Jin Zhehao, Kwon Moonhyuk, Lee Ah-Reum, Ro Dae-Kyun, Wungsintaweekul Juraithip, Kim Soo-Un
Department of Agricultural Biotechnology and Research Institute for Agricultural Sciences, Seoul National University, Seoul 08826, Republic of Korea.
Department of Agricultural Biotechnology and Research Institute for Agricultural Sciences, Seoul National University, Seoul 08826, Republic of Korea; Department of Biological Sciences, University of Calgary, Calgary, Canada.
Arch Biochem Biophys. 2018 Jan 15;638:35-40. doi: 10.1016/j.abb.2017.12.011. Epub 2017 Dec 14.
To identify terpene synthases (TPS) responsible for the biosynthesis of the sesquiterpenes that contribute to the characteristic flavors of black pepper (Piper nigrum), unripe peppercorn was subjected to the Illumina transcriptome sequencing. The BLAST analysis using amorpha-4,11-diene synthase as a query identified 19 sesquiterpene synthases (sesqui-TPSs), of which three full-length cDNAs (PnTPS1 through 3) were cloned. These sesqui-TPS cDNAs were expressed in E. coli to produce recombinant enzymes for in vitro assays, and also expressed in the engineered yeast strain to assess their catalytic activities in vivo. PnTPS1 produced β-caryophyllene as a main product and humulene as a minor compound, and thus was named caryophyllene synthase (PnCPS). Likewise, PnTPS2 and PnTPS3 were, respectively, named cadinol/cadinene synthase (PnCO/CDS) and germacrene D synthase (PnGDS). PnGDS expression in yeast yielded β-cadinene and α-copaene, the rearrangement products of germacrene D. Their k/K values (20-37.7 s mM) were comparable to those of other sesqui-TPSs. Among three PnTPSs, the transcript level of PnCPS was the highest, correlating with the predominant β-caryophyllene biosynthesis in the peppercorn. The products and rearranged products of three PnTPSs could account for about a half of the sesquiterpenes in number found in unripe peppercorn.
为了鉴定负责合成对黑胡椒（Piper nigrum）特征风味有贡献的倍半萜的萜烯合酶（TPS），对未成熟的胡椒籽进行了Illumina转录组测序。以紫穗槐-4,11-二烯合酶为查询序列进行的BLAST分析鉴定出19种倍半萜合酶（倍半TPS），其中克隆了三个全长cDNA（PnTPS1至3）。这些倍半TPS cDNA在大肠杆菌中表达以产生用于体外测定的重组酶，并且也在工程酵母菌株中表达以评估它们在体内的催化活性。PnTPS1以β-石竹烯为主要产物，以葎草烯为次要化合物，因此被命名为石竹烯合酶（PnCPS）。同样，PnTPS2和PnTPS3分别被命名为杜松醇/杜松烯合酶（PnCO/CDS）和吉马烯D合酶（PnGDS）。PnGDS在酵母中的表达产生了吉马烯D的重排产物β-杜松烯和α-可巴烯。它们的k/K值（20-37.7 s mM）与其他倍半TPS相当。在三个PnTPS中，PnCPS的转录水平最高，这与胡椒籽中主要的β-石竹烯生物合成相关。三个PnTPS的产物和重排产物约占未成熟胡椒籽中发现的倍半萜数量的一半。
