The success of forward metabolic engineering depends on a thorough understanding of the behaviour of a heterologous metabolic pathway within its host. We have recently described CRI-SPA, a high-throughput gene editing method enabling the delivery of a metabolic pathway to all strains of the Saccharomyces cerevisiae knock-out library. CRI-SPA systematically quantifies the effect of each modified gene present in the library on product synthesis, providing a complete map of host:pathway interactions. In its first version, CRI-SPA relied on the colour of the product betaxanthins to quantify strains synthesis ability. However, only a few compounds produce a visible or fluorescent phenotype limiting the scope of our approach. Here, we adapt CRI-SPA to onboard a biosensor reporting the interactions between host genes and the synthesis of the colourless product cis-cis-muconic acid (CCM). We phenotype >9,000 genotypes, including both gene knock-out and overexpression, by quantifying the fluorescence of yeast colonies growing in high-density agar arrays. We identify novel metabolic targets belonging to a broad range of cellular functions and confirm their positive impact on CCM biosynthesis. In particular, our data suggests a new interplay between CCM biosynthesis and cytosolic redox through their common interaction with the oxidative pentose phosphate pathway. Our genome-wide exploration of host:pathway interaction opens novel strategies for improved production of CCM in yeast cell factories.

中文翻译：

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影响酵母中顺式-顺式-粘康酸产生的全基因组宿主-途径相互作用


正向代谢工程的成功取决于对其宿主内异源代谢途径行为的透彻理解。我们最近描述了 CRI-SPA，这是一种高通量基因编辑方法，能够将代谢途径递送到酿酒酵母敲除文库的所有菌株。CRI-SPA 系统地量化文库中存在的每个修饰基因对产物合成的影响，提供宿主：通路相互作用的完整图谱。在第一个版本中，CRI-SPA 依靠产品 betaxanthins 的颜色来量化菌株的合成能力。然而，只有少数化合物产生可见或荧光表型，限制了我们方法的范围。在这里，我们将 CRI-SPA 调整为载入生物传感器，报告宿主基因之间的相互作用和无色产物顺式-顺式-粘康酸 （CCM） 的合成。我们通过定量在高密度琼脂阵列中生长的酵母菌落的荧光，对 >9,000 基因型进行表型分析，包括基因敲除和过表达。我们确定了属于广泛细胞功能的新型代谢靶标，并证实了它们对 CCM 生物合成的积极影响。特别是，我们的数据表明，CCM 生物合成和胞质氧化还原之间通过与氧化磷酸戊糖途径的共同相互作用存在新的相互作用。我们对宿主：通路相互作用的全基因组探索为改善酵母细胞工厂中 CCM 的生产开辟了新的策略。