Biosynthesis of Limonene Using Acetate as the Carbon Source in Metabolically Engineered Escherichia coli

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 EfMVASA110G 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.