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Cell Mol Life Sci

Title:		Design of an improved universal signal peptide based on the alpha-factor mating secretion signal for enzyme production in yeast
Author(s):		Aza P; Molpeceres G; de Salas F; Camarero S;
Address:		"Centro de Investigaciones Biologicas Margarita Salas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain. Centro de Investigaciones Biologicas Margarita Salas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain. susanacam@cib.csic.es"
Journal Title:		Cell Mol Life Sci
Year:		2021
Volume:		20210309
Issue:		7
Page Number:		3691 - 3707
DOI:		10.1007/s00018-021-03793-y
ISSN/ISBN:		1420-9071 (Electronic) 1420-682X (Print) 1420-682X (Linking)
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 alpha-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 alpha-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 alpha-factor preproleader to improve recombinant enzyme production in S. cerevisiae through two parallel engineering strategies: a bottom-up design over the native alpha-factor preproleader (alpha(nat)) and a top-down design over the fittest evolved signal peptide obtained in our lab (alpha(9H2) 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 (Aalpha9D, Aalpha20T, Lalpha42S, Dalpha83E) contained in the final optimised leader (alpha(OPT)), 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 alpha(OPT) leader fused to the target protein"
Keywords:		Hydrolases/genetics/*metabolism Mating Factor/genetics/*metabolism Oxidoreductases/genetics/*metabolism Protein Precursors/genetics/*metabolism Protein Sorting Signals/*genetics Recombinant Proteins/genetics/metabolism Saccharomyces cerevisiae/genetics/gr;
Notes:		"MedlineAza, Pablo Molpeceres, Gonzalo de Salas, Felipe Camarero, Susana eng 792070/BBI-JU/ BIO2017-86559-R/Ministerio de Ciencia e Innovacion/ Switzerland 2021/03/10 Cell Mol Life Sci. 2021 Apr; 78(7):3691-3707. doi: 10.1007/s00018-021-03793-y. Epub 2021 Mar 9"