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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 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.

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).

Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel. This work was supported by the Comunidad de Madrid Synergy CAM Project Y2018/BIO-4738-EVOCHIMERA-CM, the PID2019-106166RB-100-OXYWAVE and the PID2019-106370RB-I00-PZymes grants from the Spanish Ministry of Science and Innovation and the CSIC Project PIE-201580E042. AB-N thanks the PhD Programme in Molecular Biosciences, Doctoral School, Universidad Autónoma de Madrid.

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).

© 2020 by the authors., 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., This work was supported by PAPIIT IN209116 and IN214619 and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA. J.R. was granted the scholarship 404380 awarded by CONACYT-Mexico for his Ph.D. studies.

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.

Antecedentes y Objetivos: Los humedales de la costa central de Lima, Perú, están siendo fuertemente impactados de forma negativa por actividad antrópica, debido a que se han establecido asentamientos humanos en las cercanías de estos frágiles ecosistemas. Sarcocornia neei es una Amarantácea halófita, que habita estos humedales, con potencial de ser usada como alimento; está siendo desplazada por la actividad humana y aún se desconocen aspectos básicos de su biología. Por lo tanto, nuestro objetivo fue conocer la ecofisiología de esta especie bajo condiciones de invernadero y de laboratorio, viendo la posibilidad de ser cultivada fuera de su ambiente natural.Métodos: Las plantas de S. neei fueron colectadas tanto en el Humedal de Ventanilla como en el de Paraíso, Lima, Perú. Para la prueba de germinación se usaron 600 semillas sometidas a diferentes concentraciones de NaCl (0 M, 0.1 M, 0.3 M y 0.58 M), con tres repeticiones por 15 días. También se plantaron esquejes en sustrato orgánico y adicionando las mismas concentraciones de sal, con tres repeticiones por cuatro meses. Al final de este periodo se realizaron cortes histológicos y también se hicieron extractos de proteínas.Resultados clave: Se obtuvo una mejor germinación en 0.3 M de NaCl. No hubo diferencias significativas en el crecimiento de los esquejes. Se encontraron variaciones histológicas en los tallos dependiendo de los tratamientos y no hubo diferencias significativas en la concentración total de proteínas, aunque sí se encontró una sobreexpresión de proteínas de bajo peso molecular en el tratamiento de 0.58 M de NaCl.Conclusiones: Los resultados muestran que esta especie podría ser cultivada en terrenos salinos y usada en la alimentación humana o de animales, o como especie promisoria en la descontaminación de suelos salinos contaminados con plomo.

Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion., This study is based upon work funded by and the Spanish Government projects BIO2013-43407-R-DEWRY and BIO2016- 79106-R-Lignolution. BG-F was supported by a FPI national fellowship BES-2014-068887.

In plasma-driven biocatalysis, enzymes are employed to carry out reactions using species generated by non-thermal plasmas as the precursors. We have previously demonstrated that this is feasible in principle, but that the approach suffers from the short lifetime of the biocatalyst under operating conditions. In this work, protection strategies were investigated to prevent the dielectric barrier discharge plasma-induced inactivation of biocatalysts, using recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO), one of the most promising enzymes for plasma-driven biocatalysis. Treatment in oxygen-free atmospheres did not provide any advantage over treatment in synthetic air, indicating that the detrimental reactive species did not originate from oxygen in the plasma phase. Chemical scavengers were employed to eliminate undesired reactive species, without any long-term effect on enzyme lifetime. Similarly, chaperones, including the known stress response proteins Hsp33, CnoX, and RidA did not increase the lifetime of rAaeUPO. Immobilization of the biocatalyst proved effective in preserving enzyme activity. The residual activity of rAaeUPO after plasma treatment strongly depended on the specific immobilization support. Essentially complete protection for at least 15 min of plasma exposure was achieved with an epoxy-butyl-functionalized carrier. This study presents new insights into plasma-protein interactions and plots a path forward for protecting biocatalytic proteins from plasma-mediated inactivation.

