Targeted Engineering of Cyclooctat-9-en-7-ol Synthase: A Stereospecific Access to Two New Non-natural Fusicoccane-Type Diterpenes

Authors

  • Christian Görner,

    1. Fachgebiet für Industrielle Biokatalyse, Zentrum für Weiße Biotechnologie, Department für Chemie, Technische Universität München, Lichtenberg Str. 4, 85748 Garching (Germany), Fax: (+49) 89-289-13255
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  • Ina Häuslein,

    1. Fachgebiet für Industrielle Biokatalyse, Zentrum für Weiße Biotechnologie, Department für Chemie, Technische Universität München, Lichtenberg Str. 4, 85748 Garching (Germany), Fax: (+49) 89-289-13255
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  • Patrick Schrepfer,

    1. Fachgebiet für Industrielle Biokatalyse, Zentrum für Weiße Biotechnologie, Department für Chemie, Technische Universität München, Lichtenberg Str. 4, 85748 Garching (Germany), Fax: (+49) 89-289-13255
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  • Dr. Wolfgang Eisenreich,

    1. Lehrstuhl für Biochemie, Zentrum für Isotopolog Studien, Department für Chemie, Technische Universität München, Lichtenberg Str. 4, 85748 Garching (Germany)
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  • Prof. Dr. Thomas Brück

    Corresponding author
    1. Fachgebiet für Industrielle Biokatalyse, Zentrum für Weiße Biotechnologie, Department für Chemie, Technische Universität München, Lichtenberg Str. 4, 85748 Garching (Germany), Fax: (+49) 89-289-13255
    • Fachgebiet für Industrielle Biokatalyse, Zentrum für Weiße Biotechnologie, Department für Chemie, Technische Universität München, Lichtenberg Str. 4, 85748 Garching (Germany), Fax: (+49) 89-289-13255

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Abstract

The structural diversity of bioactive diterpenes is due to variations in their macrocyclic carbon skeletons. The chemical synthesis of these macrocycles is challenging. However, the bacterial diterpene synthase cyclooctat-9-en-7-ol synthase (CotB2) generates a complex macrocycle in a single step with geranylgeranyl diphosphate as an aliphatic substrate. This study investigates the catalytic mechanisms of the native and mutant CotB2, with a focus on identifying new carbon macrocycles. The combination of in silico modelling, targeted diterpene cyclase engineering and structural elucidation by using GC–MS, HRMS and NMR analysis resulted in the identification of new terpene olefins. CotB2 mutants produced two new non-natural fusicoccane-type macrocycles with potential bioactivities and the monocyclic compound cembrene. The observed product pattern allowed insights into the mechanistic features of CotB2. Applied strategies enable new consolidated synthesis of natural and non-natural terpenoid bioactives.

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