EP3472309A2 - Prényltransférases stilbénoïdes provenant de plantes - Google Patents

Prényltransférases stilbénoïdes provenant de plantes

Info

Publication number
EP3472309A2
EP3472309A2 EP17814218.8A EP17814218A EP3472309A2 EP 3472309 A2 EP3472309 A2 EP 3472309A2 EP 17814218 A EP17814218 A EP 17814218A EP 3472309 A2 EP3472309 A2 EP 3472309A2
Authority
EP
European Patent Office
Prior art keywords
resveratrol
arachidin
peanut
ahr3
prenylated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP17814218.8A
Other languages
German (de)
English (en)
Other versions
EP3472309A4 (fr
Inventor
Luis Fabricio Medina-Bolivar
Tianhong Yang
Keithanne Mockaitis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arkansas State University
Original Assignee
Arkansas State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Arkansas State University filed Critical Arkansas State University
Priority claimed from PCT/US2017/037995 external-priority patent/WO2017218967A2/fr
Publication of EP3472309A2 publication Critical patent/EP3472309A2/fr
Publication of EP3472309A4 publication Critical patent/EP3472309A4/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)

Definitions

  • the present invention relates generally to prenylated stilbenoids and the production of prenylated stilbenoids.
  • phytoalexins A substantial part of non-host defense responses in many plants is the pathogen-induced production of secondary metabolites, generally termed phytoalexins, that locally restrict disease progression due to bioactivities toxic to the pathogen (reviewed in Ahuja et al., 2012).
  • Peanut or groundnut (Arachis hypogaed) tissues mount a defense against infection by the soil fungus Aspergillus flavus and other pathogens by overproducing stilbene derivatives around sites of wounding and elicitor perception (Sobolev, 2013).
  • the present invention is related to prenylated stilbenoids and a method of producing the prenylated stilbenoids. Harnessing the inducible bioproduction capabilities of the peanut hairy root culture system, we have newly produced a prenylated stilbenoid, i.e., arachidin-5, and have demonstrated that the prenyl moiety on peanut prenylated stilbenoids is derived from a plastidic biosynthesis pathway. We have characterized for the first time plant membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots.
  • stilbenoid prenyltransferase genes With multidisciplinary approaches, we have isolated stilbenoid prenyltransferase genes and comprehensively characterized their functionality as stilbenoid prenyltransferase via a transient expression system in Nicotiana benthamiana and stable expression in tobacco plants and hairy roots. Moreover, we have observed the enzymatic degradation of exogenous resveratrol by peanut hairy root tissue, an observation that will lead to elucidation of further mechanisms governing phytoalexin accumulation in plants.
  • metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway.
  • microsomal fraction-derived prenyltransferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor.
  • the microsomal fraction also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid produced for the first time in this study.
  • stilbenoid prenyltransferase genes from peanut and confirmed their functionality as stilbenoid prenyltransferase via a transient expression system in Nicotiana benthamiana and stable expression in tobacco plants and hairy roots.
  • prenyltransferases AhR4DT-l catalyzes a key reaction involved in the biosynthesis of prenylated stilbenoids, in which resveratrol is prenylated at its C-4 position to form arachidin-2, while another, AhR3'DT- 1, was able to add the prenyl group to C-3' of resveratrol.
  • Each of these prenyltransferases has a high specificity for stilbenoid substrates, and their subcellular location in the plastid was confirmed by fluorescence microscopy. Structure analysis of the prenylated stilbenoids suggest that these two prenyltransferase activities represent the first committed steps in the biosynthesis of a large number of prenylated stilbenoids and their derivatives in peanut.
  • Stilbenoids are phenylpropanoid compounds that accumulate in response to biotic and abiotic stresses in a small number of higher plant families including grape (Vitaceae), pine (Pinaceae) and peanut (Fabaceae). These compounds serve as phytoalexins and provide protection to the host plant against various microbial pathogens (Ahuja et al., 2012).
