TY - JOUR
T1 - Combinatorial Alkali‐Responsive Hybrid Promoters as Tools for Heterologous Protein Expression in Saccharomyces cerevisiae
AU - Zekhnini Abdarahoul, Abdelghani
AU - Casamayor, Antonio
AU - Ariño, Joaquín
PY - 2025/9/17
Y1 - 2025/9/17
N2 - 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.
AB - 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.
KW - Saccharomyces cerevisiae
KW - alkaline pH response
KW - hybrid promoters
KW - laccase
KW - protein expression
KW - Gene Expression Regulation, Fungal
KW - Promoter Regions, Genetic
KW - Gene Expression
KW - Alkalies/metabolism
KW - Recombinant Proteins/genetics
KW - Saccharomyces cerevisiae/genetics
KW - Saccharomyces cerevisiae Proteins/genetics
KW - Green Fluorescent Proteins/genetics
KW - Culture Media/chemistry
UR - https://doi.org/10.1111/1751-7915.70213
UR - https://www.scopus.com/pages/publications/105016384562
UR - https://www.mendeley.com/catalogue/a9f9af91-8a56-3c73-b587-d438cd89e4cb/
U2 - 10.1111/1751-7915.70213
DO - 10.1111/1751-7915.70213
M3 - Article
C2 - 40960086
SN - 1751-7907
VL - 18
JO - Microbial Biotechnology
JF - Microbial Biotechnology
IS - 9
M1 - e70213
ER -