Combinatorial Alkali-Responsive Hybrid Promoters as Tools for Heterologous Protein Expression in Saccharomyces cerevisiae
Zekhnini, Abdelghani (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Casamayor Gracia, Antonio (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Ariño Carmona, Joaquín (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Data:	2025
Descripció:	14 pàg.
Resum:	The demand for strong and easily inducible promoters to produce heterologous proteins in Saccharomyces cerevisiae has attracted considerable attention in the last years. In this organism, alkalinisation triggers a wide and well-characterised transcriptional response that includes activation of the calcium-dependent calcineurin-Crz1 and the phosphate-responsive PHO pathways. Here, we present the construction of random libraries containing multiple combinations of Crz1- and Pho4-binding sequences, and we show that these elements are able to promote efficient expression of GFP by simple addition of KOH to the medium. The expression in Crz1 or Pho4-deficient cells allowed us to define the relative contribution of these elements to GFP production. We also show that the addition of a single copy of a 60-bp fragment of the ENA1 promoter containing an Stp1/2 site further enhances expression. Finally, we demonstrate that these constructs drive strong expression of secretable laccase, an enzyme of industrial interest in processing lignin biopolymers, and that the level of expression can be adjusted by modifying the pH of the medium. In conclusion, our work presents a novel expression system whose induction is simple, cheap, and easy to monitor, and that could be an attractive alternative to current expression platforms for both research and industrial protein production purposes.
Ajuts:	Agència de Gestió d'Ajuts Universitaris i de Recerca 2023/PROD-00006 Agencia Estatal de Investigación PID2020-113319RB-I00 Agencia Estatal de Investigación PID2023-150535OB-I00
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, i la comunicació pública de l'obra, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original. No es permet la creació d'obres derivades.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Saccharomyces cerevisiae ; Alkaline pH response ; Hybrid promoters ; Laccase ; Protein expression ; Gene Expression Regulation, Fungal ; Promoter Regions, Genetic ; Gene Expression ; Alkalies/metabolism ; Recombinant Proteins/genetics ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics ; Green Fluorescent Proteins/genetics ; Culture Media/chemistry
Publicat a:	Microbial biotechnology, Vol. 18 Núm. 9 (2025) , p. e70213, ISSN 1751-7915

DOI: 10.1111/1751-7915.70213
PMID: 40960086

14 p, 4.1 MB

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