Triptonide (TN) is a diterpenoid natural product with significant antitumor [1], [2], immunosuppressive [3], [4], and anti-inflammatory activities [5], [6], derived from the traditional medicinal plant Tripterygium wilfordii. Its unique triepoxide structure is the key functional group responsible for its pharmacological activity [7], [8], [9]. However, traditional plant extraction methods are limited by low yields, high costs, and environmental pressures, making it difficult to meet clinical demands [10], [11]. Chemical synthesis can achieve structural modification, but it involves cumbersome steps and challenges in controlling regio-selectivity [12], [13]. In contrast, microbial heterologous synthesis strategies based on synthetic biology offer advantages such as green sustainability and scalability, making them an important direction for natural product production [14], [15]. However, the enzymatic mechanisms underlying the key oxidative steps in the biosynthesis pathway of TN have not been fully elucidated, limiting the development of efficient cell factories.

Cytochrome P450 play a central role in the post-modification of terpenoid skeletons. Recent studies have revealed that CYP82D213 is a key enzyme in the biosynthesis of TN, responsible for catalyzing the formation of tri-epoxides at the C7-C8, C9-C11, and C12-C13 positions of the precursor compound triptophenolide [16]. The regio-selectivity of epoxidation directly determines the activity and toxicity of the product [8], but how CYP82D213 precisely controls the insertion site of the oxygen atom remains unclear, particularly which amino acid residues determine its regional selectivity. These uncertainties have hindered progress in rationally designing optimizations of its catalytic efficiency and specificity.

Understanding and controlling the regioselectivity of P450 enzymes is fundamental to synthetic biology and enzyme engineering. Mechanistic insights are essential to reconstruct functional biosynthetic pathways in microbes, ultimately guiding the rational design of these enzymes for industrial applications. In this study, we functionally characterize CYP82D213 as the tri-epoxidase in TN biosynthesis and address its catalytic inefficiency through a multi-pronged engineering approach. We confirmed its activity in tobacco and yeast, then systematically engineer its active site, functional motifs, and surface residues. We identify key structural determinants for substrate binding and propose a dynamic catalytic mechanism for epoxidation. By combining beneficial mutations, we obtained the highly improved mutant CYP82D213H425Q/L459M/T365R, laying the foundation for the efficient microbial production of TN and related bioactive diterpenoids.