Biosynthesis of Limonene Using Acetate as the Carbon Source in Metabolically Engineered <i>Escherichia coli</i>

Limonene is a high-value monoterpenoid used in food, pharmaceutical, and fragrance applications. To overcome limitations of conventional production, this study developed a biosynthesis platform using acetate as the sole carbon source. Acetate is an ideal nonfood substrate that can be sustainably derived from industrial waste streams, syngas, and CO₂ reduction. Initially, the heterologous synthetic pathway was constructed to yield 0.13 mg/L limonene from acetate. By introducing the mutated <i>EfMVAS</i>^A110G to enhance catalytic efficiency, along with promoter engineering and substitution of the synthesis substrate, the production titer was enhanced to 49.24 mg/L. Subsequent host optimization through NADPH regeneration and pathway knockout, combined with fermentation optimization, increased the titer to 167.45 mg/L. Ultimately, whole-cell biocatalysis achieved 450.72 mg/L, establishing the highest reported acetate-based limonene titer. Overall, this study establishes a framework for biosynthesis of high-value terpenoids from nonfood feedstocks and demonstrates acetate as a promising carbon source for advanced bioproduction systems.