Molecular cloning and functional characterization of three terpene synthases from unripe fruit of black pepper (Piper nigrum)

• Published in: Archives of biochemistry and biophysics 2018-01, Vol.638, p.35-40
• Main Authors: Jin, Zhehao, Kwon, Moonhyuk, Lee, Ah-Reum, Ro, Dae-Kyun, Wungsintaweekul, Juraithip, Kim, Soo-Un
• Format: Article
• Language: English
• Subjects: Black pepper; Cadinol/cadinene synthase; Caryophyllene synthase; Germacrene D synthase; Next-generation sequencing; Piper nigrum
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2017.12.011

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 kcat/Km values (20–37.7 s−1 mM−1) 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. [Display omitted] •Transcriptome of unripe fruit of Piper nigrum, black pepper plant, was analyzed.•Three sesquiterpene synthases were predicted from transcriptome database and cloned.•They were identified as caryophyllene synthase, cadinol/cadinene synthase, and germacrene D synthase.•Their kinetic parameters and tissue distribution were determined.