PROJECT SUMMARY The overall objective of this work is to develop and apply a droplet microfluidic system to facilitate rapid engineering of enzymes to synthesize drug molecules. Development of new medicines requires synthesis of complex molecules from initial lead compounds for testing. Once a drug is identified, efficient synthesis is needed for clinical trials and ultimately widespread production. Traditionally such syntheses utilize metal-based catalysts. Enzyme and cell-based systems offer numerous potential advantages including greater selectivity in installing functional groups, greener reactions, more efficient catalysts, and low toxicity. Creating biocatalysts with the desired selectivity requires enzyme engineering. The potential of enzyme engineering is seen in the awarding of a Nobel Prize in 2018 and uptake by pharmaceutical manufacturers. Enzyme engineering requires creation and isolation of thousands of enzyme variants, incubation of substrates with variants, screening of the variants for reaction activity, and identification of variants for further mutation and evolution. Current methods for engineering that use robotics, well plates, and liquid chromatography-mass spectrometry are time and resource intensive thus limiting the use in medicinal chemistry. Droplet microfluidics has potential to profoundly improve enzyme engineering through greater speed and substantially reduced materials requirements. In these methods, individual enzyme variants are encapsulated into droplets, screened for product formation, and sorted based on signal. The low volumes required (< 10 nL/reaction) and high-throughput (over 1000 samples/s) are dramatic improvements over current well-plate methods. However, early demonstrations of enzyme engineering by droplet microfluidics are impractical for development of biocatalysts due to a reliance on fluorescence detection in screening. We propose to create droplet microfluidic enzyme engineering systems that utilize mass spectrometry (MS)-based detection, offering the potential for label-free and information-rich screens at high throughput. In preliminary work, we have developed “mass-activated droplet sorting” (MADS) which can sort enzymes expressed in vitro based on their activity detected by MS. We will build on this achievement to create a versatile system with advanced analytical measurements for enzyme engineering. Many enzyme engineering protocols call for expressing the variants in microbes, therefore we will develop tools to allow individual microbe strains to be grown in single droplets and sorted by MS. Prior work has relied on direct analysis of droplets by MS; however, this precludes separations of isomers and can be vulnerable to matrix effects on signal. We will expand analytical options by interfacing droplets to rapid LC-MS and ion mobility-MS to offer separations of isomers and matrix before MS detection. The system will be used to engineer pyridoxal phosphate-dependent enzymes for the diver...