Combinatorial Alkali-Responsive Hybrid Promoters as Tools for Heterologous Protein Expression in Saccharomyces cerevisiae.

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.