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The ability of some white rot basidiomycetes to remove lignin selectively from wood indicates that low molecular weight oxidants have a role in ligninolysis. These oxidants are likely free radicals generated by fungal peroxidases from compounds in the biodegrading wood. Past work supports a role for manganese peroxidases (MnPs) in the production of ligninolytic oxidants from fungal membrane lipids. However, the fatty acid alkylperoxyl radicals initially formed during this process are not reactive enough to attack the major structures in lignin. Here, we evaluate the hypothesis that the peroxidation of fatty aldehydes might provide a source of more reactive acylperoxyl radicals. We found that Gelatoporia subvermispora produced trans-2-nonenal, trans-2-octenal, and n-hexanal (a likely metabolite of trans-2,4-decadienal) during the incipient decay of aspen wood. Fungal fatty aldehydes supported the in vitro oxidation by MnPs of a nonphenolic lignin model dimer, and also of the monomeric model veratryl alcohol. Experiments with the latter compound showed that the reactions were partially inhibited by oxalate, the chelator that white rot fungi employ to detach Mn3+ from the MnP active site, but nevertheless proceeded at its physiological concentration of 1 mM. The addition of catalase was inhibitory, which suggests that the standard MnP catalytic cycle is involved in the oxidation of aldehydes. MnP oxidized trans-2-nonenal quantitatively to trans-2-nonenoic acid with the consumption of one O2 equivalent. The data suggest that when Mn3+ remains associated with MnP, it can oxidize aldehydes to their acyl radicals, and the latter subsequently add O2 to become ligninolytic acylperoxyl radicals.IMPORTANCEThe biodegradation of lignin by white rot fungi is essential for the natural recycling of plant biomass and has useful applications in lignocellulose bioprocessing. Although fungal peroxidases have a key role in ligninolysis, past work indicates that biodegradation is initiat

Aryl-alcohol oxidases (AAOs) are members of the glucose-methanol-choline oxidase/dehydrogenase (GMC) superfamily. These extracellular flavoproteins have been described as auxiliary enzymes in the degradation of lignin by several white-rot basidiomycetes. In this context, they oxidize fungal secondary metabolites and lignin-derived compounds using O2 as an electron acceptor, and supply H2O2 to ligninolytic peroxidases. Their substrate specificity, including mechanistic aspects of the oxidation reaction, has been characterized in Pleurotus eryngii AAO, taken as a model enzyme of this GMC superfamily. AAOs show broad reducing-substrate specificity in agreement with their role in lignin degradation, being able to oxidize both nonphenolic and phenolic aryl alcohols (and hydrated aldehydes). In the present work, the AAOs from Pleurotus ostreatus and Bjerkandera adusta were heterologously expressed in Escherichia coli, and their physicochemical properties and oxidizing abilities were compared with those of the well-known recombinant AAO from P. eryngii. In addition, electron acceptors different from O2, such as p-benzoquinone and the artificial redox dye 2,6-Dichlorophenolindophenol, were also studied. Differences in reducing-substrate specificity were found between the AAO enzymes from B. adusta and the two Pleurotus species. Moreover, the three AAOs oxidized aryl alcohols concomitantly with the reduction of p-benzoquinone, with similar or even higher efficiencies than when using their preferred oxidizing-substrate, O2. IMPORTANCE In this work, quinone reductase activity is analyzed in three AAO flavooxidases, whose preferred oxidizing-substrate is O2. The results presented, including reactions in the presence of both oxidizing substrates-benzoquinone and molecular oxygen-suggest that such aryl-alcohol dehydrogenase activity, although less important than its oxidase activity in terms of maximal turnover, may have a physiological role during fungal decay of lignocellulose

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.

