Design of an improved universal signal peptide based on the \u03b1-factor mating secretion signal for enzyme production in yeast

Pablo Aza,Gonzalo Molpeceres,Susana Camarero,Felipe De Salas

doi:10.1007/s00018-021-03793-y

Pablo Aza, Gonzalo Molpeceres + Show 2 more

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https://doi.org/10.1007/s00018-021-03793-y

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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.

Highlights

Enzyme heterologous expression has been a target of interest in protein research for the last decades
MFα1 gene structure consists of a signal sequence, known as α-factor preproleader, fused to four copies of the 13-residue α-factor protein, each of them preceded by a spacer peptide of 6–8 amino acids [Lys-Arg-(Glu/Asp-Ala)2–3]
Mutations Aα9D, Fα48S, Sα58G, Gα62R were added during the directed evolution of Pycnoporus cinnabarinus laccase (PcL) for functional expression in S. cerevisiae [32], and Aα87T during the evolution of PM1 laccase (PM1L [33]); all accumulated in the leader sequence of 7D5 chimeric laccase after DNA shuffling of evolved PcL and PM1L [44]

Summary

Introduction

Enzyme heterologous expression has been a target of interest in protein research for the last decades. The yeast Saccharomyces cerevisiae has served as an expression system for eukaryotic proteins since it meets the essential protein post-translational demands, such as formation of disulfide bonds or glycosylation [1, 2] and, besides, its simple growth requirements, low average production time and well-known genome facilitate its manipulation [3, 4]. Pablo Aza and Gonzalo Molpeceres are contributed to this manuscript. MFα1 gene structure consists of a signal sequence, known as α-factor preproleader, fused to four copies of the 13-residue α-factor protein, each of them preceded by a spacer peptide of 6–8 amino acids [Lys-Arg-(Glu/Asp-Ala)2–3]. Pre α-Factor Preproleader Pro α-Factor copy I.

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