21 p.-4 fig.-1 tab.-10 fig. supl.-2 tab. supl., Dye-decolorizing peroxidase (DyP) from Auricularia auricula-judae and versatile peroxidase (VP) from Pleurotus eryngii oxidize the three mononitrophenol isomers. Both enzymes have been overexpressed in Escherichia coli and in vitro activated. Despite their very different three-dimensional structures, the nitrophenol oxidation site is located at a solvent-exposed aromatic residue in both DyP (Trp377) and VP (Trp164), as revealed by liquid chromatography coupled to mass spectrometry and kinetic analyses of nitrophenol oxidation by the native enzymes and their tryptophan-less variants (the latter showing 10–60 fold lower catalytic efficiencies)., This work was supported by the INDOX European project (KBBE-2013-7-613549) and the BIO2017-86559-R project of the Spanish Ministry of Economy, Industry and Competitiveness, co-financed by FEDER funds.

5 p.-6 fig., To investigate how ligninolytic peroxidases acquired the uniquely high redox potential they show today, their ancestors were resurrected and characterized. Unfortunately, the transient Compounds I (CI) and II (CII) from peroxide activation of the enzyme resting state (RS) are unstable. Therefore, the reduction potentials (E°') of the three redox couples (CI/RS, CI/CII and CII/RS) were estimated (for the first time in a ligninolytic peroxidase) from equilibrium concentrations analyzed by stopped-flow UV/Vis spectroscopy. Interestingly, the E°' of rate-limiting CII reduction to RS increased 70 mV from the common peroxidase ancestor to extant lignin peroxidase (LiP), and the same boost was observed for CI/RS and CI/CII, albeit with higher E°' values. A straightforward correlation was found between the E°' value and the progressive displacement of the proximal histidine Hϵ1 chemical shift in the NMR spectra, due to the higher paramagnetic effect of the heme Fe3+ . More interestingly, the E°' and NMR data also correlated with the evolutionary time, revealing that ancestral peroxidases increased their reduction potential in the evolution to LiP thanks to molecular rearrangements in their heme pocket during the last 400 million years., This work has been funded by the EnzOx2 (BBI‐PPP‐2015‐2‐720297; www.enzox2.eu) EU project, and the CTQ2015‐71290‐R, BFU2016‐77835‐R and BIO2017‐86559‐R projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds.

31 páginas.- 14 figuras.- 92 referencias.- Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/jof7060426/s1 .- Acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes. © 2021 by the authors. Licensee MDPI, Basel, Switzerland., This research was funded by the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Science and Innovation (co-financed by FEDER funds) to F.J.R.-D. and S.C.; and by the Consejo Superior de Investigaciones CientÍficas (PIE-202120E019 to A.T.M. and PIE-201620E081 to A.G.). The work conducted by the JGI, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. DOE under contract DE-AC02-05CH11231. Part of this material is also based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER) under the Early Career Award Program to D.S. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication

24 p.-6 fig.-8 tab., Agaricomycetes fungi responsible for decay of wood and other lignocellulosic substrates constitute a valuable source of lignin-degrading enzymes. Among these enzymes, laccases (multi-copper oxidases) present remarkable biotechnological potential as environmentally friendly biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the only requirement. Laccases from saprotrophic Agaricales species have been much less studied than laccases from Polyporales, despite the fact that the former fungi are excellent sources of laccases. Here, the gene of a novel laccase of Agrocybe pediades, that is secreted by the fungus during lignocellulose degradation, was synthesised de novo and expressed in Saccharomyces cerevisiae using an improved signal peptide previously obtained and enzyme directed evolution. The characterization of the new laccase variants provided new insights on the contribution of different amino acid residues to modulate laccase production, catalytic activity or optimal pH. The selected double-mutated variant also showed interesting properties as a biocatalyst, such as the ability to oxidise a wide range of substrates, including high-redox potential mediators and recalcitrant organic dyes, improved activity at neutral pH and high tolerance to inhibitors. Finally, we demonstrate the existence of three N-glycosylation sites in the laccase and their distinct effect on the secretion or catalytic activity of the enzyme., This work was supported by the GENOBIOREF (BIO2017–86559-R) project of the Spanish Ministry of Science & Innovation (co-financed by FEDER funds), and by the European Project WoodZymes funded by the Bio Based Industries Joint Undertaking, under the European Union’s Horizon 2020 research and innovation program (Grant Agreement H2020-BBI-JU-792070).

