(67e) Kinetically-Controlled RNA Aptamer Biosensors for High-Throughput Production Phenotype Screening

A major bottleneck in the biological production of biochemicals from renewable feedstocks is the lack of high throughput tools to screen for small molecule production in the generation, optimization, and industrialization of microbial strains. We have developed a new computational pipeline to design kinetically-controlled RNA based sensors that enable the rapid implementation of high-throughput screens for secreted biochemicals and production phenotypes. The biosensors combine in vitro selected RNA aptamers for molecular recognition with DNA strand displacement for signal generation, and require only standard laboratory equipment such as a qPCR machine or a plate reader. Currently, the RNA biosensor platform has a throughput of at least 128 HPLCs, and because it is not hardware-centric, can screen the same number of samples at a fraction of the cost. We validated our new approach by the construction of 38 novel devices for the aromatic amino acid derivative p-amino-phenylalanine (p-AF), a precursor in an engineered styrene derivative production pathway. We have developed a set of p-AF biosensors with EC₅₀ values between 1 - 0.5 mM, ideal for screening production of p-AF producing E.coli strains with titers as low as 100 mg/L. In this presentation, I will discuss these results and on-going efforts to demonstrate industrially relevant synthetic biology applications including production titer screens of genetic variants, biocatalysis validation of heterologous pathway enzymes, and media optimization for increased productivity and industrialization of microbial strains.