(394d) Modular and Integrative Activity Reporters Enhance Biochemical Studies in the Yeast ER.

Proteases are ubiquitous enzymes that regulate cellular processes. When dysregulated, proteases are involved in disease onset and progression. Therefore, there is a lot of interest in investigating and modulating disease-associated proteases for fundamental research and therapeutic applications. Recently, it was shown that one can model and characterize protease activity by transforming plasmid-based activity reporters in S. cerevisiae (1). This system couples yeast endoplasmic reticulum sequestration with yeast surface display to measure and engineer protease activity by flow cytometry and fluorescence-activated cell sorting. Although they have been successful, these plasmid-based systems yield a significant population of non-displaying cells and suffer from transcriptional noise stemming from variations in plasmid copy numbers and intrinsic transcriptional noise (2). These two drawbacks lower the dynamic range and reproducibility of catalytic activity measurements on the yeast surface, especially when comparing relative activity levels. To remedy this, we present an integration-based platform that empowers protease activity engineering in yeast (Figure 1A). Integration of our transcriptional cassettes, both fully or partially, yielded significant improvements in signal noise and coefficient of variance (Figure 1B). We attribute this largely due to the uniform expression of the integrated cassettes and the corresponding removal of non-displaying cell populations that were present in our plasmid-based system. We observed a 7.5-fold and 8.4-fold changes in tobacco etch virus protease (TEVp) for the plasmid and integrative systems, respectively, when enzyme expression is under a moderately-strong inducible promoter (Figure 1C). TEVp activity increased 30-fold when the driving promoter was swapped for a strong galactose-inducible configuration, with the trend remaining consistent for both systems. This approach to studying protease activity in yeast via integration was confirmed on papain-like protease (PLpro) and, in principle, can be applied to any active protease within a yeast-based system. We envision that this platform can be tailored to efficiently engineer and profile proteases while also harboring the potential for drug discovery of protease-targeting molecules.

References: (1) Denard CA, et al., ACS Synth. Biol. 2021. (2) Lee ME, et al., ACS Synth. Biol. 2015.