Ro 48 8071

See Supplemental Text for details of cholesterol extraction. Ketoconazole and clotrimazole were obtained from the National Cancer Institute Developmental Therapeutics Program and Ro 48-8071 was from MedChem Express. Lovastatin and Simvastatin were obtained from Cayman Chemical.

Engineered yeasts producing squalene or mogrol were incubated overnight in 3 mL YPD medium at 30 °C with shaking at 250 rpm as seed cultures. One milliliter seed culture was used to inoculate 50 mL of YPD medium with or without 30 μg mL⁻¹ terbinafine (Aladdin, China) for producing squalene, or 50 μg mL⁻¹ Ro 48-8071 (MedChemExpress, USA) for producing mogrol^{44 (link)}. After 6 days of cultivation, 10 mL culture of the squalene producer HCS1 strain was collected by centrifugation, and extracted with 1 mL n-hexane after shattering the cells by zirconia beads (5 mm) in an MP Fastprep-24 5 G cell disrupter (MP Biomedicals, USA). The sample was separated by centrifugation, and the upper n-hexane phase was collected for gas chromatography-mass spectrometry (GC-MS) analysis. The mogrol-producer strains HCM1 to HCM3 were cultivated for 2 days, 2 mL culture was collected by centrifugation, and extracted with 500 μL acetone. The crude extract was evaporated and resuspended in 0.2 mL methanol for analysis using liquid chromatography-high resolution mass spectrometry (LC-HRMS). The tetracistron-containing strain K4 was cultured in SC medium at 30 °C with shaking at 250 rpm for 24 h. Total 2 mL culture was collected by centrifugation, and extracted with 300 μL acetone and 700 μL methanol. After filtration, the samples were analyzed for the production of β-carotene and phytoene by LC-HRMS/MS.