  • Resveratrol studied compound in the stilbene family has attracted great attention in the scientific community, not only because of its important role for disease defense in plants (Ahuja et al., 2012), but also because of numerous bioactivities including anticancer, cardioprotective, antioxidant, anti-inflammatory and neuroprotective properties in human cell culture and in vivo, although the extent of its bioavailability has been questioned (Gambini et al., 2015; Tome-Carneiro et al., 2013; Baur and Sinclair, 2006).
  • resveratrol is synthesized in peanut, along with stilbenoids conjugated to a prenyl group, a modification not common in other stilbene-producing plants (Aguamah et al., 1981; Cooksey et al., 1988; Sobolev et al., 2006).
  • the prenylation of the stilbene backbone is the primary feature that contributes to the diversity of these peanut secondary metabolites.
  • Prenylated stilbenoids have shown equivalent or enhanced bioactivities relative to non-prenylated forms, such as resveratrol, in in vitro studies (Huang et al., 2010; Chang et al., 2006; Sobolev et al., 2011).
  • the prenylated stilbenoids arachidin-1 and arachidin-3 showed favorable metabolic profiles when compared to their non- prenylated analogs piceatannol and resveratrol (Brents et al., 2012).
  • arachidin-1 and arachidin-3 exhibited specific bioactivities not found in their non-prenylated forms, such as inhibiting the replication of rotavirus in HT29.f8 cells (Ball et al., 2015). Also, prenylated stilbenoids showed higher affinity to human cannabinoid receptors when compared to non- prenylated stilbenoids (Brents et al., 2012).
  • Prenyltransferase(s) responsible for these stilbenoid modifications is/are crucial for the biosynthesis of peanut bioactive compounds of interest.
  • no gene encoding a stilbenoid prenyltransferase has been identified in plants.
  • we developed a peanut hairy root culture system to serve as a platform for sustainable production of prenylated stilbenoids (Yang et al., 2015; Condori et al., 2010). Leveraging this system more recently, we characterized biochemically the first stilbenoid-specific prenyltransferase activity from the microsomal fraction of these elicited cultures (Yang et al., 2016).
  • This prenyltransferase utilizes plastid-derived dimethylallyl pyrophosphate (DMAPP) to prenylate resveratrol or piceatannol into arachidin-2 or arachidin-5, respectively, and shares several features in common with flavonoid prenyltransferases reported in other legume species.
  • DMAPP plastid-derived dimethylallyl pyrophosphate
  • RNAseq data assemblies designed to capture elicitor-induced mRNAs, and used these sequences to clone potential prenyltransferase cDNAs.
  • the first transcripts to be identified as encoding stilbenoid-specific prenyltransferase enzymes. These catalyze two distinct dimethylallylation reactions in the biosynthesis of prenylated stilbenoids. Functionalities of the enzymes are uncovered using a transient expression system of Agrobacterium-mfiltr&ted Nicotiana benthamiana leaves, and their subcellular location is proposed from fluorescence microscopy imaging in particle- bombarded onion epidermal cells. We further use available genome assemblies of the two diploid progenitors of A. hypogaea to align the prenyltransferase transcripts and estimate their genomic structure.
  • Figure 1 shows A, Structure of stilbene backbone and main prenylation patterns found on peanut prenylated stilbenoids.
  • B Chemical structures of stilbenoids identified in elicited peanut hairy roots: (a) resveratrol; (b) piceatannol; (c) arachidin-2; (d) arachidin-5; (e) arachidin-3; (f) arachidin-1. All compounds are shown in their trans isomers.
  • Figure 2 shows a HPLC chromatogram (UV 320 nm) of ethyl acetate extract from peanut hairy root culture medium after 72 h post treatment with 100 ⁇ methyl jasmonate and 9 g/L methyl- -cyclodextrin.
  • a arachidin-5 derivative
  • b arachidin-2 derivative.
  • Figure 3 shows effects of mevastatin and clomazone on the production of stilbenoids in elicited peanut hairy root culture.
