Abstract：
Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.
摘要：
冰片,樟脑,乙酸冰片酯和乙酸冰片酯是广泛用于医学的极有前途的单萜类化合物，风味,食物,和化学应用。二磷酸冰片酯（BPP）是这些单萜生物合成的常见前体。尽管在多种植物中已鉴定出催化香叶酰二磷酸（3GPP）环化为BPP的二磷酸冰片酯合酶（BPPS），负责BPP水解以产生冰片的酶尚未被报道。这里,我们进行了体外和体内功能表征，以鉴定来自W.villosa的Nudix水解酶WvNUDX24，特异性催化BPP水解生成磷酸冰片酯（BP），然后BP在磷酸酶的作用下形成冰片。亚细胞定位实验表明，BPP的水解可能发生在细胞质中。此外，定点诱变实验表明，对于WvNUDX24的水解活性，有四个关键残基（R84，S96，P98和G99）。此外,磷脂酸磷酸酶（PAP）的功能鉴定表明，WvPAP5和WvPAP10能够水解香叶基香叶基香叶基二磷酸（GGMP）和法尼基二磷酸（FPP）生成香叶基香叶基磷酸（GGP）和法尼基磷酸（FP），分别,但不能水解BPP，3GPP,和橙基二磷酸（NPP）生产相应的单磷酸盐产品。这些发现强调了WvNUDX24在BPP水解生产冰片的第一步中的重要作用，并通过植物代谢工程和合成生物学为BPP相关萜类化合物的生产提供了遗传元件。
