From laccase design to application of the overexpressed biocatalyst in an industrial environment for eco-friendly synthesis of polyaniline and dyes.

Polyaniline is a conductive polymer with distinctive optical and electrical properties. Its enzymatic synthesis is an environmentally friendly alternative to the use of harsh oxidants and extremely acidic conditions. 7D5L, a high-redox potential laccase developed in our lab, is the biocatalyst of choice for the synthesis of green polyaniline (emeraldine salt) due to its superior ability to oxidize aniline and kinetic stability at the required polymerization conditions (pH 3 and presence of anionic surfactants) as compared with other fungal laccases. Doses as low as 7.6 nM of 7D5L catalyze the polymerization of 15 mM aniline (in 24 h, room temperature, 7% yield) in the presence of different anionic surfactants used as doping templates to provide linear and water-soluble polymers. Aniline polymerization was monitored by the increase of the polaron absorption band at 800 nm (typical for emeraldine salt). Best polymerization results were obtained with 5 mM sodium dodecylbenzenesulfonate (SDBS) as template. At fixed conditions (15 mM aniline and 5mM SDBS), polymerization rates obtained with 7D5L were 2.5-fold the rates obtained with commercial Trametes villosa laccase. Moreover, polyaniline yield was notably boosted to 75% by rising 7D5L amount to 0.15 μM, obtaining 1g of green polyaniline in 1L-reaction volume. The green polymer obtained with the selected system (7D5L/SDBS) holds excellent electrochemical and electro-conductive properties displayed in water-dispersible nanofibers, which is advantageous for the nanomaterial to be readily cast into uniform films for different applications.

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.

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.

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae 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 S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae 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 S. cerevisiae 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.