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Abstract
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.DatabaseModel data are available in the PMDB database under the accession number PM0080780.
Highlights
Enzyme stability is often the limiting factor in the development of commercial enzymes [1,2]
The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies
We discovered that the tetrabutylammonium salt of farnesyl pyrophosphate (FPP) (TBA-FPP), which could be prepared readily and economically from farnesol, was an effective substrate for each of the sesquiterpene synthases we tested
Summary
Enzyme stability is often the limiting factor in the development of commercial enzymes [1,2]. Valuable terpenes have always been extracted directly from the plants that natively produced them, but in the last decade, there have been substantial efforts put into obtaining high-value sesquiterpenes via microbial fermentation Such microbial platforms require TSs that are expressed and active at fermentation temperatures. Another approach developed a presilphiperfolan-8b-ol synthase towards thermostability using a directed evolution approach, leading to an enzyme that retained full activity up to 50 °C (with a T50 value of 54 °C) [19] Both approaches for generating thermostable TSs could be informed by model examples from nature that retain activity up to high temperatures. We demonstrate that one of the enzymes, RoseRS_3509, has a higher turnover rate than any other bacterial TS, and that these two enzymes can be used as the first model for thermostability in this class of enzymes
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