23 p.-6 fig.-5 tab., The functional diversity of the New Caledonian mangrove sediments was examined,observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments,collected beneath either Avicennia marina or Rhizophora stylosa trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the R. stylosa area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51–100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in Pichia pastoris. The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical–chemical properties, its ability to oxidize diverse phenolic substrates,and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 ºC, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi., This research was funded by the ECosphère Continentale et Côtière MicroBien program (EC2CO, 2017–2019) and by the Agence Nationale de la Recherche (project PeroxiDiv ANR-RF- 2015-01). D.L. and F.J.R-D. were supported by H2020 BBI-JU (https://www.bbi-europe.eu) EnzOx2 (H2020-BBI-PPP-2015-2-720297; https://www.enzox2.eu) project and GENOBIOREF (BIO2017-86559- R) project of the Spanish Ministry of Science & Innovation (co-financed by FEDER funds).

20 p.-9 fig.-2 tab., A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin., This research was funded by Projects RTI2018-093683-Β-I00 and BIO2017-86559-R, co-financed by the Spanish Ministry of Science and Innovation and FEDER-EU funds, S2018/EMT-4459, from Comunidad de Madrid.

23 p.-7 fig.-4 tab., We aim to clarify the ligninolytic capabilities of dye-decolorizing peroxidases (DyPs) from bacteria and fungi, compared to fungal lignin peroxidase (LiP) and versatile peroxidase (VP). With this purpose, DyPs from Amycolatopsis sp., Thermomonospora curvata, and Auricularia auricula-judae, VP from Pleurotus eryngii, and LiP from Phanerochaete chrysosporium were produced, and their kinetic constants and reduction potentials determined. Sharp differences were found in the oxidation of nonphenolic simple (veratryl alcohol, VA) and dimeric (veratrylglycerol-β- guaiacyl ether, VGE) lignin model compounds, with LiP showing the highest catalytic efficiencies (around 15 and 200 s−1·mM−1 for VGE and VA, respectively), while the efficiency of the A. auricula-judae DyP was 1–3 orders of magnitude lower, and no activity was detected with the bacterial DyPs. VP and LiP also showed the highest reduction potential (1.28–1.33 V) in the rate-limiting step of the catalytic cycle (i.e., compound-II reduction to resting enzyme), estimated by stopped-flow measurements at the equilibrium, while the T. curvata DyP showed the lowest value (1.23 V). We conclude that, when using realistic enzyme doses, only fungal LiP and VP, and in much lower extent fungal DyP, oxidize nonphenolic aromatics and, therefore, have the capability to act on the main moiety of the native lignin macromolecule., This work has been funded by the H2020 BBI-JU (https://www.bbi-europe.eu, accessed on 4 March 2021) EnzOx2 (H2020-BBI-PPP-2015-2-720297; https://www.enzox2.eu, accessed on 4 March 2021) project, the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Science & Innovation (co-financed by FEDER funds), and the CSIC project PIE-201620E081.

19 p.-6 fig., As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates -namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases- we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf-litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes, but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways, and mapped the appearance of the different enzyme types in a time-calibrated species tree., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (BIO2017-86559-R to F.J.R.-D., S.C. and A.T.M., BIO2015-7369-JIN to J.B, and AGL2014-55971-R to A.G.P and L.R., projects cofinanced by FEDER funds); National Science Foundation (grant 1457721 to D.C.); Bundesministerium für Bildung und Forschung (CEFOX 031B0831B to H.K.); Deutsche Forschungsgemeinschaft (Biodiversity-Exploratories BLD-MFD-HZG III, KE 1742/2-1 to H.K.); Consejo Superior de Investigaciones Científicas (PIE-201620E081 to A.T.M.); and the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01), the Region Lorraine, the European Regional Development Fund, and the Plant–Microbe Interfaces Scientific Focus Area in the Genomic Science Program, U.S. DOE Office of Science to F.M. The work conducted by the JGI, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. DOE under contract DE-AC02-05CH11231.

