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The generation of enantiodivergent biocatalysts for C-H oxyfunctionalizations 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 the 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., BT/Biocatalysis

[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.

[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.

Fungal high-redox-potential laccases (HRPLs) are multi-copper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (E-T1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased E-T1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic highredox-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 E-T1 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 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 E-T1, 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.

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] Electrochemical characterization of the GreeDo variant of a high redox potential fungal laccase obtained by laboratory evolution together with computer-guided mutagenesis, in comparison to its parental variety (the OB-1 mutant), is presented. Both laccases, when immobilized on graphite electrodes either by direct physical adsorption or covalently attached via gold nanoparticles, were capable of both non-mediated and mediator-based bioelectroreduction of molecular oxygen at low overpotentials. GreeDo exhibited higher open circuit potential values and onset potentials for oxygen bioelectroreduction compared to OB-1. However, even though in homogeneous catalysis GreeDo outperforms OB-1 in terms of turnover numbers and catalytic efficiency, when exposed to high redox potential substrates, direct electron transfer based bioelectrocatalytic currents of GreeDo and OB-1 modified electrodes were similar. High operational stability of freely diffusing GreeDo and also the immobilized enzyme in the acidic electrolyte was registered, in agreement with high storage stability of GreeDo in acidic solutions.

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.

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.