A Photoclick‐Based High‐Throughput Screening for the Directed Evolution of Decarboxylase OleT

Chemistry - A European Journal, vol. 27, pp. 954–958

Abstract

Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases.

Authors 8

1. Ulrich Markel Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

2. Pia Lanvers Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

3. Daniel F. Sauer Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

4. Malte Wittwer Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

5. Gaurao V. Dhoke Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

6. Mehdi D. Davari Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

7. Johannes Schiffels Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

8. Ulrich Schwaneberg corresponding Aachen Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI—Leibniz Institute for Interactive Materials Forckenbeckstraße 50 52074 Aachen Germany

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany