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

• Published in: Chemistry : a European journal 2021-01, Vol.27 (3), p.954-958
• Main Authors: Markel, Ulrich, Lanvers, Pia, Sauer, Daniel F., Wittwer, Malte, Dhoke, Gaurao V., Davari, Mehdi D., Schiffels, Johannes, Schwaneberg, Ulrich
• Format: Article
• Language: English
• Subjects: Assaying; Chemistry; Communication; Communications; decarboxylase; Decarboxylation; Directed evolution; Enzymes; Evolution; Fatty acids; high-throughput screening; Hydrogen peroxide; P450; photoclick chemistry; Reductases; Saturation mutagenesis; Screening; Styrene; Substrate preferences; Substrates
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.202003637

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. Enzymatic decarboxylation is synthetically important, but so far, directed evolution of decarboxylases was impeded by the lack of a high‐throughput screening (HTS). Here, a simple photoclick‐based HTS was developed and used to evolve the decarboxylase OleT toward the acceptance of small aromatic substrates. The improved enzyme variants efficiently convert small aromatic substrates while the parent enzyme favors long‐chain fatty acids.