Search

2,5-Furandicarboxylic acid (FDCA) is listed as one of the top building blocks by the US Department of Energy due to its ability to polymerise forming polyesters. Indeed, it is condensed with ethylene glycol to produce poly(ethylenefurandicarboxylate) (PEF). PEF has similar, if not superior, characteristics to those of widely used poly(ethyleneterephthalate) (PET) and the advantage of being renewable and biodegradable: FDCA is a derivative of 5-hydroxymethylfurfural (HMF), itself a product of dehydration of hexoses in plant biomass. The recent years have witnessed intense research in the field of the biocatalytic conversion of HMF into FDCA. Enzymes are favoured by their mild operational conditions and, generally, high product selectivity. Our group has focused on oxidoreductases to accomplish the three oxidation steps that lead from HMF to FDCA. The last of the oxidations, the conversion of 5-formylfurancarboxylic acid to FDCA, constitutes the bottleneck of the process. On the one hand, enzymatic cascades involving aryl-alcohol oxidases (AAO) and unspecific peroxygenases (UPO) were developed. AAO oxidises HMF and related compounds to give rise to intermediates and H 2O2, while UPO catalyses oxygenation to FDCA at the expense of H2O2. Moreover, optimization of the reaction led to complete oxidation of HMF using AAO alone thanks to the action of catalase which in this case eliminates deleterious H2O2. On the other, the discovery of hydroxymethylfurfural oxidases (HMFO), that catalyse the three abovementioned oxidations, paved the way for the improvement of the process. Since then, reactions with HMFO have been optimized and new enzymes of this class have been discovered and characterized. A main aim of current FURENPOL project, in collaboration with BSC (www.bsc.es), is to discover novel HMFOs and to engineer them to improve the FDCA yield in order to ultimately scale up the enzymatic conversion of biobased HMF to FDCA.

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

Las enzimas que degradan la lignina son de interés biotecnológico para las biorrefinerías de la lignocelulosa [1]. En concreto, las lignina peroxidasas (LiPs) han recibido una atención especial por su capacidad para oxidar directamente este polímero aromático [2]. Hasta ahora, estas enzimas solo se habían identificado en basidiomicetos del orden Polyporales, donde se incluyen la mayoría de las especies degradadoras de madera.Tras la secuenciación del genoma de Agrocybe pediades, caracterizado por crecer sobre hojarasca, identificamos la primera enzima ligninolítica de esta familia (ApeLiP) en un hongo del orden Agaricales [3]. La secuencia de ApeLiP se clonó, expresó en cuerpos de inclusión en Eschericchia coli y replegó in vitro, obteniéndose así la enzima activa. Una vez purificada demostramos su capacidad para oxidar compuestos modelo de lignina fenólicos y no fenólicos. Esta actividad se asoció a un triptófano catalítico (Trp166) expuesto al solvente (identificado en la estructura de ApeLiP obtenida mediante difracción de rayos-X) tras verificar la pérdida de la capacidad oxidativa en la variante Trp166Ala. Diferentes estudios, incluyendo la determinación de su potencial redox y estabilidad a pH, determinaron que las propiedades de ApeLiP son compatibles con su capacidad para oxidar y modificar estructuralmente la lignina, tal como se demostró utilizando lignina real mediante espectroscopía de flujo detenido y 2D-NMR. Este trabajo demuestra que no solo los Polyporales degradadores de madera, sino también los hongos Agaricales formadores de setas, tienen enzimas de enorme relevancia tanto para el reciclado del carbono en la naturaleza como para la modificación biotecnológica de la lignina.

Ancestral enzyme “resurrection” allows studying evolutionary hypotheses otherwise impossible to be tested. Here, we target fungal peroxidases playing a key role in lignin degradation, a central process for carbon recycling in land ecosystems. Ligninolytic peroxidases are secreted by wood-rotting fungi (order Polyporales), whose origin was established in the Carboniferous period associated to the appearance of these enzymes [1]. The lignin peroxidase (LiP) family includes the most efficient extant ligninolytic peroxidases. To reconstruct their evolution from the common ancestor of Polyporales peroxidases (CaPo), sequences in representative fungal genomes were curated, their phylogeny established by RAxML, and ancestral nodes reconstructed by PAML and heterologously expressed in Escherichia coli [2]. First, we found that the high reduction-potential that characterizes LiPs today was not inherited from CaPo but progressively increased through evolution. Secondly, CaPo, and its immediate progeny, were recognized as manganese peroxidases, a second peroxidase family acting on phenolic lignin via Mn(III) chelates. Interestingly, we found that the production of more recalcitrant lignin by angiosperms correlated with the appearance of a solvent-exposed catalytic tryptophan in ancestral peroxidases resulting in a shift from softwood to hardwood lignin preference, as revealed by kinetic and 2D-NMR analyses [3]. Moreover, this lignin-oxidizing tryptophan is a convergent trait appearing not only in LiP ancestors but also in the evolutionary line leading to versatile peroxidases [2] a minor third peroxidase family in Polyporales. 1. Floudas D; Binder M; Riley R; Barry K; Blanchette RA; Henrissat B; Martínez AT et al. (2012) The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336, 1715-9 2. Ayuso-Fernández I; Ruiz-Dueñas FJ; Martínez AT (2018) Evolutionary convergence in lignin degrading enzymes. PNAS 115, 6428-33 3. Ayuso-Fernández

Ancestral enzyme resurrection shows that lignindegrading peroxidases improved their activity and switched their substrate preferences during evolution with the rise of a surface catalytic tryptophan. This residue appeared coincidently with angiosperm radiation, characterized by more complex and less phenolic lignin due to incorporation of a new syringyl (S) unit. This way, fungal evolution followed that of lignin, pointing to fascinating coevolution between fungal saprotrophs and their plant hosts. Crucial events in ligninolytic peroxidase evolution coincided with plant lignin appearance and angiosperms diversification

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Lignin-like fractions isolated from several ancestral plants –including moss, lycophyte, horsetail, fern, cycad, and gnetophyte species– were structurally characterized by pyrolysis-GC/MS and 2D-NMR spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the moss and horsetail lignin samples, and the 2D-NMR experiments displayed intense correlations signals of guaiacyl (G) units in fern and cycad lignins, along with smaller amounts of p-hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte and the gnetophyte also incorporated significant amounts of syringyl (S) units, characteristic of angiosperms, which appeared much later in plant evolution. Remarkably, the HSQC spectra also revealed the presence of flavonoid compounds never reported before in natural lignins.

