RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes

Abstract

Synthetic biology and metabolic engineering seek to re-engineer microbes into "living foundries" for the production of high value chemicals. Through a "design-build-test" cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production.

Fig. 1

Overview of RAPID Screening. RNA-aptamers-in-droplets (RAPID) screening uses analyte-responsive RNA aptamers grafted to the Spinach aptamer backbone to detect analyte concentrations in microdroplets. The aptamer is co-encapsulated with a member of a yeast mutant library and incubated to produce the molecule of interest and develop a fluorescence signal. Droplets then flow through a microfluidic device and are sorted based on fluorescence using dielectrophoresis. Improved variants are recovered and the evolution cycle can be repeated if desired

Fig. 2

Establishing a panel of Spinach-based aptamers that can detect analytes in droplets. A panel of Spinach-based aptamer sensors was tested across a variety of small molecule and protein targets. Aptamers were synthesized through in vitro transcription and incubated with or without the analyte of interest, and fluorescence was measured. Bar graphs represent the mean of three replicates (error bars depict s.d.). On the right, solutions of dye with RNA aptamer or with both RNA aptamer and analyte were encapsulated and the resulting microdroplets were imaged using fluorescence microscopy. The negative (RNA alone) and positive (RNA and analyte) droplets were mixed together for simultaneous imaging of contrast. All images were acquired under the same magnification, and representative images from multiple independent trial runs are depicted. Sequences of all aptamers used in this study can be found in Supplementary Table 1

Fig. 3

RAPID screening for the improvement of tyrosine production through an evolved Aro4p. The RAPID screening approach was used to identify novel mutations in aro4 that improve tyrosine production by yeast. Libraries were constructed through mutagenizing the proposed regulatory region of Aro4p (residues 191–263) a–c and through whole-gene mutagenesis of the best previously reported variant aro4-K229L d–f. Histograms of droplet fluorescence pre-sort and post-sort (re-encapsulated) demonstrate enrichment through the process (a, d). Isolated and re-transformed clones randomly selected from the pre- and post-sort populations were quantified for tyrosine production with the sorted clones having a 1.9- and 5.8-fold increase in secretion compared to the unsorted clones for Aro4p and K229L clones, respectively (b, e). Crystal structures of Aro4p are derived from PDB 1OF6 and marked with the identified mutations (c, f). Error bars represent standard error of biological triplicates. Tyrosine production was increased nearly 28-fold over the wild-type production using this approach

Fig. 4

RAPID screening for the improvement of streptavidin protein secretion through an evolved secretory tag. The RAPID screening approach was used to identify mutations in the α-mating factor (αMF) secretory leader fused to streptavidin in yeast. a Histograms of droplet fluorescence pre-sort and post-sort (re-encapsulated) demonstrate enrichment through the process. b Isolated and re-transformed clones randomly selected from the pre- and post-sort populations were quantified for streptavidin production. Error bars represent 95% confidence intervals of biological triplicates. Protein secretion from an individual clone was increased nearly threefold over wild-type production using this approach, and there was also twofold increase in secretion between the mean of the sorted and unsorted clones