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.

多底物萜烯合酶 (TPS) 与与单一底物反应的典型 TPS 不同。尽管已经报道了少数多底物 TPS 的体外活性，但其中大多数的体内表征尚未得到很好的研究。在这里，来自Cananga odorata的新 TPS ，CoTPS5，属于 TPS-f 亚家族，在体外和体内都进行了功能表征。CoTPS5 与多种链长的异戊二烯焦磷酸底物反应，作为多底物 TPS。它催化香叶基焦磷酸酯形成( E )-β-罗勒烯、( E , E )-α-法呢烯和α-弹簧烯，( E, E )-法呢基焦磷酸酯和香叶基香叶基焦磷酸酯，分别。在本氏烟草中瞬时表达后，CoTPS5 定位于细胞质并仅产生 ( E , E )-α-法呢烯。然而，质体靶向 CoTPS5 在本氏烟草中的表达导致了所有三种化合物的生物合成，( E )-β-罗勒烯、( E , E )-α-法呢烯和 α-springene。类似地，过表达质体靶向 CoTPS5 的转基因拟南芥植物表现出稳定和可持续的 ( E )-β-罗勒烯生产， ( E , E)-α-法呢烯和α-弹簧烯。此外，它们的产生不影响转基因拟南芥植物的生长和发育。我们的结果表明，将多底物 TPS 重定向到不同的细胞内隔室可能是证明多底物 TPS体内活性的有效方法，从而允许在植物中同时产生多种萜类化合物。