Droplet microfluidics for directed evolution of biocatalysts

Abstract: Biocatalysts, biologically derived catalysts, are of great importance for a wide range of industrial applications. They are used in the production of for example foods, pharmaceuticals and biofuels. Improving biocatalysts commonly relies on directed evolution, i.e. mutagenesis to form diverse variants followed by functional screening in an iterative fashion.Droplet microfluidics is an emerging technology that can be applied for high throughput screening. A key feature of droplet microfluidics is the ability to encapsulate discrete objects, such as single cells, in picoliter-sized droplets at rates of over 1000 cells per second. Each droplet serves as a reaction vessel, analogous to a microwell, where a single clone can be screened.In this thesis, droplet microfluidics is employed for directed evolution of biocatalysts. In paper I, a multiplexed droplet microfluidic method for characterization of enzyme variants is presented and validated by measuring the kinetics of β-galactosidase inhibited by IPTG. In paper II-III, a method for directed evolution of cells with improved production of industrially important enzymes is presented. Two rounds of directed evolution yielded improved strains. The strains had up to 6 times increased enzyme expression levels and whole-genome sequencing revealed 300 mutations, many of which mapped to the protein secretory pathway. In Paper IV, a method for directed evolution of enzyme variants under conditions lethal to host cells is developed. The method is used to screen for α-amylase variants with improved activity or stability at pH4. In Paper V, a method to screen cyanobacteria cell factories is developed and we show that the method can enrich for a strain with high production of L-lactate. In Paper VI, the metabolism of yeast cells encapsulated in microfluidic droplets is studied and found to depend on the choice of emulsion incubation device.Taken together, droplet microfluidics is a promising technology for directed evolution of biocatalysts with the potential to vastly increase throughput and cut costs. The technology could pave the way for process customized biocatalysts and help replace polluting processes with sustainable green chemistry.