Work

Multiplexed Metabolic and Protein Engineering for D‐Limonene Overproduction in Saccharomyces cerevisiae

Year: 2025

Type: article

Abstract: ABSTRACT 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.

Source: Biotechnology and Bioengineering

DOI: 10.1002/bit.70133