Heterologous expression of CrMATE1 increases flux of intermediates in monoterpenoid indole alkaloid pathway

Catharanthus roseus is the source of many pharmacologically valuable monoterpenoid indole alkaloids (MIAs), yet their low abundance can often constrain investigations into new bioactivities. Heterologous reconstruction of MIA pathways in microbial hosts offers a scalable solution, but yields can be compromised by low product titers, by-product formation, and a lack of pathway orthogonality. To address these challenges, we evaluated the functional deployment of two C. roseus transporters in engineered yeast. CrMATE1 imports secologanin into the vacuole, where it is condensed with tryptamine to form strictosidine, the precursor of nearly all MIAs, which is exported by CrNPF2.9. We incorporated both transporters in engineered yeast and confirmed their tonoplast localization. Yeast substrate uptake assays verified CrMATE1 as a selective importer of secologanin. CrMATE1 expression in a de novo ajmalicine-producing strain markedly enhanced product titers almost four-fold, suggesting that vacuolar import is a critical bottleneck. Indeed, supplementation with secologanin further highlighted the role of CrMATE1 in guiding flux towards end-state titers. In a minimal ajmalicine pathway, starting with STR catalysis, the inclusion of CrMATE1 had a greater positive impact than CrNPF2.9, while their co-expression enhanced titers additively, up to five-fold. Relocating strictosidine formation to the cytosol had a negative impact on titers, recapitulating the importance of transporter-mediated compartmentalization. Finally, CrMATE1 overexpression in C. roseus petals modified the metabolic profile towards enrichment of branch-pathway MIAs, including akuammicine and serpentine. Therefore, transporters can play a role in both plant and microbial-based metabolic engineering, enhancing the production of high-value metabolites.