Understanding lignin biodegradation by saprotrophic fungi has attached an increasing interest during the last decades, as they secrete an array of oxidative enzymes that may be exploited for this polymer revalorization in biorefineries [1]. Among these enzymes, lignin peroxidases (LiPs) stand as the most efficient oxidizing lignin, being capable of acting towards the major non-p-her:¡olic moiety of its structure [2]. Until now, LiPs had been only identified and studied in white-rot Polyporales, and nothing was known about their presence in Agaricales and about the contribution of these fungi to lignin degradation in nature. Genomic studies of the grass-litter fungus Agrocybepediades revealed the existence of a lignin peroxidase in this Agaricales species, being the first LiP identified out of wood-rotting polypores [3]. Crystal structure and biochemical characterization of this enzyme (ApeLiP hereafter) revealed several features similar to other well-known ligninolytic peroxidases from Polyporales. Thus, ApeLiP contains a conserved heme-pocket and a solvent exposed tryptophan as catalytic sites. Characterization of a variant lacking the surface tryptophan showed that this is the oxidation site for non-phenolic lignin model compounds and other high redox potential substrates, also playing a role in phenolic substrate oxidation. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, it was d'emonstrated that ApeLiP can also act on real lignin. The reduction potentials of its catalytic-cycle intermediates, estimated by stopped-flow in equilibrium reactions, showed similar activation by H20 2, but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Finally, ApeLiP is stable from acidic to basic pH, a relevant feature for its biotechnological application considering its different optima for oxidation of phenolic and non phenolic compounds. These studies prove that not only wood-rottingPolyporales, but al so Agaricales mu, [1] Martínez AT, Camarero S, Ruiz-Dueñas FJ, Martínez MJ (2018). In: Beckham GT, editor. Lignin valorization: Emerging approaches. Cambridge: RSC; pp. 199-225. [2] Sáez-Jlménez V, Baratto MC, Pogni R, Rencoret J, Gutiérrez A, Santos JI, et al (2015). J BiolChem. 290: 23201-23213 [3] Ruiz-Dueñas FJ, Barrasa JM, Sánchez-García M, Camarero S, Miyauchi S, Serrano A, et al (2021). Mol Biol Evol.;38: 1428-1446

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.

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).

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

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.

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.

[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 spectro, 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, 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 lignin, 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 ligninolíti, 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.

[Background] Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of P. brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide., [Results] We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners.

In nature, white-rot fungi and some bacteria secrete powerful oxidative enzymes such as peroxidases and laccases to break down lignin and lignin products. These enzymes, together with different chemical mediators, form a chemo-enzymatic system for delignifying biomass so that the microbes can gain access to the polysaccharides. Ligninolytic oxidoreductases have been widely studied in terms of their unique reaction mechanisms and industrial applicability (largely related to production and stability issues). In the context of an integrated biorefinery, both ligninolytic organisms and ligninolytic enzymes may have a role to play for delignification and lignin valorization. This chapter reviews microbial degradation of lignin from an enzymatic perspective, discusses the recent discoveries and advances in a global context including genomic and structure/function information, and provides a perspective on the potential for its utilization in different green chemistry transformations.

Nuestro grupo de investigación, coordinado por los Dres. Ángel T. Martínez y Mª Jesús Martínez, estáintegrado en el Departamento de Biología Medioambiental y en la Unidad IPSBB (Integrated Protein Science for Biomedicine and Biotechnology) del Centro de Investigaciones Biológicas (CIB) de Madrid, uno de los centros pluridisciplinares de mayor tradición en el área de Biología en España. El principal objetivo de nuestro trabajo es aplicar microorganismos, principalmente hongos filamentosos, y sus enzimas extracelulares, en procesos industriales destinados a la obtención de combustibles, materiales y productos químicos a partir de recursos vegetales renovables, tratando de contribuir al desarrollo sostenible de nuestra sociedad. Estas actividades se concretan en varios proyectos de investigación, estructurados en torno a dos grandes líneas que abordan la bioconversión de distintos componentes de la biomasa vegetal. Los componentes de la pared celular sobre los que trabajamos, por ser muy abundantes en la naturaleza, son los polisacáridos (celulosa y hemicelulosa) y la lignina., Los primeros son transformados gracias a la acción concertada de varias glicosil hidrolasas (principalmente endoglucanasas, endoxilanasas y gluco y xilosidasas, junto con LPMOs –lytic polysaccharide monooxygenases-), mientras que la degradación del polímero de lignina se lleva a cabo gracias a la intervención de un complejo sistema enzimático extracelular en el que las oxidorreductasas ligninolíticas (principalmente peroxidasas y lacasas) y otras enzimas auxiliares (GMC oxidasas/deshidrogenasas) juegan un papel fundamental. Los lípidos son otros de los componentes importantes en muchos residuos urbanos e industriales procedentes de biomasa vegetal y también pueden ser modificados y valorizados mediante el empleo de lipasas y esterol esterasas.

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly “fueling” electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron tra