可调节的T7促进器对T7RNAP的正交性,用于cis-Aconitate脱碳酶演化通过基编辑器和从Itaconic Acid生物传感器进行选
1Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan.
ACS synthetic biology
|September 26, 2023
概括
这项研究介绍了dT7-Muta,一种新的酶进化平台,使用脱氨酶融合的T7RNA聚合酶 (T7RNAP) 和修饰的T7促进剂. 它可以同时进行DNA修改和选,以改善酶功能.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 酶工程是什么? 酶工程是什么?
背景情况:
- 脱氨酶融合的T7RNA聚合酶 (T7RNAP) 在体内提供酶进化,同时进行DNA修饰和选.
- 了解T7RNAP/T7促进器系统对于优化突变发生效率至关重要.
研究的目的:
- 建立一个新的平台,dT7-Muta,通过调整T7RNAP和T7促进体结合效率来进行基因突变发生.
- 使用dT7-Muta平台展示酶进化的概念证明.
主要方法:
- 在T7促销器区域 (上游,中游,下游) 设计了退化的子,以创建不同强度 (S,M,W) 的变体 (dT7s).
- 通过改变sfGFP突变体的光分布和选itaconate诱导的生物传感器,评估Aspergillus terreus cis-aconitate脱碳酶 (AtCadA) 突变体的突变效率.
- 利用流细胞计测量来选突变物种.
主要成果:
- dT7-Muta平台表现出成功的突变发生,导致sfGFP和AtCadA突变的光分布更广泛.
- 特定的dT7促进体变体 (CW,DS,UW) 提高了突变效率.
- 获得了最高的光和伊塔康酸标位 (860AU). 和0.51 g/L) 与使用T7,UW和DS促进体的AtCadA突变体.
结论:
- 该dT7-Muta平台通过调节T7RNAP-T7促进体结合,为脱氨酶基突变发生提供了一种新的方法.
- 改变T7促进体结合效率是推动酶进化努力的可行策略.
- 这个平台有潜力用于指导进化和蛋白质工程的未来应用.
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