The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800, Kongens Lyngby, Denmark.
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800, Kongens Lyngby, Denmark.
Metab Eng. 2021 Mar;64:52-63. doi: 10.1016/j.ymben.2021.01.007. Epub 2021 Jan 16.
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.
合成生物学使通过重组微生物生产小分子成为可能，可应用于制药、食品和材料领域。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有限了解，工程改造具有挑战性。在这里，我们描述了一种用于全基因组转运蛋白敲除的方法，该方法与代谢物生物传感器相结合，可用于鉴定影响酵母酿酒酵母中特定目标代谢产物生产的转运蛋白。我们将该方法应用于研究外源性化合物顺,顺-粘康酸（CCM）、原儿茶酸（PCA）和甜菜黄素的运输。我们发现了 22 种影响 CCM 或 PCA 生产的转运蛋白。12 折药物:H(+)反向转运蛋白（DHA1）家族 Tpo2p 的转运蛋白进一步在非洲爪蟾表达实验中被证实可摄取 CCM 和 PCA。我们还鉴定出三种参与甜菜黄素运输的转运蛋白（Qdr1p、Qdr2p 和 Apl1p）。总之，所描述的方法可用于细胞工厂中小分子的高通量转运蛋白鉴定。
