Journal
METABOLIC ENGINEERING
Volume 61, Issue -, Pages 397-405Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2020.08.002
Keywords
Multi-substrate terpene synthase; CoTPS5; (E)-beta-ocimene/(E,E)-alpha-farnesene/alpha-springene synthase; Plastid-targeting; Redirection of terpene synthase; Metabolic engineering; Ylang ylang
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Funding
- National Research Foundation, Prime Minister's Office, Singapore under its Synthetic Biology Research and Development Programme [SBP-P3]
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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)-beta-ocimene, (E,E)-alpha-farnesene and alpha-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)-alpha-farnesene. However, expression of plastid-targeted CoTPS5 in N. benthamiana resulted in biosynthesis of all three compounds, (E)-beta-ocimene, (E,E)-alpha-farnesene and alpha-springene. Similarly, transgenic Arabidopsis plants overexpressing plastid-targeted CoTPS5 showed stable and sustainable production of (E)-beta-ocimene, (E,E)-alpha-farnesene and alpha-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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