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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.

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ¿-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation (A77L, Fett variant) found by structural studies and saturation mutagenesis near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity as determined by GC/MS. While molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was overexpressed in Pichia pastoris at 0.4 g/L-1 operating a fed-batch bioreactor and used for the preparative synthesis of 1.4 g of (¿-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee for the S-enantiomer.

[EN] The generation of enantiodivergent biocatalysts for C-H oxyfunctionalisations is ever more important in modern synthetic chemistry. Here, we have applied the FuncLib algorithm based on phylogenetic and Rosetta calculations to design a diverse repertoire of active, stable and enantiodivergent fungal peroxygenases. 24 designs, each carrying 4-5 mutations in the catalytic core, were expressed functionally in yeast and benchmarked against characteristic model compounds. Several designs were active and stable in a range of temperature and pH, displaying unprecedented enantiodivergence, changing regioselectivity from alkyl to aromatic hydroxylation, and increasing catalytic efficiencies up to 10-fold, with 15-fold improvements in total turnover numbers over parental enzyme. We find that this dramatic functional divergence stems from beneficial epistasis among the mutations and an extensive reorganization of the heme channel. Our work demonstrates that FuncLib can rapidly design highly functional libraries enriched in enantioselective peroxygenases not seen in nature for a range of biotechnological applications.

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. © 2023 The Authors. ChemCatChem published by Wiley-VCH GmbH.

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 × 10−2 g L−1 h−1 © 2023 The Royal Society of Chemistry.

Mol-scale oxyfunctionalization of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using an unspecific peroxygenase is reported. Using AaeUPO from Agrocybe aegerita and simple H2O2 as an oxidant, cyclohexanol concentrations of more than 300 mM (>60% yield) at attractive productivities (157 mM h-1, approx. 15 g L-1 h-1) were achieved. Current limitations of the proposed biooxidation system have been identified paving the way for future improvements and implementation. © 2023 The Authors. Published by American Chemical Society

Fungal unspecific peroxygenases (UPOs) are among the most promising biocatalysts in synthetic chemistry. The kaleidoscope of oxyfunctionalization reactions that are of industrial and environmental relevance offer UPOs the opportunity to provide solutions to many processes. However, there are important shortcomings that must first be overcome in order to convert UPOs into applied biocatalysts, which include their general poor levels of heterologous functional expression, the presence of unwanted peroxidase activity and overoxidation reactions, as well as the oxidative inactivation provoked by the co-substrate, hydrogen peroxide. The specific engineering of these proteins, and that of the reactions in which they participate, is being used to tackle these principal challenges, with a view to generate a portfolio of highly active, selective and stable variants. In this review, we will discuss some of the most recent developments that will convert UPOs into true industrial biocatalysts, focusing on the different trends currently being followed in this fascinating field of research. © 2023 Elsevier B.V.

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. © 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.

[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.

[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.

[EN] Peroxygenases require a controlled supply of HO to operate efficiently. Here, we propose a photocatalytic system for the reductive activation of ambient O to produce HO which uses the energy provided by visible light more efficiently based on the combination of wavelength-complementary photosensitizers. This approach was coupled to an enzymatic system to make formate available as a sacrificial electron donor. The scope and current limitations of this approach are reported and discussed.

[EN] There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.

Fungal high-redox-potential laccases (HRPLs) are multicopper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (ET1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased ET1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic high-redox-potential mediators with different oxidation routes and chemical natures, while computer-aided evolution experiments were run in parallel to guide benchtop mutagenesis, without compromising protein stability. Through this strategy, the ET1 of the evolved HRPL increased from 740 to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic constants for high-redox-potential mediators were markedly improved and were then successfully tested within laccase mediator systems (LMSs). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects, and they were rationalized at the atomic level. Together, this study represents a proof-of-concept of the joint elevation of the ET1, redox mediator activity, and stability in an HRPL, making this versatile biocatalyst a promising candidate for future LMS applications and for the development of bioelectrochemical devices.

[EN] High-redox potential laccases (HRPLs) from white-rot fungi are versatile biocatalysts whose practical use is highly dependent on their thermostability. In this work, an evolved HRPL variant was subjected to structure-guided evolution to improve its thermostability. We first selected several surface flexible loops in the laccase structure by inspecting them through molecular dynamics and an analysis of B-factors. The resulting segments were grouped into three MORPHING (Mutagenic Organized Recombination Process by Homologous In vivo Grouping) blocks, which were constructed in Saccharomyces cerevisiae and explored at high temperatures. This evolution process gave rise to a double mutant that showed a half-life at 70°C enhanced by 31 min with an optimum temperature for activity of 75°C and similar kinetic parameters. The Ser264Lys and Ser356Asn mutations modified the contacts established between these residues and those that surround them, altering the surface loops and thereby the enzyme properties.

