Chemicals and culture conditions

The phytosterols substrate used is a sterol mixture contained (by weight percentage) 51.7% β-sitosterol, 27.2% stigmasterol, 17.1% campesterol, and 4.0% brassicasterol (COFCO Tech Bioengineering Co., Ltd., Tianjin). Standards AD and ADD were purchased from Sigma-Aldrich Co. (USA). All chemical solvents and salts were of analytical grade or higher. The cultivation and bioconversion of microorganisms and the preparation and analysis of transformation products, were performed following the procedures described by Shen et al. [23]. The minimal medium contained (g/L): glucose 10, MgSO₄ 0.5, K₂HPO₄ 0.5, (NH₄)₂HPO₄ 3.5, citric acid 2, and ammonium iron citrate 0.05 with pH of 7.2. Experiments were conducted under different culture conditions with phytosterols (5 g/L) or soybean oil (16%) and Tween 80 (0.5%) in minimal medium. Different concentrations of NA were added to the medium at the start of fermentation. In the phytosterols-free medium, the growth of cell was measured through optical density. However, the cell growth in the phytosterols-contained culture broth is difficult to measure by using this method. In this study, the cell growth measurement in the medium with phytosterols was conducted following the method by Meyers et al. [24]. The amount of protein was related to the dry cell weight (DCW) obtained using an adequate calibration curve: DCW (mg/mL) = 2.10 ×  protein (mg/mL) - 0.17(R² = 0.99). The growth of cell was represented by DCW. All experiments were performed in triplicate and the data were statistically analyzed by one-way ANOVA.