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Wood is the main renewable material on Earth and is largely used as building material and in paper-pulp manufacturing. This review describes the composition of lignocellulosic materials, the different processes by which fungi are able to alter wood, including decay patterns caused by white, brown, and soft-rot fungi, and fungal staining of wood. The chemical, enzymatic, and molecular aspects of the fungal attack of lignin, which represents the key step in wood decay, are also discussed. Modern analytical techniques to investigate fungal degradation and modification of the lignin polymer are reviewed, as are the different oxidative enzymes (oxidoreductases) involved in lignin degradation. These include laccases, high redox potential ligninolytic peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase), and oxidases. Special emphasis is given to the reactions catalyzed, their synergistic action on lignin, and the structural bases for their unique catalytic properties. Broadening our knowledge of lignocellulose biodegradation processes should contribute to better control of wood-decaying fungi, as well as to the development of new biocatalysts of industrial interest based on these organisms and their enzymes. La madera es el principal material renovable en la Tierra y es utilizada en gran parte como material de construcción y en la fabricación de celulosa. Esta revisión describe la composición de los materiales lignocelulósicos y diversos procesos de alteración de la madera por parte de hongos, como el deterioro causado por los llamados hongos de podredumbre blanca, de podredumbre parda y de podredumbre blanda y por los hongos cromógenos o manchadores de la madera. También se tratan los aspectos químicos, enzimáticos y moleculares del ataque a la lignina por los hongos, que es clave en el deterioro de la madera. Se describen las técnicas analíticas modernas para investigar la degradación y la modificación del polímero de la lignina causadas por hongos, así como las diversas enzimas oxidativas (oxidoreductasas) que intervienen en la degradación de la lignina. Entre éstas se encuentran lacasas, peroxidasas ligninolíticas de alto potencial redox (lignina-peroxidasa, manganeso-peroxidasa y peroxidasas versátiles) y las oxidasas. Se destacan las reacciones catalizadas, su acción sinérgica sobre la lignina, y las bases estructurales de sus exclusivas propiedades catalíticas. Un mejor conocimiento de los procesos de biodegradación de la lignocelulosa debería contribuir a un mejor control de los hongos descomponedores de la madera, así como al desarrollo de nuevos biocatalizadores de interés industrial basados en estos organismos y sus enzimas. A madeira é o principal material renovável na Terra e é utilizada em grande parte como material de construção e na fabricação de celulose. Esta revisão descreve a composição dos materiais lignocelulósicos e diversos processos de alteração da madeira por parte de fungos, como a deterioração causada pelos chamados fungos de podridão branca, de podridão parda e de podridão mole e pelos fungos emboloradores ou manchadores da madeira. Também se tratam os aspectos químicos, enzimáticos e moleculares do ataque à lignina pelos fungos, que é chave na deterioração da madeira. Se descrevem as técnicas analíticas modernas para investigar a degradação e a modificação do polímero da lignina causadas por fungos, assim como as diversas enzimas oxidativas (oxidoreductasas) que intervêm na degradação da lignina. Entre estas se encontram lacasas, peroxidasas ligninolíticas de alto potencial redox (lignina-peroxidasa, manganeso-peroxidasa e peroxidasas versáteis) e as oxidasas. Se destacam as reações catalisadas, sua ação sinérgica sobre a lignina, e as bases estruturais de suas exclusivas propriedades catalíticas. Um melhor conhecimento dos processos de biodegradação da lignocelulosa deveria contribuir para um melhor controle dos fungos descomponedores da madeira, assim como ao desenvolvimento de novos biocatalisadores de interesse industrial baseados nestes organismos e suas enzimas.

13 p.-4 fig.-2 tab., [Background]: Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility, [Results]: Two water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis) were chemically characterized and used to estimate single electron-transfer rates to the H2O2-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies (k 3app values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~20–30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used., [Conclusions]: We demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer., This work was supported by the INDOX (KBBE-2013-613549 to ATM) and EnzOx2 (H2020-BBI-PPP-2015-RIA-720297 to ATM) EU projects, and the NOESIS (BIO2014-56388-R to FJR-D), BIORENZYMERY (AGL2014-53730-R to AG) and LIGNIN (CTQ2014-60764-JIN to JR) projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

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).

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).

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.-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.

