Microbial biosynthesis of plant natural products (PNPs) can facilitate access to valuable medicinal compounds and derivatives. Such efforts are challenged by metabolite transport limitations, which arise when complex plant pathways distributed across organelles and tissues are reconstructed in unicellular hosts without concomitant transport machinery. We recently reported an engineered yeast platform for production of the tropane alkaloid (TA) drugs hyoscyamine and scopolamine, in which product accumulation is limited by vacuolar transport. Here, we demonstrate that alleviation of transport limitations at multiple steps in an engineered pathway enables increased production of TAs and screening of useful derivatives. We first show that supervised classifier models trained on a tissue-delineated transcriptome from the TA-producing plant Atropa belladonna can predict TA transporters with greater efficacy than conventional regression- and clustering-based approaches. We demonstrate that two of the identified transporters, AbPUP1 and AbLP1, increase TA production in engineered yeast by facilitating vacuolar export and cellular reuptake of littorine and hyoscyamine. We incorporate four different plant transporters, cofactor regeneration mechanisms, and optimized growth conditions into our yeast platform to achieve improvements in de novo hyoscyamine and scopolamine production of over 100-fold (480 μg/L) and 7-fold (172 μg/L). Finally, we leverage computational tools for biosynthetic pathway prediction to produce two different classes of TA derivatives, nortropane alkaloids and tropane N-oxides, from simple precursors. Our work highlights the importance of cellular transport optimization in recapitulating complex PNP biosyntheses in microbial hosts and illustrates the utility of computational methods for gene discovery and expansion of heterologous biosynthetic diversity.

植物天然产物（PNP）的微生物生物合成可以促进有价值的药用化合物和衍生物的获取。这些努力受到代谢物运输限制的挑战，当分布在细胞器和组织中的复杂植物途径在单细胞宿主中重建而没有伴随的运输机器时，就会出现代谢物运输限制。我们最近报道了一种用于生产托烷生物碱（TA）药物天仙碱和东莨菪碱的工程酵母平台，其中产物积累受到液泡运输的限制。在这里，我们证明了在工程化途径的多个步骤中减轻运输限制可以增加 TA 的产量并筛选有用的衍生物。我们首先表明，在来自产生 TA 的植物颠茄的组织描绘转录组上训练的监督分类器模型可以比传统的基于回归和聚类的方法更有效地预测 TA 转运蛋白。我们证明了两种已鉴定的转运蛋白Ab PUP1 和Ab LP1 通过促进水泡碱和天仙子胺的液泡输出和细胞再摄取来增加工程酵母中 TA 的产量。我们将四种不同的植物转运蛋白、辅因子再生机制和优化的生长条件整合到我们的酵母平台中，以将天仙碱和东莨菪碱的从头产量提高超过 100 倍 (480 μg/L) 和 7 倍 (172 μg/L) 。最后，我们利用生物合成途径预测的计算工具，从简单的前体生产两类不同类型的 TA 衍生物：去甲托烷生物碱和托烷N-氧化物。我们的工作强调了细胞运输优化在微生物宿主中重现复杂的 PNP 生物合成中的重要性，并说明了计算方法在基因发现和扩展异源生物合成多样性方面的实用性。