14 páginas.- 6 figuras.- 3 tablas.- referencias.- The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021.740923/full#supplementary-material, In the present work, lignin-like fractions were isolated from several ancestral plants –including moss (Hypnum cupressiforme and Polytrichum commune), lycophyte (Selaginella kraussiana), horsetail (Equisetum palustre), fern (Nephrolepis cordifolia and Pteridium aquilinum), cycad (Cycas revoluta), and gnetophyte (Ephedra fragilis) species– and structurally characterized by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the H. cupressiforme, P. commune and E. palustre “lignins,” where they were practically absent. Additional structural information on the other five samples was obtained from 2D-NMR experiments displaying intense correlations signals of guaiacyl (G) units in the fern and cycad lignins, along with smaller amounts of p-hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte S. kraussiana and the gnetophyte E. fragilis were not only composed of G- and H-lignin units but they also incorporated significant amounts of the syringyl (S) units characteristic of angiosperms, which appeared much later in plant evolution, most probably due to convergent evolution. The latter finding is also supported by the abundance of syringol derivatives after the Py-GC/MS analyses of these two samples. Regarding lignin structure, β−O−4′ alkyl-aryl ethers were the most abundant substructures, followed by condensed β−5′ phenylcoumarans and β−β′ resinols (and dibenzodioxocins in the fern and cycad lignins). The highest percentages of alkyl-aryl ether structures correlated with the higher S/G ratio in the S. Kraussiana and E. fragilis lignin-like fractions. More interestingly, apart from the typical monolignol-derived lignin units (H, G and S), other structures, assigned to flavonoid compounds never reported before in natural lignins (such as amentoflavone, apigenin, hypnogenol B, kaempferol, and naringenin), could also be identified in the HSQC spectra of all the lignin-like fractions analyzed. With this purpose, in vitro synthesized coniferyl-naringenin and coniferyl-apigenin dehydrogenation polymers were used as standards. These flavonoids were abundant in H. cupressiforme appearing as the only constituents of the moss lignin-like fraction (including 84% of dimeric hypnogenol B) and their abundance decreased in those of S. Kraussiana (with amentoflavone and naringenin representing 14% of the total aromatic units), and the two ancient gymnosperms (0.4–1.2%) and ferns (0–0.7%)., This work was funded by the Spanish Ministry of Industry, Economy and Competitiveness projects BIO2017-86559-R, AGL2017-83036-R, and PID2020-118968RB-I00, cofinanced by FEDER funds, by the Junta de Andalucía project P20-00017, and by the CSIC projects PIE-202120E019 and PIE-202040E185. We would like to acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

1 p., Las lignina peroxidasas (LiPs) han recibido especial atención en el contexto de las biorrefinerías de lignocelulosa por su capacidad para oxidar directamente la lignina.Hasta ahora, estas enzimas solo se habían identificado en basidiomicetos del orden Polyporales, que incluye la mayoría de las especies degradadoras de madera.Tras la secuenciación del genoma de Agrocybe pediades [5] identificamos la primera enzima ligninolítica de esta familia en un hongo del orden Agaricales (ApeLiP) degradador de hojarasca. Su estructura cristalográfica reveló que contiene un grupo hemo como centro activo y un triptófano catalítico posicionado en su superficie (Trp166). La caracterización bioquímica de la enzima nativa y de una variante carente de dicho triptófano confirmó que este residuo está involucrado en la oxidación directa de sustratos aromáticos no-fenólicos de alto potencial redox y de compuestos fenólicos, incluyendo monómeros y dímeros modelos de lignina y distintos colorantes. Además, mediante espectroscopía de flujo detenido y 2D-NMR se confirmó que este residuo es responsable de la oxidación directa de lignina real, tanto de angiospermas como de gimnospermas, con una eficiencia igual o superior al de otras peroxidasas ligninolíticas conocidas. La caracterización fue complementada con el estudio de los potenciales de reducción de los intermediarios del ciclo catalítico y con estudios de estabilidad. Así, se observó una activación por peróxido similar a la de otras peroxidasas, pero un menor potencial redox en el paso limitante de la reacción comparado con otras LiPs, así como una alta estabilidad a pH básico. Todos estos estudios demuestran que en hongos Agaricales también existen enzimas relevantes para el reciclado del carbono en la naturaleza que además pueden aprovecharse para su aplicación biotecnológica.

23 páginas.- 7 figuras.- 3 tablas.- 81 referencias.- : The following are available online at https://www.mdpi.com/article/10.3390/antiox10091446/s1 .- Support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Lignin biodegradation has been extensively studied in white-rot fungi, which largely belong to order Polyporales. Among the enzymes that wood-rotting polypores secrete, lignin peroxidases (LiPs) have been labeled as the most efficient. Here, we characterize a similar enzyme (ApeLiP) from a fungus of the order Agaricales (with ~13,000 described species), the soil-inhabiting mushroom Agrocybe pediades. X-ray crystallography revealed that ApeLiP is structurally related to Polyporales LiPs, with a conserved heme-pocket and a solvent-exposed tryptophan. Its biochemical characterization shows that ApeLiP can oxidize both phenolic and non-phenolic lignin model-compounds, as well as different dyes. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, we demonstrate that ApeLiP can also act on real lignin. Characterization of a variant lacking the above tryptophan residue shows that this is the oxidation site for lignin and other high redox-potential substrates, and also plays a role in phenolic substrate oxidation. The reduction potentials of the catalytic-cycle intermediates were estimated by stopped-flow in equilibrium reactions, showing similar activation by H2O2, but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Unexpectedly, ApeLiP was stable from acidic to basic pH, a relevant feature for application considering its different optima for oxidation of phenolic and nonphenolic compounds., This research was funded by the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Science & Innovation (co-financed by FEDER funds) to F.J.R.-D. and A.T.M.; and by the Consejo Superior de Investigaciones Científicas PIE-202120E019 to A.T.M., and the SusPlast pltform to F.J.R.-D., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