10 p.-6 fig., Lentinula is a broadly distributed group of fungi that contains the cultivated shiitake mushroom, L. edodes. We sequenced 24 genomes representing eight described species and several unnamed lineages of Lentinula from 15 countries on four continents. Lentinula comprises four major clades that arose in the Oligocene, three in the Americas and one in Asia–Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes of L. edodes from China that were previously published as raw Illumina reads and added them to our dataset. Lentinula edodes sensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest of L. edodes s. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase (lecsl) and γ-glutamyl transpeptidase (leggt), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified in Lentinula. Paralogs of both genes that are unique to Lentinula (lecsl 3 and leggt 5b) are coordinately up-regulated in fruiting bodies of L. edodes. The pangenome of L. edodes s. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation., This research was supported by the NSF awards DEB-1456588 to D.H. and DEB-1442113 to A.R., the Department of Energy award 0000238431 to D.H., the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Science and Innovation (cofinanced by FEDER funds) to F.J.R.-D., S.C., and A.T.M., grant PID2019-103901GB-I00 of the Spanish Ministry of Science and Innovation to P.F., and the NIH award R56AI146096 to A.R. J.L.S. and A.R. were supported by the HHMI and the Burroughs Wellcome Fund. The work (proposal: 10.46936/10.25585/60001048) conducted by the US Department of Energy JGI (https://ror.org/04xm1d337), a DOE Office of Science User Facility, was supported by the Office of Science of the US Department of Energy operated under contract no. DE-AC02-05CH11231.

18 p.-4 fig.-2 tab., The study of evolution is limited by the techniques available to do so. Aside from the use of the fossil record, molecular phylogenetics can provide a detailed characterization of evolutionary histories using genes, genomes and proteins. However, these tools provide scarce biochemical information of the organisms and systems of interest and are therefore very limited when they come to explain protein evolution. In the past decade, this limitation has been overcome by the development of ancestral sequence reconstruction (ASR) methods. ASR allows the subsequent resurrection in the laboratory of inferred proteins from now extinct organisms, becoming an outstanding tool to study enzyme evolution. Here we review the recent advances in ASR methods and their application to study fungal evolution, with special focus on wood-decay fungi as essential organisms in the global carbon cycling., ,, Evolution, This research was funded by the Genobioref (BIO2017-86559-R) and Lig2Plast (PID2021-126384OB-I00) projects of the Spanish Ministry of Science and Innovation (co-financed by FEDER funds); by the WoodZymes (H2020-BBI-JTI-2017-792070) and CuBE – ERC Synergy Project (H2020-ERC-2019-SyG-856446) EU projects, and by the Consejo Superior de Investigaciones Científicas project PIE-202120E019 and grant 2021AEP106, SusPlast platform, and program for the Spanish Recovery, Transformation and Resilience Plan funded by the Recovery and Resilience Facility of the European Union, established by the Regulation (EU) 2020/2094. G.M. acknowledges The Tatiana Pérez de Guzmán el Bueno Foundation for his predoctoral Environment grant. Finally, we acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

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.

238 p.-49 fig.-17 tab., [EN]Background Lignin is one of the most abundant biomaterials on Earth, and its degradation is an important biological and industrial problem. In nature, lignin recycling is essential in the carbon cycle, being white-rot fungi the main organisms able to mineralize this polymer. In the industry, the use of these fungi or their enzymatic machinery is studied for a better use of lignocellulose in the biorefinery context, as a green alternative to chemical (or physical) methods. For lignin degradation, white-rot fungi secrete different types of peroxidases: i) manganese peroxidases (MnPs), which have a Mn2+-binding site for oxidation of the metal cation to Mn3+ whose chelates diffuse in the wood and are able to oxidize the phenolic moiety of lignin; ii) lignin peroxidases (LiPs), which have a catalytic tryptophan in their surface where lignin is oxidized directly; iii) versatile peroxidases (VPs), which combine both oxidation sites in their structure. The origin of fungal wood degradation was established in the Carboniferous period associated to the production of the first ligninolytic peroxidases and, together with other geochemical factors, contributed to the end of coal accumulation. This evolutionary event is the starting point of this thesis, where the posterior