Development of Selection Method for Directed Evolution of Genetic Switches in Saccharomyces Cerevisiae

Genetic switches that turn gene expression “ON” and “OFF” in response to specific inputs are indispensable tools for metabolic engineering. Although there are hundreds of natural genetic switches in yeast, most of these systems cannot be directly used for metabolic engineering applications because of their inappropriate switching properties, such as insufficient switching sensitivity, expression intensity or basal leakiness. Directed evolution strategy is applicable to the improvement of any genetic switches in their properties. Desired switches can be obtained simply by coupling a rescuer and a conditional killer to their expression outputs. The rescuer leaves the variants to satisfy the sufficient output under “ON” states, while the conditional killer excludes the variants to exhibit the leaky output under “OFF” states. For the rapid construction of tailored genetic switches for various applications, all selection procedures should be completed by liquid handling that enables the parallel operation of multiple projects in a multi-well plate format. In this study, we report a rapid and liquid-based ON/OFF selection method in yeast S. cerevisiae to meet the wide demand for improving switching stringency, expression level, and sensitivity to the target metabolite.

In search for the robust OFF selection method that can be operated by liquid handling only, we chose the kinase activity of herpes simplex virus thymidine kinase (hsvTK) for non-natural nucleoside dP, initially used for the directed evolution of genetic switches/circuits in Escherichia coli (Y. Tashiro et al., Nucleic Acids Res., 39, e12 (2011)). However, yeast cells expressing hsvtk gene exhibited only a small loss of viability in the presence of 100 µM dP, which was over 100-fold higher concentration than the case in showing the complete loss of viability of E. coli cells expressing hsvTK. As the way to increase the dP sensitivity of yeast cells, we found that the co-expression of human equilibrative nucleoside transporter 1 (hENT1) gene significantly improved the dP sensitivity of the cells and caused the cell death with only sub-micromolar concentration of dP. Furthermore, hsvtk gene was fused to zeocin resistant gene from Streptoalloteichus hindustanus (ble) to conduct ON (zeocin) and OFF (dP) selection seamlessly. By using this fusion gene (hsvtk::ble), we completed both zeocin and dP selection with only 8-hour incubation in liquid media. Thus, we succeeded in significantly reducing the days of incubation steps using the conventional methods on solid media. After zeocin selection, the viability of hsvTK::Ble-expressing cells was 5 x 10⁵ higher than the value of the control cells (not-expressing hsvTK::Ble). In contrast, after dP-selection the viability of hsvTK::Ble-expressing cells got 1 x 10^-4 lower than the value of the control cells.

Finally, we further fused the hsvtk::ble gene with a fluorescent protein gene (monomeric Umikinoko-Green), establishing the novel selection system which enabled the ON/OFF selection and subsequent characterization of genetic switches by the rapid and seamless operations using only liquid handling. Having established the selection/characterization scheme, we will demonstrate the promising, high-quality selections to isolate new yeast genetic switches with varied switching properties.