Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae.

Pablo Aza,Felipe De Salas,David Rodríguez-Escribano,Iñigo De La Fuente,Susana Camarero,Gonzalo Molpeceres

doi:10.3390/ijms22031157

Abstract

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.

Highlights

Laccases are multicopper oxidases that catalyse the oxidation of lignin phenols, aromatic amines and other various organic and inorganic compounds by reducing oxygen to water
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
Directed evolution in S. cerevisiae of fungal laccases fused to the α-factor preproleader resulted in mutated laccase coding sequence (CDS) and mutated α leader sequences that notably promoted enzyme secretion by the yeast [12,28,32,33]

Summary

Introduction

Laccases are multicopper oxidases that catalyse the oxidation of lignin phenols, aromatic amines and other various organic and inorganic compounds by reducing oxygen to water. Laccases are usually composed of three structural cupredoxin-like domains (D1–D3) each folded into a Greek key β-barrel topology [1]. Four copper ions (namely T1–T3) are located in two catalytic sites. The mononuclear T1 (blue copper) is located in D3 and serves as the primary electron acceptor site for substrate oxidation; the mononuclear T2 and binuclear T3 copper ions form the trinuclear cluster at the interface between D1 and D3 and function as electron acceptors from T1 prior to the reduction of oxygen [2,3,4]. Most basidiomycete laccases are monomeric glycoproteins carrying short mannoseenriched glycans linked mostly through N-glycosylation, which accounts for 5–25% of the Mw of the secreted enzyme. The number of putative N-glycosylation sites can vary among laccase sequences (according to the presence of the consensus Asn-X-Thr/Ser amino acid sequence), on average fungal laccases are N-glycosylated in 2–5 sites [5,6,7]

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