Dna-linked enzyme-coupled assays

Abstract

Traditional enzyme characterization methods are low-throughput, and therefore limit engineering efforts in synthetic biology and biotechnology. Here we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification. DLEnCA is generalizable for many enzymatic reactions, and here we adapt it for glucosyltransferases, methyltransferases, and oxidoreductases. The assay utilizes cell free transcription/translation systems to produce enzymes of interest, while UDP-Glucose and T4-β-glucosyltransferase are used to modify DNA, which is detected post-reaction using qPCR or similar means of DNA analysis. For monitoring methyltransferases, consumption of SAM is observed by coupling to EcoRI methyltransferase. For monitoring oxidoreductases, consumption of NADH is observed by coupling to Taq orDNA ligase. OleD and two glucosyltransferases fromwere used to verify the assay's generality toward glucosyltransferases. Two methyltransferases from human andwere used to verify the assay's generality towards methyltransferases. We show DLEnCA's utility by mapping out the substrate specificity for these enzymes and observing the multiple steps of a biosynthetic pathway.