RNA-Aptamer Sensors for Phenotype Screening of Engineered Microbial Pathways

The recent years have provided tremendous improvements in microbial pathway engineering through the design of complex control circuits, however industrial relevant titers are still difficult to obtain. One major bottleneck in pathway engineering is screening for small molecule production since only a small fraction of engineered pathway products (chromophores or fluorophores) can be screened using traditional high throughput methods. Here, we present an RNA-aptamer based sensor system to detect secreted metabolites from engineered pathways in a high throughput and cost efficient manner. Taking inspiration from the many examples of naturally occurring kinetically controlled RNA-based sensors, such as riboswitches, we have engineered a set of RNA-aptamer based biosensors that sense and respond to p-amino-phenylalanine (p-AF) co-transcriptionally. To our knowledge, this is the first time that an RNA-aptamer based sensor has been engineered deliberately to function under kinetic control. These sensors were screened in silico from approximately 1.5E5 unique sequences that each contain the same parental RNA aptamer sequence. We have developed a set of p-AF sensors that exhibit EC₅₀ values between 1 – 0.5 mM, ideal for screening the production of p-AF producing E. coli strains with titers as low as 100 mg/L. Currently, we can detect p-AF in cell culture supernatant, and we are working towards optimizing a high throughput assay to screen p-AF production strain variants. To validate our computational pipeline and demonstrate the utility of this method, we are also generating sensors for tyrosine, another small molecule product that has been produced using the core of the engineered p-AF pathway.