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10 páginas.- 4 figuras.- 2 tablas.- 30 referencias.- Supporting information for this article is given via a link at the end of the document https://onlinelibrary.wiley.com/doi/10.1002/anie.202217372, he hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer.The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer., This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2, the I+D+I PID2019-106166RB-I00-OXYWAVE Spanish project funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/, the PID2020-118968RB-I00 LILI project from the Spanish MCIN/AEI/10.13039/501100011033, the ‘Comunidad de Madrid’ Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CMand the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. PGS thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and to the Ministry of Science and Innovation for her contract as part of PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR.DGP thanks Juan de la Cierva Incorporación contract Ref nº: IJC2020-043725-I, funded by MCIN / AEI / 10.13039/501100011033, and the EUNextGenerationEU/PRTR program. The authors thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline.

Deadwood represents an important structural component of forest ecosystems, where it provides diverse niches for saproxylic biota. Although wood-inhabiting prokaryotes are involved in its degradation, knowledge about their diversity and the drivers of community structure is scarce. To explore the effect of deadwood substrate on microbial distribution, the present study focuses on the microbial communities of deadwood logs from 13 different tree species investigated using an amplicon based deep-sequencing analysis. Sapwood and heartwood communities were analysed separately and linked to various relevant wood physico-chemical parameters. Overall, Proteobacteria, Acidobacteria and Actinobacteria represented the most dominant phyla. Microbial OTU richness and community structure differed significantly between tree species and between sapwood and heartwood. These differences were more pronounced for heartwood than for sapwood. The pH value and water content were the most important drivers in both wood compartments. Overall, investigating numerous tree species and two compartments provided a remarkably comprehensive view of microbial diversity in deadwood.

Nitrogen deposition can strongly affect biodiversity, but its specific effects on terrestrial microbial communities and their roles for ecosystem functions and processes are still unclear. Here, we investigated the impacts of N deposition on wood-inhabiting fungi (WIF) and their related ecological functions and processes in a highly N-limited deadwood habitat. Based on high-throughput sequencing, enzymatic activity assay and measurements of wood decomposition rates, we show that N addition has no significant effect on the overall WIF community composition or on related ecosystem functions and processes in this habitat. Nevertheless, we detected several switches in presence/absence (gain/loss) of wood-inhabiting fungal OTUs due to the effect of N addition. The responses of WIF differed from previous studies carried out with fungi living in soil and leaf-litter, which represent less N-limited fungal habitats. Our results suggest that adaptation at different levels of organization and functional redundancy may explain this buffered response and the resistant microbial-mediated ecosystem function and processes against N deposition in highly N-limited habitats.

Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less‐activated hydrocarbons, by transferring peroxide‐borne oxygen. We investigated a cell‐free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg−1. Although the well‐known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5‐epoxide of testosterone in β‐configuration and 16α‐hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.

Fil: Carabajal, Maira Lia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Micologia y Botanica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micologia y Botanica; Argentina

Peroxygenasen katalysieren eine Vielzahl stereoselektiver Oxyfunktionalisierungen unter Verwendung von Wasserstoffperoxid als einzigem Oxidationsmittel. Allerdings werden Peroxygenasen auch durch H2O2 irreversibel inaktiviert. Fur robuste Peroxygenase-Reaktionen ist also eine effiziente In-situ-Bereitstellung von H2O2 unabdingbar. Hier beschreiben wir eine neuartige Enzymkaskade zur reduktiven Aktivierung von Luftsauerstoff unter Verwendung von Methanol als stochiometrischem Reduktionsmittel. Am Beispiel der stereoselektiven Hydroxylierung von Ethylbenzol zu (R)-1-Phenylethanol zeigen wir die effiziente Nutzung aller in Methanol gespeicherten Reduktionsaquivalente zur reduktiven Aktivierung von O2.

Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions.

