Sclareol, a diterpene alcohol, is widely utilized in the perfume industry, particularly as a substitute for natural ambergris. Microbial synthesis of sclareol serves as an alternative way to produce this valuable and marketable compound; however, it was still challenged by ineffective expression of heterogeneous enzymes, insufficient activity of key enzymes, byproduct accumulation, scale-up, especially downstream separation, etc. In this study, sclareol biosynthesis was established in Yarrowia lipolytica by coexpressing the truncated exogenous sclareol synthase genes tSsTPS and tSsLPPS. Through semirational mutagenesis of the exogenous enzyme tSsTPS, the mutant tSsTPS (V325I) was screened, exhibiting a 70% increase in catalytic activity. Multicopy optimization of the genes tSsTPS and tSsLPPS enabled the strain to produce 348. 6 mg/L sclareol. Regulating the supply and consumption of GGPP further improved the titer of sclareol to 1565. 4 mg/L. Furthermore, the efflux capacity of various transporters for sclareol was evaluated. Among them, the ABC transporter YALI1C28310p increased extracellular sclareol production by 8. 6-fold compared to the control without the solvent extractant. Additionally, citrate overflow metabolism in Y. lipolytica was eliminated by knocking out CEX1, achieving a final sclareol titer of 3307. 3 mg/L in shake flasks and 13. 9 g/L in a 5 L bioreactor without solvent overlay by fed-batch fermentation, both representing the highest reported sclareol titer to date. This achievement establishes a robust de novo biosynthesis platform for sclareol in Y. lipolytica, which can be broadly adapted for synthesizing other natural terpenoids.
Huang et al. (2026) studied this question.