Validation of an insertion-engineered isoprene synthase as a strategy to functionalize terpene synthases

Terpene synthases are biotechnologically-relevant enzymes with a variety of applications. However, they are typically poor catalysts and have been difficult to engineer. Structurally, most terpene synthases share two conserved domains (alpha- and beta-domains). Some also contain a third domain containing a second active site (gamma-domain). Based on the three-domain architecture, we hypothesized that alpha beta terpene synthases could be engineered by insertion of a heterologous domain at the site of the gamma-domain (an approach we term "Insertion-engineering terpene synthase"; Ie-TS). We demonstrate that by mimicking the domain architecture of alpha beta gamma terpene synthases, we can redesign isoprene synthase (ISPS), an alpha beta terpene synthase, while preserving enzymatic activity. Insertion of GFP or a SpyCatcher domain within ISPS introduced new functionality while maintaining or increasing catalytic turnover. This insertion-engineering approach establishes that the gamma-domain position is accessible for incorporation of additional sequence features and enables the rational engineering of terpene synthases for biotechnology.