Utilizing RNA aptamers for small molecule biosensing in engineered biosynthetic pathways

Abstract

Chemical production from renewable feedstocks using engineered microbes will play an important role in advancing sustainable technological solutions to climate change and other environmental challenges. Biosensors are already playing a critical role in production screening and dynamic genetic control to reduce the time and costs and increase titers in engineered biosynthetic pathways. RNA aptamers are an increasingly utilized tool for metabolite biosensing but matching the sensitivity of RNA aptamer-based devices to the titers from engineered pathways is challenging and unpredictable. This thesis addresses that problem, modeling the energetic contributions of metabolite characteristics towards RNA aptamer binding affinities to predict pathways and molecules more amenable to RNA aptamer biosensing. We present tools and approaches for selecting RNA aptamers and using those aptamers to build functional, responsive RNA biosensors. This thesis details a label-free in vitro selection for an RNA aptamer binding the metabolite p-aminocinnamic acid. This work presents, for the first time, an approach to predict binding affinities of RNA aptamers towards small molecule targets from the chemical properties of the small molecules. We demonstrate the utility of kinetic aptamer ribosensors in reporting titers of the small molecule p-aminophenylalanine in supernatants from engineered cells. This thesis also details the first demonstration of engineering metastable RNA folding states to encode time using multistate co-transcriptional RNA design. Collectively, this work provides tools for assessing the suitability of RNA aptamers for sensing applications in metabolic engineering while simplifying the process of in vitro selections for small molecule aptamers and providing a battery of tools for tuning the sensitivities of kinetic co-transcriptional RNA biosensors.