Here we present a droplet microfluidics workflow for cell factory screening by yield of an intracellular product from isogenic microcolonies, i.e. minimal cell populations, encapsulated in picoliter droplets. This allows us to utilize all the benefits of droplet microfluidic screening in terms of throughput, but based on the signal from a population average, rather than the noisy single cell signal. We demonstrate microcolony sorting by integrated droplet fluorescence area of encapsulated E. coli, optimize triglyceride (TG) microcolony assay in droplets and apply the microcolony screening concept to analyze triglyceride (TG) production in S. cerevisiae.

Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.

Bioindustry is expanding to an increasing variety of food, chemical and pharmaceutical products, each requiring rapid development of a dedicated cell factory and bioprocess. Microfluidic tools are, together with tools from synthetic biology and metabolic modeling, being employed in cell factory and bioprocess development to speed up development and address new products. Recent examples of microfluidics for bioprocess development range from integrated devices for DNA assembly and transformation, to high throughput screening of cell factory libraries, and micron scale bioreactors for process optimization. These improvements act to improve the biotechnological engineering cycle with tools for building, testing and evaluating cell factories and bioprocesses by increasing throughput, parallelization and automation.

The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell screening in droplets, as cell metabolic state directly affects production yields in cell factories. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format has an impact on cell proliferation and metabolite production. Furthermore, we engineered a new better oxygenated droplet incubation format, with retained droplet stability and size.

Cellular heterogeneity in isogenic cell populations is a major obstacle for single-cell screening campaigns, as the phenotype of individual cells might differ drastically from the mean, leading to large overlaps between productivity assessments of populations. At the other end of the spectrum, isogenic bulk assays provide a more accurate picture of a strain’s capacity at production scale, but suffers from low throughput and high reagent consumption.

Here, we present a screening format for cell factory variant libraries, aiming at combining the advantages of single-cell screening and bulk assay formats. Using microfluidic droplets, we compartmentalize yeast cell producer candidates, culture them to form isogenic microcolonies and sort colonies at higher throughput than bulk experiments to assess the genetic potential more accurately than in a single-cell screening format. To this end, we developed a fluorescence area-based sorting method that integrates the fluorescence signal from the entire fluorescence profile of a droplet and bases the sorting decision on that integrated fluorescence area. We validate the concept by sorting droplet microcolonies of fluorescent protein expressing Escherichia coli. Finally, we successfully sorted encapsulated iso-genic microcolonies of a low-producing and a high-producing strain of Saccharomyces cerevisiae by Triacylglycerol (TAG) production at 220 Hz, enriching the high-producing strain 4.45-fold.