Enhanced Biosynthesis of Sclareol in <i>Yarrowia lipolytica</i> via Systematic Metabolic Engineering

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 <i>Yarrowia lipolytica</i> by coexpressing the truncated exogenous sclareol synthase genes <i>tSsTPS</i> and <i>tSsLPPS</i>. 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 <i>tSsTPS</i> and <i>tSsLPPS</i> 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 YALI1_C28310p increased extracellular sclareol production by 8.6-fold compared to the control without the solvent extractant. Additionally, citrate overflow metabolism in <i>Y. lipolytica</i> was eliminated by knocking out <i>CEX1</i>, 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 <i>Y. lipolytica</i>, which can be broadly adapted for synthesizing other natural terpenoids.