evolution of ligninolytic peroxidases is analyzed using ancestral enzyme resurrection. Aims i. Ancestral sequence reconstruction of fungal peroxidases using the phylogeny of Polyporales peroxidases and the PAML software. The sequences of interest and their molecular models will be evaluated to select those ancestral enzymes that will be studied in detail. ii. Resurrection of the selected ancestral enzymes for their biochemical characterization. Model substrates will be used and both the kinetic constants and stability parameters will be obtained. iii.Optimization of reduction potential measurements to evaluate the redox potential of ligninolytic peroxidases through evolution, including stopped-flow spectrophotometry at redox equilibrium, spectroeloectrochemical titration and protein NMR. iv. Characterization of ancestral and extant peroxidases using lignosulfonates from gymnosperms and angiosperms as water-soluble lignin models. Stopped-flow spectrophotometry will be used to analyze transient-state kinetics, and 2D-NMR to evaluate the modification of the polymer in long-term treatments. Results Chapter 1. Experimental recreation of the evolution of lignin degrading enzymes from the Jurassic to date In this chapter is presented the first resurrection study of ligninolytic enzymes, already published in the literature. Using PAML and the phylogeny of class-II peroxidases from Polyporales (where most wood-rotting fungi are included) the ancestors in the lineage leading to the most efficient ligninolytic enzymes, LiPs, are obtained. Using simple model compounds, the different oxidation sites and their change through evolution are characterized, showing the exploration of new strategies for lignin oxidation. Likewise, the stability of ancestral enzymes is analyzed, showing that their stability to acid pHs, at which ligninolytic peroxidases act in nature, increases when the catalytic tryptophan for direct lignin oxidation appears. Chapter 2. Evolutionary convergence in lignin degrading enzymes In the second chapter a different lineage leading to a distant clade from LiPs is analyzed. This clade includes extant VPs, which also have the direct lignin oxidation site. To evaluate if the appearance of this site is due to duplication events of an ancestor already containing it in its structure, or due to evolutionary convergence appearing twice in different lineages, the ancestors of interest are resurrected. The results show that convergence took place in wood-rotting fungi evolution for a direct lignin oxidation, due to the production of similar types of enzymes through time. It is also showed that during LiPs evolution, the catalytic tryptophan environment became more negative, which could explain their higher efficiency oxidizing model substrates. The results also show convergence in the stability properties of the enzymes in both lineages, being more stable to acidic pHs after the appearance of the catalytic tryptophan., Chapter 3. Redox potential increased during the evolution of enzymes degrading recalcitrant lignin Ligninolytic peroxidases are characterized by their uniquely high reduction potential. This allows them to oxidize multiple substrates including lignin. In this chapter, it is evaluated how the redox potential changed through evolution. To calculate reduction potentials of ancestral and extant enzymes, stopped-flow spectrophotometric measurements at redox equilibrium and spectroelectrochemical titration are employed, obtaining the values of reduction potential of all the catalytic cycle couples and the Fe2+/Fe3+ pair. The results show that there is an increase in the redox potential of ligninolytic peroxidases through evolution, a fact correlated with the displacement of the signal of the proximal histidine observed using protein NMR. This indicates that an structural change in the heme proximal side, with alterations in the geometry of the proximal histidine – heme iron bond, would be involved in the modification of redox potential in evolution. Chapter 4. Peroxidase evolution in white-rot fungi follows wood lignin evolution in plants In the last chapter the linage to LiPs is characterized using two types of water-soluble lignin: lignosulfonates from gymnosperms and angiosperms. With stopped-flow spectrophotometry the transient-state kinetic constants in lignin oxidation are obtained and using 2D-NMR it is evaluated how the polymer is modified through evolution in long-term treatments of both lignosulfonates using extant and resurrected enzymes. After time calibration of the peroxidases phylogeny, it has been concluded that there was a switch in the substrate preference when the solvent exposed catalytic tryptophan appeared in the surface of ligninolytic peroxidases. This way, there was a change from a better oxidation of gymnosperm lignin, more basal, to a preference for