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

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.

Design of an improved universal signal peptide based on the α-factor mating secretion signal for enzyme production in yeast

“…While both construction gave detectable laccase activity, α 9H2 leader provided significantly higher laccase activity levels than α nat leader, roughly twofold for PK2 and 12-fold for ApL ( Fig. S2), confirming previous results obtained with ApL [46]. Due the superiority of α 9H2 , it was used as upper reference leader in this study.…”

“…In a previous work, we proved the capability of the evolved α 9H2 leader [42] to improve the secretion by S. cerevisiae of diverse laccases compared to other evolved signal peptides [46]. The α 9H2 leader differs from the native α-factor preproleader, α nat leader from now on (Fig.…”

“…We present here the designing of an optimised version of the α-factor preproleader from S. cerevisiae to improve the production of fungal enzymes by the yeast. The α 9H2 leader developed in our lab [42] was selected as reference signal peptide because this mutated leader significantly improves the secretion by S. cerevisiae of several laccases compared with other evolved α leaders obtained in our lab [46], or with the α nat leader (Invitrogen) as shown here for PK2 and ApL. Thus, we studied the effect of the mutations accumulated in α 9H2 leader sequence through its evolution pathway as well as other mutations of the α-factor preproleader selected (and eventually lost) during successive laccase directed evolution campaigns [32,33,42,44] Two engineering pathways of the signal peptide were carried out: a bottom-up designing strategy on α nat leader and a top-down one on α 9H2 leader using PK2 and ApL laccases as model enzymes.…”

“…The secretory potential of α OPT leader was evaluated for the production by S. cerevisiae of other fungal oxidoreductases like two more laccases from Pleurotus eryngii (PeL) [46] and Pycnoporus cinnabarinus (PcL) [32], an aryl-alcohol oxidase from P. eryngii (AAO) [47] and a versatile peroxidase (VP) from P. eyringii [48]. Besides, we assayed it with fungal hydrolases such as two β-glycosidases (BGL2 and BGL3) from Talaromyces amestolkiae [49,50] and a sterol esterase (OPE) from Ophiostoma piceae [51].…”

“…I. Agrocybe pediades laccase [46], Pleurotus eryngii laccase (with two mutations to facilitate its functional expression) [46]. PK2 laccase [42], Pycnoporus cinnabarinus laccase [32], P. eryngii versatil peroxidase [48], P. eryngii aryl-alcohol oxidase [47], Talaromyces amestolkiae β-glucosidases [49,50] and Ophiostoma piceae sterol esterase [51] were obtained from our collection of enzymes at CIB.…”