(142e) RNA-Aptamer-in-Droplet (RAPID) High-Throughput Screening for Secretory Phenotypes

Synthetic biology tools have been used to rewire a wide range of microorganisms to create living foundries for the biorenewable production of high value chemicals. This engineering process often comprises a long and costly design-build-test cycle in which pools or libraries of very large numbers of variants must be constructed and tested to optimize performance. Currently, library generation capacity often greatly exceeds the rate of high throughput testing and screening. Overcoming this mismatch is especially challenging for secretory phenotypes, as the secreted product must be physically entrapped with the responsible producer cell and a biosensor module to enable identification and isolation of improved variants. Well plate assays are flexible and achieve this goal with limited throughput, but droplet microfluidic techniques can enable ultrahigh-throughput screening for secretory production. Here, we present an approach we term RNA Aptamer In Droplet (RAPID) screening, a method that greatly expands the scope and generality of high throughput microfluidic screening. Using in vitro transcribed RNA aptamers, we synthetically transduce target product concentration into fluorescence, allowing modular biosensor construction, fast measurement, and rapid screening of millions of variants in droplets. We apply the RAPID approach by enhancing secreted production of a small molecule metabolite (tyrosine) and a recombinant protein (streptavidin) from yeast. In these case studies, we isolate mutants with improvements exceeding 20-fold for metabolite production. Our proof-of-concept results demonstrate that RAPID screening affords a more general approach to massively enhancing screening power for secretory phenotypes.