Production of multiple terpenes of different chain lengths by subcellular targeting of multi-substrate terpene synthase in plants |
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Institution: | 1. State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, PR China;2. Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming, 650201, PR China;3. University of Chinese Academy of Sciences, Beijing, 100049, PR China;1. Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN 37996, USA;2. Department of Biochemistry, Max Planck Institute for Chemical Ecology, Hans-Knoell-Strasse 8, D-07745 Jena, Germany;3. Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA;4. College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China |
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Abstract: | Multi-substrate terpene synthases (TPSs) are distinct from typical TPSs that react with a single substrate. Although in vitro activity of few multi-substrate TPSs have been reported, in vivo characterization has not been well investigated for most of them. Here, a new TPS from Cananga odorata, CoTPS5, belonging to TPS-f subfamily was functionally characterized in vitro as well as in vivo. CoTPS5 reacted with multiple prenyl-pyrophosphate substrates of various chain lengths as a multi-substrate TPS. It catalyzed the formation of (E)-β-ocimene, (E,E)-α-farnesene and α-springene from geranyl pyrophosphate, (E,E)-farnesyl pyrophosphate and geranylgeranyl pyrophosphate, respectively. Upon transient expression in Nicotiana benthamiana, CoTPS5 localized to cytosol and produced only (E,E)-α-farnesene. However, expression of plastid-targeted CoTPS5 in N. benthamiana resulted in biosynthesis of all three compounds, (E)-β-ocimene, (E,E)-α-farnesene and α-springene. Similarly, transgenic Arabidopsis plants overexpressing plastid-targeted CoTPS5 showed stable and sustainable production of (E)-β-ocimene, (E,E)-α-farnesene and α-springene. Moreover, their production did not affect the growth and development of transgenic Arabidopsis plants. Our results demonstrate that redirecting multi-substrate TPS to a different intracellular compartment could be an effective way to prove in vivo activity of multi-substrate TPSs and thereby allowing for the production of multiple terpenoids simultaneously in plants. |
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Keywords: | Multi-substrate terpene synthase CoTPS5 Plastid-targeting Redirection of terpene synthase Metabolic engineering Ylang ylang |
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