Monoterpene Indole Alkaloids (MIAs) are a diverse group of plant natural products (PNPs) with significant pharmacological potential, including anti-cancer and pain-relieving effects. The heterologous production of MIAs in engineered hosts has gained increasing attention as an alternative to overcome the limitations of traditional extraction methods or chemical synthesis. One key problem in producing MIAs in heterologous hosts is the elucidation of the target biosynthesis pathways. Although the universal upstream pathways for MIA synthesis are well elucidated, a significant challenge remains in the discovery of the pathways downstream of the central MIA scaffold strictosidine. In this study, we have successfully characterized STR (strictosidine synthase) and SGD (strictosidine β-D-glucosidase) genes from a less-studied medicinal plant, Kratom (Mitragyna speciosa), which encode for the enzymes responsible for producing MIA precursors, using yeast as the platform. We further achieved a higher-titer production of these precursors, which can serve as substrates for the validation of downstream pathway gene candidates. In addition, we developed a transient expression system in Nicotiana benthamiana through agroinfiltration for rapid screening of putative cytochrome P450s (CYPs) in MIA biosynthetic pathways. These findings reveal the potential of yeast and N. benthamiana as platforms for MIA-related gene expression and functional validation, providing a foundation for further discovery and optimization of MIA pathways using heterologous hosts.