Unspecific peroxygenases (UPOs) are extracellular fungal enzymes of biotechnological interest as self-sufficient (and more stable) counterparts of cytochrome P450 monooxygenases, the latter being present in most living cells. Expression hosts and structural information are crucial for exploiting UPO diversity (over eight thousand UPO-type genes were identified in sequenced genomes) in target reactions of industrial interest. However, while many thousands of entries in the Protein Data Bank include molecular coordinates of P450 enzymes, only 19 entries correspond to UPO enzymes, and UPO structures from only two species (Agrocybe aegerita and Hypoxylon sp.) have been published to date. In the present study, two UPOs from the basidiomycete Marasmius rotula (rMroUPO) and the ascomycete Collariella virescens (rCviUPO) were crystallized after sequence optimization and Escherichia coli expression as active soluble enzymes. Crystals of rMroUPO and rCviUPO were obtained at sufficiently high resolution (1.45 and 1.95 angstrom, respectively) and the corresponding structures were solved by molecular replacement. The crystal structures of the two enzymes (and two mutated variants) showed dimeric proteins. Complementary biophysical and molecular biology studies unveiled the diverse structural bases of the dimeric nature of the two enzymes. Intermolecular disulfide bridge and parallel association between two alpha-helices, among other interactions, were identified at the dimer interfaces. Interestingly, one of the rCviUPO variants incorporated the ability to produce fatty acid diepoxides-reactive compounds with valuable cross-linking capabilities-due to removal of the enzyme C-terminal tail located near the entrance of the heme access channel. In conclusion, different dimeric arrangements could be described in (short) UPO crystal structures.

Terminal alkenes are among the most attractive starting materials for the synthesis of epoxides, which are essential and versatile intermediate building blocks for the pharmaceutical, fla-voring, and polymer industries. Previous research on alkene epoxidation has focused on the use of several oxidizing agents and/or different enzymes, including cytochrome P450 monooxygenases, as well as microbial whole-cell catalysts that have several drawbacks. Alternatively, we explored the ability of unspecific peroxygenases (UPOs) to selectively epoxidize terminal alkenes. UPOs are attractive biocatalysts because they are robust extracellular enzymes and only require H2O2 as cosub-strate. Here, we show how several UPOs, such as those from Cyclocybe (Agrocybe) aegerita (AaeUPO), Marasmius rotula (MroUPO), Coprinopsis cinerea (rCciUPO), Humicola insolens (rHinUPO), and Daldinia caldariorum (rDcaUPO), are able to catalyze the epoxidation of long-chain terminal alkenes (from C12:1 to C20:1) after an initial optimization of several reaction parameters (cosolvent, cosub-strate, and pH). In addition to terminal epoxides, alkenols and other hydroxylated derivatives of the alkenes were formed. Although all UPOs were able to convert and epoxidize the alkenes, nota-ble differences were observed between them, with rCciUPO being responsible for the highest sub-strate turnover and MroUPO being the most selective with respect to terminal epoxidation. The potential of peroxygenases for epoxidizing long-chain terminal alkenes represents an interesting and green alternative to the existing synthesis technologies. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Lignins released in the black liquors of kraft pulp mills are an underutilised source of aromatics. Due to their phenol oxidase activity, laccases from ligninolytic fungi are suitable biocatalysts to depolymerise kraft lignins, which are characterised by their elevated phenolic content. However, the alkaline conditions necessary to solubilise kraft lignins make it difficult to use fungal laccases whose activity is inherently acidic. We recently developed through enzyme-directed evolution high-redox potential laccases active and stable at pH 10. Here, the ability of these tailor-made alkaliphilic fungal laccases to oxidise, demethylate, and depolymerise eucalyptus kraft lignin at pH 10 is evidenced by the increment in the content of phenolic hydroxyl and carbonyl groups, the methanol released, and the appearance of lower molecular weight moieties after laccase treatment. Nonetheless, in a second assay carried out with higher enzyme and lignin concentrations, these changes were accompanied by a strong increase in the molecular weight and content of β–O–4 and β–5 linkages of the main lignin fraction, indicating that repolymerisation of the oxidised products prevails in one-pot reactions. To prevent it, we finally conducted the enzymatic reaction in a bench-scale reactor coupled to a membrane separation system and were able to prove the depolymerisation of kraft lignin by high-redox alkaliphilic laccase.

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

Epoxides of vegetable oils and free and methylated fatty acids are of interest for several industrial applications. In the present work, refined rapeseed, sunflower, soybean, and linseed oils, with very different profiles of mono- and poly-unsaturated fatty acids, were saponified and transesterified, and the products treated with wild unspecific peroxygenases (UPOs, EC 1.11.2.1) from the ascomycete Chaetomium globosum (CglUPO) and the basidiomycete Marasmius rotula (MroUPO), as well as with recombinant UPO of the ascomycete Humicola insolens (rHinUPO), as an alternative to chemical epoxidation that is non-selective and requires strongly acidic conditions. The three enzymes were able of converting the free fatty acids and the methyl esters from the oils into epoxide derivatives, although significant differences in the oxygenation selectivities were observed between them. While CglUPO selectively produced "pure" epoxides (monoepoxides and/or diepoxides), MroUPO formed also hydroxylated derivatives of these epoxides, especially in the case of the oil hydrolyzates. Hydroxylated derivatives of non-epoxidized unsaturated fatty acids were practically absent in all cases, due to the preference of the three UPOs selected for this study to form the epoxides. Moreover, rHinUPO, in addition to forming monoepoxides and diepoxides of oleic and linoleic acid (and their methyl esters), respectively, like the other two UPOs, was capable of yielding the triepoxides of alpha-linolenic acid and its methyl ester. These enzymes appear as promising biocatalysts for the environmentally friendly production of reactive fatty-acid epoxides given their self-sufficient monooxygenase activity with selectivity toward epoxidation, and the ability to epoxidize, not only isolated pure fatty acids, but also complex mixtures from oil hydrolysis or transesterification containing different combinations of unsaturated (and saturated) fatty acids.

Unspecific peroxygenases (UPOs) are fungal secreted counterparts of the cytochrome P450 monooxygenases present in most living cells. Both enzyme types share the ability to perform selective oxygenation reactions. Moreover, the Marasmius rotula UPO (MroUPO) catalyzes reactions of interest compared with the previously described UPOs, including formation of reactive epoxy fatty acids. To investigate substrate epoxidation, the most frequent positions of oleic acid at the MroUPO heme channel were predicted using binding and molecular dynamics simulations. Then, mutations in neighbor residues were designed aiming at modulating the enzyme epoxidation vs hydroxylation ratio. Both the native (wild-type recombinant) MroUPO and the mutated variants were expressed in Escherichia coli as active enzymes, and their action on oleic and other fatty acids was investigated by gas chromatography−mass spectrometry in combination with kinetic analyses. Interestingly, a small modification of the cannel shape in the I153T variant increased the ratio between epoxidized oleic acid and its additionally hydroxylated derivatives. A fully opposite effect was attained with the double I153F/S156F variant that completely abolished the ability of the MroUPO to epoxidize oleic acid (while no activity was detected for the I153V variant). The rationale for these results was revealed by the substrate positioning in the above computational simulations, which predict a shorter distance between the oleic acid double bond and the oxygen atom of the peroxide-activated heme (compound I) in the I153T variant than in the native enzyme, promoting epoxidation. In contrast, the I153F/S156F double mutation fully prevents the approach of oleic acid in the bent conformation required for double-bond epoxidation, although its (sub)terminal hydroxylation was predicted and experimentally confirmed. The I153T mutation also increased the UPO selectivity on polyunsaturated fatty acid epoxidation, strongly reducing the ratio

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.

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.

Selective oxyfunctionalizations of aliphatic compounds are difficult chemical reactions, where enzymes can play an important role due to their stereo- and regio-selectivity and operation under mild reaction conditions. P450 monooxygenases are well-known biocatalysts that mediate oxyfunctionalization reactions in different living organisms (from bacteria to humans). Unspecific peroxygenases (UPOs), discovered in fungi, have arisen as “dream biocatalysts” of great biotechnological interest because they catalyze the oxyfunctionalization of aliphatic and aromatic compounds, avoiding the necessity of expensive cofactors and regeneration systems, and only depending on H2O2 for their catalysis. Here, we summarize recent advances in aliphatic oxyfunctionalization reactions by UPOs, as well as the molecular determinants of the enzyme structures responsible for their activities, emphasizing the differences found between well-known P450s and the novel fungal peroxygenases. © 2021 The Authors

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete., We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources.The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signaling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and Lytic Polysaccharide Monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition.Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