Specific oxyfunctionalization chemistry requires a delicate balance between reactivity and selectivity, and therefore still represents a major challenge in organic chemistry. Cytochrome P450 monooxygenases (P450s) are widely considered as catalysts that solve this dilemma. Here, the reactive Compound I is embedded within a defined three-dimensional protein structure, thus enabling regio-, chemo-, and enantiospecific oxyfunctionalization reactions even on nonactivated hydrocarbons. Their practical application, however, is limited to whole-cell biotransformations because of their cofactor dependency and their complex molecular architecture. However, microbial transformations are not always straightforward to perform and suffer from intrinsic disadvantages such as reactant metabolization and toxicity, and often low productivities. Great hope has been placed on the protein superfamily of heme/thiolate peroxidases to solve both challenges, the cofactor dependency as well as the complicated molecular architecture of P450s by using the hydrogen peroxide shunt. Here, the catalytically active oxoferryl species is formed directly from H2O2 instead of from reductive activation of O2. Seemingly, this allows simple circumvention of the abovementioned challenges and enables practical oxyfunctionalization procedures. Chloroperoxidase (CPO) from Caldariomyces fumago (Leptoxyphium fumago) represents the archetype of such heme/thiolate peroxidases. In fact, thus far CPO is the only peroxidase used for oxyfunctionalization reactions. Unfortunately, CPO is active in sulfoxidation reactions only, whereas its performance in hydroxylation of C H bonds and epoxidation reactions drops by several orders of magnitude. Generally, CPO only performs a few hundred turnovers prior to loss of catalytic activity (see below), and nonactivated hydrocarbons such as cyclohexane are not converted at all by CPO. Obviously, this disqualifies CPO as a broadly applicable catalyst for oxyfunctionalization chemistry. However, with more and more genome sequences becoming available, today, more than 100 putative heme/ thiolate CPO analogues can be found in nucleotide databases (Scheme 1). Hence, there is a wealth of potential alternatives to CPO that are waiting to be discovered, and they potentially have more suitable catalytic properties for oxyfunctionalization. Recently, a novel peroxidase from the basidomycetous fungus Agrocybe aegerita (AaeAPO=Agrocybe aegerita aromatic peroxygenase) has been isolated and characterized. AaeAPO specifically converts aromatic hydrocarbons into the corresponding phenols and has recently been classified as an “unspecific peroxygenase” in the E.C. nomenclature (E.C.1.11.2.1). Herein we report that AaeAPO is an active and versatile catalyst for enantiospecific hydroxylation and epoxidation reactions. Under nonoptimized reaction conditions AaeAPO performance (in terms of turnover number) exceeds that reported for CPO by at least two orders of magnitude. Furthermore, we demonstrate that AaeAPO, in contrast to CPO, can also hydroxylate nonactivated C H bonds. Since AaeAPO, like all heme-dependent enzymes, is easily inactivated by H2O2, strict control over the in situ Scheme 1. Simplified phylogenetic tree of the heme/thiolate superfamily. The names marked in bold refer to fungal species whose heme/ thiolate enzymes have been purified and characterized. The other terms belong to taxonomic units of the fungal kingdom with putative CPOor APO-like proteins found in databases.

Fungal peroxygenases are novel extracellular heme-thiolate biocatalysts that are capable of catalyzing the selective monooxygenation of diverse organic compounds, using only H2O2 as a cosubstrate. Little is known about the physiological role or the catalytic mechanism of these enzymes. We have found that the peroxygenase secreted by Agrocybe aegerita catalyzes the H2O2-dependent hydroxylation of linear alkanes at the 2-position and 3-position with high efficiency, as well as the regioselective monooxygenation of branched and cyclic alkanes. Experiments with n-heptane and n-octane showed that the hydroxylation proceeded with complete stereoselectivity for the (R)-enantiomer of the corresponding 3-alcohol. Investigations with a number of model substrates provided information about the route of alkane hydroxylation: (a) the hydroxylation of cyclohexane mediated by H218O2 resulted in complete incorporation of 18O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n-hexane-1,1,1,2,2,3,3-D7 showed a large intramolecular deuterium isotope effect [(kH/kD)obs] of 16.0 ± 1.0 for 2-hexanol and 8.9 ± 0.9 for 3-hexanol; and (c) the hydroxylation of the radical clock norcarane led to an estimated radical lifetime of 9.4 ps and an oxygen rebound rate of 1.06 × 1011 s−1. These results point to a hydrogen abstraction and oxygen rebound mechanism for alkane hydroxylation. The peroxygenase appeared to lack activity on long-chain alkanes (> C16) and highly branched alkanes (e.g. tetramethylpentane), but otherwise exhibited a broad substrate range. It may accordingly have a role in the bioconversion of natural and anthropogenic alkane-containing structures (including alkyl chains of complex biomaterials) in soils, plant litter, and wood.

Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient.

Agrocybe aegerita peroxidase (AaP) is a versatile extracellular biocatalyst that can oxygenate aromatic com- pounds. Here, we report on the selective oxidation of pyridine (PY) yielding pyridine N-oxide as sole product. Using H2 18 O2 as co-substrate, the origin of oxygen was confirmed to be the per- oxide. Therefore, AaP can be regarded as a true peroxygenase transferring one oxygen atom from peroxide to the substrate. To our best knowledge, there are only two types of enzymes oxi- dizing PY at the nitrogen: bacterial methane monooxygenase and a few P450 monooxygenases. AaP is the first extracellular enzyme and the first peroxidase that catalyzes this reaction, and it converted also substituted PYs into the corresponding N-oxides. � 2008 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved.

