High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase

An easy assay: A synthetic substrate enables a colorimetric screen for terpene synthase cyclization activity, thereby facilitating the engineering of these enzymes. By using directed evolution, the thermostability of a sesquiterpene synthase was increased without the loss of other properties. The te...

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Veröffentlicht in:	Angewandte Chemie International Edition 2013-05, Vol.52 (21), p.5571-5574
Hauptverfasser:	Lauchli, Ryan, Rabe, Kersten S., Kalbarczyk, Karolina Z., Tata, Amulya, Heel, Thomas, Kitto, Rebekah Z., Arnold, Frances H.
Format:	Artikel
Sprache:	eng
Schlagworte:	Alkyl and Aryl Transferases - chemistry Alkyl and Aryl Transferases - genetics Catalysis Colorimetry Cyclization directed evolution Directed Molecular Evolution Enzymes Evolution high-throughput screening High-Throughput Screening Assays Methanol - chemistry Molecular Structure Mutation Optimization protein engineering Screening Screens Stabilization Substrate Specificity Terpenes Terpenes - chemical synthesis Terpenes - chemistry thermostability Vinyl Compounds - chemistry
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Beschreibung
Zusammenfassung:	An easy assay: A synthetic substrate enables a colorimetric screen for terpene synthase cyclization activity, thereby facilitating the engineering of these enzymes. By using directed evolution, the thermostability of a sesquiterpene synthase was increased without the loss of other properties. The technique also enabled rapid optimization of conditions for expression and stabilization in lysate of another terpene synthase. PPO=diphosphate.
ISSN:	1433-7851 1521-3773
DOI:	10.1002/anie.201301362