  • A HPLC chromatograms (UV 340 nm) of ethyl acetate extracts from peanut hairy root cultures after 48-hour treatment with (a) 100 ⁇ methyl jasmonate (MeJA) and 9 g/L methyl-P-cyclodextrin (CD); (b) 100 ⁇ MeJA, 9 g/L CD, and 10 ⁇ mevastatin; (c) 100 ⁇ MeJA, 9 g/L CD, and 10 ⁇ clomazone and (d) 100 ⁇ MeJA, 9 g/L CD, and 100 ⁇ clomazone.
  • MeJA methyl jasmonate
  • CD methyl-P-cyclodextrin
  • the asterisks above bars represent significant difference when compared to the group treated with MeJA and CD alone (*, j? ⁇ 0.05; **, _p ⁇ 0.01; ***, pO.001; ****, p ⁇ 0.0001; n.s., not significant; n.d., not detected ); and the asterisks above the connecting line represent significant difference between 48 hour and 72 hour treatments (*, p ⁇ 0.05; ***, p ⁇ 0.001; ****, p ⁇ 0.0001; n.s., not significant).
  • Statistical analyses were performed by two-way ANOVA with Dunnett's and Sidak's multiple-comparisons test respectively. .
  • Figure 4 shows enzymatic degradation of resveratrol by crude cell-free protein extract from non-elicited peanut hairy root.
  • HPLC chromatograms UV 320 nm of ethyl acetate extracts from the incubation mixtures contained 1 mM resveratrol with (A) 50 ⁇ g crude cell-free protein extract for 30 min; (B) 50 ⁇ g heat-denatured crude cell-free protein extract for 30 min; (C) 50 ⁇ g crude cell-free protein extract and additional 5 mM DTT for 120 min. All reactions were done in a pH 7.6 Tris-HCl buffer. (D) Close-up (3.92-7.19 min) of chromatogram shown in (A).
  • Figure 5 shows resveratrol prenyltransferase activity in microsomal fraction of elicited peanut hairy root.
  • HPLC chromatograms UV 320 nm of (A) purified arachidin-2; (B) ethyl acetate extract of 500 ⁇ reaction mixture containing 30 ⁇ g microsomal fraction, 100 ⁇ resveratrol, 300 ⁇ DMAPP, 10 mM MgCl 2 and 5 mM DTT
  • C ethyl acetate extract of 500 ⁇ reaction mixture containing heat-denatured 30 ⁇ g microsomal fraction, 100 ⁇ resveratrol, 300 ⁇ DMAPP, 10 mM MgCl 2 and 5 mM DTT. All reactions were done for 60 min in a pH 9.2 Tris-HCl buffer.
  • Figure 6 shows substrate specificity of resveratrol prenyltransferase in microsomal fraction of elicited peanut hairy root.
  • A Chemical structures of prenyl acceptors used for substrate specificity analysis and their prenylated products: stilbenoids (al, resveratrol, a2 arachidin-2; bl, piceatannol, b2, arachidin-5; cl, pinosylvin, c2, chiricanine-A, d, oxyresveratrol and e, pterostilbene), flavanone (e, naringenin), flavone (f, apigenin) and isoflavone (g, genistein).
  • B Prenylation activity of microsomal fraction with various prenyl acceptors. Values are the average of 2 replicates and error bars represent standard deviation (n.d., not detected).
  • Figure 7 shows a table of detected stilbenoids.
  • Figure 8 shows a table of resveratrol prenyltransferase activity.
  • Figure 9 shows enzymatic characterization of AhR4DT transiently expressed in Nicotiana benthamiana leaf.
  • Figure 10 shows enzymatic characterization of resveratrol prenyltransferase transiently expressed in Nicotiana benthamiana leaf.
  • HPLC chromatograms UV 320 nm of ethyl acetate extraction of 1000 ⁇ reaction mixture of 200 ⁇ resveratrol, 600 ⁇ DMAPP, 10 mM MgCl 2 and 10 mM DTT incubated with 50 ⁇ crude protein of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring (A) pBIBKan-lOkl ; (B) pBIBKan-4el; (C) pBIBKan-4elO and (D) pBIBKan-5m3 binary vectors in a pH 9.2 Tris-HCl buffer for 40 min.
