Department of Biology, University of British Columbia-Okanagan, 3333 University Way, Kelowna, British Columbia, V1V 1V7, Canada.
Planta. 2011 Apr;233(4):685-96. doi: 10.1007/s00425-010-1332-5. Epub 2010 Dec 17.
En route to building genomics resources for Lavandula, we have obtained over 14,000 ESTs for leaves and flowers of L. angustifolia, a major essential oil crop, and identified a number of previously uncharacterized terpene synthase (TPS) genes. Here we report the cloning, expression in E. coli, and functional characterization of β-phellandrene synthase, LaβPHLS. The ORF--excluding the transit peptide--for this gene encoded a 62.3 kDa protein that contained all conserved motifs present in plant TPSs. Expression in bacteria resulted in the production of a soluble protein that was purified by Ni-NTA agarose affinity chromatography. While the recombinant LaβPHLS did not utilize FPP as a substrate, it converted GPP (the preferred substrate) and NPP into β-phellandrene as the major product, with K (m) and k (cat) of 6.55 μM and 1.75 × 10(-2) s(-1), respectively, for GPP. The LaβPHLS transcripts were highly abundant in young leaves where β-phellandrene is produced, but were barely detectable in flowers and older leaves, where β-phellandrene is not synthesized in significant quantities. This data indicate that β-phellandrene biosynthesis is transcriptionally and developmentally regulated. We also cloned and expressed in E. coli a second TPS-like protein, LaTPS-I, that lacks an internal stretch of 73 amino acids, including the signature DDxxD divalent metal binding motif, compared to other plant TPSs. The recombinant LaTPS-I did not produce detectable products in vitro when assayed with GPP, NPP or FPP as substrates. The lack of activity is most likely due to the absence of catalytically important amino acid residues within the missing region.
在为薰衣草构建基因组资源的过程中，我们已经获得了超过 14000 个薰衣草属植物 angustifolia 的叶片和花朵的 ESTs，这是一种主要的精油作物，并鉴定了一些以前未被描述的萜烯合酶（TPS）基因。在这里，我们报告了 β-水芹烯合酶，LaβPHLS 的克隆、在大肠杆菌中的表达和功能特征。该基因的 ORF——不包括转运肽——编码一个 62.3 kDa 的蛋白质，其中包含所有存在于植物 TPS 中的保守基序。在细菌中的表达导致产生一种可溶的蛋白质，该蛋白质通过 Ni-NTA 琼脂糖亲和层析进行纯化。虽然重组 LaβPHLS 不以 FPP 作为底物，但它将 GPP（首选底物）和 NPP 转化为 β-水芹烯作为主要产物，GPP 的 K（m）和 k（cat）分别为 6.55 μM 和 1.75×10(-2) s(-1)。LaβPHLS 的转录物在产生 β-水芹烯的幼叶中高度丰富，但在花和老叶中几乎检测不到，在花和老叶中，β-水芹烯没有大量合成。这些数据表明，β-水芹烯的生物合成受到转录和发育的调控。我们还在大肠杆菌中克隆并表达了另一种 TPS 样蛋白，LaTPS-I，与其他植物 TPS 相比，它缺少一段 73 个氨基酸的内部序列，包括特征性的 DDxxD 二价金属结合基序。当用 GPP、NPP 或 FPP 作为底物进行体外测定时，重组 LaTPS-I 没有产生可检测的产物。缺乏活性很可能是由于缺失区域内缺乏催化重要的氨基酸残基所致。
