Suchergebnisse

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. ; Peer reviewed

24 p.-6 fig.-8 tab. ; Agaricomycetes fungi responsible for decay of wood and other lignocellulosic substrates constitute a valuable source of lignin-degrading enzymes. Among these enzymes, laccases (multi-copper oxidases) present remarkable biotechnological potential as environmentally friendly biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the only requirement. Laccases from saprotrophic Agaricales species have been much less studied than laccases from Polyporales, despite the fact that the former fungi are excellent sources of laccases. Here, the gene of a novel laccase of Agrocybe pediades, that is secreted by the fungus during lignocellulose degradation, was synthesised de novo and expressed in Saccharomyces cerevisiae using an improved signal peptide previously obtained and enzyme directed evolution. The characterization of the new laccase variants provided new insights on the contribution of different amino acid residues to modulate laccase production, catalytic activity or optimal pH. The selected double-mutated variant also showed interesting properties as a biocatalyst, such as the ability to oxidise a wide range of substrates, including high-redox potential mediators and recalcitrant organic dyes, improved activity at neutral pH and high tolerance to inhibitors. Finally, we demonstrate the existence of three N-glycosylation sites in the laccase and their distinct effect on the secretion or catalytic activity of the enzyme. ; This work was supported by the GENOBIOREF (BIO2017–86559-R) project of the Spanish Ministry of Science & Innovation (co-financed by FEDER funds), and by the European Project WoodZymes funded by the Bio Based Industries Joint Undertaking, under the European Union's Horizon 2020 research and innovation program (Grant Agreement H2020-BBI-JU-792070). ; 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

31 páginas.- 14 figuras.- 92 referencias.- Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/jof7060426/s1 .- Acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). ; Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. ; This research was funded by the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Science and Innovation (co-financed by FEDER funds) to F.J.R.-D. and S.C.; and by the Consejo Superior de Investigaciones CientÍficas (PIE-202120E019 to A.T.M. and PIE-201620E081 to A.G.). The work conducted by the JGI, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. DOE under contract DE-AC02-05CH11231. Part of this material is also based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER) under the Early Career Award Program to D.S. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, ; Peer reviewed