(Received 17 October 2000/Accepted 12 April 2001) The sources of ligninocellulose that occur in various forms in nature are so vast that they can only be compared to those of water. The results of several, more recent experiments showed that laccase probably possesses the big ability for "lignin-barrier" breakdown of ligninocellulose. The degradation of this compound is currently understood as an enzymatic process mediated by small molecules, therefore, this review will focus on the role of these mediators and radicals working in concert with enzymes. The fungi having a versatile machinery of enzymes are able to attack directly the "lignin- barrier" or can use a multienzyme system including "feed-back" type enzymes allowing for simulta- neous transformation of lignin and carbohydrate compounds.

The depolymerization of humic acids (HAs) obtained from low-rank coal (lignite) to fulvic acids (FAs) was investigated in a cell-free system (in vitro) using manganese peroxidase (MnP) from the white-rot fungus Clitocybula dusenii b11. MnP was produced in surface cultures of C. dusenii which were induced with manganese (II) ions (Mn2+, 300 microM). The optimum conditions for the action of MnP were determined by varying following parameters of the enzyme assay: i) concentration of Mn2+, ii) concentration of hydrogen peroxide (H2O2), iii) pH value and iv) temperature. Optimum parameters determined were used in subsequent in vitro depolymerization studies of humic acids. For that purpose, following parameters of the reaction mixture were additionally varied: concentration of HAs, concentration of the thiol mediator glutathione (GSH), presence and concentration of organic solvents. As the result, following parameters were found to be optimal for the MnP-catalyzed in vitro depolymerization of HAs into low-molecular weight FAs (MnP activity 0.12 U/ml): 250 micrograms/ml HAs, 1 mM MnCl2, 46 microM/min H2O2 (continuously supplied by glucose oxidase), 600 microM GSH, 4% (v/v) N,N-dimethylformamide (DMF), pH 4.0, and 37 degrees C.

The in vitro-oxidation of the three- and four-ring polycyclic aromatic hydrocarbons (PAH) anthracene, phenanthrene, pyrene and fluoranthene by preparations of extracellular lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lacc) of the white rot fungus Nematoloma frowardii and by mushroom tyrosinase (Tyr) and horseradish peroxidase (HRP) was investigated. LiP transformed 58.6% of anthracene and 34.2% of pyrene, whereas 31.5% of anthracene and 11.2% pyrene were oxidized by MnP. In the presence of the mediating substances veratryl alcohol (for LiP), GSH (for MnP), and ABTS (for Lacc, Tyr, HRP), the conversion of PAH was enhanced in most cases. Inclusion of PAH-derivatives, known as intermediates or potential dead-end-products of microbial PAH metabolism, in the in vitro-oxidation studies, demonstrated that the hydroxylated PAH metabolites served as substrates for all oxidoreductases tested, whereas PAH-quinones and oxo-metabolites were not transformed.

Preparations of extracellular manganese peroxidase from the white-rot fungus Nematoloma frowardii and the litter decaying fungus Stropharia rugosoannulata converted rapidly the main intermediates of the explosive 2,4,-trinitrotoluene--the aminonitrotoluenes. In a cell-free system, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,6-diamino-4-nitrotoluene were degraded without formation of identifiable metabolites. Radioactive experiments using a complex mixture of uniform ring-labeled 14C-TNT reduction products demonstrated the partial direct mineralization of these compounds by manganese peroxidase. The extent of aminonitrotoluene conversion as well as the release of 14CO2 from TNT reduction products were considerably enhanced in the presence of thiols like reduced glutathione or the amino acid L-cystein, which probably act as secondary mediators.

Polycyclic aromatic hydrocarbons (PAHs), like phenanthrene (three ring PAH) and pyrene (four ring PAH) are widely distributed pollutants in terrestrial and aquatic ecosystems (BLUMER 1976). The ability of wood-decaying fungi to mineralize PAHs seems to be con- nected with the activity of ligninolytic enzymes, since mineralization in liquid or soil cultu- res has mostly been described for wood-decaying fungi like the white-rot fungi

Methyl-substituted phenols are natural components of coal tar and mineral oil. Therefore waste streams from coal and oil manufacturing industry contain large amounts of these compounds. In addition p-cresol is formed during the degradation process of tyrosin in bacteria under anaerobic conditions (D' ARI and BARKER 1985). The microbial metabolism of cresols, especially of p-cresol, has been intensively studied. Two separate catabolic sequences have been described forp-cresol: i) via 4-methylcatechol and preservation of the methyl group and ii) via oxidation of the methyl group with protocatechuate as the intermediate. In the first pathway, an additional hydroxyl group is introduced to form 4-methylcatechol as substrate for ring fission dioxygenases (BAYLY et al. 1966). In most cases bacteria use the metu-cleava- ge for splitting the aromatic ring of 4-methylcatechol (DAGLEY and PATEL 1957); the only procaryote that has been clearly shown to use ortho-fission enzymes for producti- ve degradation of 4-methylcatechol appears to be Gordona rubra (MILLER and CAIN 1968). Other bacteria (e.g. Pseudomonas spec. B 13, Bacillus pumilus) cometabolize me- thylcatechols via ortho-ring fission and formation of methylated muconolactones as dead- end products (KNACKMUSS er al. 1976, GUNTHER

The conversion and enantioselectivity of peroxygenase-catalyzed benzylic hydroxylation and epoxidation reactions depend strongly on the structure of starting compounds.