angiosperm lignin, more recent and complex, coincident approximately with the origin of this type of plants in evolution., Conclusions Ancestral enzyme resurrection allowed the analysis of ligninolytic peroxidase evolution in wood-rotting fungi. In this thesis, it is demonstrated that during evolution these enzymes explored several strategies for lignin degradation, converging in distant lineages the ability to oxidize the polymer directly in a solvent-exposed tryptophan. With this acquisition, together with the stabilization to acidic pHs, the increase in the negative charge surrounding the tryptophan and the boost of reduction potentials through evolution, the enzymes acquired the unique properties they have today. Likewise, the use of soluble lignin shows a change in the substrate preference from gymnosperms to angiosperms wood lignin, concomitant with the rise of the catalytic tryptophan., [ES]Objetivos i. Reconstrucción de secuencias de peroxidasas fúngicas usando la filogenia de las peroxidasas ligninolíticas de Polyporales y el software PAML. Se evaluarán las secuencias de interés y los modelos tridimensionales de sus estructuras para seleccionar aquellas enzimas ancestrales que se estudiarán en detalle. ii. Resurrección de las enzimas ancestrales seleccionadas para su posterior caracterización bioquímica. Para ello se usarán sustratos modelo y se estudiarán tanto sus constantes cinéticas como su estabilidad. iii. Puesta a punto de técnicas para medir y evaluar el potencial redox de las peroxidasas ligninolíticas a lo largo de su evolución, incluyendo espectrometría de flujo detenido (stopped-flow) en equilibrio redox, titulación espectroelectroquímica y NMR de proteínas. iv. Estudio de la actividad de las peroxidasas ancestrales y actuales usando lignosulfonatos de gimnospermas y angiospermas como modelos de lignina soluble en agua. Para ello se realizarán estudios cinéticos de los estados transitorios de las enzimas mediante espectrofotometría de stopped-flow, y análisis de NMR bidimensional (2D-NMR) de los productos de reacción en los que se evaluará la modificación de los polímeros en tratamientos prolongados. Resultados Capítulo 1: Recreación experimental de la evolución de enzimas degradadoras de lignina desde el Jurásico hasta hoy En este capítulo se presenta el primer estudio de resurrección de enzimas ligninolíticas realizado y ya recogido en la literatura. Usando PAML y la filogenia de las peroxidasas de clase II de los Polyporales (donde se incluyen la mayoría de hongos degradadores de la madera) se obtienen ancestros en el linaje que conduce a las enzimas ligninolíticas más eficientes que existen actualmente, las LiPs. Usando sustratos modelo simples, se caracterizan los distintos sitios de oxidación en las enzimas y se comprueba cómo han ido cambiando a lo largo de la evolución, explorando nuevas estrategias para la oxidación de lignina. Así mismo, se analiza la estabilidad de las enzimas ancestrales y actuales, viendo que su estabilidad a pHs ácidos, donde actúan en la naturaleza, aumenta cuando aparece el triptófano catalítico para la oxidación directa de la lignina. Capítulo 2: Convergencia evolutiva en enzimas degradadoras de lignina En este segundo capítulo se analiza otro linaje evolutivo que lleva a un clado de enzimas diferenciado y distante con respecto a las LiPs estudiadas en el capítulo anterior. En este clado se incluyen VPs actuales, que también poseen el sitio de oxidación directa de la lignina. Para evaluar si la aparición de dicho sitio se debió a eventos de duplicación desde un ancestro que lo contenía en su estructura, o se produjo como consecuencia de un proceso de convergencia evolutiva apareciendo dos veces en distintos linajes, se resucitan los ancestros correspondientes. Los resultados muestran que se produjo convergencia evolutiva en hongos degradadores de madera para la oxidación directa de la lignina debido a la producción de enzimas similares durante la evolución. Se muestra también que durante la evolución de las LiPs se produjo un aumento de carga negativa alrededor del triptófano, lo que podría explicar su mayor eficacia oxidando sustratos modelo. Los resultados también muestran que se produjo convergencia en las propiedades de las enzimas de ambos linajes, siendo más estables a pHs ácidos tras la aparición del triptófano catalítico., Capítulo 3: El potencial redox de las enzimas que degradan lignina aumentó durante la evolución Las peroxidasas ligninolíticas se caracterizan por su alto potencial redox. Esto les permite oxidar múltiples sustratos entre los que se encuentra la lignina. En este capítulo se analiza cómo cambió el potencial redox de estas enzimas a lo largo de la evolución. Para calcular los potenciales de reducción de las enzimas ancestrales y actuales se utilizan medidas espectrofotométricas de stopped-flow en