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Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture

BACKGROUND: Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris sub...

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Autores principales: Caniard, Anne, Zerbe, Philipp, Legrand, Sylvain, Cohade, Allison, Valot, Nadine, Magnard, Jean-Louis, Bohlmann, Jörg, Legendre, Laurent
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3520730/
https://www.ncbi.nlm.nih.gov/pubmed/22834731
http://dx.doi.org/10.1186/1471-2229-12-119
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author Caniard, Anne
Zerbe, Philipp
Legrand, Sylvain
Cohade, Allison
Valot, Nadine
Magnard, Jean-Louis
Bohlmann, Jörg
Legendre, Laurent
author_facet Caniard, Anne
Zerbe, Philipp
Legrand, Sylvain
Cohade, Allison
Valot, Nadine
Magnard, Jean-Louis
Bohlmann, Jörg
Legendre, Laurent
author_sort Caniard, Anne
collection PubMed
description BACKGROUND: Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. RESULTS: Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. CONCLUSIONS: SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.
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spelling pubmed-35207302012-12-13 Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture Caniard, Anne Zerbe, Philipp Legrand, Sylvain Cohade, Allison Valot, Nadine Magnard, Jean-Louis Bohlmann, Jörg Legendre, Laurent BMC Plant Biol Research Article BACKGROUND: Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. RESULTS: Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. CONCLUSIONS: SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes. BioMed Central 2012-07-26 /pmc/articles/PMC3520730/ /pubmed/22834731 http://dx.doi.org/10.1186/1471-2229-12-119 Text en Copyright ©2012 Caniard et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Caniard, Anne
Zerbe, Philipp
Legrand, Sylvain
Cohade, Allison
Valot, Nadine
Magnard, Jean-Louis
Bohlmann, Jörg
Legendre, Laurent
Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture
title Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture
title_full Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture
title_fullStr Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture
title_full_unstemmed Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture
title_short Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture
title_sort discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in salvia sclarea (l.) and their relevance for perfume manufacture
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3520730/
https://www.ncbi.nlm.nih.gov/pubmed/22834731
http://dx.doi.org/10.1186/1471-2229-12-119
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