[EN] Continuous low-level supply or in situ generation of hydrogen peroxide (H2O2) is essential for the stability of unspecific peroxygenases, which are deemed ideal biocatalysts for the selective activation of C-H bonds. To envisage potential large scale applications of combined catalytic systems the reactions need to be simple, efficient and produce minimal by-products. We show that gold-palladium nanoparticles supported on TiO2 or carbon have sufficient activity at ambient temperature and pressure to generate H2O2 from H2 and O2 and supply the oxidant to the engineered unspecific heme-thiolate peroxygenase PaDa-I. This tandem catalyst combination facilitates efficient oxidation of a range of C-H bonds to hydroxylated products in one reaction vessel with only water as a by-product under conditions that could be easily scaled.

Cellulases catalyze the hydrolysis of cellulose. Improving their catalytic efficiency is a long-standing goal in biotechnology given the interest in lignocellulosic biomass decomposition. Although methods based on sequence alteration exist, improving cellulases is still a challenge. Here we show that Ancestral Sequence Reconstruction can “resurrect” efficient cellulases. This technique reconstructs enzymes from extinct organisms that lived in the harsh environments of ancient Earth. We obtain ancestral bacterial endoglucanases from the late Archean eon that efficiently work in a broad range of temperatures (30–90 °C), pH values (4–10). The oldest enzyme (~2800 million years) processes different lignocellulosic substrates, showing processive activity and doubling the activity of modern enzymes in some conditions. We solve its crystal structure to 1.45 Å which, together with molecular dynamics simulations, uncovers key features underlying its activity. This ancestral endoglucanase shows good synergy in combination with other lignocellulosic enzymes as well as when integrated into a bacterial cellulosome.

[EN] Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5- formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.

[EN] Oxidation of primary alcohols by aryl-alcohol oxidase (AAO), a flavoenzyme that provides H2O2 to fungal peroxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary aryl-alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.

Oxidation of primary alcohols by aryl-alcohol oxidase (AAO), a flavoenzyme that provides H2O2 to fungal peroxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary aryl-alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.

Fungal laccases are biotechnologically relevant enzymes that are capable of oxidizing a wide array of compounds, using oxygen from the air and releasing water as the only by product. The laccase structure is comprised of three cupredoxin domains sheltering two copper centers -the T1Cu site and the T2/T3 trinuclear Cu cluster- connected to each other through a highly conserved internal electron transfer pathway. As such, the generation of laccase chimeras with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. Here, we have obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with 70 % protein sequence identity. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computationally selected SCHEMA-RASSP blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated, with multiple recombination events simultaneously occurring between and within the SCHEMA blocks. The resulting library was screened at high temperature identifying a collection of thermostable chimeras with considerable sequence diversity which varied from their closest parent homolog by 46 amino acids on average. The most thermostable variant increased its half-life of thermal inactivation at 70°C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acidic pH. The two catalytic copper sites spanned different SCHEMA blocks, shedding light on the recognition of specific residues involved in substrate oxidation. In summary, this case-study through comparison with previous laccase engineering studies, highlights the benefits of bringing together computationally-guided recombination and in vivo shuffling as an invaluable strategy for laccase evolution, which can be translated to other enzyme systems.

[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.

Because of their minimal requirements, substrate promiscuity and product selectivity, fungal peroxygenases are now considered to be the jewel in the crown of C–H oxyfunctionalization biocatalysts. In this work, the crystal structure of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å. Notable differences were detected between the evolved and native peroxygenase from Agrocybe aegerita, including the presence of a full N-terminus and a broader heme access channel due to the mutations that accumulated through directed evolution. Further mutagenesis and soaking experiments with a palette of peroxygenative and peroxidative substrates suggested dynamic trafficking through the heme channel as the main driving force for the exceptional substrate promiscuity of peroxygenase. Accordingly, this study provides the first structural evidence at an atomic level regarding the mode of substrate binding for this versatile biocatalyst, which is discussed within a biological and chemical context.

The invention relates to an unspecific peroxygenase of the Agrocybe aegerita fungus, obtained by means of directed molecular evolution to facilitate the functional expression thereof in an active, soluble and stable form. The peroxygenase described in the invention shows a significant improvement in the functional expression thereof, improved monooxygenase activity and reduced peroxidase activity, in relation to the monooxygenase and peroxidase activities showed by the unspecific wild-type peroxygenase of A. aegerita. The peroxygenase of the invention is useful in chemical processes, including industrial transformations such as the selective oxyfunctionalisation of carbon-hydrogen bonds of various organic compounds.

