基于光的屏幕用于工程酶,与化三相关联
Kevin B Reed1, Simon d'Oelsnitz2, Sierra M Brooks
1McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States.
ACS synthetic biology
|March 27, 2024
概括
这项研究引入了一种多功能转录因子 (TF) 生物传感器系统,用于有效选素酶和相关生物催化剂. 这一进步加速了通过定向进化生产化化学品的酶的发现.
科学领域:
- 生物技术和合成生物学
- 酶工程和定向进化研究
- 代谢工程和生物生产
背景情况:
- 定向进化对于酶发现至关重要,但往往受到低通量选方法的阻碍.
- 转录因子 (TF) 生物传感器通过将酶活性与记者基因表达联系起来,为高通量选提供了一个潜在的解决方案.
- 开发可以充当激活器和抑制器的多功能TF生物传感器是扩大其适用性的关键.
研究的目的:
- 为了证明可重构的转录因子 (TF) 生物传感器系统的可行性,用于定向进化.
- 为了验证特定TF生物传感器对酶酶的高通量选的实用性.
- 建立一个生物传感器导向的进化方案,用于识别参与化化学生产的酶.
主要方法:
- 专门设计的5-或6-三甲特异性TF生物传感器,用于*in vivo*检测化三甲.
- 生物传感器在两个不同的酸酶特异性-三胺积累屏幕中的验证.
- 重构TF电路以应对基质耗尽,以选下游酶变体.
主要成果:
- 通过使用TF生物传感器,成功地从混合池中以100%的效率分离了5-三甲酶,XsHal.
- 以100%的效率生成了6-三甲酸酶,Th-Hal和分离的功能变体的目标库.
- 创建了一个高通量生物传感器导向进化方案的原型,用于选杂交的化酸转化酶.
结论:
- 开发的TF生物传感器系统显著提高了选化酶和相关酶的吞吐量和准确性.
- 这项工作代表了快速生物生产有价值的化化学品的重大进展.
- 可重构TF系统为未来生物催化酶发现和工程工作提供了一个强大的平台.
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