Engineering Seminar

Abstract: Proteases and other post-translational modification (PTM) enzymes are uniquely powerful yet hard to engineer and reprogram because their most important properties (activity, specificity, inhibition mode) are difficult to measure at a library scale. In our lab, we use Saccharomyces cerevisiae as a programmable “organelle reactor,” exploiting compartmental sequestration (especially the ER) and quantitative cell-surface readouts to translate otherwise invisible biochemical events into selectable phenotypes. This strategy builds principles we’ve outlined for yeast sequestration as a general route to study and engineer PTM enzymes, and it has enabled a family of high-dynamic-range platforms that couple enzyme activities to high-throughput selection. Against this backdrop, I will highlight four vignettes of our research in the past few years: (i) PERRC, where tunable ER retention provides defined operating conditions for protease evolution; (ii) HARP, which directly isolates inhibitory macromolecules from antibody-derived libraries by linking ER protease inhibition to a selectable surface phenotype; (iii) our extension of these ideas to sortases with HEROS(S), enabling parallel evolution/profiling of ligase specificity and discovery of nucleophile–substrate pairs that mitigate reversibility; and (iv) a scalable yeast model to functionally interrogate clinically observed SRP54 variants in an ER-targeting pathway that remains difficult to study by other means. Together, these approaches establish a generalizable framework for engineering and interrogating complex enzymatic functions in living cells, enabling advances in enzyme design, inhibitor discovery, and functional genomics.

Bio: Dr. Denard received his B.S. degree in Chemical Engineering from North Carolina State University. He then moved to the Chemical and Biomolecular Engineering department at the University of Illinois at Urbana-Champaign, where he obtained his PhD under the tutelage of Prof. Huimin Zhao. As a Dow Chemical fellow, he focused his PhD thesis on developing cooperative one-pot chemoenzymatic reactions. Dr. Denard later completed a postdoctoral fellowship in the lab of Prof. Brent Iverson at the University of Texas at Austin, establishing high-throughput platforms to engineer the substrate specificity of proteases, enabling their use as protein therapeutics. Dr. Denard joined the University of Florida Chemical Engineering department as an Assistant Professor in January 2020. His research focuses on protein engineering for therapeutic and synthetic biology applications. Dr. Denard is the recipient of the NIH MIRA and NSF CAREER awards.