The use and integration of enzymatic processes for the biotransformation of biomass within the biorefinery framework creates the need to confirm whether these novel production systems are in the route to environmental sustainability. In this study, the environmental profiles of the production of two oxidative enzymes, hydroxymethylfurfural oxidase (HMFO) from Methylovorus and unspecific peroxygenase (UPO) from Chaetomium globosum (CglUPO) for the enzymatic production of FDCA as precursor of bioplastics were analyzed. Laboratory-scale experiments allowed the identification of the consumption of energy, with over 80% share in every impact category for HMFO and chemicals and energy in CglUPO as primary hotspots of the systems. The results are transposed for HMFO when laboratory inventories were extrapolated to full scale processing, showing that impacts are attributed not only to energy demand but also to the use of chemicals required for the formulation of the culture medium. In terms of process units, the fermenter, where enzyme production takes place, corresponds to the stage that contributes the most to the environmental impacts, with a 57% share, followed by the downstream separation scheme (37%). Extrapolation of laboratory data to full-scale also represented a change in the relative difference of the impact per functional unit of 45% for CgIUPO. The endpoint damage categories showed a significant reduction in their full-scale impacts to about half the burden. The analysis of the outcomes of the uncertainty analysis showed that the resource depletion category had the least dispersion of data, while the level of uncertainty is more relevant for human health, as it takes into account the combined effect of a larger number of impact categories and the processes involved. This study shows that, although being bio-based catalysts, the production of enzymes involves several steps which may incur in environmental impact. Thus, it is recommended that enzymes are carefull

Background: 2,5-Furandicarboxylic acid (FDCA) is a precursor for green plastics due to its structural similarity to terephthalic acid, a common precursor of oil-derived polymers, and its potential production from sugars obtained from plant biomass. Hydroxymethylfurfural oxidase (HMFO) has been reported as a promising biocatalyst for FDCA production since it can convert bio-based 5-hydroxymethylfurfural (HMF) into FDCA building block. This three-step oxidation reaction occurs through the diformylfuran and 2,5-formylfurancarboxylic acid (FFCA) intermediates. Several efforts have been made for the development of HMFO variants that increase FDCA yields by improving their activities over the reaction intermediates. However, there is still limited insight into how operational conditions can influence these enzymatic reactions. The setup of optimal reaction conditions would enable to understand potential problems hampering the effective industrial production of this bioplastic precursor using HMFO as biocatalyst., Results: In this work, several parameters affecting the performance of Methylovorus sp HMFO oxidizing HMF have been analyzed for the wild-type enzyme, and its V367R and W466F single variants, V367R/W466F double variant, and I73V/H74Y/G356H/V367R/T414K/A419Y/A435E/W466F (8BxHMFO) octuple variant. Our results show how the oxidation of HMF by HMFO enzymes is highly influenced by pH, with different optimal pH values for the different improved variants. Moreover, the enzymes are not stable at high hydrogen peroxide concentrations and their activity is inhibited by the FFCA intermediate in a pH-dependent way. These limitations can be efficiently overcome with the addition of catalase to the reaction medium, which removes the hydrogen peroxide formed during the oxidations, and the controlled dosage of the substrate to limit the amount of FFCA accumulated in the reaction. The different behavior of wild-type HMFO and its variants against pH, hydrogen peroxide and FFCA highlights the importance of considering each variant as an individual enzyme with its own operational conditions for an eventual industrial FDCA production., Conclusions: This work provides information of those parameters that condition a high production of FDCA by HMFO. Unraveling these factors allowed to increase the FDCA yields by using the most stable enzymes at their optimal pH for HMF oxidation, removing the peroxide with catalase, and avoiding FFCA accumulation by controlling substrate and/or enzyme concentration. These above findings will be useful when planning a future scale-up of these conversions and will provide new viewpoints for the design of HMFO variants that render a more effective performance during HMF conversion into FDCA.

Epoxide metabolites from n-3 and n-6 polyunsaturated fatty acids arouse interest thanks to their physiological and pharmacological activities. Their chemical synthesis has significant drawbacks, and enzymes emerge as an alternative with potentially higher selectivity and greener nature. Conversion of eleven eicosanoid, docosanoid, and other n-3/n-6 fatty acids into mono-epoxides by fungal unspecific peroxygenases (UPOs) is investigated, with emphasis on the Agrocybe aegerita (AaeUPO) and Collariella virescens (rCviUPO) enzymes. GC-MS revealed the strict regioselectivity of the n-3 and n-6 reactions with AaeUPO and rCviUPO, respectively, yielding 91%-quantitative conversion into mono-epoxides at the last double bond. Then, six of these mono-epoxides were obtained at mg-scale, purified and further structurally characterized by 1H, 13C and HMBC NMR. Moreover, chiral HPLC showed that the n-3 epoxides were also formed (by AaeUPO) with total S/R enantioselectivity (ee > 99%) while the n-6 epoxides (from rCviUPO reactions) were formed in nearly racemic mixtures. The high regio- and enantioselectivity of several of these reactions unveils the synthetic utility of fungal peroxygenases in fatty acid epoxidation.

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.