Silica gel p60

Equal volume of ethyl acetate (ACS reagent grade; J.T. Baker) and approximate 10 g of 0.5 mm glass beads (BioSpec) were added to 400 mL of induced TS-expressing yeast culture in a 2 L flask. The flask was shaken at 20 °C overnight. The culture was centrifuged at 2700 × g for 10 min. The pellet was discarded, and the supernatant (organic and aqueous layers) was extracted with 400 mL of ethyl acetate twice in a 2 L separatory funnel. Additional centrifugation of the supernatant might be helpful for the separation of the two phases. Organic phases were combined and dried using rotary evaporation. Flash chromatography was carried out using a 7 cm diameter column loaded with 20 g of P60 silica gel (SiliCycle) with hexane (HPLC grade; VWR) as the mobile phase. Isocratic elution (100% hexane) was performed and eluent was collected in 5 mL fractions. Fractions were analyzed by GCMS and those containing taxadiene were combined and dried by rotary evaporation to yield 15.5 mg of taxadiene (1) as colorless oil (39 mg/L). Iso-taxadiene was co-purified with 1 in a 1:10 ratio as indicated by NMR peak integration. The ¹H and ¹³C NMR data of taxadiene (1) are reported in Supplementary Figs. 11–13.

The synthesis of taxadien-5α-ol (2) is adapted from a previously described method^{30 (link)}. Tert-BuOOH (21.3 μL of 98% purity, 0.114 mmol; Sigma–Aldrich) was added to a solution of SeO₂ (3.3 mg, 0.030 mmol; Strem) in CH₂Cl₂ (50 μL; Sigma–Aldrich) and stirred for 0.5 h. Subsequently, purified taxadiene (1) (15.5 mg, 0.0570 mmol) in CH₂Cl₂ (200 μL) was added to the solution and allowed to stir for 2.5 h at room temperature. The resulting yellow solution was concentrated on a rotary evaporator under reduced pressure to yield yellow crude oil. Flash chromatography was carried out using a 2 mL glass Pasteur pipette loaded with 200 mg of P60 silica gel (SiliCycle) with hexane (HPLC grade; VWR) as the initial mobile phase. The column was first eluded with 100% hexane to yield unreacted 1 (21% recovery), then with hexane-diethyl ether (Fischer Chemical) in a ratio of 5:1 to yield 2.3 mg of an over-oxidized ketone product (TLC R_f = 0.63 when eluded with hexane-Et₂O 5:1 and stained with KMnO₄; 14% isolated yield) and 1.8 mg of 2 (TLC R_f = 0.36 when eluded with hexane-Et₂O 5:1 and stained with KMnO₄; 11% isolated yield). Fractions were collected in 200 μL volume. The ¹H NMR data of synthetic taxadien-5α-ol (2) are reported and compared to 2 heterologously produced in E. coli to confirm its identity (Supplementary Figs. 14–15).

NR chloride (beta form) was a gift from ChromaDex Company. Sodium dithionate was purchased from VWR, sodium hydrogen carbonate was purchased from Aldrich, silica gel (P60, 40–63 μm, 60 Å) was purchased from SiliCycle, and basic alumina (50–200 μm, 60 Å, pH 8) was purchased from Acros. Methanol (99.9%, Certified ACS, Fisher), acetone (99.8% Certified ACS, Fisher), sodium hydroxide (certified ACS, Fisher Chemical), hexanes (≥98.5%, GR ACS), ethyl acetate (EtOAc, >99.9%, certified ACS) and silica gel 60 F254 Coated Aluminum-Backed TLC Sheets were purchased from EMD Millipore (Billerica, MA, USA). Deuterated water and dimethyl sulfoxide (DMSO-d6, D, 99.9%) were purchased from Cambridge Isotope Laboratories, Inc.