equilibrio redox y titulación espectroelectroquímica, obteniéndose los valores de potencial redox de todas las parejas del ciclo catalítico y del par Fe2+/Fe3+. Los resultados muestran que se produjo un aumento del potencial redox en las peroxidasas ligninolíticas a lo largo de la evolución, un hecho que se ha podido correlacionar con el desplazamiento observado de la señal correspondiente a la histidina proximal usando NMR de proteínas. Esto indica que un cambio estructural en el entorno proximal del hemo, con alteraciones en la geometría del enlace histidina proximal – hierro del hemo, estaría implicado en la modificación del potencial redox a lo largo de la evolución. Capítulo 4: La evolución de las peroxidasas de los hongos de podredumbre blanca sigue la evolución de la lignina de la madera en plantas En este último capítulo se caracteriza el linaje de las LiPs usando dos tipos de lignina soluble en agua: lignosulfonatos de gimnospermas y de angiospermas. Mediante espectrofotometría de stopped-flow se obtienen las constantes de estado transitorio para la oxidación de lignina, y usando 2D-NMR se analiza cómo se modifica el polímero a lo largo de la evolución en base a tratamientos prolongados de ambos lignosulfonatos con enzimas actuales y resucitadas. Tras la calibración temporal de la filogenia de las peroxidasas, se ha podido concluir que hubo un cambio en la preferencia de sustrato cuando apareció el triptófano catalítico en la superficie de las peroxidasas ligninolíticas. De este modo, se pasó de una mejor oxidación de lignina de gimnospermas, más basal, a una preferencia por la lignina de angiospermas, más reciente y compleja, coincidiendo aproximadamente con el origen de este tipo de plantas en la evolución., Conclusiones Usando la resurrección de enzimas ancestrales se ha analizado la evolución de las peroxidasas ligninolíticas de los hongos degradadores de la madera. En esta tesis se demuestra que durante la evolución de estas enzimas se exploraron varias estrategias para la degradación de lignina, convergiendo en linajes distintos la capacidad de oxidar directamente este polímero en un triptófano expuesto en la superficie de la proteína. Con esta adquisición, junto con su estabilización a pHs ácidos, el incremento en la carga negativa alrededor del triptófano y el aumento del potencial redox a lo largo de la evolución, las enzimas adquirieron las propiedades únicas que tienen hoy día. Así mismo, empleando ligninas solubles se muestra el cambio en la preferencia de sustrato experimentado por las peroxidasas ligninolíticas, de lignina de gimnospermas a lignina de angiospermas, coincidente con la aparición del triptófano catalítico., Ha sido financiada por las becas (FPI, Ref BES-2012-053513) y Ref EEBB-I-15-10267) ; los proyectos europeos (INDOX, Ref KBBE-2013-613549) y H2020- BBI-PPP-2015-720297) Y los proyectos del Plan Nacional (NOESIS, Ref BIO2014-56388-R) y (GENOBIOREF, Ref BIO2017-86559-R)

212 p.-36 fig.-10 tab.-anexo., [EN] Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. They are produced by white-rot fungi, the main organisms being able to mineralize lignin in nature. Lignin is the second most abundant polymer in nature. Due to its aromatic and heterogeneous nature, it is very recalcitrant and its degradation is difficult. White-rot fungi secrete a battery of oxidoreductases to the extracellular medium that, together with low molecular redox mediators, attack the lignin and achieve its degradation. Ligninolytic peroxidases play a key role in this process oxidizing the aromatic units of lignin. Ligninolytic peroxidases can be classified into three families: lignin peroxidases (LiP), manganese peroxidases (MnP) and versatile peroxidases (VP). VPs are characterized by the presence of three catalytic sites: i) the catalytic tryptophan, a solvent exposed residue that forms a protein radical trough a long range electron transfer from the heme, able to oxidize high redox potential compounds; ii) the manganese oxidation site, able to oxidize Mn2+ to Mn3+, which acts as a diffusible oxidizer, and iii) the main heme access channel where low redox potential compounds are oxidized. Among the different ligninolytic peroxidases, VP shows a special interest since it gathers the catalytic properties of LiP, MnP and generic peroxidases and, in addition, the enzyme does not need redox mediators to develop its activity. For these reasons, the VP from Pleurotus eryngii (isoenzyme VPL2) was the enzyme selected and studied in this thesis., The above catalytic properties allow VP to oxidize not only lignin but also other aromatic phenolic and non-phenolic (high or low redox potential) compounds, dyes and pesticides. The oxidative reactions VP and other ligninolytic peroxidases catalyze led to the break of lignin bonds or to the formation of new ones by radical condensation, or even