2.6. Functional characterization of Aa7DR 1 in S. cerevisiae Yeast lacks the 7DR gene, and Δ 5,7 -eposterol can be used as a substrate for the heterologous 7DR enzyme (Fig. 1). The two Aa7DRs were heterologously expressed in S. cerevisiae for functional characterization. Aa7DR1 and Aa7DR2 were cloned into the expression vectors pESC-HIS-Aa7DR1 and pESC-HIS-Aa7DR2, then were transformed into S. cerevisiae strain Cen.pk2-1D. Compared with the empty-vector-transferred control, the yeast strain expressing Aa7DR1 yielded two prominent products (peaks 1 and 2) at comparable levels (Fig. 6A). While there were no significant differences between pESC-His-Aa7DR2 and the control. To determine the structural features of Aa7DR1 products a large-scale fermentation (7 L) was used to accumulate enough products. The products were isolated and purified using open silica gel column and semi-preparative reversed-phase high performance liquid chromatography. The structures of p1 were confirmed as ergosta-5,22-dien-3β- ol by 1 H and 13 C-NMR data (Supplemental Figures S6 and S 7; Supplemental Table S6). Product p2 was identified as campesterol by 1 H-NMR and comparison with the standard. We determined Aa7DR1 possessed 7- dehydrocholesterol reductive function to reduce Δ 5,7 -eposterol and ergosterol at Δ 7 position. The indirect product campesterol (p2) was further catalyzed by endogenous gene erg4 on ostreasterol (Fig. 5).

Published as part of Wang, Yunpeng, Xiong, Wenbo, Chen, Yidu, Zhu, Min, Liang, Jincai, Li, Yu, Huang, Jia, Huang, Liufang, Liu, Zhongqiu, Ji, Aijia & Duan, Lixin, 2021, Transcriptomic investigation of the biochemical function of 7 - dehydro- cholesterol reductase 1 from the traditional Chinese medicinal plant Anemarrhena asphodeloides Bunge, pp. 1-9 in Phytochemistry (112954) (112954) 192 on page 4, DOI: 10.1016/j.phytochem.2021.112954, http://zenodo.org/record/8257226