Fig. 1.

Cellular metabolite-transport limitations in yeast engineered for de novo production of TAs. TA biosynthesis is distributed across five metabolic modules (I through V) and five cellular compartments: mitochondrion, peroxisome, ER, vacuole, and cytosol. The central esterification reaction assembling the TA scaffold from tropine and PLA glucoside is catalyzed by littorine synthase (AbLS) with N-terminally fused fluorescent protein DsRed in the vacuole. For this reaction, substrates (tropine and PLA glucoside) must be imported to the vacuole lumen, and product (littorine) must be exported to the cytosol. Principal starting materials and products are shown for each module. Black arrows represent one or more biosynthetic steps (reference SI Appendix, Fig. S3 for complete pathway); gray arrows indicate metabolite transport steps critical to pathway flux and product accumulation. Charges (“+,” positive; no symbol, uncharged) of key intermediates and products relevant to transport are indicated. Protein names: NtJAT1, Nicotiana tabacum jasmonate-inducible alkaloid transporter 1; NtMATE2, N. tabacum multidrug and toxin extrusion transporter 2; AbPUP1, A. belladonna purine uptake permease-like transporter 1; AbLP1, A. belladonna lactose permease-like transporter 1; PdrXp, yeast pleiotropic drug resistance transporters.