Abstract 1513 Identifying serine hydrolase activity in E. coli utilizing gene knockout strains

Serine hydrolases compose a diverse enzyme family capable of acting as catalysts for various reactions including esterase, thioesterase, epoxidase, and amidase activities. With this diverse reactivity, serine hydrolases have found utility in a wide array of biocatalytic applications such as organic synthesis and the development of prodrugs. The identification of novel and unique serine hydrolase activity in Escherichia coli is of interest because results could potentially be exploited to create and discover prodrugs, specifically for targeted antimicrobial resistance. This project focused on fifty-six E. coli gene knockouts thought to possibly possess serine hydrolase activity based on past activity-based protein profiling screens, bioinformatic analysis of serine hydrolase signatures in E. coli, and past publications suggesting potential serine hydrolase activity. Whole cell lysates were collected from all fifty-six knockout strains and a normal parent strain at various time points along the standard E. coli logarithmic growth scale. In-gel fluorescence imaging based on native-PAGE separation of whole cell lysates were employed to measure esterase activity against variable fluorogenic ester substrates. After pinpointing multiple esterases connected with the measured in gel activity, esterase activity was confirmed via heterologous expression and purification of the identified proteins followed by detailed enzymatic characterization across a variety of fluorogenic ester substrates. Promising combinations of ester substrates and E. coli knockouts, including E. coli proteins yjfP and tesA, have been confirmed to possess high esterase activity under logarithmic growth conditions and may therefore be useful in future biocatalytic applications.