Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 638, Issue -, Pages 35-40Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2017.12.011
Keywords
Black pepper; Cadinol/cadinene synthase; Caryophyllene synthase; Germacrene D synthase; Next-generation sequencing; Piper nigrum
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Funding
- Cooperative Research Program for Agriculture Science & Technology Development (Next Generation Biogreen 21 Program) [PJ0110552017]
- National Science and Engineering Research Council of Canada (NSERC) [RGPIN-2017-04874]
- Basic Science Research Program through the National Research Foundation (NRF) of Republic of Korea - Ministry of Education [2017R1A6A3A03003409]
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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 beta-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 beta-cadinene and alpha-copaene, the rearrangement products of germacrene D. Their K-cat/K-m 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 beta-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.
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