A photoclick‐based high‐throughput screening for the directed evolution of decarboxylase OleT
Ulrich Markel, Pia Lanvers, Daniel F. Sauer, Malte Wittwer, Gaurao V. Dhoke, Mehdi D. Davari, Johannes Schiffels, Ulrich Schwaneberg, Chemistry – A European Journal, 2020, DOI: 10.1002/chem.202003637
Copyright: © BIO VI
In this study, we present a photoclick assay that enables the high-throughput screening of decarboxylase variants in directed evolution.
Despite the synthetic importance of enzymatic oxidative decarboxylation, the directed evolution of decarboxylases is still hampered by the lack of high-throughput screening. In this study, we present a simple photoclick assay for the detection of styrenyl decarboxylation products. In a proof‐of‐principle study, the assay was applied for the directed evolution of the decarboxylase OleT. Our assay is compatible with both potential OleT operation modes, including the direct use of hydrogen peroxide as the enzyme’s co‐substrate or the use of a reductase partner protein. The screening of saturation mutagenesis libraries identified two enzyme variants that altered the substrate preference of the enzyme from long-chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to accelerate the directed evolution of OleT and other decarboxylases.Copyright: © Chemistry – A European Journal