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

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.PMID:33687500 | DOI:10.1007/s00018-021-03793-y
Source: Cellular and Molecular Life Sciences : CMLS - Category: Cytology Authors: Source Type: research