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Pedro Almodóvar foi, desde o primeiro momento, creador de propostas de renovación formal, estética, temática e social do panorama cinematográfico español. Acompañado doutros nomes da Movida madrileña, inseriuse neste sistema cultural pouco despois do inicio da transición democrática, época idónea para o xurdimento dunha figura destas características grazas á busca por parte do Estado español de exportar unha imaxe propia que fose capaz de liberarse do pasado franquista. Esta e outras coordenadas son as que analiza Mario de la Torre-Espinosa en Almodóvar y la cultura: del tardofranquismo a la Movida, traballo no que desenvolve a necesaria tarefa de ter en consideración o conxunto de elementos que permitiron o nacemento da carreira artística de Almodóvar, obxectivo para o que bota man da teoría dos polisistemas fundada por Itamar Even-Zohar.

A peroxygenase-catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita (AaeUPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h−1), catalyst performance (up to 84 s−1 and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme-substrate interaction puts a basis for the mechanistic understanding of AaeUPO selectivity.

Unspecific peroxygenases (UPOs) are promising biocatalysts for oxyfunctionalisation reactions, owing to their simplicity of handling, stability and robustness. A limitation of using UPOs on a large scale is their deactivation in the presence of even rather modest concentrations of H2O2, requiring a constant and controlled supply of low amount of H2O2. Herein, we report an organometallic complex [Cp*Ir(pica)NO3] {pica=picolinamidate=κ2-pyridine-2-carboxamide ion (−1)} 1 capable of efficiently regenerating FMNH2 from FMN (TOF=350 h−1, 298 K), driven by NaHCOO; FMNH2, in turn, spontaneously reacts with O2 leading to H2O2. After having studied the compatibility of 1 with the UPO from Agrocybe aegerita (rAaeUPO PaDa-I) and individuated the best experimental conditions, we applied such a hybrid catalytic tandem in some hydroxylation, epoxidation and sulfoxidation reactions. Best performances were obtained by using a 1/rAaeUPO molar ratio of 50. TONs for the biocatalyst of up to 18933 were obtained for the transformation of ethylbenzene derivatives into (R)-1-phenylethanols (ee>99 %). 1/rAaeUPO was found to oxidise also cis-methyl styrene (TON=13488), leading exclusively (1R,2S)-cis-methyl styrene oxide (ee>99 %), cyclohexane (TON=1634) and thioanisole (TON=1369).

Enzyme immobilization is the key to an intensified bioprocess that allows recycling of the heterogenized enzyme and/or continuous biocatalytic production. In this communication, we present a case study for enzyme immobilization in a novel, 3D printable synthetic hydrogel and its use in continuous oxidation reactions. Immobilization resulted in an average immobilization yield of 6.1% and continuous synthesis was run for 24 hours with a space-time yield of 3.1

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.

Biocatalytic oxidation reactions of toluene derivates to the corresponding aldehydes are typically challenged by regio- and chemoselectivity issues. In this contribution we address both challenges by a combined reactant- and reaction engineering approach. We demonstrate that the peroxygenase-catalysed transformation of ring-substituted toluenes proceeds highly regioselectively in benzylic position. Furthermore, neat reaction conditions not only enable attractive product concentrations (up to 185 mM) but also result in highly chemoselective oxidations to the aldehyde level.

Utilisation of fatty acids generally relies on pre-existing functional groups such as the carboxylate group or C=C-double bonds. Addition of new functionalities into the hydrocarbon part opens up new possibilities for fatty acid valorisation. In this contribution we demonstrate the synthetic potential of a peroxygenase mutant AaeUPO−Fett for selective fatty acid oxyfunctionalisation. The ω-1 hydroxy fatty acid (esters) produced are further transformed into lactones, alcohols, esters and amines via multi-enzyme cascades thereby paving the way for new fatty acid valorisation pathways.

