Synthetic biology enables the production of small molecules by recombinant microbes for pharma, food, and materials applications. The secretion of products reduces the cost of separation and purification, but it is challenging to engineer due to the limited understanding of the transporter proteins' functions. Here we describe a method for genome-wide transporter disruption that, in combination with a metabolite biosensor, enables the identification of transporters impacting the production of a given target metabolite in yeast Saccharomyces cerevisiae. We applied the method to study the transport of xenobiotic compounds, cis,cis-muconic acid (CCM), protocatechuic acid (PCA), and betaxanthins. We found 22 transporters that influenced the production of CCM or PCA. The transporter of the 12-spanner drug:H(+) antiporter (DHA1) family Tpo2p was further confirmed to import CCM and PCA in Xenopus expression assays. We also identified three transporter proteins (Qdr1p, Qdr2p, and Apl1p) involved in betaxanthins transport. In summary, the described method enables high-throughput transporter identification for small molecules in cell factories.

合成生物学能够通过重组微生物生产小分子，用于制药、食品和材料应用。产物的分泌降低了分离和纯化的成本，但由于对转运蛋白功能的了解有限，工程设计具有挑战性。在这里，我们描述了一种全基因组转运蛋白破坏的方法，该方法与代谢物生物传感器相结合，能够识别影响酿酒酵母中给定目标代谢物产生的转运蛋白。我们应用该方法研究了外源化合物、顺式粘康酸 (CCM)、原儿茶酸 (PCA) 和甜菜青素的转运。我们发现了 22 个影响 CCM 或 PCA 生产的转运蛋白。12-spanner药物的转运蛋白：H(+)逆向转运蛋白(DHA1)家族Tpo2p在非洲爪蟾表达测定中进一步证实可导入CCM和PCA 。我们还鉴定了参与甜菜青素转运的三种转运蛋白（Qdr1p、Qdr2p 和 Apl1p）。总之，所描述的方法能够对细胞工厂中的小分子进行高通量转运蛋白识别。