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

Abstract

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.

Tailor-made alkaliphilic and thermostable fungal laccases for industrial wood processing

“…Finally, in order to improve the production of the last evolved extremophilic variant Mol3, that was diminished compared to C-LeB (Table 4 ), the CDS of Mol3 was fused to an optimized signal peptide based on the α-factor pre-proleader, which has been recently designed to enhance the secretion of fungal enzymes by the yeast [ 29 ]. Production of Mol3 laccase with the α OPT leader (Additional File 1 : Fig.…”

“…Production of Mol3 laccase with the α OPT leader (Additional File 1 : Fig. S7) doubled the levels obtained with the evolved α 9H2 leader [ 27 ] that was used so far for the rest of the variants developed in this work due to its preceding demonstrated capability to increase laccase secretion by the yeast [ 29 ]. In addition, Komagataella pastoris has been used as host for scaling up the production of some of the extremophilic variants evaluated in the application tests (see below).…”

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications

“…Likewise, SS from other genes have been evaluated, for example, the SS of Rhizopus oryzae glucoamylase or the SS of Aspergillus niger α-amylase ( Moon et al, 2013 ; Inokuma et al, 2014 ). Recent studies on directed evolution of signal peptides have shown that changes in the hydrophobic core of the SS significantly impacts protein secretion ( Mateljak et al, 2017 ; Barrero et al, 2018 ; Aza et al, 2021 ). It is worth mentioning that SS modifications can affect the protein secretion pathway, as discussed later.…”

Saccharomyces cerevisiae cell surface display technology: Strategies for improvement and applications

“…The extracellular activity of BGL1 was 1.3-fold and 1.9-fold higher than that of the Rhizopus oryzae glucoamylase (GLUASP) and S. cerevisiae α-mating pheromone (MFα1SP), respectively ( Inokuma et al, 2016 ). The design mutations of the a-factor preproleader of the MFα1SP have the opportunity to obtain enhanced signal peptides for the secretion of a variety of fungal enzymes ( Aza et al, 2021 ). In addition, some researchers proposed that the fusion of the target protein with a secretion-enhancing peptide cassette and rational optimization of the original peptide could enhance the secretion of the heterologous proteins ( Cho et al, 2022 ).…”