Resting phenol-grown mycelia of the fungus Penicillium frequentans strain Bi 7/2 were shown to be capable of metabolizing various monohalogenated phenols as well as 3,4-dichlorophenol. 2,4.dichlorophenol could be metabolized in the presence of phenol as cosubstrate. In the first degradation step the halogenated phenols were oxidized to the corresponding halocatechols. Halocatechols substituted in para-position (4-halocatechols) were further degraded under formation of 4-carboxymethylenbut-2-en-4-olide. A partial dehalogenation took place splitting the ring system. 3-Halocatechols were cleaved to 2-halomuconic acids as dead end metabolites without a dehalogenation step. Dichlorophenols were only transformed to the corresponding catechols. In addition 3,5-dichloro-catechol was O-methylated to give two isomers of dichloroguiacol. The halogenated catechols with the exception of 4-fluorocatechol partly polymerized oxidatively in the culture fluid to form insoluble dark-brown products. The degradation of halophenols are due to the action of unspecific intracellular enzymes responsible for phenol catabolism (phenol hydroxylase, catechol-1,2-dioxygenase, muconate cycloisomerase I).

Aromatic peroxygenases are extracellular fungal biocatalysts that selectively oxidize a variety of organic compounds. We found that the peroxygenase of the fungus Agrocybe aegerita (AaeAPO) catalyzes the H2O2-dependent hydroxylation of diverse flavonoids. The reactions proceeded rapidly and regioselectively yielding preferentially monohydroxylated products, e.g., from flavanone, apigenin, luteolin, flavone as well as daidzein, quercetin, kaempferol, and genistein. In addition to hydroxylation, O-demethylation of fully methoxylated tangeretin was catalyzed by AaeAPO. The enzyme was merely lacking activity on the quercetin glycoside rutin, maybe due to sterical hindrance by the bulky sugar substituents. Mechanistic studies indicated the presence of epoxide intermediates during hydroxylation and incorporation of H2O2-derived oxygen into the reaction products. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of flavonoid metabolites.

The Penicillium strain Bi 7/2 utilized phenol, catechol, resorcinol, hydroquinone, pyrogallol, hydroxyhydroquinone, phloroglucinol, m- and p-cresol, orcinol, 4-methylcatechol. 4-methoxy-phenol, 4-aminophenol, benzyl alcohol, benzoic acid, 2-, 3- and 4-hydroxybenzoic acid, anthranilic acid, protocatechuic acid and gallic acid as sole sources of carbon and energy. The central metabolites catechol, protocatechuic acid and hydroxyquinone could be determined by HPLC with diode-array detection. Pathways for the degradation of aromatic substances were proposed.

Introduction Genesis and Stability of Humic Substances General Aspects of the Degradation of HS Aerobic Degradation of HS Methods to Estimate Microbial Degradation of HS Bacteria Fungi Anaerobic Transformation of HS Outlook and Perspectives Keywords: humic substances; humic acid; fulvic acid; melanoidin; degradation; decomposition; actinomycetes; heterotrophic bacteria; microfungi; basidiomycetes; Streptomyces; Arthrobacter; Penicillium frequentans; Phanerochaete chrysosporium; Trametes versicolor; Nematoloma frowardii; Geobacter metallireducens; Geothrix fermentans; Wolinella succinogenes; Propionibacterium freundenreichii; lignin peroxidase; manganese peroxidase; laccase; electron acceptor; electron donor; redox mediator; interspecies electron transfer; respiration

Introduction Historical Outline Modification of Hard Coal Bacteria Fungi Bioconversion of Brown Coal Mechanisms: Solubilization, Depolymerization, Utilization Organisms Solubilization of Brown Coal Depolymerization of Brown Coal by Oxidative Enzymes Anaerobic and other Approaches to Convert Coal Outlook and Perspectives Patents Keywords: hard coal; brown coal; lignite; low-rank coal; humic acid; fulvic acid; humin; solubilization; depolymerization; bacteria; microfungi; basidiomycetes; chelators; alkaline substances; oxidative enzymes; manganese peroxidase; lignin peroxidase; laccase; esterase