  • Figure 11 shows the development of transgenic tobacco plants expressing a stilbenoid- specific prenyltransferase from peanut.
  • A, B Regenerated shoots from the Agrobacterium inoculation sites.
  • C Rooting of transgenic tobacco plant.
  • Figure 12 shows a sequence view of the gene and protein sequence of stilbenoid-specific prenyltransferase AhR4DT-9bl3 (renamed as AhR4DT-l; GenBank accession No. KY565244) involved in the biosynthesis of arachidin-2.
  • Figure 13 shows a sequence view of the gene and protein sequence of stilbenoid prenyltransferase AhRPT-4el (Renamed as AhR3'DT-2; GenBank Accession No. KY565246).
  • Figure 14 shows a sequence view of the gene and protein sequence of stilbenoid prenyltransferase AhRPT-4elO (Renamed as AhR3'DT-3; GenBank Accession No. KY565247).
  • Figure 15 shows a sequence view of the gene and protein sequence of stilbenoid prenyltransferase AhRPT-5m3 (Renamed as AhR3'DT-4; GenBank AccesionNo. KY565248).
  • FIG 16 shows a sequence view of the gene and protein sequence of stilbenoid prenyltransferase AhRPT-lOkl (AC1) (Renamed as AhR3'DT-l; GenBank AccesionNo. KY565245).
  • FIG. 17 shows 1H NMR analysis of arachidin-5. ! H NMR was recorded at 400 MHz in acetone-d 6 on a Bruker AV-400 NMR spectrometer.
  • Figure 18 shows 13 C NMR analysis of arachidin-5. 13 C NMR was recorded at 100 MHz in acetone-d 6 on a Bruker AV-400 NMR spectrometer.
  • Figure 19 shows effect of mevastatin on the production of stilbenoids in elicited peanut hairy root culture.
  • HPLC chromatograms UV 340 nm of ethyl acetate extracts from peanut hairy root cultures after 48-hour treatment with (A) 100 ⁇ methyl jasmonate (MeJA) and 9 g/L methyl- -cyclodextrin (CD); (B) 100 ⁇ MeJA, 9 g/L CD, and 1 ⁇ mevastatin; (C) 100 ⁇ MeJA, 9 g/L CD, and 10 ⁇ mevastatin and (D) 100 ⁇ MeJA, 9 g/L CD, and 100 ⁇ mevastatin.
  • MeJA methyl jasmonate
  • CD methyl- -cyclodextrin
  • Figure 20 shows effect of clomazone on the production of stilbenoids in elicited peanut hairy root culture.
  • HPLC chromatograms UV 340 nm of ethyl acetate extracts from peanut hairy root cultures after 48-hour treatment with (A) 100 ⁇ methyl jasmonate (MeJA) and 9 g/L methyl- -cyclodextrin (CD); (B) 100 ⁇ MeJA, 9 g/L CD, and 1 ⁇ clomazone; (C) 100 ⁇ MeJA, 9 g/L CD, and 10 ⁇ clomazone and (D) 100 ⁇ MeJA, 9 g/L CD, and 100 ⁇ clomazone.
  • MeJA methyl jasmonate
  • CD methyl- -cyclodextrin
  • Figure 21 shows effect of clomazone on the production of stilbenoids in non-elicited peanut hairy root culture.
  • HPLC chromatograms UV 340 nm of ethyl acetate extracts from peanut hairy root cultures after 48-hour treatment with (A) 100 ⁇ methyl jasmonate and 9 g/L methyl-P-cyclodextrin; (B) 10 ⁇ clomazone; (C) 50 ⁇ clomazone and (D) 100 ⁇ clomazone.
  • Figure 22 shows comparison of stilbenoid yields from the culture medium and root tissue.
