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 (α
nat
) and a top-down design over the fittest evolved signal peptide obtained in our lab (α
9H2
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 (α
OPT
), 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 α
OPT
leader fused to the target protein.
Multicopper oxidases (MCOs) share a common catalytic mechanism of activation by oxygen and cupredoxin-like folding, along with some common structural determinants. Laccases constitute the largest ...group of MCOs, with fungal laccases having the greatest biotechnological applicability due to their superior ability to oxidize a wide range of aromatic compounds and lignin, which is enhanced in the presence of redox mediators. The adaptation of these versatile enzymes to specific application processes can be achieved through the directed evolution of the recombinant enzymes. On the other hand, their substrate versatility and the low sequence homology among laccases make their exact classification difficult. Many of the ever-increasing amounts of MCO entries from fungal genomes are automatically (and often wrongly) annotated as laccases. In a recent comparative genomic study of 52 basidiomycete fungi, MCO classification was revised based on their phylogeny. The enzymes clustered according to common structural motifs and theoretical activities, revealing three novel groups of laccase-like enzymes. This review provides an overview of the structure, catalytic activity, and oxidative mechanism of fungal laccases and how their biotechnological potential as biocatalysts in industry can be greatly enhanced by protein engineering. Finally, recent information on newly identified MCOs with laccase-like activity is included.
Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by ...one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed.
Saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a fungal laccase previously engineered in the lab. Mutant libraries were screened using sinapic acid ...as a model substrate, and those mutants presenting increased activity were selected for exploring the oxidation of lignin-derived phenols. The latter comprised a battery of phenolic compounds of interest due to their use as redox mediators or precursors of added-value products and their biological activity. The new laccase variants were investigated in a multi-screening assay and the structural determinants, at both the substrate and the protein level, for the oxidation of the different phenols are discussed. Laccase activity greatly varied only by changing one or two residues of the enzyme pocket. Our results suggest that once the redox potential threshold is surpassed, the contribution of the residues of the enzymatic pocket for substrate recognition and binding strongly influence the overall rate of the catalytic reaction.
Multi-copper oxidases (MCO) share a common molecular architecture and the use of copper ions as cofactors to reduce O2 to H2O, but show high sequence heterogeneity and functional diversity. Many new ...emerging MCO genes are wrongly annotated as laccases, the largest group of MCOs, with the widest range of biotechnological applications (particularly those from basidiomycete fungi) due to their ability to oxidise aromatic compounds and lignin. Thus, comprehensive studies for a better classification and structure-function characterisation of MCO families are required. Laccase-ferroxidases (LAC-FOXs) constitute a separate and unexplored group of MCOs with proposed dual features between laccases and ferroxidases. We aim to better define this cluster and the structural determinants underlying putative hybrid activity. We performed a phylogenetic analysis of the LAC-FOXs from basidiomycete fungi, that resulted in two subgroups. This division seemed to correlate with the presence or absence of some of the three acidic residues responsible for ferroxidase activity in Fet3p from Saccharomyces cerevisiae. One of these LAC-FOXs (with only one of these residues) from the fungus Heterobasidion annosum s. l. (HaLF) was synthesised, heterologously expressed and characterised to evaluate its catalytic activity. HaLF oxidised typical laccase substrates (phenols, aryl amines and N-heterocycles), but no Fe (II). The enzyme was subjected to site-directed mutagenesis to determine the key residues that confer ferroxidase activity. The mutated HaLF variant with full restoration of the three acidic residues exhibited efficient ferroxidase activity, while it partially retained the wide-range oxidative activity of the native enzyme associated to laccases sensu stricto.
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Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement.
is a preferred host for ...engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of
alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in
of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of
cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.
Xylanases can boost pulp bleachability in Elemental Chlorine Free (ECF) processes, but their industrial implementation for producing bleached kraft pulps is not straightforward. It requires enzymes ...to be active and stable at the extreme conditions of alkalinity and high temperature typical of this industrial process; most commercial enzymes are unable to withstand these conditions. In this work, a novel highly thermo and alkaline-tolerant xylanase from Pseudothermotoga thermarum was overproduced in E. coli and tested as a bleaching booster of hardwood kraft pulps to save chlorine dioxide (ClO2) during ECF bleaching. The extremozyme-stage (EXZ) was carried out at 90 °C and pH 10.5 and optimised at lab scale on an industrial oxygen-delignified eucalyptus pulp, enabling us to save 15% ClO2 to reach the mill brightness, and with no detrimental effect on paper properties. Then, the EXZ-assisted bleaching sequence was validated at pilot scale under industrial conditions, achieving 25% ClO2 savings and reducing the generation of organochlorinated compounds (AOX) by 18%, while maintaining pulp quality and papermaking properties. Technology reproducibility was confirmed with another industrial kraft pulp from a mix of hardwoods. The new enzymatic technology constitutes a realistic step towards environmentally friendly production of kraft pulps through industrial integration of biotechnology.
The laccase from the ascomycete
Myceliophthora thermophila 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 biocatalyst also showed good operational stability, maintaining 84% of its initial activity after 17 cycles of oxidation of ABTS. The immobilized laccase was applied to the decolorization of six synthetic dyes (Reactive Black 5, Acid Blue 25, Methyl Orange, Remazol Brilliant Blue B, Methyl Green and Acid Green 27) with or without the redox mediator 1-hydroxybenzotriazole. The laccase immobilized in Sepabeads EC-EP3 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.
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.
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