1 p., Los hongos Agaricomicetes responsables de la descomposición de la madera y de otros sustratos lignocelulósicos constituyen una valiosa fuente de enzimas degradadoras de lignina. Entre estas, las lacasas (oxidasas multicobre) presentan un notable potencial biotecnológico como biocatalizadores dada su capacidad de oxidar una amplia gama de compuestos aromáticos utilizando como único requisito el oxígeno. Las lacasas de especies saprótrofas de Agaricales, a pesar de ser excelentes fuentes de estas enzimas, han sido mucho menos estudiadas que las lacasas de Poliporales. En este trabajo, el gen de una nueva lacasa de Agrocybe pediades, que es secretada por el hongo durante la degradación de la lignocelulosa, fue sintetizado de novo y expresado en Saccharomyces cerevisiae utilizando evolución dirigida de la enzima y un péptido señal previamente mejorado. La caracterización de las nuevas variantes de lacasa obtenidas proporcionó nuevos conocimientos sobre la contribución de diferentes residuos de la enzima para modular la producción, actividad catalítica o el pH óptimo de las lacasas. La variante seleccionada, con dos mutaciones de diferencia con la lacasa nativa, mostró propiedades muy interesantes como biocatalizador, como la capacidad de oxidar una amplia gama de sustratos, incluyendo mediadores de alto potencial redox y colorantes orgánicos recalcitrantes, además de mayor actividad a pH neutro y alta tolerancia a ciertos inhibidores. Por último, demostramos la existencia de tres sitios de N-glicosilación en la lacasa y su distinto efecto sobre la secreción o la actividad catalítica de la enzima.

15 p.-6 fig.-3 tab., Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen’s genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives., This project was financially supported by Academy of Finland (Grant No. 307580). The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. A.T.M., S.C., F.J.R.D. and A.S. were funded by the EnzOx2 (BBI-PPP-2015-2-720297; www.enzox2.eu) EU project, and the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Economy, Industry and Competitiveness,co-financed by FEDER funds.

14 p.-8 fig.-1 tab., White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here we combined comparative genomics,transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood decaying activity within the Basidiomycota genus Pycnoporus. We observed strong conservation in the genome structures and the repertoires of protein coding genes across the four Pycnoporus species described to date,despite the species having distinct geographic distributions. We further analyzed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin.The co-occurrence in the exo-proteomes of H2O2 producing enzymes with H2O2 consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 LPMO gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood decaying process., This work was supported by The French National Agency for Research (ANR-14-CE06-0020). The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The work by CSIC was supported by the Ministerio de Ciencias, Innovación y Universidades de España (BIO2017-86559-R).

6 p.-5 fig., The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degradation, an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period associated with the appearance of these enzymes. These first peroxidases were not able to degrade lignin directly and used diffusible metal cations to attack its phenolic moiety. The phylogenetic analysis of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are included, suggests that later in evolution these enzymes would have acquired the ability to degrade nonphenolic lignin using a tryptophanyl radical interacting with the bulky polymer at the surface of the enzyme. Here, we track this powerful strategy for lignin degradation as a phenotypic trait in fungi and show that it is not an isolated event in the evolution of Polyporales. Using ancestral enzyme resurrection, we study the molecular changes that led to the appearance of the same surface oxidation site in two distant peroxidase lineages. By characterization of the resurrected enzymes, we demonstrate convergent evolution at the amino acid level during the evolution of these fungi and track the different changes leading to phylogenetically distant ligninolytic peroxidases from ancestors lacking the ability to degrade nonphenolic lignin., This work was supported by the IndOx (KBBE-2013-613549, https://www.indoxproject.eu) and EnzOx2 (H2020-BBI-PPP-2015-2-720297, https://www.enzox2.eu) European Union projects and the BIO2014-56388-R and BIO2017-86559-R Spanish projects. The work conducted by JGI was supported by the Office of Science of the US Department of Energy under Contract DE-AC02-05CH11231.

