Despite intensive research in recent years, the biosynthetic route to costunolide in sunflower so far remained obscured. Additional P450 sequences from public sunflower transcriptomic database were screened to search for candidate enzymes which are able to introduce the 6α-hydroxy-group required for the esterification with the carboxy group of germacarane A acid, the final step in costunolide formation. CYP71BL9, a new P450 enzyme from sunflower was shown to catalyze this hydroxylation, hence being identified as HaCOS. Phylogentically, HaCOS is closer related to HaG8H than to any other known costunolide synthase in Asteraceae.The enzyme was successfully employed to reconstruct the sunflower biosynthesis of costunolide in transformed tobacco. Contrary, in yeast, only minor amounts of sesquiterpene lactone was produced, while 5-hydroxyfarnesylic acid was formed instead. HaCOS in combination with HaG8H produced 8β-hydroxycostunolide (eupatolide) in transformed plants, thus indicating that sunflower possesses two independent modes of eupatolide synthesis via HaCOS and via HaES. The lack of HaCOS expression and of costunolide in trichomes suggests that the enzyme triggers the costunolied synthesis of the inner tissues of sunflower and might be linked to growth regulation processes.

中文翻译：

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CYP71BL9，向日葵木香内酯合成的缺失环节

尽管近年来进行了深入研究，但迄今为止，向日葵中木香内酯的生物合成途径仍不清楚。对来自公共向日葵转录组数据库的其他 P450 序列进行了筛选，以寻找能够引入 6α-羟基基团的候选酶，该基团是与木香木炭酸的羧基酯化所需的，这是木香内酯形成的最后一步。CYP71BL9 是一种来自向日葵的新 P450 酶，经证实可催化这种羟基化作用，因此被鉴定为 HaCOS。在系统发育上，HaCOS 与 HaG8H 的亲缘关系比与菊科中任何其他已知木香内酯合成酶的关系更密切。该酶已成功用于重建向日葵转化烟草中木香内酯的生物合成。相反，在酵母中，仅产生少量倍半萜内酯，而5-羟基法呢酸则形成。HaCOS 与 HaG8H 结合在转化植物中产生了 8β-羟基木香内酯（eupatolide），因此表明向日葵具有通过 HaCOS 和通过 HaES 两种独立的 eupatolide 合成模式。毛状体中缺乏 HaCOS 表达和木香内酯表明该酶触发了向日葵内部组织的木香内酯合成，并且可能与生长调节过程有关。