The novel formate oxidase from Aspergillus oryzae (AoFOx) is a useful catalyst to promote H2O2-dependent oxyfunctionalisation reactions. In this contribution we exploit the substrate promiscuity of AoFOx to fully oxidise methanol and formaldehyde to CO2 and drive peroxygenase-catalysed stereoselective oxyfunctionalisation reactions. The highly atom efficient H2O2 generation system also enabled high catalytic turnover of the peroxygenase production enzyme.

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride-based deep eutectic solvents serving as co-solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15 mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.

Propranolol is a widely used beta-blocker that is metabolized by human liver P450 monooxygenases into equipotent hydroxylated human drug metabolites (HDMs). It is paramount for the pharmaceutical industry to evaluate the toxicity and activity of these metabolites, but unfortunately, their synthesis has hitherto involved the use of severe conditions, with poor reaction yields and unwanted byproducts. Unspecific peroxygenases (UPOs) catalyze the selective oxyfunctionalization of C–H bonds, and they are of particular interest in synthetic organic chemistry. Here, we describe the engineering of UPO from Agrocybe aegerita for the efficient synthesis of 5′-hydroxypropranolol (5′-OHP). We employed a structure-guided evolution approach combined with computational analysis, with the aim of avoiding unwanted phenoxyl radical coupling without having to dope the reaction with radical scavengers. The evolved biocatalyst showed a catalytic efficiency enhanced by 2 orders of magnitude and 99% regioselectivity for the synthesis of 5′-OHP. When the UPO mutant was combined with an H2O2 in situ generation system using methanol as sacrificial electron donor, total turnover numbers of up to 264 000 were achieved, offering a cost-effective and readily scalable method to rapidly prepare 5′-OHP., This work was supported by the European Union Project H2020-BBI-PPP-2015-2-720297-ENZOX2 and the COST Action CM1303 Systems Biocatalysis, the Spanish Government Projects BIO2016-79106-R-Lignolution and CTQ2016-70138-R, and the CSIC Project PIE-201580E042.

Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere’s carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering. Precambrian and extant form II Rubiscos were subjected to an ensemble of directed evolution strategies aimed at improving thermostability. The most recent ancestor of proteobacteria -dating back 2.4 billion years- was uniquely tolerant to mutagenic loading. Adaptive evolution, focused evolution and genetic drift revealed a panel of thermostable mutants, some deviating from the characteristic trade-offs in CO2-fixing speed and specificity. Our findings provide a novel approach for identifying Rubisco variants with improved catalytic evolution potential., This work was supported by the REPSOL Research contracts Rubolution (RC020401120018), Rubolution 2.0 (RC 020401140042), the CSIC project PIE-201780E043 and the Australian Research Council grant CE140100015.

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., This work was funded by the European Union [(Bioenergy-FP7-PEOPLE-2013-ITN-607793], the CSIC [project PIE-201580E042], and the Spanish Ministry of Economy, Industry and Competitiveness [projects BIO2013-43407-R.DEWRY and BIO2016-79106-R. LIGNOLUTION]., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

[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., Financial support by the European Research Council (ERC Consolidator Grant No. 648026) is gratefully acknowledged.

9 p.-8 fig.-4 tab., 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., This work was supported by the INDOX ( KBBE-2013-7-613549) EU project and by the BIO2017-86559-R (GenoBioref), CTQ2016-79138-R and BIO2016-79106-R projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds., We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI

An increasing number of biocatalytic oxidation reactions rely on H2O2 as a clean oxidant. The poor robustness of most enzymes towards H2O2, however, necessitates more efficient systems for in situ H2O2 generation. In analogy to the well‐known formate dehydrogenase to promote NADH‐dependent reactions, we here propose employing formate oxidase (FOx) to promote H2O2‐dependent enzymatic oxidation reactions. Even under non‐optimised conditions, high turnover numbers for coupled FOx/peroxygenase catalysis were achieved., The authors gratefully acknowledge funding by the European Research Commission (ERC consolidator grant, No. 648026), the European Union (H2020‐BBI‐PPP‐2015‐2‐1‐720297), the Netherlands Organisation for Scientific Research (VICI grant No. 724.014.003), the National Science Foundation (NSF) of the United States (grant IIP‐1540017) and the Comunidad de Madrid Synergy CAM ProjectOrganisation for Scientific Research.

[EN] Photoenzymatic cascades can be used for selective oxygenation of C−H-Bonds under mild conditions circumventing the hydrogen peroxide mediated peroxygenase inactivation via in situ HO generation. Here, we report the “on demand” production of hydrogen peroxide via methanol assisted reduction of molecular oxygen using UV-illuminated titanium dioxide (Aeroxide P25) combined with the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanole catalyzed by the Unspecific Peroxygenase from Agrocybe Aegerita. For the application of the system it is important to understand the influence of the reaction parameters to be able to optimize the system. Therefore, we systematically investigated product formation and enzyme inactivation as well as ROS formation (HO, OH and O) applying different light intensities and temperatures. As a result, Turnover Numbers up to 220 000, photonic efficiencies up to 13.6 % and production rates up to 0.9 mM h were achieved., SRdB, BOB and JZB gratefully acknowledge financial support by German Research Foundation (DFG, Grant No: BL 1425/1-1). FH thanks the European Research Council (ERC Consolidator GrantNo. 648026) for financial support.

[EN] Aryl alcohol oxidase (AAO) is a fungal flavoenzyme capable of oxidizing aromatic primary alcohols into their correspondent aldehydes through a stereoselective hydride abstraction. Unfortunately, this enzyme does not act on secondary benzyl alcohols in racemic mixtures due to the strict control of substrate diffusion and positioning at the active site restricted to primary benzyl alcohols. Here we describe the engineering of AAO from Pleurotus eryngii to oxidize chiral benzyl alcohols with high enantioselectivity. The secondary benzyl alcohol oxidase was remodeled at the active site through four cycles of structure‐guided evolution, including a final step of in vivo site‐directed recombination to address the positive epistatic interactions between mutations. The final variant, with five substitutions and a renovated active site, was characterized at biochemical and computational level. The mutational sculpting helped position the bulkier (S)‐1‐(p‐methoxyphenyl)‐ethanol, improving the mutant's catalytic efficiency by three orders of magnitude relative to the native enzyme while showing a high enantioselectivity (ee >99%). As a promising candidate for racemic resolution, this evolved secondary benzyl alcohol oxidase maintained its natural stereoselective mechanism while displaying activity on several secondary benzyl alcohols., This research was supported by the EU project FP7‐KBBE‐2013‐7‐613549‐INDOX, by the Spanish Government projects BIO2016‐79106‐R‐Lignolution, CTQ2016‐74959‐R (MINECO/FEDER, EU) and by the Comunidad de Madrid project Y2018/BIO4738‐EVOCHIMERA.

Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae so that neutral mutations were simply added and recombined in vivo. With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded and active UPO variants were generated. After only 8 rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with modifications in peroxidative and peroxygenative activities, kinetic thermostability and enhanced tolerance to organic solvents. With an average 4.6 substitutions introduced per clone, neutral mutations covered roughly 10% of the protein sequence. As such, this study opens new avenues of UPO design by bringing together neutral genetic drift and DNA recombination in vivo., This work was supported by the European Union [FP7-KBBE-2013-7-613549-INDOX; H2020-BBI-PPP-2015-2-720297-ENZOX2], the CSIC [project PIE-201580E042], and the Spanish Ministry of Economy, Industry and Competitiveness [projects BIO2013-43407-R.DEWRY and BIO2016-79106-R. LIGNOLUTION].

