Department of Bioengineering, Stanford University, Stanford, CA, USA.
Department of Biological Science, Boise State University, Boise, ID, USA.
Nat Commun. 2021 Mar 4;12(1):1437. doi: 10.1038/s41467-021-21716-0.
Biosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.
生物传感器是工程化生物系统的关键组成部分，为测量和作用于活细胞中的大型生化空间提供了一种手段。然而，生成小分子感应元件并将其集成到活体生物传感器中一直具有挑战性。在这里，使用适配体偶联核酶文库和核酶再生方法，新的体外 RNA 生物传感器快速进化（DRIVER）能够对生物传感器进行多路复用发现。通过在液体处理系统上完全自动化的 DRIVER 和高通量表征（CleaveSeq），我们针对六种小分子鉴定和验证了生物传感器，其中包括五种以前没有发现适配体的小分子。DRIVER 进化的生物传感器可直接应用于调节酵母中的基因表达，显示出高达 33 倍的激活比。DRIVER 生物传感器还应用于检测来自多酶生物合成途径的代谢产物的产生。这项工作展示了 DRIVER 作为一种可扩展的工程化生物传感器的流水线，在生物制造、诊断、治疗和合成生物学方面具有广泛的应用。
