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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. ; This research was supported by the Spanish Government project BIO2016-79106-R287 Lignolution and the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA.

Versatile peroxidase (VP) secreted by white-rot fungi is involved in the degradation of lignin within land ecosystems. With a broad substrate scope and minor requirements, VP is an extremely attractive blueprint to be designed by the directed evolution tool-box. In recent years, improved VP variants have been generated that meet industrial requirements in terms of improved heterologous functional expression and activity, as well as stability at high temperatures or in the presence of strong inhibitors. This review describes the making of VP by directed evolution, addressing the most important findings and challenges that were faced, along with the future prospects for this research field. ; European UnionFP7-KBBE-2013-7-613549-INDOXCOST Action CM1303 Spanish Government BIO2016-79106-R- Lignolution Seventh Framework Programme10.13039/100011102FP7-KBBE-2013-7-613549-INDOXEuropean Cooperation in Science and Technology10.13039/501100000921CM1303 Systems BiocatalysisThis work was supported by grants from the European Union [FP7-KBBE-2013-7-613549-INDOX], the COST Action [CM1303 Systems Biocatalysis] and the Spanish Government [BIO2016-79106-R-Lignolution]. ; Peer reviewed

Special Issue: Thematic Issue: From complex waste to plastic value. ; Laccases are multicopper containing enzymes capable of performing one electron oxidation of a broad range of substrates. Using molecular oxygen as the final electron acceptor, they release only water as a by‐product, and as such, laccases are eco‐friendly, versatile biocatalysts that have generated an enormous biotechnological interest. Indeed, this group of enzymes has been used in different industrial fields for very diverse purposes, from food additive and beverage processing to biomedical diagnosis, and as cross‐linking agents for furniture construction or in the production of biofuels. Laccases have also been studied intensely in nanobiotechnology for the development of implantable biosensors and biofuel cells. Moreover, their capacity to transform complex xenobiotics makes them useful biocatalysts in enzymatic bioremediation. This review summarizes the most significant recent advances in the use of laccases and their future perspectives in biotechnology. ; We recognize the much appreciated financial support of the EU (projects FP7‐KBBE‐2013‐7‐613549‐INDOX, FP7‐People‐2013‐ITN‐607793 and COST Action CM1303 Systems Biocatalysis) and the Spanish Government (BIO2013‐43407‐R‐DEWRY). ; Peer reviewed

[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) ; Peer Reviewed

[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. ; This study is based upon work funded by and 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 under grant agreement nº 886567 (BIZENTE project). B.J.G.F was supported by a FPI national fellowship BES-2014-068887. ; 1. A consensus design to identify consensus ancestral mutations 2. SCHEMA-RASPP computation for homologous in vivo recombination to generate laccase chimeras 3. Computer-guided evolution to enhance the laccase redox potential and the activity toward high-redox potential mediators 4. Ancestral sequence reconstruction for the directed evolution of a resurrected basidiomycete laccase toward initiators

[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. ; This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA.

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. ; Peer reviewed

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). ; Peer Reviewed

Modern biocatalysis is developing new and precise tools to improve a wide range of production processes, which reduce energy and raw material consumption and generate less waste and toxic side-products. Biocatalysis is also achieving new advances in environmental fields from enzymatic bioremediation to the synthesis of renewable and clean energies and biochemical cleaning of "dirty" fossil fuels. Among the obvious benefits of biocatalysis the major hurdles hindering the exploitation of the great repertoire of enzymatic processes are, in many cases, the high production costs and the low yields obtained. This article will discuss these issues, pinpointing specific new advances in recombinant DNA techniques for future biocatalyst development, as well as drawing the attention of the biotechnology scientific community to the active pursuing and developing of Environmental Biocatalysis, covering from remediation with enzymes to novel green processes. ; This material is based upon work founded by European Union project MERG-CT-2004-505242 "BIOMELI", Spanish Ministry of Education and Science projects BIO2002-00337, VEM2004-08559 and CTQ2005-08925-C02-02/PPQ, Comunidad de Madrid project GR/AMB/0690/2004, CSIC Project 200580M121 and the company ViaLactiaBiosciences Ltd. (New Zealand). ; Peer reviewed

12 páginas, 6 figuras ; [Background] Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory. ; [Results] A blood-active laccase (ChU-B mutant) fused to the native/evolved α-factor prepro-leader was cloned under the control of two different promoters (PAOX1 and PGAP) and expressed in Pichia pastoris. The most active construct, which contained the PAOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the α-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers. ; [Conclusions] The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications. ; This study is based upon a work funded by European Union Projects (grant numbers NMP4-SL-2009-229255-3D-Nanobiodevice, FP7-KBBE-2010-4-26537-Peroxicats and COST Action CM0701) and a Spanish National Project (Evofacel, BIO2010-19697) and by the Austrian Science Fund (FWF, P25148-B20). We thank Prof. S. Shleev from Malmö University (Sweden) for carrying out the measurements of the laccase activity in human plasma and blood. D.M.M. is grateful to the CSIC for a JAE fellowship. ; Peer reviewed

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]. ; Peer reviewed

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]. ; Peer reviewed

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. ; Peer reviewed

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. ; Peer reviewed

The Myceliophthora thermophila laccase was covalently immobilized on polymethacrylate-based polymers (Sepabeads EC-EP3 and Dilbeads NK) activated with epoxy groups. The enzyme immobilized on Sepabeads EC-EP3 exhibited notable activity (203 U/g) along with remarkably improved stability towards pH, temperature and storage time, but no increased resistance to organic solvents. In addition, the immobilized laccase also showed good operational stability, maintaining 84% of its initial activity after 17 cycles of oxidation of ABTS. The immobilized biocatalyst was applied to the decolorization of six synthetic dyes. Immobilized laccase retained 41% activity in the decolorization of Methyl Green in a fixed-bed reactor after five cycles. The features of these biocatalysts are very attractive for their application on the decolorization of dyes in the textile industry in batch and continuous fixed-bed bioreactors. To our knowledge, this is the first report on immobilization of laccase on Sepabeads carriers and its efficient dyes decolorization. ; We thank Drs. Moreno Daminati and Paolo Caimi (Resindion) and Vyasa Rajasekar (DilComplex) for providing us Sepabeads EC-EP3 and Dilbeads NK polymers, respectively. We are grateful to Ramiro Martínez (Novozymes A/S, Spain) for DeniLite II S samples. This material is based upon work founded by Spanish MEC (Projects VEM2004-08559 and CTQ2005-08925-C02-02/PPQ); European Union (Project NMP2-CT-2006-026456) and CSIC (Project 200580M121). Spanish MEC is also thanked for the post-doctoral fellowship (SB2004-0011) of Dr. A. Kunamneni and for the Ramon y Cajal contracts of Drs. S. Camarero and M. Alcalde. ; This material is based upon work financed by Spanish MEC (Projects VEM2004-08559 and CTQ2005-08925-C02-02/PPQ); European Union (Project NMP2-CT-2006-026456) and CSIC (Project 200580M121). Spanish MEC is also thanked for the post-doctoral fellowship (SB2004-0011) of Dr. A. Kunamneni and for the Ramon y Cajal contracts of Drs. S. Camarero and M. Alcalde. ; Peer reviewed