Ingeniería metabólica y de proteínas multiplexada para la sobreproducción de D-limoneno en Saccharomyces cerevisiae

d-limonene, a monocyclic monoterpene compound, is widely used in biopesticides, food additives, and pharmaceuticals. In this study, we integrated metabolic and enzyme engineering approaches to develop a high-producing Saccharomyces cerevisiae platform for d-limonene biosynthesis. Initial analysis showed that the previously used d-limonene synthase (LS) from Mentha spicata exhibited insufficient catalytic efficiency for effective conversion of geranyl pyrophosphate into d-limonene. To overcome this limitation, we reconstructed the biosynthetic pathway by screening a more efficient LS from Citrus limon (ClLS), thereby producing 2.80 mg/L of d-limonene. Subsequently, metabolic engineering strategies, including precursor flux optimization, downregulation of the squalene pathway, and modification of off-pathway genes, redirected carbon flux toward monoterpene biosynthesis, achieving a d-limonene titer of 888.27 mg/L. Further enhancement was obtained through semi-rational protein engineering of CltLS, generating the mutant CltLS^H520F/Q471K, which increased d-limonene production by 0.84-fold relative to the parental enzyme. To improve substrate channeling, the previously reported ERG20^{F96W/N127W/K197G} variant was co-assembled with CltLS^H520F/Q471K using a protein scaffold, resulting in 2080.47 mg/L of d-limonene in shake-flask fermentation. Finally, scale-up fed-batch fermentation in a 5 L bioreactor produced 5.96 g/L of d-limonene. Collectively, these results establish a robust and scalable microbial platform for industrial d-limonene production and provide a versatile framework for the biosynthesis of other valuable monoterpenes.