Co-Overexpression of Geissoschizine Synthase and Catharanthine Synthase Increases Catharanthine Biosynthesis in Catharanthus roseus Hairy Roots

Plant-based platforms remain vital for producing anticancer vinca alkaloids. However, one major challenge in plant-based metabolic engineering is that target metabolite yields are often hindered by the presence of multiple competing pathway branches that disperse metabolic flux. To overcome these bottlenecks, we first implemented a competitive branch-point diversion strategy by co-overexpressing geissoschizine synthase and catharanthine synthase under an inducible promoter in Catharanthus roseus hairy roots. This targeted intervention successfully shifted metabolic partitioning, driving a peak relative increase of 67% in catharanthine accumulation at the 96 h time point, while significantly reducing competing by-products such as ajmalicine, serpentine, and tabersonine. Targeted qPCR analysis revealed a transient upregulation of core monoterpene indole alkaloid biosynthetic genes and a coordinated suppression of some competing branch genes. Despite this effective downstream diversion, overall yield enhancement was constrained by tight regulation, notably the downregulation of BIS transcriptional activators, which restricts the supply of upstream terpenoid precursors. Furthermore, untargeted metabolomic profiling highlighted a widespread metabolic shifts featuring the significant enrichment of phenylalanine-derived secondary pathways. Ultimately, this study demonstrates the efficacy of competitive branch-point diversion for target alkaloid enrichment, while highlighting the complex regulatory dynamics and broad metabolic alterations within the C. roseus network.