Aryl‐alcohol oxidase (AAO) plays a fundamental role in the fungal ligninolytic secretome, acting as a supplier of H2O2. Despite its highly selective mechanism of action, the presence of this flavooxidase in different biotechnological settings has hitherto been hampered by the lack of appropriate heterologous expression systems. We recently described the functional expression of the AAO from Pleurotus eryngii in Saccharomyces cerevisiae by fusing a chimeric signal peptide (preαproK) and applying structure‐guided evolution. Here, we have obtained an AAO secretion variant that is readily expressed in S. cerevisiae and overproduced in Pichia pastoris. First, the functional expression of AAO in S. cerevisiae was enhanced through the in vivo shuffling of a panel of secretion variants, followed by the focused evolution of the preαproK peptide. The outcome of this evolutionary campaign—an expression variant that accumulated 4 mutations in the chimeric signal peptide, plus two mutations in the mature protein‐ showed 350‐fold improved secretion (4.5 mg/L) and was stable. This secretion mutant was cloned into P. pastoris and fermented in a fed‐batch bioreactor to enhance production to 25 mg/L. While both recombinant AAO from S. cerevisiae and P. pastoris were subjected to the same N‐terminal processing and had a similar pH activity profile, they differed in their kinetic parameters and thermostability. The strong glycosylation observed in the evolved AAO from S. cerevisiae underpinned this effect, since when the mutant was produced in the glycosylation‐deficient S. cerevisiae strain Δkre2, its kinetic parameters and thermostability were comparable to its poorly glycosylated P. pastoris recombinant counterpart., This research was supported by the EU project FP7-KBBE-2013-7- 613549-INDOX and by the Spanish Government project BIO2016- 79106-R-Lignolution.

[EN] The ligninolytic enzyme consortium is one of the most-efficient oxidative systems found in nature, playing a pivotal role during wood decay and coal formation. Typically formed by high redox-potential oxidoreductases, this array of enzymes can be used within the emerging lignocellulose biorefineries in processes that range from the production of bioenergy to that of biomaterials. To ensure that these versatile enzymes meet industry standards and needs, they have been subjected to directed evolution and hybrid approaches that surpass the limits imposed by nature. This Opinion article analyzes recent achievements in this field, including the incipient groundbreaking research into the evolution of resurrected enzymes, and the engineering of ligninolytic secretomes to create consolidated bioprocessing microbes with synthetic biology applications., European Commission projects Indox-FP7-KBBE-2013-7-613549; Bioenergy-FP7-PEOPLE-2013-ITN-607793; Cost Action CM1303 Systems Biocatalysis; and the National projects Evofacel (BIO2010-19697), Dewry (BIO201343407-R], and Cambios (RTC-2014-1777-3).

[EN] Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate >green catalysts>. This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts., EU (FP7-KBBE-2013-7-613549-INDOX, FP7-People2013-ITN-607793 and COST-Action CM1303 Systems Biocatalysis) and the Spanish Government (BIO2010-19697-EVOFACEL, BIO2013-43407-RDEWRY and CAMBIOS-RTC-2014-1777-3)

Special Issue: Thematic Issue: From complex waste to plastic value., Although the functional expression of fungal laccases in Saccharomyces cerevisiae has proven to be complicated, the replacement of signal peptides appears to be a suitable approach to enhance secretion in directed evolution experiments. In this study, twelve constructs were prepared by fusing native and evolved α‐factor prepro‐leaders from S. cerevisiae to four different laccases with low‐, medium‐ and high‐redox potential (PM1L from basidiomycete PM1; PcL from Pycnoporus cinnabarinus; TspC30L from Trametes sp. strain C30; and MtL from Myceliophthora thermophila). Microcultures of the prepro‐leader:laccase fusions were grown in selective expression medium that used galactose as both the sole carbon source and as the inducer of expression so that the secretion and activity were assessed with low‐ and high‐redox potential mediators in a high‐throughput screening context. With total activity improvements as high as sevenfold over those obtained with the native α‐factor prepro‐leader, the evolved prepro‐leader from PcL (αPcL) most strongly enhanced secretion of the high‐ and medium‐redox potential laccases PcL, PM1L and TspC30L in the microtiter format with an expression pattern driven by prepro‐leaders in the order αPcL > αPM1L ~ αnative. By contrast, the pattern of the low‐redox potential MtL was αnative > αPcL > αPM1L. When produced in flask with rich medium, the evolved prepro‐leaders outperformed the αnative signal peptide irrespective of the laccase attached, enhancing secretion over 50‐fold. Together, these results highlight the importance of using evolved α‐factor prepro‐leaders for functional expression of fungal laccases in directed evolution campaigns., This work was supported by the European Union projects (Bioenergy‐FP7‐PEOPLE‐2013‐ITN‐607793; H2020‐BBI‐PPP‐2015‐2‐720297‐ENZOX2) the COST Action [CM1303 Systems Biocatalysis] and the Spanish Government [BIO2016‐79106‐R‐Lignolution].

Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C–H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values. The full ensemble of segments (43% of UPO sequence) was subjected to focused evolution by the Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) method in Saccharomyces cerevisiae. Five independent mutant libraries were screened in terms of P:p ratio and thermostability. We identified several variants that harbored substitutions at positions 120 and 320 with a strong enhancement in the P:p ratio albeit at the cost of stability. The most thermostable mutant of this process (S226G with an increased T50 of 2°C) was subjected to further combinatorial saturation mutagenesis on Thr120 and Thr320 yielding a collection of variants with modified P:p ratio and recovered stability. Our results seem to indicate the coexistence of several oxidation sites for peroxidative and peroxygenative activities in UPO., This work was supported by the European Union [FP7-KBBE-2013-7-613549-INDOX, H2020-BBI-PPP-2015-2-720297-ENZOX2], the COST Action [CM1303 Systems Biocatalysis] and the Spanish Government [BIO2016-79106-R-Lignolution].

The directed evolution of ancestral ‐resurrected‐ enzymes can give a new twist in protein engineering approaches towards more versatile and robust biocatalysts.

Directed evolution is a robust approach to tailor enzymes with improved attributes. One round of laboratory evolution comprises three essential steps: (1) the generation of diversity by random mutagenesis and/or DNA recombination of the parental genes, (2) cloning and functional expression of the mutant library in a suitable host, and (3) selection or screening for the desired feature. This pocket manual of directed evolution provides an overview of the current methods and protocols used for steps (1) to (3), while giving clever tips and tricks to circumvent common hurdles that appear during creation of the mutant library and its exploration. The new research trends in the evolutionary field (genetic drift, circular permutation, de novo enzyme design, metagenomics, and ancestral resurrection) are also mentioned briefly.

The ligninolytic enzymatic consortium produced by white-rot fungi is one of the most efficient oxidative systems found in nature, with many potential applications that range from the production of 2nd generation biofuels to chemicals synthesis. In the current study, two high redox potential oxidoreductase fusion genes (laccase -Lac and versatile peroxidase -Vp-) that had been evolved in the laboratory were re-assembled in Saccharomyces cerevisiae. First, cell viability and secretion were assessed after co-transforming the Lac and Vp genes into yeast. Several expression cassettes were inserted in vivo into episomal bi-directional vectors in order to evaluate inducible promoter/terminator pairs of different strengths in an individual and combined manner. The synthetic white-rot yeast model harboring Vp(GAL1/CYC1)-Lac(GAL10/ADH1) displayed up to 1000 and 100 Units per L of peroxidase and laccase activity, respectively, representing a suitable point of departure for future synthetic biology studies. © 2014 Landes Bioscience. Do not distribute., This work was supported by European Commission Projects (Peroxicats-FP7-KBBE-2010-4-26537; Indox-FP7-KBBE-2013-7-613549; COST-Action CM1303: Systems Biocatalysis) and the National Project (Evofacel-cel-BIO2010-19697)

