High-throughput (HT) platforms for enzyme discovery and characterization

We use HT functional genomics, proteomics, and structural approaches in silico for systematically identifying and predicting the functions of enzymes involved in nucleic acid modification. Genes predicted to encode enzymes with relevant activities such as cytosine methylation, hydroxymethylation, or base hypermodification are screened for functionality using a golden-gate multiple gene expression module that relies on the endogenous E. coli metabolome as a potential substrate pool. Activities are subsequently reconstituted in vitro using HT methods for protein expression, isolation, and biochemical characterization that we are currently developing in the lab. In addition to HT condition optimization and end-point confirmation of enzyme activity, we aim to implement HT kinetics to inform us on reaction intermediates, side-products, and rates of reaction steps. This information is typically invisible in traditional end-point HT approaches but is essential for mechanistic dissection of enzyme function and for molecular understanding of the enzyme’s modus operandi.