Selective rewiring of Met4 ubiquitin regulation enhances S-adenosylmethionine accumulation in sake yeast

S-Adenosylmethionine (SAM) is a central sulfur-containing metabolite that serves as a universal methyl-group donor and a high-value molecule for food and pharmaceutical applications. In Saccharomyces cerevisiae, sulfate assimilation into methionine and SAM is coordinately governed by the transcription factor Met4, whose activity is controlled by Met30-dependent ubiquitination. This feedback represses sulfur-metabolic gene expression under methionine-replete conditions and limits metabolic input into the methionine-SAM branch. While metabolic engineering has often targeted individual enzymatic steps to enhance SAM accumulation, direct tuning of this transcriptional hub remains underexplored. Here, we engineered a sake yeast strain expressing a Met4 K163RΔInh variant combining a Lys163-to-Arg substitution with deletion of the Met30-interacting inhibitory region. The engineered Met4 remained predominantly nuclear during methionine supplementation and maintained elevated expression of sulfur-assimilation genes under methionine-replete conditions. Metabolite profiling under methionine supplementation revealed increased abundance of methionine/SAM-cycle-related metabolites relative to the control strain, consistent with enhanced output into the methionine/SAM-linked branch. Even in nutrient-rich medium, the Met4 K163RΔInh strain expanded the intracellular SAM pool compared with the parental strain. Together, selective rewiring of Met4 ubiquitin control provides a transcription-factor-centric route to construct SAM-accumulating sake yeast strains and complements enzyme-level pathway engineering for sulfur-derived metabolite production.