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1 p., Saccharomyces cerevisiae desempeña un papel importante como sistema de expresión heteróloga de proteínas debido a su fácil manipulación, bajos requerimientos y capacidad para realizar las modificaciones postraduccionales en las proteínas. Una de las estrategias más utilizadas para incrementar la secreción es el uso de péptidos señal que determinan la vía de secreción.Históricamente, la secuencia pre-prolíder de la feromona factor α de S. cerevisiae ha jugado un papel importante en la producción de proteínas recombinantes. El uso de este péptido señal en combinación con la evolución dirigida de enzimas ha permitido conseguir la difícil expresión de lacasas fúngicas en S. cerevisiae, obteniendo diferentes secuencias señales evolucionadas.Sin embargo, el diseño de un péptido señal optimizado, de carácter universal, que incremente la producción de diversas enzimas es un desafío pendiente.En este estudio, se aplicaron dos estrategias paralelas de ingeniería del pre-prolíder del factor α para mejorar la producción de enzimas analizando el efecto de mutaciones acumuladas en dicha secuencia a lo largo de sucesivas rondas de evolución dirigida y sus posibles interacciones epistáticas. Ambos enfoques coincidieron en el efecto sinérgico de cuatro mutaciones contenidas en el péptido señal optimizado final, que mejoraron notablemente la secreción de varias oxidorreductasas e hidrolasas fúngicas. Además, se proporcionan directrices adicionales para mejorar aún más la producción heteróloga de una enzima en particular mediante la mutagénesis saturada combinatorial de dos posiciones del péptido señal fusionado a la proteína de interés. Los resultados obtenidos ofrecen un importante avance para la producción de biocatalizadores de interés industrial., Spanish project BIO2017-86559-R. WoodZymes project funded by the Bio Based Industries Joint Undertaking (JU) under grant agreement No 792070

32 p.-5 fig., Fungal laccases are multicopper oxidases with high catalytic versatility and low catalytic requirements (only O2 of the air is required for activation). Their high redox potential, especially of certain basidiomycete laccases, significantly increases their oxidation capacity compared to bacterial laccases. These characteristics together provide these enzymes with great potential for applications as biocatalysts in a range of synthetic or degrading reactions. We review here some of the main properties and biotechnological applications of fungal laccases, underlining their overexpression limitations for industrial scale application. We focus on the use of laccases as biocatalysts for organic synthesis, with special emphasis on polymer synthesis.

12 p.-12 fig.-1 tab., Fungal laccases can play an important role as biocatalysts in organic chemistry to replace chemical synthesis. In a previous work we synthesized conductive polyaniline using a high-redox potential laccase from our collection of recombinant fungal variants. Still, the oxidation of aniline is hindered by the reaction conditions (low pH and presence of anionic surfactants). Thus, we tackle here the directed evolution of the enzyme assisted by computational simulation aiming at improving aniline oxidation at the required polymerization conditions while maintaining the enzyme's substrate promiscuity. Simultaneously, its secretion by the host used for the engineering (Saccharomyces cerevisiae) was enhanced. Then, the improved laccase variant was overproduced in the industrial host Aspergillus oryzae and assayed for one-pot synthesis of polyaniline and naphtol-derived dyes whose textile dyeing properties were verified in an industrial environment. Finally, modification of its C-terminal tail further enhanced laccase stability by flexibilization of the region. The resulting biocatalyst displays noticeable stability at high temperature and extreme pH while shows improved k(cat) values on the different substrates tested. Moreover, it is remarkably produced in S. cerevisiae at rates not formerly reported in the literature. These facts, together with the overexpression in A. oryzae opens new scenarios for its further development and application., This work has been funded by the INDOX EU project (KBBE- 2013-7-613549), the Spanish projects BIO2017-86559-R and CTQ2016-79138-R and the BBI JU project WoodZymes (H2020-BBI-JU-792070). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

13 p.-7 fig.-2 tab, Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding., This work has been funded by the INDOX EU project (KBBE-2013-7-613549), the Spanish projects BIO2017-86559-R and CTQ2016-79138-R, the BBI JU project WoodZymes (H2020-BBI-JU-792070), the H2020-iNEXT grant numbers 1676 and ISCIII.

10 p.-4 fig.-3 tab., The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2-9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50-70 degrees C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times., This work was funded by the Spanish National Projects NOESIS (BIO2014-56388-R) and GENOBIOREF (BIO2017-86559-R) and the European Project WoodZymes (H2020-BBI-JU-792070).

10 p.- 7 fig., The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2–9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50–70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times., This work was funded by the Spanish National Project NOESIS (BIO2014-56388-R) and the European Projects INDOX (KBBE-2013-7-613549) and ENZOX2 (H2020-BBI-PPP-2015-2-720297).

17 p.-7 fig., Saccharomyces cerevisiae plays an important role in the heterologous expression of an array of proteins due to its easy manipulation, low requirements and ability for protein post-translational modifications. The implementation of the preproleader secretion signal of the α-factor mating pheromone from this yeast contributes to increase the production yields by targeting the foreign protein to the extracellular environment. The use of this signal peptide combined with enzyme-directed evolution allowed us to achieve the otherwise difficult functional expression of fungal laccases in S. cerevisiae, obtaining different evolved α-factor preproleader sequences that enhance laccase secretion. However, the design of a universal signal peptide to enhance the production of heterologous proteins in S. cerevisiae is a pending challenge. We describe here the optimisation of the α-factor preproleader to improve recombinant enzyme production in S. cerevisiae through two parallel engineering strategies: a bottom-up design over the native α-factor preproleader (αnat) and a top-down design over the fittest evolved signal peptide obtained in our lab (α9H2 leader). The goal was to analyse the effect of mutations accumulated in the signal sequence throughout iterations of directed evolution, or of other reported mutations, and their possible epistatic interactions. Both approaches agreed in the positive synergism of four mutations (Aα9D, Aα20T, Lα42S, Dα83E) contained in the final optimised leader (αOPT), which notably enhanced the secretion of several fungal oxidoreductases and hydrolases. Additionally, we suggest a guideline to further drive the heterologous production of a particular enzyme based on combinatorial saturation mutagenesis of positions 86th and 87th of the αOPT leader fused to the target protein., This work has been funded by the Spanish project BIO2017-86559-R, and the WoodZymes project funded by the Bio Based Industries Joint Undertaking (JU) under grant agreement No 792070. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and the Bio Based Industries Consortium.