Lysine analogue-driven adaptive evolution produces lysine-overproducing wine yeast strains

Lysine, though a poor nitrogen source for Saccharomyces cerevisiae, is rapidly consumed during wine fermentation and has been linked to enhanced oxidative and sulfur dioxide resistance. To investigate and harness this phenotype, the industrial wine strain P14 was subjected to adaptive laboratory evolution under increasing concentrations of the toxic lysine analogue S-aminoethyl cysteine (AEC). The resulting evolved isolates accumulated up to tenfold more intracellular lysine, displayed elevated reduced glutathione, and exhibited substantially decreased ROS levels while maintaining robust fermentation performance. Whole-genome sequencing revealed extensive genomic rearrangements and recurrent mutations associated with lysine biosynthesis. Among these, the LYS21^V127F mutation emerged as the major driver of lysine overproduction and AEC resistance. CRISPR/Cas9 reconstruction of this allele in multiple wine yeasts confirmed its causal role and conferred a competitive advantage during fermentation. These findings demonstrate the effectiveness of AEC-driven evolution for generating lysine-overproducing wine yeasts and highlight LYS21 feedback-resistance as a key metabolic innovation.