[EN] Directed evolution is a powerful strategy to tailor enzymes with improved attributes. The use of laboratory evolution is becoming more refined, whereby computational and experimental approaches are being combined so that more effective libraries can be created, producing enzymes with greater biotechnological potential while notably reducing the demands on screening. This chapter summarizes the most recent findings from our laboratory to tailor fungal high-redox potential laccases by bringing together computational approaches with in vitro and in vivo methods for library creation. We focus on four recent case studies of laccase engineering in which different computational algorithms were applied at both the gene and protein levels.

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.

[EN] Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.

[EN] Discovery and testing of new bioactive compounds are becoming emergent fields in contemporary chemistry. All metabolites formed above 10% of the parent drug should be tested in terms of safety; therefore it is fundamental to produce high amounts of them. Unspecific peroxygenases (UPOs, EC 1.11.2.1) are stable and extracellular heme-thiolate enzymes considered by many as the generational relief of P450s, acting on drugs as “extracellular livers”. These enzymes have been studied before for the synthesis of human drug metabolites (HDMs) presenting a wide range of substrate conversion. Several UPO mutant libraries were constructed and screened in search for improved peroxygenase activity and diminished peroxidase activity during this given biotransformation. The final mutant, SoLo, carried one single mutation (F191S) and showed a catalytic efficiency for the conversion of propranolol enhanced by two orders of magnitude together with 99% regioselectivity in the synthesis of the true HDM 5´-hydroxypropranolol (5´-OHP). This mutant together with other evolved AaeUPO variants were further tested with other drugs, unveiling the importance of the amino acids lining the heme channel. Additionally, UPO fusion enzymes were designed by linking the evolved UPO to aryl-alcohol oxidase (AAO, EC 1.1.3.7). After testing several orientations, signal sequences and peptide linkers, five constructions of UPO_AAO were functionally expressed in yeast and characterized biochemically. The best fusion was tested achieving 62,145 TTNs for HDM dextrorphan synthesis. This fusion represents a self-sufficient system to produce HDMs in a preparative manner from newly discovered drugs as well as to be applied in cascade reactions where both AAO and UPO partners could interact., [ES] El descubrimiento y evaluación de nuevos compuestos bioactivos constituye un campo emergente dentro de la química contemporánea. Todos los metabolitos formados a partir de un fármaco en una proporción superior al 10% deben ser evaluados en términos de seguridad, por lo que su producción para tales estudios se considera prioritaria. Sin embargo, su síntesis química se encuentra generalmente asociada con bajos rendimientos y procesos muy complejos, por lo que están surgiendo nuevos procesos enzimáticos como potencial solución. En este sentido, las peroxigenasas inespecíficas (UPOs, EC 1.11.2.1) son enzimas hemotioladas estables y extracelulares que son consideradas por muchos como el relevo generacional de las citocromo P450 monooxigenasas, pudiendo actuar sobre los fármacos como “hígados extracelulares”. Estas enzimas han sido estudiadas con anterioridad para la síntesis de metabolitos humanos de fármacos (HDMs), presentando una gran diversidad en cuanto a conversión de sustrato. En la presente Tesis Doctoral, se ha estudiado la ingeniería de la UPO de Agrocybe aegerita (AaeUPO) i) para la producción eficiente de HDMs del beta-bloqueante propranolol, ii) para la exploración de variantes enzimáticas para futuros estudios de síntesis de HDMs, y iii) para la creación de un sistema autosuficiente para este propósito basado en la tecnología de proteínas de fusión. Para lograr la producción eficiente de HDMs de propranolol, se llevó a cabo evolución dirigida guiada por la estructura de la proteína, conjugando un sistema de expresión en Saccharomyces cerevisiae junto con un robusto método de cribado de alto rendimiento. Se construyeron y evaluaron diversas genotecas buscando un incremento en la actividad peroxigenasa y una disminución en la actividad peroxidasa para esta transformación en particular. El mutante final, SoLo, presentó una única mutación (F191S) y mostró una eficiencia catalítica para la conversión del propranolol aumentada en dos órdenes de magnitud junt

[EN] Fungal peroxygenases are deemed emergent biocatalysts for selective C¿H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal aryl-alcohol oxidase that supplies H2O2in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl alcohol oxidase fusions in the pharmaceutical and chemical sectors.

Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.