The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) functions in the shikimate pathway which is responsible for the production of aromatic amino acids and precursors of other essential secondary metabolites in all plant species. EPSPS is also the molecular target of the herbicide glyphosate. While some plant EPSPS variants have been characterized with reduced glyphosate sensitivity and have been used in biotechnology, the glyphosate insensitivity typically comes with a cost to catalytic efficiency. Thus, there exists a need to generate additional EPSPS variants that maintain both high catalytic efficiency and high glyphosate tolerance. Here, we create a synthetic yeast system to rapidly study and evolve heterologous EPSP synthases for these dual traits. Using known EPSPS variants, we first validate that our synthetic yeast system is capable of recapitulating growth characteristics observed in plants grown in varying levels of glyphosate. Next, we demonstrate that variants from mutagenesis libraries with distinct phenotypic traits can be isolated depending on the selection criteria applied. By applying strong dual-trait selection pressure, we identify a notable EPSPS mutant after just a single round of evolution that displays robust glyphosate tolerance (K i of nearly 1 mM) and improved enzymatic efficiency over the starting point (~2.5 fold). Finally, we show the crystal structure of corn EPSPS and the top resulting mutants and demonstrate that certain mutants have the potential to outperform previously reported glyphosate-resistant EPSPS mutants, such as T102I and P106S (denoted as TIPS), in whole-plant testing. Altogether, this platform helps explore the trade-off between glyphosate resistance and enzymatic efficiency.

中文翻译：

---

使用合成酵母选择系统进化双性状 EPSP 合酶变体


5-烯醇丙酮莽草酸-3-磷酸合酶 (EPSPS) 在莽草酸途径中发挥作用，该途径负责所有植物物种中芳香族氨基酸和其他重要次生代谢物前体的产生。 EPSPS 也是除草剂草甘膦的分子靶标。虽然一些植物 EPSPS 变体具有降低草甘膦敏感性的特点，并已用于生物技术，但草甘膦不敏感性通常会降低催化效率。因此，需要产生额外的EPSPS变体，以保持高催化效率和高草甘膦耐受性。在这里，我们创建了一个合成酵母系统来快速研究和进化针对这些双重性状的异源 EPSP 合酶。使用已知的 EPSPS 变体，我们首先验证我们的合成酵母系统能够重现在不同草甘膦水平下生长的植物中观察到的生长特征。接下来，我们证明可以根据所应用的选择标准从具有不同表型性状的诱变文库中分离出变体。通过施加强大的双性状选择压力，我们仅经过一轮进化就鉴定出了一个显着的 EPSPS 突变体，该突变体表现出强大的草甘膦耐受性（K i 接近 1 mM），并且酶效率较起始点提高（约 2.5 倍）。最后，我们展示了玉米 EPSPS 的晶体结构和顶部产生的突变体，并证明某些突变体在全株测试中具有优于先前报道的抗草甘膦 EPSPS 突变体的潜力，例如 T102I 和 P106S（表示为 TIPS）。总而言之，该平台有助于探索草甘膦抗性和酶效率之间的权衡。