Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes

The terpenoid family of natural products is being targeted for heterologous microbial production as a cheaper and more reliable alternative to extraction from plants. The key enzyme responsible for diversification of terpene structure is the class‐I terpene synthase (TS), and these often require engineering to improve properties such as thermostability, robustness and catalytic activity before they are suitable for industrial use. Improving thermostability typically relies on screening a large number of mutants, as there are no naturally thermostable TSs described upon which to base rational design decisions. We have characterized the first examples of natural TSs exhibiting thermostability, which catalyse the formation of the sesquiterpene τ‐muurolol at temperatures up to 78 °C. We also report an enzyme with a k (cat) value of 0.95 s(−1) at 65 °C, the highest k (cat) recorded for a bacterial sesquiterpene synthase. In turn, these thermostable enzymes were used as a model to inform the rational engineering of another TS, with the same specificity but low sequence identity to the model. The newly engineered variant displayed increased thermostability and turnover. Given the high structural homology of the class‐I TS domain, this approach could be generally applicable to improving the properties of other enzymes in this class. DATABASE: Model data are available in the PMDB database under the accession number PM0080780.