Combinatorial metabolic engineering of Yarrowia lipolytica for high-level production of the plant-derived diterpenoid sclareol.

Background: Sclareol, a diterpene alcohol derived from Salvia sclarea, is primarily used in the synthesis of ambrox, an alternative to the expensive spice ambergris. However, commercial production of sclareol from plant extraction is costly and environmentally problematic, limiting its scalability. Recent advances in synthetic biology have enabled the construction of efficient cell factories for sclareol synthesis, offering a more sustainable solution.

Results: In this study, we engineered Yarrowia lipolytica to produce sclareol by integrating genes encoding (13E)-8α-hydroxylabden-15-yl diphosphate synthase (LPPS) and sclareol synthase (SCS). Sclareol titers were further enhanced through the fusion of SsSCS and SsLPPS proteins, as well as multi-copy gene integration. To increase the precursor geranylgeranyl diphosphate (GGPP), we overexpressed various geranylgeranyl diphosphate synthases (GGS1), resulting in significant accumulation of GGPP. Additionally, optimization of the mevalonate pathway, coupled with the downregulation of lipid synthesis and upregulation of lipid degradation, directed more acetyl CoA towards sclareol production.

Conclusions: In this study, we reprogrammed the metabolism of Y. lipolytica by combinatorial metabolic engineering with a sclareol titer of 2656.20 ± 91.30 mg/L in shake flasks. Our findings provide a viable strategy for utilizing Y. lipolytica as a microbial cell factory to produce sclareol.