White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for green-chemistry applications due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative directed evolution protocols have been applied to improve their expression, activity and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express: first, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3-4 subtle mutations that change the polarity, solvation and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles. These stable and versatile HRPLs are a step towards the generation of an effective lignin-degrading consortium of enzymes that can be secreted from yeast. More broadly, the stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.

Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations., Püllmann et al developed a modular Golden Gate-based secretion system, which enabled production and one-step purification of active fungal unspecific peroxygenases (UPOs) in yeast. Their system was applied to an enantioselective conversion on a preparative scale and may be used in the future for other genes of interest that are suitable for production in yeast.

The ganglioneuroma is a benign primitive neuroectodermal tumor, which derives from the ganglia of the sympathetic system and is composed of mature Schwann cells, ganglion cells, and nerve fibers. A rare neck tumor, in a female pediatric patient with a history of cervical tumor mass for two years is presented. In the imaging studies performed, a heterogeneous solid lesion with punctate calcifications was diagnosed, compressing adjacent bone and vascular structures. To obtain histology, an ultrasound-guided core needle biopsy was performed, and the diagnosis of cervical ganglioneuroma was informed by pathological anatomy. Due to the low incidence of ganglioneuroma as a tumor variety, and in turn, the rare cervical location in a small percentage of patients, the present research aims to enrich the national and international scientific literature, providing a new case that serves as the basis for future research, respecting the ethical principles of health research.

The combination of heterogeneous catalysis and biocatalysis into one-pot reaction cascades is a potential approach to integrate enzymatic transformations into existing chemical infrastructure. Peroxygenases, which can achieve clean C-H activation, are ideal candidates for incorporation into such tandem systems; however a constant supply of low-level hydrogen peroxide (H2O2) is required. The use of such enzymes at industrial scale will likely necessitate the in situ generation of the oxidant from cheap and widely available reactants. We show that combing heterogeneous catalysts (AuxPdy/TiO2) to produce H2O2 in situ from H2 and air, in the presence of an evolved unspecific peroxygenase from Agrocybe aegerita (PaDa-I variant) yields a highly active cascade process capable of oxidizing alkyl and alkenyl substrates under mild conditions. When alkenes such as styrene are subjected to this tandem oxidation process, divergent reaction pathways are observed due to the competing hydrogenation of the alkene by palladium rich nanoparticles in the presence of H2. Each pathway presents opportunities for value added products. Product selectivity was highly sensitive to the rate of reduction compared to hydrogen peroxide delivery. Here we show that some control over product selectivity may be exerted by careful selection of nanoparticle composition.

[EN] In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)- or (S)-phenylethanols using the (R)-selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)-selective ADH from Rhodococcus ruber (ADH-A). In a one-pot two-step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity., European Research Commission (ERC consolidator grant,No. 648026),the NetherlandsOrganizationfor ScientificResearch(VICIgrant,No. 724.014.003)and the GuangzhouElite Project.

The use of neat reaction media, that is the avoidance of additional solvents, is the simplest and the most efficient approach to follow in biocatalysis. Here, we show that unspecific peroxygenase from Agrocybe aegerita (AaeUPO) can hydroxylate the neat model substrate cyclohexane. H 2O 2 was photocatalytically generated in situ by nitrogen-doped carbon nanodots (N−CNDs) and UV LED illumination. AaeUPO entrapment in alginate beads increased enzyme stability and facilitated the reaction in neat cyclohexane. N−CNDs absorption in beads containing AaeUPO created a 2-in-1 heterogeneous photobiocatalyst that was active for up to seven days under reaction conditions and produced cyclohexanol, 2.5 mM. To increase productivity, the bead size and the photocatalyst-to-enzyme ratio have been identified as promising targets for optimisation.

