Engineering a Tyrosol-Responsive Dynamic System in Saccharomyces cerevisiae for Adaptive Regulation of Hydroxytyrosol Biosynthesis

Static metabolic regulation during microbial synthesis of polyphenolic compounds leads to tyrosol accumulation, inducing cellular toxicity and growth inhibition. Dynamic regulation effectively alleviates the imbalance between cell growth and production; yet, a tyrosol-responsive dynamic system for yeast adaptive regulation remains unreported. In this study, endogenous tyrosol-responsive promoters were mined via transcriptomics, and the optimal promoter P YOR153W was identified. Subsequently, a novel tyrosol-responsive biosensor composed of the responsive promoter and CRISPR/dSpCas9-VPR was designed to dynamically regulate green fluorescent protein (GFP) expression and hydroxytyrosol biosynthesis, respectively. This strategy successfully enhanced hydroxytyrosol production by 1.53-fold with negligible tyrosol accumulation. Furthermore, this dynamic system improved strain robustness during longer yeast fermentation periods, and the hydroxytyrosol titer increased to 4.26 g/L in a 5 L bioreactor. Our findings establish the first tyrosol-responsive biosensor in yeast and provide a framework for the efficient biosynthesis of high-value tyrosol derivatives.