High-Throughput Analysis and Evolution of Stereoselective Enzymes using Flow Cytometry

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Enzymes are among the most efficient catalysts known, and they are responsible for nearly all the reactions that make life possible. The capability of enzymes to catalyze chemical transformations can also be harnessed to provide environmentally benign routes for the synthesis of pharmaceuticals and other high-value chemicals. However, naturally occurring enzymes must be honed to function in these non-natural reactions. Key to augmenting enzyme capabilities is the development of fast, high-fidelity sensing tools that measure the function of enzymes in large numbers. With support from the Chemical Measurement and Imaging Program in the Division of Chemistry and partial co-funding from the Biosensing Program in the Division of Chemical, Bioengineering, Environmental and Transport Systems at NSF, Dr. Jennifer Heemstra at Emory University uses a high-speed sequential platform to measure the function of millions of different modified versions of naturally occurring enzymes generated by her team, thereby allowing for identification of those enzymes best suited to important non-natural reactions. This project is developing a user-friendly platform that can be widely applied to discover new enzymes for constructing these high-value chemicals. Integrated with this research, Dr. Heemstra develops and broadly disseminates multi-media resources that help to prepare the future workforce in science, technology, engineering, and mathematics. Specifically, these resources provide information and advice on professional skills, including oral and written communication, pioneering new research ideas, promoting diversity and inclusion, and professional networking. These resources are freely available to the general public and utilized by students and researchers nationwide.Dr. Jennifer Heemstra at Emory University is using enantiomeric DNA biosensors to enable high-throughput enantiopurity measurement as a broadly applicable approach to biocatalyst discovery. DNA biosensors are powerful in their ability to transduce the presence of a specific small-molecule target into a dose-dependent fluorescence output. Dr. Heemstra utilizes sensors comprised of each of the two enantiomers of DNA to quantify the concentrations of the two enantiomers of a target small molecule that are produced as a result of an enzymatic reaction. This analysis is integrated with droplet microfluidic technology to enable screening and sorting by flow cytometry. As a result, large libraries of enzyme variants can be rapidly screened, and those having the desired levels of stereoselectivity isolated, thereby accelerating the discovery of new biocatalysts for the synthesis of pharmaceuticals and other high-value chemicals. This research is integrated with an undergraduate curriculum in which students learn how to read and analyze the primary literature and craft original research proposals, as well as publicly available resources to promote the professional development of early-career researchers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.