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Significance Our work highlights the importance of addressing metabolite transport limitations in parallel with strain and culture optimization for production of plant-derived medicines and derivatives in engineered microbes. We show that supervised classification strategies can outperform conventional approaches when searching plant transcriptomes for metabolite transporters. Our discovery of two vacuolar tropane alkaloid (TA) exporters provides insight into the spatial organization associated with biosynthesis of these molecules. We illustrate how differences in transporter specificity and mechanisms can be leveraged to control accumulation of pathway products in an enhanced yeast-based TA production platform. By engineering this platform for production of TA derivatives using enzymes evolved for alkaloid detoxification, we reveal sources of biocatalysts for expanding biosynthetic diversity in heterologous hosts.

Engineering cellular metabolite transport for biosynthesis of computationally predicted tropane alkaloid derivatives in yeast