Gene identification and characterization of enzymes involved in alkaloid and sesquiterpenoids biosyntheses in Polygonum tinctorium L. and Piper nigrum L.

학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2018. 2. 김수언.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 (PnMCHLPnPKS1 and 2Pn4CL3Part I: Isolation and functional studies of indole synthase from Polygonum tinctorium L. 1 Introduction 2 Polygonum tinctorium L. 2 Indole biosynthetic pathway 2 Indole synthase 6 The purposes of research 10 Materials and Methods 11 Plant material and growth conditions 11 Bacterial strains and culture media 11 Plasmids 11 Enzyme and chemicals 12 Oligonucleotides 12 Genomic DNA, total RNA isolation and cDNA synthesis 14 Isolation of IGL and UTR sequences 14 Complementation assay in E. coli ΔtrpA ΔtnaA 15 QRT-PCR 15 Determination of subcellular localization 15 Bioinformatics analyses 16 Results and Discussion 17 Cloning of IGLs 17 Analysis of UTR sequences 22 Complementation of E. coli ΔtnaA ΔtrpA by PtIGL-short 28 PtIGL transcript levels among plant organs 32 Change in IGL transcription upon BTH treatment 32 Intracellular localization of PtIGLs 38 Part II: Cloning and functional analysis of three sesquiterpene synthases identified by transcriptome sequencing of peppercorn 43 Introduction 44 Piper nigrum L. 44 Transcriptome sequencing 44 Black pepper sesquiterpenoids 44 The purposes of this study 45 Materials and Methods 46 Plant material and growth conditions 46 Bacterial, yeast strains and culture media 46 Enzyme and chemicals 47 Oligonucleotides 47 Total RNA isolation and cDNA preparation 49 Isolation of sesqui-TPSs 49 Yeast transformation and fermentation 50 Transient expression in N. benthamiana 50 Heterologous expression and in-vitro assay 50 Steady-state kinetics 51 GC-MS analysis 52 QRT-PCR 52 Bioinformatics analyses 53 Results and Discussion 54 Transcriptome analysis 54 Screening of sesqui-TPS 56 Analysis of sesquiterpenes in pepper fruit 62 Functional analyses of sesqui-TPSs 67 Kinetic parameters 74 Catalytic mechanism 74 Phylogenetic analysis 78 PnTPS transcript levels among pepper organs 82 Part III: Isolation of genes putatively involved in piperine biosynthesis in Piper nigrum L. 87 Introduction 88 The piperine 88 The purposes of this study 88 Materials and Methods 90 Bacterial, yeast strains and culture media 90 Enzyme and chemicals 90 Oligonucleotides 91 Isolation of genes putatively involved in piperine biosynthesis 94 Yeast PAD1, FDC1 disruption mutant 94 Heterologous expression and protein purification 95 In-vitro assay 96 In-vivo bioconversion assay by co-transformants 96 Analysis of metabolites 100 GC-MS analysis 100 LC-MS analysis 101 QRT-PCR 101 Bioinformatics analyses 102 Results and Discussion 103 Metabolites profiling 103 Search for genes involved in piperine biosynthesis 106 Side chain extension of phenylpropenate 108 PnMCHL, an enzyme for 3,4-MDCA-specific side chain cleavage 114 Piperate:Coenzyme A ligation by Pn4CL3 126 Piperidine-piperoyltransferase 130 Conclusion 134 Supplementary Data 138 References 154 Abstract in Korean 160 Curriculum Vitae 162 Publications and Patents 163Docto