indirectly to oxygenation reactions. All these reactions are of interest from a biotechnological point of view. On the other hand, ligninolytic peroxidases use hydrogen peroxide as substrate, which is cheap and of easy acquisition. Therefore, the use of these enzymes as biocatalysts in different industrial processes is very attractive as a sustainable and ecofriendly alternative to the conventional chemical methods. However, in spite of the high potential of ligninolytic peroxidases, they are not applicable as they are produced by natural organisms, since they do not present a proper selectivity and/or compatibility with the conditions under which the industrial processes are carried out. For these reasons, an optimization of the catalytic properties and stability of these enzymes is necessary previous to their biotechnological use. Among the drawbacks that limit the application of these peroxidases, the relative low stability towards pH, temperature and H2O2, or the insufficient production levels are found. The low stability of ligninolytic peroxidases towards H2O2 (their natural co-substrate) is one of the reasons delaying the development of applications based on these fungal enzymes. H2O2 is involved in enzyme inactivation by a mechanism-based process described as a suicide inactivation. This irreversible oxidative inactivation is produced in absence of reducing substrates or when the enzyme is exposed to a high excess of peroxide, and it is a consequence of multiple oxidization events affecting different components of the enzyme, including amino acids and the porphyrin ring. The relatively low pH stability of this and other fungal peroxidases is another drawback for their application. Under alkaline or even neutral conditions, ligninolytic peroxidases lose the structural calcium ions, which leads to a relaxation of the structure and the hexacoordination of the heme iron, resulting in an inactive enzyme. In a similar way, at acidic pH, the heme environment of these enzymes is affected being the interaction between the heme iron and the proximal histidine broken and the enzyme inactivated., On the other hand, although the catalytic properties of VP have been thoroughly studied, there are still catalytic aspects that are not clearly understood. Taking all this into account, studying the bases that regulate the stability and the catalytic properties of ligninolytic peroxidases is of interest for the future design of useful biocatalysts. 2. Aims The main aims of the present doctoral thesis were: i) the design of a VP with more adequate properties for its biotechnological application, and ii) the acquisition of an in-depth knowledge about the structural determinants that govern the catalysis and the stability of the enzyme. With this in mind, the following issues were addressed: i) The study of the oxidative inactivation of VP, and the factors that influence this process, as well as the design of a VP with improved H2O2-stability. ii) The design of a VP variant with optimized pH stability through the transfer of stabilizing structural motifs found in other peroxidases (Fernández-Fueyo et al. Biotechnology for Biofuels 2014, 7:2). iii) The analysis of the evolved variant 2-1B, obtained by directed evolution of VP (García-Ruiz et al. Biochem. J. (2012) 441, 487– 498), to determine which structural determinants generated by the mutations introduced improved its catalytic and stability properties. iv) To obtain an in-depth knowledge of the function of the catalytic tryptophan of VP, characterize the electron transfer between the peroxidase and the lignin, and study the ability of VP to oxidize technical lignins. Thanks to the structural-function information existing about VP and other ligninolytic peroxidases, and the availability of the crystal structure of VP, the optimization of VP using a rational approach has been possible. In this way, several VP variants were designed and produced and their properties were evaluated., The oxidative inactivation of VP by H2O2 was studied and different strategies were evaluated with the aim of improving its H2O2 stability. The studies performed demonstrated that VP is easily inactivated by H2O2 and that, during the inactivation process, the four methionine residues of VP are oxidized to methionine derivatives. Substitution of 3.1 Study and improvement of the oxidative stability of VP these residues, located in a sensitive region of the enzyme (near the heme cofactor and the catalytic tryptophan), rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy was developed consisting in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine (His47) with the aim of modifying the reactivity of the enzyme with H2O2 and consequently, slowing down the inactivation process. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with the slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III (FeIII-O2.