For more than thirty years, biotechnology has borne witness to the power of directed evolution in designing molecules of industrial relevance. While scientists all over the world discuss the future of molecular evolution, dozens of laboratory-designed products are being released with improved characteristics in terms of turnover rates, substrate scope, catalytic promiscuity or stability. In this review we aim to present the most recent advances in this fascinating research field that are allowing us to surpass the limits of nature and apply newly gained attributes to a range of applications, from gene therapy to novel green processes. The use of directed evolution in non-natural environments, the generation of catalytic promiscuity for non-natural reactions, the insertion of unnatural amino acids into proteins or the creation of unnatural DNA, is described comprehensively, together with the potential applications in bioremediation, biomedicine and in the generation of new bionanomaterials. These successful case studies show us that the limits of directed evolution will be defined by our own imagination, and in some cases, stretching beyond that., The laboratory of MA gratefully acknowledges the financial support received from the EU (FP7-KBBE-2013-7-613549-INDOX, FP7-People-2013-ITN-607793 and COST-Action CM1303 Systems Biocatalysis) and the Spanish Government (BIO2010-19697-EVOFACEL, BIO2013-43407-R-DEWRY and CAMBIOS-RTC-2014-1777-3) projects.

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., We thank Prof. Ed Bayer’s group for kindly providing the plasmids used in the minicellulosome constructs. Research was supported by the Basque Government grant ELKARTEK to R.P.-J, and also partly by Ministry of Economy and Competitiveness (MINECO) grant BIO2016-77390-R, BFU2015-71964 to R.P.-J., BIO2016-74875-P to J. A.G., and CTQ2015-65320-R and RYC-2016-19590 to D.D.S.; European Commission grant CIG Marie Curie Reintegration program FP7-PEOPLE-2014 to R.P.-J, and European Commission grant NMP-FP7 604530-2 (CellulosomePlus), and the ERA-IB EIB.12.022 grant (FiberFuel) funded by the MINECO (PCIN-2013-011-C02-01) to M.C.-V. We also thank Fundación Repsol and Gipuzkoako Foru Aldundia for financial support.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP (versatile peroxidase) expressed in Saccharomyces cerevisiae [García-Ruiz, González-Pérez, Ruiz-Dueñas, Martínez and Alcalde (2012) Biochem. J. 441, 487–498]. 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120 h of incubation at pH 9 compared with complete inactivation of the native enzyme after only 1 h). The latter is highly demanded for biorefinery applications. In the present study we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were expressed in Escherichia coli, and their alkaline stability and biochemical properties were determined. In addition, the crystal structures of 2-1B and one of the intermediate variants were solved and carefully analysed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections between haem and helix B (where the distal histidine residue is located), and formation of new electrostatic interactions, that avoided the hexa-co-ordination of the haem iron. These new structural determinants stabilized the haem and its environment, helping to maintain the structural enzyme integrity (with penta-co-ordinated haem iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305 in the proximal side, prompted by the Q202L mutation, further enhanced the stability., This work was supported by HIPOP [grant number BIO2011-26694], NOESIS [grant number BIO2014-56388-R] and DEWRY [grant number BIO2013-43407-R] projects of the Spanish Ministry of Economy and Competitiveness (MINECO), and by the PEROXICATS [grant number KBBE-2010-4-265397] and INDOX [grant number KBBE-2013-7-613549] European projects. V.S.-J. and F.J.R.-D. are grateful for the financial support of an FPI fellowship and a Ramón y Cajal contract of the Spanish MINECO, respectively.

Enzymatic production of C–N heteropolymeric dyes at alkaline pHs is an attractive process for the textile industry. In this work, we have designed a fungal laccase by directed evolution so that it may be used at alkaline pHs for the synthesis of C–N heteropolymeric dyes (C–N polydye) from catechol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA). Firstly, several medium- and high-redox potential fungal laccases from previous laboratory evolution campaigns were benchmarked for the synthesis of the C–N polydye at pH 8.0, choosing an alkaline laccase mutant from Myceliophthora thermophila as the departure point for further engineering. Mutant libraries were then constructed, expressed in Saccharomyces cerevisiae and screened using a high-throughput colorimetric assay for the detection of the C–N polydye. By combining directed and focused molecular evolution, a novel, strongly expressed alkaline laccase variant was identified. This laccase was secreted at 37 mg/L and its catalytic efficiency for the oxidation of catechol and 2,5-DABSA at pH 8.0 was enhanced 3.5-fold relative to that of the wild-type, promoting the synthesis of the C–N polydye at basic pHs. While the improved expression was mostly the result of accumulating mutations that favor the yeast’s codon usage together with the recovery of a secretion mutation, the enhanced C–N polydye synthetic activity of the mutant laccase was dependent on the alkaline mutations it inherited. Readily secreted, this laccase mutant would appear to be a valuable platform for organic synthesis at basic pHs., This research was supported by the European Commission funded FP7-KBBE-2013-7-613549-INDOX project, a COST-Action CM1303 and by the Spanish Government (grants: BIO2013-43407-R-DEWRY and CAMBIOS-RTC-2014-1777-3).

