Growth amplification in ultrahigh-throughput microdroplet screening increases sensitivity of clonal enzyme assays and minimizes phenotypic variation

Microfluidic ultrahigh-throughput screening of enzyme activities provides information on libraries with millions of variants in a day. Each individual library member is represented by a recombinant single cell, compartmentalised in an emulsion droplet, in which an activity assay is carried out. Key to the success of this approach is the precision and sensitivity of the assay. Assay quality is most profoundly challenged when initially weak, promiscuous activities are to be enhanced in early rounds of directed evolution or when entirely novel catalysts are to be identified from metagenomic sources. Implementation of measures to widen the dynamic range of clonal assays would increase the chances of finding and generating new biocatalysts. Here, we demonstrate that the assay sensitivity and DNA recovery can be improved by orders of magnitude by growth of initially singly compartmentalised cells in microdroplets. Homogeneous cell growth is achieved by continuous oxygenation and recombinant protein expression is regulated by diffusion of an inducer from the oil phase. Reaction conditions are adjusted by directed droplet coalescence to enable full control of buffer composition and kinetic incubation time, creating level playing field conditions for library selections. The clonal amplification multiplies the product readout because more enzyme is produced per compartment. At the same time, phenotypic variation is reduced by measuring monoclonal populations rather than single cells and recovery efficiency is increased. Consequently, this workflow increases the efficiency of lysate-based microfluidic enzyme assays and will make it easier for protein engineers to identify or evolve new enzymes for applications in synthetic and chemical biology. Successful screening of enzyme libraries in functional metagenomics and directed evolution becomes more likely after uniform cell growth in droplets amplifies genotype and phenotype.