Double emulsion microdroplets for high-throughput yeast single-cell analysis
Saccharomyces cerevisiae is the most successful eukaryotic organism acting as a cell factory for producing a wide range of value-added products in an economical and environmental-friendly manner. To acquire desirable phenotypes (e.g., high biomass and metabolite productivity, and high acid tolerance) for large-scale industrial production, directed evolution has been widely used, by mimicking the natural selection of rare variants from random mutant libraries. However, traditional directed evolution of variants is performed in bulk, obscuring cell-to-cell heterogeneity and slowing evolution efficiency. Fluorescence-activated cell sorting (FACS) can perform high-throughput single-cell analyses; however, it can only analyse biomarkers that are intracellular or expressed on cell surface.
The advent of droplet microfluidics opens avenues for the analysis of individual cells encapsulated within picoliter microdroplets, based on extracellular activities (e.g., secreted protein production). Particularly, water-in-oil-in-water (w/o/w) double emulsions (DEs) are compatible with commercial flow cytometric instruments, allowing the characterisation and selection of single cells exhibiting desirable properties. Although there exist a few studies on the use of the integrated DE and FACS approach for directed evolution of enzymes in microorganisms, several practical challenges have prevented its widespread adoption and applications. Therefore, the thesis aims to optimise DE-FACS methods in differing aspects, for a high-throughput directed evolution of enzymes in yeast.
First, the use of a density-matching reagent, OptiPrepTM, for acquiring high-efficiency single-cell encapsulation in droplets has been quantitatively characterised. OptiPrepTM creates a neutral buoyance of cell suspension, making distribution of cell numbers in droplets fit closely to the Poisson distribution. In addition, there was no obvious decrease in cell viability after 24 h cultivation when different concentrations of OptiPrepTM of different concentrations were used.
To investigate yeast single-cell growth and physiological behaviours in droplets, yeast single cell cultures were compared with corresponding bulk cultures using different cell strains and environmental conditions. It was found that S. cerevisiae single cells in droplets have a similar growth rate to that of bulk cultures in the exponential phase and can reveal subpopulation behaviours obscured in bulk cultures. Moreover, the effect of acids on cell growth, and these effects of potassium ion and mutation on cell resistance to PA at the single-cell level were studied, showing similar trends to bulk cultures.
To achieve a rapid, simple, and inexpensive generation of monodisperse DEs, a method to prepare PDMS devices with local wettability has been developed. A convenient hand-held corona treater that can generate local corona discharge was used to render microchannels hydrophilic at target regions. In addition, a specific customised channel (i.e., a serpentine narrow channel as a plasma resistor) was designed to prevent the diffusion of corona discharge to unwanted regions. With this approach, I achieved the generation of DEs with multiple structures and morphologies. Furthermore, I demonstrated the abilities of generated DEs for microgel synthesis and yeast single cell culture and its downstream flow cytometric screening.
Lastly, the improved DE-FACS platform technology was applied to screening and selection of extracellular enzymatic activities in yeast cells. As a proof of concept, an artificial library of wild type and cellulase-secreting mutant cells were mixed with a ratio of 1:1 and encapsulated in DEs. Mutant cells showing a high level of extracellular enzymatic activity were successfully selected by FACS, with an accuracy of up to 92%.
In summary, this thesis has studied different approaches to improving the performance of integrated DE-FACS platform technology for single-cell analysis. These results have shown great potential for directed evolution of extracellular enzymes in yeast for industrial production.