The white rot basidiomycete Gelatoporia subvermispora produces fatty aldehydes that enable fungal manganese peroxidases to degrade recalcitrant lignin structures.

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 initiated by smaller, as yet unidentified oxidants that can infiltrate the substrate. Here, we present evidence that the peroxidase-catalyzed oxidation of naturally occurring fungal aldehydes may provide a source of ligninolytic free radical oxidants.

Depolymerisation of Kraft Lignin by Tailor-Made Alkaliphilic Fungal Laccases.

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 ß-O-4 and ß-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.

Expanding the Physiological Role of Aryl-Alcohol Flavooxidases as Quinone Reductases.

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 by the reduction of quinones (and phenoxy radicals) from lignin degradation, preventing repolymerization. Moreover, the resulting hydroquinones would participate in redox-cycling reactions for the production of hydroxyl free radical involved in the oxidative attack of the plant cell-wall. Hydroquinones can also act as mediators for laccases and peroxidases in lignin degradation in the form of semiquinone radicals, as well as activators of lytic polysaccharide monooxygenases in the attack of crystalline cellulose. Moreover, reduction of these, and other phenoxy radicals produced by laccases and peroxidases, promotes lignin degradation by limiting repolymerization reactions. These findings expand the role of AAO in lignin biodegradation.

A global phylogenomic analysis of the shiitake genus Lentinula.

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.

Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements.

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 Å, 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 α-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.

Engineering Collariella virescens Peroxygenase for Epoxides Production from Vegetable Oil.

Vegetable oils are valuable renewable resources for the production of bio-based chemicals and intermediates, including reactive epoxides of industrial interest. Enzymes are an environmentally friendly alternative to chemical catalysis in oxygenation reactions, epoxidation included, with the added advantage of their potential selectivity. The unspecific peroxygenase of Collariella virescens is only available as a recombinant enzyme (rCviUPO), which is produced in Escherichia coli for protein engineering and analytical-scale optimization of plant lipid oxygenation. Engineering the active site of rCviUPO (by substituting one, two, or up to six residues of its access channel by alanines) improved the epoxidation of individual 18-C unsaturated fatty acids and hydrolyzed sunflower oil. The double mutation at the heme channel (F88A/T158A) enhanced epoxidation of polyunsaturated linoleic and α−linolenic acids, with the desired diepoxides representing > 80% of the products (after 99% substrate conversion). More interestingly, process optimization increased (by 100-fold) the hydrolyzate concentration, with up to 85% epoxidation yield, after 1 h of reaction time with the above double variant. Under these conditions, oleic acid monoepoxide and linoleic acid diepoxide are the main products from the sunflower oil hydrolyzate.

Novel Fatty Acid Chain-Shortening by Fungal Peroxygenases Yielding 2C-Shorter Dicarboxylic Acids.

Unspecific peroxygenases (UPOs), the extracellular enzymes capable of oxygenating a potpourri of aliphatic and aromatic substrates with a peroxide as co-substrate, come out with a new reaction: carbon-chain shortening during the conversion of fatty acids with the well-known UPOs from Coprinopsis cinerea (rCciUPO) and Cyclocybe (Agrocybe) aegerita (AaeUPO). Although a pathway (Cα-oxidation) for shortening the hydrocarbon chain of saturated fatty acids has already been reported for the UPO from Marasmius rotula (MroUPO), it turned out that rCciUPO and AaeUPO shorten the chain length of both saturated and unsaturated fatty acids in a different way. Thus, the reaction sequence does not necessarily start at the Cα-carbon (adjacent to the carboxyl group), as in the case of MroUPO, but proceeds through the subterminal (ω-1 and ω-2) carbons of the chain via several oxygenations. This new type of shortening leads to the formation of a dicarboxylic fatty acid reduced in size by two carbon atoms in the first step, which can subsequently be further shortened, carbon by carbon, by the UPO Cα-oxidation mechanism.

Enzymatic Epoxidation of Long-Chain Terminal Alkenes by Fungal Peroxygenases.

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, flavoring, 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 cosubstrate. 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, cosubstrate, 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, notable differences were observed between them, with rCciUPO being responsible for the highest substrate 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.

Ancestral sequence reconstruction as a tool to study the evolution of wood decaying fungi.

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.

Regioselective and Stereoselective Epoxidation of n-3 and n-6 Fatty Acids by Fungal Peroxygenases.

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.

New Insights on Structures Forming the Lignin-Like Fractions of Ancestral Plants.

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, ß-O-4' alkyl-aryl ethers were the most abundant substructures, followed by condensed ß-5' phenylcoumarans and ß-ß' 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%).

Agaricales Mushroom Lignin Peroxidase: From Structure-Function to Degradative Capabilities.

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.

Optimizing operational parameters for the enzymatic production of furandicarboxylic acid building block.

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.

A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material.

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.

Comparing Ligninolytic Capabilities of Bacterial and Fungal Dye-Decolorizing Peroxidases and Class-II Peroxidase-Catalases.

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-ß- 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.

Advances in enzymatic oxyfunctionalization of aliphatic compounds.

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.

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

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 signalling 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.

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity.

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.

Author Correction: Genome sequencing of Rigidoporus microporus provides insights on genes important for wood decay, latex tolerance and interspecific fungal interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reaction mechanisms and applications of aryl-alcohol oxidase.

Aryl-alcohol oxidases (AAO) constitute a family of FAD-containing enzymes, included in the glucose-methanol-choline oxidase/dehydrogenase superfamily of proteins. They are commonly found in fungi, where their eco-physiological role is to produce hydrogen peroxide that activates ligninolytic peroxidases in white-rot (lignin-degrading) basidiomycetes or to trigger the Fenton reactions in brown-rot (carbohydrate-degrading) basidiomycetes. These enzymes catalyze the oxidation of a plethora of aromatic, and some aliphatic, polyunsaturated alcohols bearing conjugated primary hydroxyl group. Besides, the enzymes show activity on the hydrated forms of the corresponding aldehydes. Some AAO features, such as the broad range of substrates that it can oxidize (with the only need of molecular oxygen as co-substrate) and its stereoselective mechanism, confer good properties to these enzymes as industrial biocatalysts. In fact, AAO can be used for different biotechnological applications, such as flavor synthesis, secondary alcohol deracemization and oxidation of furfurals for the production of furandicarboxylic acid as a chemical building block. Also, AAO can participate in processes of interest in the wood biorefinery and textile industries as an auxiliary enzyme providing hydrogen peroxide to ligninolytic or dye-decolorizing peroxidases. Both rational design and directed molecular evolution have been employed to engineer AAO for some of the above biotechnological applications.