쪽(Polygonum tinctorium L.)과 후추(Piper nigrum L.)에서 알칼로이드와 세스퀴테펜 생합성효소의 유전자 동정과 특성

Abstract

PnNAT6 and 7). The genes were expressed in E. coli to obtain proteins for in-vitro assay. In addition, they were expressed in the engineered yeast and E. coli to directly produce the expected products or to effect bioconversion in-vivo. In particular, Part II fully describes sesqui-TPSs mentioned above. PnTPS1 produced caryophyllene as a major product and minor humulene, and thus was named caryophyllene synthase (PnCPS). Likewise, PnTPS2 and PnTPS3 were named cadinol/cadinene synthase (PnCO/CDS) and germacrene D synthase (PnGDS). PnGDS expression in yeast system yielded β-cadinene and α-copaene also found in pepper extract. They were verified as rearrangement products of germacrene D not found in pepper. Part III describes transcriptome-based gene mining for elucidation of piperine biosynthesis. At first, P. nigrum 3,4-methylenedioxycinnamic acid (MDCA) hydratase-lyase (PnMCHL) responsible for conversion of MDCA to piperonal, was identified and described. Piperonylic acid, possibly an oxidation product from piperonal in¬-planta, could undergo 2×C2 extension in the side chain to arrive at C6C5 carbon skeleton of piperic acid, as opposed to the common belief that piperic acid skeleton would be the results of one C2 extension from MDCA. Also described is 3,4-methylenedioxyphenyl-specific 4-coumaroyl-coenzyme A ligase (Pn4CL3) which converted piperic acid into piperoyl-CoA. Finally, two clones coding enzymes for transfer of piperoyl-CoA to piperidine are described.

This thesis presents enzymes involved in biosynthesis of plant alkaloids, indigo and piperine from Polygonum tinctorium L. and Piper nigrum L., respectively, and those in sesquiterpene synthesis in P. nigrum. The first chapter presents indole synthase from P. tinctorium. Indigo is an old natural blue dye produced by plants such as P. tinctorium. The first key step in plant indigoid biosynthesis is the production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit homologs, PtIGL-short and -long forms on genome of the plant contained two genes each coding for IGL. The short and the long forms respectively encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate, signifying the production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant. It was tentatively named PtTSA. PtTSA is transported to the chloroplast as predicted by 42 amino acid residues of targeting sequence, whereas PtINS is localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to five-fold higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA significantly enhanced in the plant. The second and the third chapter respectively focuses on sesquiterpene synthesis imparting characteristic peppery bouquet and piperine alkaloid biogenesis responsible for pungent taste of black pepper. The unripe peppercorn was submitted to transcriptome analysis utilizing the Illumina next-generation sequencing (NGS). Compared with gene cloning based on rapid amplification of cDNA ends (RACE)-PCR, NGS technology offers more cost-effective and time-saving alternative to identify specific gene by collecting massive sequencing data. Using Local tBLASTn routine against query genes with similar biochemical functions, I have found three full-length of sesquiterpene synthase (sesqui-TPS) clones (PnTPS1 through 3) and four kinds of enzymes putatively involved in piperine biosynthesis (PnMCHL

PnPKS1 and 2

Pn4CL3