Coupling cell growth to protein secretion enables pooled screening at single-cell resolution in bioreactors

Preserving the genotype-phenotype linkage is critical in high-throughput screens exploring large parameter spaces. Screening pooled libraries for recombinant protein secretion is therefore particularly difficult, since protein diffusion into the medium disrupts this linkage. Existing methods impose significant restrictions on the environment of the screened cells. Here, we present AutoGrowth, a growth-based screening strategy linking growth rate to secretion via a single-cell biosensor. We demonstrated the proportional modulation of growth rate by secretion and validated the approach on five model proteins. As proof of concept, we screened a signal peptide library in bioreactors for improved secretion of a single-chain variable fragment by coupling AutoGrowth with deep sequencing. Additionally, through spike-in experiments, we showed that AutoGrowth can identify favorable variants diluted at ratios as low as one in hundreds of millions in three days. Our growth-based strategy provides unprecedented throughput potential and ensures selection of genetic variants presenting advantageous secretion and fitness capabilities.