Ligninolytic peroxidases are involved in natural wood decay in strict acid environments. Despite their biotechnological interest, these high-redox potential enzymes are not functional at basic pH due to the loss of calcium ions that affects their structural integrity. In this study, we have built catalytic activity at basic pH in a versatile peroxidase (VP) previously engineered for thermostability. By using laboratory evolution and hybrid approaches, we designed an active and highly stable alkaline VP while the catalytic bases behind the alkaline activation were unveiled. A stabilizing mutational backbone allowed the pentacoordinated heme state to be maintained, and the new alkaline mutations hyperactivated the enzyme after incubation at basic pHs. The final mutant oxidises substrates at alkaline pHs both at the heme channel and at the Mn2+ site, while the catalytic tryptophan was not operational under these conditions. Mutations identified in this work could be transferred to other ligninolytic peroxidases for alkaline activation., This research was supported by the European Commission project FP7-KBBE-2013-7-613549-INDOX, the COST-Action CM1303 and by the Spanish government (grants: BIO2013-43407-R-DEWRY and CAMBIOS-RTC-2014-1777-3).

12 páginas, 5 figuras, 2 tablas -- PAGS nros. 487-498, The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21 mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43 min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability

β-Fructofuranosidases can divert their hydrolytic activity towards transglycosylation for the synthesis of high value-added products, including prebiotic fructooligosaccharides (FOS). A directed evolution strategy has been employed to enhance the transferase rate of the β-fructofuranosidase (SoINV) from the Schwanniomyces occidentalis yeast for the production of β-(2→6)-linked FOS. To screen for transferase activity of the SoINV functionally expressed in Saccharomyces cerevisiae, a high-throughput screening protocol based on two colorimetric assays was validated (with coefficient of variance below 11%). Mutagenic libraries were constructed by error-prone PCR and clones showing higher glucose:fructose ratio with respect to the parental type were identified. Further analysis by anion-exchange chromatography coupled with pulsed amperometric detection helped to identify mutants with improved yields for the synthesis of β-(2→6) fructooligosaccharides. Selected mutants displayed transferase initial rates enhanced ∼2-fold over parent type, reaching production levels up to 47 g/L after 48 h of reaction for the formation of 6-kestose.

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution.

A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved (509)VSG(511) tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The K(m)(O)(2) value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the (509)VSG(511) tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.

SummaryFungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase—R2 variant—was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.

Fungal laccases are useful for several remarkable transformations, such as bioremediation of polycyclic aromatic hydrocarbons (PAHs), synthesis of phenolic-based resins, oxidation of lignin derivatives and others. Most of these substrates are barely water-soluble, and although polar organic co-solvents may be added to enhance their solubility, transformation rates dramatically decrease due to the negative effect of organic solvents on the protein structure. Laccase from Myceliophthora thermophila variant T2 (MtLT2) has been submitted to laboratory evolution in Saccharomyces cerevisiae with the aim of improving activity and stability in organic co-solvents. Some 4500 clones created by random mutagenesis were screened in two rounds of directed evolution. Libraries were explored under increasing concentrations of acetonitrile and ethanol, and several mutants with improved features were purified and further characterised. Turnover rates of MtLT2 in 30% (v/v) acetonitrile and 50% (v/v) ethanol were increased u...

Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (~8. mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217. mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production., EU (FP7-KBBE-2013-7-613549-INDOX, FP7-People-2013-ITN-607793, COST-Action CM1303 Systems Biocatalysis) and the Spanish Government (BIO2010-19697-EVOFACEL, BIO2013-43407-R-DEWRY and CAMBIOS-RTC-2014-1777-3 projects).

[EN] Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating -factor and the K1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: prepro-AAO > preproK-AAO > preKpro-AAO > preproK-AAO. The chimeric preproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the Kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability., This study was supported by the European Commission projects Indox FP7-KBBE-2013-7-613549 and Cost-Action CM1303-Systems Biocatalysis and by the National Projects Dewry (BIO201343407-R) and Cambios (RTC-2014-1777-3).

Combinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hot-spot residues discovered by directed enzyme evolution or performing semi-rational studies. CSM coupled to a reliable high-throughput screening assay -coefficient of variance below 10%- has been used to enhance turnover rates in the fungal laccase variant T2 from Myceliophthora thermophila. The influence of the highly conserved pentapeptide 509-513 on the redox potential of blue-copper containing enzymes is well described. We focused combinatorial saturation mutagenesis in residues Ser510 and Leu513. Libraries were constructed in Saccharomyces cerevisiae by in vivo overlap extension -IVOE- of the PCR products. This methodology provides a simple manner to build CSM libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. After exploring more than 1,700 clones, mutant (7E1) with approximately 3-fold higher kinetics than parent type was found. 7E1 showed one synonymous mutation (L513L, CGT/TTG) and one beneficial mutation S510G (TCG/GGG) that can not be achieved by conventional error-prone PCR techniques. Mutation S510G seems to affect the C-terminal plug, which modulates the transit of water and oxygen to the trinuclear copper cluster.

The synthesis of methyl alpha-D-glucooligosaccharides, using sucrose as glucosyl donor and methyl alpha-D-glucopyranoside as acceptor, was studied with dextransucrase from Leuconostoc mesenteroides NRRL B-1299. The enzyme was immobilized by entrapment in alginate. By NMR and mass spectrometry we identified three homologous series (S1-S3) of methyl alpha-D-glucooligosaccharides. Series S2 and S3 were characterized by the presence of alpha(1-2) linkages, in combination with alpha(1-6) bonds. Two parameters, sucrose to acceptor concentration ratio (S/A) and the total sugar concentration (TSC) determined the yield of methyl alpha-D-glucooligosaccharides. The maximum concentration achieved of the first acceptor product, methyl alpha-D-isomaltoside, was 65 mM using a S/A 1:4 and a TSC of 336 g l-1. When increasing temperature, a shift of selectivity towards compounds containing alpha(1-2) bonds was observed. The formation of leucrose as a side process was very significant (reaching values of 32 g l-1) at high sucrose concentrations., We are very grateful to Profs. P. Monsan and M. Remaud-Simeon (INSA, Toulouse) for providing us with dextransucrase. We thank Prof. A. Cortés (ICP, CSIC) for helpful comments. We thank Gobierno Vasco and Instituto Danone for research fellowships. This work was supported by the Spanish Education and Science Ministry (Project BIO2004-03773-C04-01).

Under aerobic conditions, the enzyme γ-hexachlorocyclohexane dechlorinase (LinA) from Sphingomonas paucimobilis UT26 catalyses the elimination of chlorine atoms from the molecule of γ-hexachlorocyclohexane (γ-HCH) or lindane, a recalcitrant pesticide that is still widely used. In its native metabolic context, LinA starts the biodegradation process of lindane by transforming γ-HCH to 1,2,4 trichlorobenzene (TCB), a less persistent chemical. In an attempt to generate an improved version of this enzyme to be used in lindane bioremediation schemes, we have run an experimental evolution procedure on LinA, using Escherichia coli as the surrogate host. One round of random mutagenesis and subsequent screening for improved dechlorination in vivo sufficed to yield one mutant enzyme (LinAT10), bearing a single substitution C132R, that displayed a two-fold enhanced expression and three-fold enhanced solubility of the enzyme compared to the wild type protein. This resulted in a biological product with a six-fold incre...