-), a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme., [ES] Las pLaeroxidasas ligninolíticas son enzimas que tienen una gran potencial biotecnológico debido a sus propiedades catalíticas que les permiten oxidar una gran variedad de sustratos recalcitrantes. Son producidas por los hongos de podredumbre blanca, los principales responsables de la degradación de la lignina en la naturaleza. La lignina es el material aromático renovable más abundante en la naturaleza y, junto con la celulosa y la hemicelulosa, compone la biomasa lignocelulósica. Estructuralmente, es un polímero aromático muy heterogéneo, y recalcitrante. Para degradar la lignina, los hongos de podredumbre blanca secretan un conjunto de oxidorreductasas al medio, entre las que se encuentran las peroxidasas ligninolíticas, que juegan un papel central en este proceso. Las peroxidasas ligninolíticas son hemo peroxidasas de alto potencial redox. Se clasifican en tres familias: lignina peroxidasas (LiP), manganeso peroxidasas (MnP) y peroxidasas versátiles (VP). Las VP se caracterizan por la presencia de tres sitios catalíticos: i) un triptófano catalítico expuesto en superficie, que puede formar un radical de proteína a través de una ruta de transferencia electrónica desde el hemo y es capaz de oxidar compuestos de alto potencial redox; ii) el sitio de oxidación de manganeso, que le permite oxidar Mn2+ a Mn3+; y iii) el canal principal de acceso al hemo en el que se oxidan compuestos de bajo potencial redox. Entre las distintas peroxidasas ligninolíticas, la VP presenta un especial interés debido a que combina las propiedades catalíticas propias de LiP, MnP y las peroxidasas genéricas y, además, no necesita de la presencia de mediadores para su actuación. Por ello, la VP, en concreto la variante alélica VPL2 de Pleurotus eryngii, fue la peroxidasa escogida para realizar los trabajos de la presente tesis doctoral., Las propiedades catalíticas de la VP y demás peroxidasas ligninolíticas les permiten oxidar compuestos aromáticos fenólicos y no fenólicos, así como diferentes tintes industriales y pesticidas. Por ello, las reacciones de oxidación que llevan a cabo estas enzimas son interesantes desde un punto de vista biotecnológico. Además, estas peroxidasas tienen la ventaja de ser autosuficientes, en el sentido de que son activadas por H2O2, un oxidante de fácil adquisición. Por tanto, la utilización de peroxidasas ligninolíticas como biocatalizadores en diferentes procesos industriales representa una alternativa sostenible y respetuosa con el medio ambiente frente al uso de métodos químicos convencionales. Sin embargo, muy pocas de ellas se comercializan y su aplicación industrial es modesta. Esto es debido, principalmente, a que no son aplicables tal cual se producen en la naturaleza, ya que no presentan una adecuada selectividad y compatibilidad con los rigurosos procesos industriales. Por ello, algunas de sus propiedades han de ser optimizadas antes de su aplicación. La estabilidad frente a H2O2 (estabilidad oxidativa) es uno de los principales obstáculos a superar para el uso biotecnológico de las peroxidasas ligninolíticas. El H2O2, necesario para el funcionamiento de las peroxidasas, es un fuerte oxidante que tiene efectos deletéreos sobre estas enzimas. La inactivación por H2O2 es un proceso irreversible, especialmente importante cuando existe un exceso de peróxido o no existe sustrato reductor. Su mecanismo no se conoce por completo, aunque se sabe que es consecuencia de diferentes reacciones de oxidación que afectan a distintos componentes de la enzima, incluyendo aminoácidos y el anillo de porfirina del cofactor. La estabilidad a pH es otro de los factores clave que a menudo necesita ser optimizado. La inactivación de las peroxidasas ligninolíticas debida al pH alcalino, o incluso neutro, se produce como consecuencia de la pérdida de los calcios estructurales responsables del mantenimiento de la correcta conformación del bolsillo del hemo. La ausencia de los calcios conduce al colapso de la estructura, lo que lleva a que el hierro del hemo quede hexacoordinado y la enzima inactiva. De igual forma, estas enzimas también se inactivan a pH ácido, aunque en este caso como consecuencia de la rotura del enlace entre el hierro del hemo y la histidina proximal. Por otra parte, aunque las propiedades de la VP y las otras peroxidasas ligninolíticas están ampliamente estudiadas, todavía hay aspectos catalíticos que no se conocen claramente., Por todo ello, es necesario comprender las bases estructurales que determinan y regulan la estabilidad y catálisis de estas enzimas para que su optimización y empleo sea una opción real. Los principales objetivos de la presente tesis fueron: i) el diseño de una VP que presente unas características más adecuadas para su uso biotecnológico, y