使用合成酵母选择系统演化双特征EPSP合成酶变体
Kevin B Reed1, Wantae Kim1, Hongyuan Lu1
1McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712.
概括
研究人员开发了一种合成酵母系统,以进化5-enolpyruvylshikimate-3-phosphate合成酶 (EPSPS) 以提高草甘耐受性和催化效率. 一个单一的进化周期产生了具有强大的除草剂耐药性和改善酶功能的突变物.
科学领域:
- 生物化学 生物化学
- 植物科学 植物科学
- 合成生物学 合成生物学
背景情况:
- 5enolpyruvylshikimate-3-phosphate合成酶 (EPSPS) 对于植物中的芳香氨基酸合成至关重要,并且是草甘除草剂的目标.
- 现有的抗草甘的EPSPS变体往往表现出减少的催化效率,需要新的变体与双特征改进.
研究的目的:
- 建立一个合成酵母系统,以快速进化EPSPS,具有高的草甘耐受性和催化效率.
- 为了确定克服除草剂耐药性和酶性能之间的典型权衡的新型EPSPS突变.
主要方法:
- 创建了一个合成酵母系统,用于研究和演化异质EPSP合成酶.
- 该系统使用已知的EPSPS变体进行了验证,以确认草甘应激下植物生长特征的总结.
- 突变性图书馆在强烈的双特征选择压力下进行了选.
主要成果:
- 合成酵母系统成功地回顾了植物对草甘的生长反应.
- 在双特征选择压力下的单一进化周期确定了一个优越的EPSPS突变.
- 与起始酶相比,进化突变体表现出强大的草甘耐受性 (Ki ~1 mM) 和酶效率增加了约2.5倍.
- 水晶结构揭示了对突变的洞察力,赋予了改进的双重特征.
结论:
- 开发的合成酵母平台对进化EPSPS有效,具有改善的草甘耐药性和催化效率.
- 确定了新的EPSPS突变,它们有可能在全植物应用中优于现有的耐药变体.
- 这个平台有助于探索EPSPS中草甘耐药性和酶效率之间的复杂关系.
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