The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-δ-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-δ-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-δ-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-δ-cadinene synthase suggested that the G helix plays a very important role in (+)-δ-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%). ©2006 Elsevier Ltd All rights reserved.
Yoshikuni, Y., Martin, V. J. J., Ferrin, T. E., & Keasling, J. D. (2006). Engineering cotton (+)-δ-cadinene synthase to an altered function: Germacrene D-4-ol synthase. Chemistry and Biology, 13(1), 91–98. https://doi.org/10.1016/j.chembiol.2005.10.016