ii) la profundización en el conocimiento de las bases estructurales que regulan la catálisis y estabilidad de la VP. Para ello, se abordaron las siguientes tareas: i) El estudio de la estabilidad oxidativa de la VP y de las bases estructurales que determinan esta propiedad, así como el diseño de una variante mutada que presente mayor resistencia oxidativa. ii) El diseño de una variante de VP con mayor estabilidad a pH mediante la transferencia de motivos estabilizantes encontrados en peroxidasas relacionadas (Fernández-Fueyo et al. Biotechnology for Biofuels 2014, 7:2). iii) El análisis de la variante VP 2-1B obtenida por evolución dirigida (García-Ruiz et al. Biochem. J. (2012) 441, 487–498) con objeto de averiguar qué determinantes estructurales generados por las mutaciones introducidas regulan sus propiedades catalíticas y estabilidad mejoradas. iv) Profundizar en el conocimiento del funcionamiento del triptófano catalítico, estudiar la transferencia electrónica entre la peroxidasa y la lignina durante la oxidación de ésta, y mostrar la capacidad de la VP para oxidar ligninas técnicas. Para realizar estas tareas, se utilizó un enfoque racional y se diseñaron diferentes variantes mutadas, que posteriormente fueron evaluadas. El estudio de la estabilidad oxidativa de la VP demostró que la enzima se inactiva fácilmente por H2O2. Durante la inactivación, las cuatro metioninas presentes en la VP se oxidaron a derivados de metionina. Teniendo en cuenta la proximidad de estos residuos a los sitios catalíticos de la enzima es probable que su oxidación provoque una alteración en la conformación de la enzima con un efecto directo sobre su actividad catalítica y estabilidad. De acuerdo con estas evidencias, la primera estrategia utilizada para mejorar la estabilidad oxidativa de la VP consistió en el diseño de variantes en las que las metioninas fueron sustituidas por residuos más resistentes a la oxidación. Asimismo, se diseñó una segunda estrategia consistente en la modificación del entorno de la histidina distal (His47) mediante la introducción de las mutaciones T45A e I103T. Los dos aminoácidos mutados se localizan por encima del área ocupada por la histidina 47, que está implicada, junto con la arginina 43, en la ruptura heterolítica del H2O2 durante la activación de la enzima. Las mutaciones se seleccionaron con la intención de provocar cambios sutiles en la posición de His47 y Arg43 que lleven a la ralentización de la velocidad de reacción con el H2O2 y, como consecuencia, a una menor inactivación. Finalmente, las dos estrategias se combinaron en la variante quíntuple T45A/I103T/M152F/M262F/M265L., Ambas estrategias dieron lugar a variantes mutadas con mayor estabilidad oxidativa. Los mejores resultados se obtuvieron con la variante quíntuple, ya que su velocidad de inactivación por H2O2 disminuyó 11 veces. Además, en presencia de un exceso de 2000 equivalentes de H2O2, la vida media de esta variante se incrementó hasta los 30 minutos, comparados con los 5 minutos de la enzima nativa. Finalmente, la mejora de la estabilidad del compuesto III (FeIII-O2.-), un intermediario catalítico relacionado con la inactivación por H2O2, parece ser la causa de las mejoras observadas asociadas a estas mutaciones. El trabajo llevado a cabo en esta tesis ha permitido profundizar en el conocimiento del mecanismo catalítico de la VP así como avanzar en el conocimiento de las bases estructurales que regulan algunas de sus propiedades como la estabilidad oxidativa, alcalina o ácida. Asimismo, se obtuvieron variantes mutadas que presentaron una mayor estabilidad frente a peróxido y pH, que son algunos de los principales factores que actualmente dificultan el uso biotecnológico de estas peroxidasas. La mejora en la estabilidad a pH ácido es especialmente importante teniendo en cuenta que la VP presenta un pH óptimo ácido para la oxidación de compuestos aromáticos de alto potencial redox. Además, el éxito de la estrategia consistente en transferir motivos estructurales estabilizadores de unas peroxidasas a otras sugiere que esta metodología se podría usar para el diseño de biocatalizadores de interés. Por otra parte, se confirmó la capacidad de la VP para degradar lignosulfonatos y se estudió el patrón de degradación de estas ligninas producido por la enzima. Además, se puso de manifiesto la transferencia electrónica que se produce entre el polímero de lignina y la peroxidasa, que fue caracterizada cinéticamente en condiciones de flujo detenido (stopped flow) y se asoció a la presencia del triptófano 164, el cual forma una radical catalíticamente activo., Beca predoctoral de Formación de Personal Investigador (FPI, Ref. BES-2009-014246). Proyectos europeos (PEROXICATS, KBBE-2010-4-265397) y (INDOX, KBBE- 2013-613549). Proyectos del plan nacional de I+D+i (HIPOP, BIO2011-26694) y (NOESIS, BIO2014-56388-R).