(12c) Microfluidic Droplets as Nanobioreactors for Screening Metabolic Engineering Libraries

Metabolic engineering has contributed significantly to the rational improvement of strains for industrial applications. In addition to enzymatic steps closely associated with the product-forming pathway, other, so-called distal genes may also impact production in a profound way due to (often unknown) kinetic and regulatory effects. Such genes are identified by combinatorial methods whereby libraries of the host strain are constructed harboring random variants of the basic strain genes, as well as random combinations of gene knock outs and over-expressions. Cells with superior properties are selected from these libraries and the specific genetic alteration identified in a process known as Inverse Metabolic Engineering. However, these approaches necessitate the use of high throughput screening methods to select the most desirable clones from these libraries of engineered strains. For many metabolic engineering libraries, the selection criterion is the production of a secreted metabolite or the consumption of a medium component. Thus, a strategy for compartmentalizing clones is necessary so that each clone grows in a separate environment allowing for the measurement of clone-specific metabolite concentrations. Traditional methods such as microwell plates for culturing and assaying can be utilized but are not sufficiently high throughput. Here, we will present our development of a high throughput screening platform which utilizes microfluidics to encapsulate yeast cells in nanoliter aqueous droplets surrounded by an immiscible fluorinated oil phase. This system contains modules for cell culturing, measurement of the metabolite of interest with a fluorescent enzymatic assay, and sorting. To demonstrate the capabilities of this platform, we screened an incoming population which contained equal numbers of two Saccharomyces cerevisiae strains and enriched for the higher xylose consuming strain by over 20 times. Furthermore, we screened a S. cerevisiae xylose transporter library for high xylose consumption and selected a mutant which consumes 5% more xylose than the library as a whole.