Unspecific peroxygenases have recently gained significant interest due to their ability to catalyse the hydroxylation of non-activated C−H bonds using only hydrogen peroxide as a co-substrate. However, the development of preparative processes has so far mostly concentrated on benzylic hydroxylations using liquid substrates. Herein, we demonstrate the application of a peroxygenase for the hydroxylation of the inert, gaseous substrate butane to 2-butanol in a bubble column reactor. The influence of hydrogen peroxide feed rate and enzyme loading on product formation, overoxidation to butanone and catalytic efficiency is investigated at 200 mL scale. The process is scaled up to 2 L and coupled with continuous extraction. This setup allowed the production of 115 mmol 2-butanol and 70 mmol butanone with an overall total turnover number (TTN) of over 15.000, thereby demonstrating the applicability of peroxygenases for preparative hydroxylation of such inert, gaseous substrates at mild reaction conditions.

Graphical abstract, Aryl-alcohol oxidase (AAO) is a fungal GMC flavoprotein secreted by white-rot fungi that supplies H2O2 to the ligninolytic consortium. This enzyme can oxidize a wide array of aromatic alcohols in a highly enantioselective manner, an important trait in organic synthesis. The best strategy to adapt AAO to industrial needs is to engineer its properties by directed evolution, aided by computational analysis. The aim of this review is to describe the strategies and challenges we faced when undertaking laboratory evolution of AAO. After a comprehensive introduction into the structure of AAO, its function and potential applications, the different directed evolution enterprises designed to express the enzyme in an active and soluble form in yeast are described, as well as those to unlock new activities involving the oxidation of secondary aromatic alcohols and the synthesis of furandicarboxylic acids.

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based

Propargylic alcohols and amines are versatile building blocks in organic synthesis. We demonstrate a straightforward enzymatic cascade to synthesize enantiomerically pure propargylic alcohols and amines from readily available racemic starting materials. In the first step, the peroxygenase from Agrocybe aegerita converted the racemic propargylic alcohols into the corresponding ketones, which then were converted into the enantiomerically pure alcohols using the (R)-selective alcohol dehydrogenase from Lactobacillus kefir or the (S)-selective alcohol dehydrogenase from Thermoanaerobacter brokii. Moreover, an enzymatic Mitsunobu-type conversion of the racemic alcohols into enantiomerically enriched propargylic amines using (R)-selective amine transaminase from Aspergillus terreus or (S)-selective amine transaminase from Chromobacterium violaceum was established. The one-pot two-step cascade reaction yielded a broad range of enantioenriched alcohol and amine products in 70-99% yield.

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use, but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel, but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wild type enzymes are functionally expressed in yeast whereas their wild type parents are not. Three of these designs exhibit substantial and useful diversity in reactivity profile and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families.

[EN] Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression in yeast of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) and their production at large scale in bioreactor. Our strategy was based on adopting secretion mutations from Agrocybe aegerita UPO mutant −PaDa-I variant− designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as PabUPOs –long-type UPOs− and that shares 65% sequence identity. After replacing the native signal peptides by the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries yielding two recombinant PabUPOs with expression levels of 5.4 and 14.1 mg/L in S. cerevisiae. These variants were subsequently transferred to P. pastoris for overproduction in fed-batch bioreactor, boosting expression levels up to 290 mg/L with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/L, with veratryl alcohol as substrate). With a broad pH activity profile, ranging from 2.0 to 9.0, these highly secreted, active and stable peroxygenases are promising tools for future engineering endeavors, as well as for their direct application in different industrial and environmental settings., This work was supported by the Comunidad de Madrid Synergy CAM Project Y2018/BIO-4738-EVOCHIMERA-CM, the Spanish Government Projects BIO2016-79106-R-Lignolution, PID2019-106166RB-100-OXYWAVE and the CSIC Project PIE- 201580E042. P. Gomez de Santos is grateful to the Ministry of Science, Innovation and Universities (Spain) for her FPI and POP contracts (BES-2017-080040).

