Scarless Genetic Engineering of <i>Saccharomyces cerevisiae</i> for Enhanced Guanosine Monophosphate Production as a Natural Flavor Enhancer

<i>Saccharomyces cerevisiae</i> and <i>Cyberlindnera jadinii</i> are widely utilized in the natural food seasoning industry as sources of flavor enhancing nucleotides such as inosine monophosphate (IMP) and guanosine monophosphate (GMP), which contribute to umami taste and support sodium reduction in food. However, wild type yeast strains produce GMP at levels that are inadequate for industrial scale applications, necessitating metabolic engineering strategies to increase production efficiency. This study employed a CRISPR-Cas9-based scarless genome engineering approach to enhance GMP biosynthesis in <i>S. cerevisiae</i> via promoter replacement. The key genes <i>IMD3</i> and <i>GUA1</i>, responsible for converting IMP to GMP, were overexpressed to redirect purine flux toward GMP production. To address precursor limitations, <i>ZWF1</i> and <i>RKI1</i>, involved in the pentose phosphate pathway, were also overexpressed. In parallel, the expression of <i>STB5</i> and <i>RAP1</i> was increased to enhance NADPH regeneration and relieve transcriptional bottlenecks. As a result, the final engineered strain SCJ-7 demonstrated a 1.77-fold increase in GMP titer and a 1.40-fold increase in GMP content during flask fermentation compared to the wild-type. In fed-batch fermentation, GMP titer was further improved by 27.6%. These findings demonstrate that combining metabolic flux enhancement with transcriptional regulation provides an effective and scalable strategy for boosting GMP production in <i>S. cerevisiae</i>, offering strong potential for industrial application in the food industry.