Harnessing CYP71 Enzymes for Terpenoid Indole Alkaloid Production: A Synthetic Biology Perspective from Discovery to Application

Abstract Terpenoid indole alkaloids (TIAs) are a class of structurally diverse natural products with important pharmacological activities. Recently, substantial progress has been made in elucidating TIA biosynthetic pathways, and these studies have revealed the central roles of CYP71 clan enzymes (CYP71s) in both the formation and the modification of the TIA scaffold. In this review, we trace the stepwise elucidation of representative TIA biosynthetic pathways and summarize the functions and catalytic principles of the CYP71s, with emphasis on the reaction types and mechanistic features that underlie their functional versatility. Nevertheless, reconstructing these extended multienzyme pathways in heterologous cell factories remains challenging, in large part because plant cytochrome P450 enzymes (P450s) frequently become rate-limiting owing to inefficient electron transfer and limited cofactor availability. We therefore review metabolic engineering strategies established for P450 systems and assess their applicability to the optimization of CYP71s and to TIA production. We further discuss artificial intelligence (AI)-assisted approaches for CYP71 discovery, functional engineering, and de novo design. Together, these strategies open new avenues for identifying high-performance CYP71s and for advancing TIA biomanufacturing.