Compartmentalized orthogonal biosynthesis for boosting limonene and oxygenated monoterpenoid production in...

Limonene and its oxygenated derivatives are valuable cyclic monoterpenoids with applications in cosmetics, pharmaceuticals, agriculture, and biofuels. These compounds are typically derived from the precursor geranyl pyrophosphate (GPP). Engineering Saccharomyces cerevisiae provides a sustainable platform for their production; however, productivity is often constrained by the bifunctional synthase ERG20, which diverts metabolic flux from GPP toward farnesyl pyrophosphate (FPP). To overcome this limitation, we developed a hybrid dual cytoplasmic-peroxisomal compartmentation strategy to reconstruct orthogonal monoterpenoid biosynthesis in S. cerevisiae using the non-competitive precursor neryl pyrophosphate (NPP). Individually engineered strains target the cytoplasm and peroxisome produced 1.5 g/L and 1.3 g/L of (+)-limonene, respectively. While the hybrid strain achieved 2.1 g/L in shake flasks and 15.2 g/L in a 5 L bioreactor, marking the highest de novo microbial limonene biosynthesis reported to date. Further introduction of a tailored P450tol enabled the highest bioproduction of (+)-perillyl alcohol (4.6 g/L) and (+)-perillic acid (0.9 g/L) in the optimized fed-batch fermentation. Additionally, enhanced limonene production also stimulated the synthesis of (+)-cis -carveol (104 mg/L) and (+)-trans -isopiperitenol (205 mg/L) via episomally expressed P450s . These endeavors highlight the efficiency of hybrid cytoplasm-peroxisome engineering of the orthogonal biosynthetic pathway for producing limonene and its derivatives, thereby opening new avenues for the biosynthesis of other valuable GPP-derived chemicals in yeast.