Azadirachtin, a limonoid isolated from the neem tree, has attracted considerable interest due to its excellent performance in pest control. Studies have also reported pharmaceutical activities of dihydroniloticin, an intermediate in azadirachtin biosynthesis, but these pharmaceutical activities could not be validated due to the limited supply. In this study, AiCYP71CD2 was first identified as involved in azadirachtin biosynthesis in neem by expressing it in Nicotiana benthamiana and yeast (Saccharomyces cerevisiae). Homology modeling and molecular docking analysis revealed that AiCYP71CD2 may exhibit a higher ability in catalyzing tirucalla-7,24-dien-3β-ol into dihydroniloticin compared with MaCYP71CD2 from Melia azedarach L. G310 was identified as the critical residue responsible for the higher catalytic ability of AiCYP71CD2. Condon-Optimized AiCYP71CD2 greatly improved the catalytic efficiency in yeast. De novo dihydroniloticin production using the novel AiCYP71CD2 was achieved by constructing the S. cerevisiae DI-3 strain, and the titer could reach up to 405 mg/L in a fermentor, which was an alternative source for dihydroniloticin.

The production of glycyrrhetinic acid (GA) and 11-oxo-β-amyrin, the major bioactive components in liquorice, was typically inhibited by P450 oxidation in Saccharomyces cerevisiae. This study focused on optimizing CYP88D6 oxidation by balancing its expression with cytochrome P450 oxidoreductase (CPR) for the efficient production of 11-oxo-β-amyrin in yeast. Results indicated that a high CPR:CYP88D6 expression ratio could decrease both 11-oxo-β-amyrin concentration and turnover ratio of β-amyrin to 11-oxo-β-amyrin, whereas a high CYP88D6:CPR expression ratio is beneficial for improving the catalytic activity of CYP88D6 and 11-oxo-β-amyrin production. Under such a scenario, 91.2% of β-amyrin was converted into 11-oxo-β-amyrin in the resulting S. cerevisiae Y321, and 11-oxo-β-amyrin production was further improved to 810.6 mg/L in fed-batch fermentation. Our study provides new insights into the expression of cytochrome P450 and CPR in maximizing the catalytic activity of P450s, which could guide the construction of cell factories in producing natural products.

Cannabidiol (CBD), the main nonpsychoactive cannabinoid in Cannabis sativa, has diverse applications in the pharmacological, food, and cosmetic industries. The long plantation period and the complex chemical structure of cannabidiol pose a great challenge on CBD supply. Here, we achieved de novo biosynthesis of cannabidiol in Saccharomyces cerevisiae. The CBD production was further enhanced by 2.53-fold through pushing the supply of precursors and fusion protein construction. Bile pigment transporter 1 (BPT1) was the most effective transporter for transferring cannabigerolic acid (CBGA) from the cytoplasm to the vacuole, which removed the physical barrier separating CBGA and its catalytic enzyme. The lowest binding energy of the CBGA–BPT1 complex confirmed a strong interaction between BPT1 and CBGA. A CBD yield of 6.92 mg/L was achieved, which was 100-fold higher than the yield generated by the starting strain. This study provides insights into high-level CBD-producing strain construction and lays the foundation for CBD supply.