Ethers can be found in the environment as structural, active or even pollutant molecules, although their degradation is not efficient under environmental conditions. Fungal unspecific heme-peroxygenases (UPO were reported to degrade low-molecular-weight ethers through an H2O2-dependent oxidative cleavage mechanism. Here, we report the oxidation of a series of structurally related aromatic ethers, catalyzed by a laboratory-evolved UPO (PaDa-I) aimed at elucidating the factors influencing this unusual biochemical reaction. Although some of the studied ethers were substrates of the enzyme, they were not efficiently transformed and, as a consequence, secondary reactions (such as the dismutation of H2O2 through catalase-like activity and suicide enzyme inactivation) became significant, affecting the oxidation efficiency. The set of reactions that compete during UPO-catalyzed ether oxidation were identified and quantified, in order to find favorable conditions that promote ether oxidation over the secondary reactions.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L–1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L–1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L-1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L-1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

Oxyfunctionalisation reactions in neat substrate still pose a challenge for biocatalysis. Here, we report an alginate-confined peroxygenase-CLEA to catalyse the enantioselective epoxidation of cis-β-methylstyrene in a solvent-free reaction system achieving turnover numbers of 96 000 for the biocatalyst and epoxide concentrations of 48 mM., Chemical Communications, 57 (47), ISSN:1359-7345, ISSN:1364-548X

Aromatic hydroxylation reactions catalyzed by heme-thiolate enzymes proceed via an epoxide intermediate. These aromatic epoxides could be valuable building blocks for organic synthesis giving access to a range of chiral trans-disubstituted cyclohexadiene synthons. Here, we show that naphthalene epoxides generated by fungal peroxygenases can be subjected to nucleophilic ring opening, yielding non-racemic trans-disubstituted cyclohexadiene derivates, which in turn can be used for further chemical transformations. This approach may represent a promising shortcut for the synthesis of natural products and APIs.

Employing the homologous DNA recombination apparatus of Saccharomyces cerevisiae as a dynamic engineering tool allows mutant libraries to be constructed in a rapid and efficient manner. Among the plethora of methods based on the yeast's splicing apparatus, site-directed recombination (SDR) is often useful to gather information from mutations discovered in directed evolution experiments. When using SDR, the target gene is divided in segments carrying the selected mutation positions so that the resulting PCR fragments show 50% mutated and 50% wild type residues at the codons of interest. The PCR products are then assembled and cloned into yeast through one-pot transformations with the help of homologous overlapping flanking regions. By screening SDR libraries, the effect of the mutations/reversions at the different positions can be rapidly sorted out in a combinatorial manner. As such, SDR can serve as the `final polishing step´ in a laboratory evolution campaign, revealing beneficial synergies among mutations and/or overriding deleterious mutations. In practice, using SDR it is possible to discern between beneficial and negative epistasis, that is, it should be possible to collect positive synergistic mutations while discarding detrimental substitutions that affect the enzyme's fitness.

Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.

Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ∼500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidation, a β–diketone initiator commonly used in vinyl polymerization reactions., This study is based upon work funded by the Spanish Government projects BIO2013-43407-R-DEWRY and BIO2016-79106-R-Lignolution and by the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 Research and Innovation programme (grant agreement no. 886567). B.J.G.-F. was supported by FPI national fellowship BES-2014-068887.

[EN] Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase-catalysed oxyfunctionali-zation reactions. Choline chloride-based NADES are of particularinterest as they can serve as solvent, enzyme-stabiliser and sacrificial electron donor for the in situ H2O2generation. This report provides the first proof-of-concept and basic character-isation of this new reaction system. Highly promising turnovernumbers for the biocatalysts of up to 200,000 have been achieved., This work was supported by the National Outstanding Youth Science Foundation of China (31725022), National Key R&D Program of China (2018YFC0311104), International Collaboration Base for Molecular Enzymology and Enzyme Engineering (2017 A050503001), and also supported by the 111 Project (B17018). F.H. gratefully acknowledges funding by the European Research Commission (ERC consolidator grant, No.648026), the European Union (H2020-BBI-PPP-2015-2-1-720297), and the Netherlands Organization for Scientific Research (VICI grant, No 724.014.003)