Engineering Cotton (+)-δ-Cadinene Synthase to an Altered Function: Germacrene D-4-ol Synthase
2006; Elsevier BV; Volume: 13; Issue: 1 Linguagem: Inglês
10.1016/j.chembiol.2005.10.016
ISSN1879-1301
AutoresYasuo Yoshikuni, Vincent J. J. Martin, Thomas E. Ferrin, Jay D. Keasling,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoThe 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%).
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