Structural determination of unknown product in β-amyrin synthase/CYP716E26 co-expressing yeast

Yeast strain SY56 was cultured in 6 L SD medium containing 2% galactose. Yeast cells were collected by centrifugation, incubated with 720 mL methanol and 180 mL 40% KOH at 80°C, and extracted with 900 mL n-hexane three times. The extracts were evaporated, and the remaining residues were applied to column chromatography on silica gel (60 N 40–50 μm) in a hexane:ethyl acetate gradient. After purification, 10 mg of an unknown compound was obtained. Nuclear magnetic resonance (NMR) spectra (¹H and ¹³C) of purified compound were reported on a JEOL-ECS400 system (400 MHz; JEOL, Tokyo, Japan) in CDCl₃. Tetramethylsilane was used as an internal standard.