Technological watch

ABSTRACTd?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 CltLSH520F/Q471K, which increased d?limonene production by 0.84?fold relative to the parental enzyme. To improve substrate channeling, the previously reported ERG20F96W/N127W/K197G variant was co?assembled with CltLSH520F/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.