6 p.-6 fig.-1 tab., A comparison of sequenced Agaricomycotina genomes suggests that efficient degradation of wood lignin was associated with the appearance of secreted peroxidases with a solvent-exposed catalytic tryptophan. This hypothesis is experimentally demonstrated here by resurrecting ancestral fungal peroxidases, after sequence reconstruction from genomes of extant white-rot Polyporales, and evaluating their oxidative attack on the lignin polymer by state-of-the-art analytical techniques. Rapid stopped-flow estimation of the transient-state constants for the 2 successive one-electron transfers from lignin to the peroxide-activated enzyme (k2app and k3app) showed a progressive increase during peroxidase evolution (up to 50-fold higher values for the rate-limiting k3app). The above agreed with 2-dimensional NMR analyses during steady-state treatments of hardwood lignin, showing that its degradation (estimated from the normalized aromatic signals of lignin units compared with a control) and syringyl-to-guaiacyl ratio increased with the enzyme evolutionary distance from the first peroxidase ancestor. More interestingly, the stopped-flow estimations of electron transfer rates also showed how the most recent peroxidase ancestors that already incorporated the exposed tryptophan into their molecular structure (as well as the extant lignin peroxidase) were comparatively more efficient at oxidizing hardwood (angiosperm) lignin, while the most ancestral “tryptophanless” enzymes were more efficient at abstracting electrons from softwood (conifer) lignin. A time calibration of the ancestry of Polyporales peroxidases localized the appearance of the first peroxidase with a solvent-exposed catalytic tryptophan to 194 ± 70 Mya, coincident with the diversification of angiosperm plants characterized by the appearance of dimethoxylated syringyl lignin units., This work was supported by the EnzOx2 (H2020-BBI-PPP-2015-2-720297, https://www.enzox2.eu) European project and the BIO2017-86559-R and AGL2017-83036-R Spanish projects. The work conducted by JGI was supported by the Office of Science of the US Department of Energy under Contract DE-AC02-05CH11231.

Comunicación oral presentada en el Lignin Gordon Research Conference Towards Viable Solutions for Lignin Valorization August 5 - 10, 2018, (Easton, MA), US

4 páginas.-- 3 figuras.-- Comunicación oral presentada en el 15th European Workshop on Lignocellulosics and Pulp June 26-29, 2018 Aveiro, Portugal, Stopped-flow spectrophotometry enables us to estimate the rate constants for lignin oxidation by ligninolytic peroxidases following the electron-transfer from the "enzyme side" (heme reduction). Using this technique and water-soluble (hardwood and softwood) lignosulfonates as substrates, we were able to demonstrate that a solvent exposed tryptophan residue is strictly required for oxidation of nonphenolic (permethylated) lignin by ligninolytic peroxidases. Simultaneously, the use of 2D-NMR spectroscopy revealed that the tryptophan-Iess variants only cause negligible modification of lignin in long-term steady-state enzymatic treatments (and null modification when applied on nonphenolic lignin, Borregaard LignoTech is acknowledged for the lignosulfonate samples, and the Public-Priva Partnership of Bio-Based Industries (www.bbi-europe.eu) for the financial support.

3 p. Jérémy Tournayre, Laurent Parry, Anne-Catherine Maurin, Julien Averous, Alain Bruhat, et al.. Identification de marques épigénétiques marqueurs de prédisposition aux maladies métaboliques. JOBIM 2018 Journées Ouvertes Biologie, Informatique et Mathématiques, Jul 2018, Marseille, France. ffhal-01930383f

Comunicación oral presentada en Lignobiotech 2018 - the 5th Symposium of Biotechnology Applied to Lignocelluloses 29 ago-1 sept (2018) Helsinki, Finlandia, Methodological limitations difficult estimation of lignin oxidation rates by Iigninolytic peroxidases. Here, lignin oxidation was seen from the "enzyme side" by estimating the reduction rates of H202 activated peroxidase compounds I and 11, using rapid spectrophotometry. Removal of Trp164 in versatile peroxidase resulted in 4-100 fold slower rate-limiting compound-II reduction by watersoluble sulfonated lignins. Moreover, when methylated lignosulfonates were used, negligible transfer was found, confirming that residual reduction by native lignin was due to its phenolic moiety. In agreement with the transient-state kinetic data, low or null structural modification of lignin was found (by NMR) during steady-state treatment with the W164S variant compared with native VP. Similar results were obtained with lignin peroxidase. Therefore, we demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer.

8 p.-6 fig.-2 tab., Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP., This work was supported by European Contract AIR2-CT93-1219 and Project BIO96-393 of the Spanish Biotechnology Programme.