  • Ethyl acetate extracts were prepared from elicited peanut hairy root cultures. HPLC chromatograms (UV 340 nm) of ethyl acetate extract from (A) culture medium and (B) lyophilized root tissue. Cultures were elicited for 48 hours with 100 ⁇ methyl jasmonate and 9 g L methyl- -cyclodextrin.
  • Figure 23 shows time course of resveratrol prenyltansferase assay without DTT and subsequent degradation of resveratrol and arachidin-2 in the reaction. Assays were done for 0 to 120 min. HPLC chromatograms (UV 320 nm) of ethyl acetate extracts from the reaction mixture of 30 ⁇ g microsomal fraction, 100 ⁇ resveratrol, 300 ⁇ DMAPP, and 10 niM MgCl 2 . Assays were done for 0, 10, 20, 30 , 45, 60 or 120 min in a pH 9.2 Tris-HCl buffer.
  • Figure 24 shows biotransformation of arachidin-2 by protein extracts from elicited peanut hairy root culture.
  • HPLC chromatograms (UV 320 nm) of ethyl acetate extracts from the 60 min reaction contained (a) 100 ⁇ arachidin-2 with 30 ⁇ g heat-denatured crude cell-free protein extract as control; (b) 30 ⁇ g crude cell-free protein extract; (c) 30 ⁇ g microsomal fraction and (d) 30 ⁇ g 156,000 g supernatant. All reactions were done in apH 9.2 Tris-HCl buffer.
  • Figure 25 shows resveratrol prenyltransferase activity.
  • Figure 26 shows pH dependency of resveratrol prenyltransferase activity. Resveratrol prenyltransferase activity at various pH values was measured using 100 mM Tris-HCl buffer at pH 7.0, 8.0, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6 and 10.
  • Figure 27 shows divalent cation requirement for resveratrol prenyltransferase activity.
  • Resveratrol prenyltransferase activity was compared in the presence of various divalent cations: Mg 2+ ,' Mn 2+ , Fe 2+ , Ca 2+ , Co 2+ , Zn 2+ , Ni + and Cu 2+ .
  • the activity in the presence of Mg 2+ is shown as 100%.
  • Figure 28 shows biochemical characterization of resveratrol prenyltransferase in microsomal fraction of elicited peanut hairy root.
  • HPLC chromatogram UV 320 nm of ethyl acetate extract of a 60 min incubation mixture containing (A) standard reaction (30 ⁇ g microsomal fraction, 100 ⁇ resveratrol, 300 ⁇ DMAPP, 10 mM MgCl 2 and 5 mM DTT in a pH 9.2 Tris-HCl buffer); (B) standard reaction without divalent cation added; (C) standard reaction with 300 ⁇ IPP instead of DMAPP and (D) standard reaction with 30 g microsomal fraction isolated from non-elicited peanut hairy root instead of 30 ⁇ g microsomal fraction isolated from elicited peanut hairy root.
  • A standard reaction (30 ⁇ g microsomal fraction, 100 ⁇ resveratrol, 300 ⁇ DMAPP, 10 mM MgCl 2 and 5 m
  • Figure 29 shows effects of (A) resveratrol and (B) DMAPP concentrations on resveratrol prenyltransferase activity. Enzymatic activity was measured with a microsomal fraction from peanut hairy roots. The apparent K m and V max values for resveratrol and DMAPP were determined with varying concentrations of resveratrol (10 - 640 ⁇ ) and of DMAPP (10 - 640 ⁇ ) respectively and calculated via nonlinear regression analysis with Michaelis-Menten equation by Graphpad Prism 6 software.
  • Figure 30 shows substrate specificity of resveratrol prenyltransferase in microsomal fraction of elicited peanut hairy root.
  • HPLC chromatograms UV 320 nm
  • ethyl acetate extracts of a 60 min incubation mixture containing 30 ⁇ g microsomal fraction with 300 ⁇ DMAPP, 10 niM MgCl 2 and 5 mM DTT, and 100 ⁇ prenyl acceptors (A, resveratrol; B, piceatannol; C, pinosylvin; D, pterostilbene; E, naringenin; F, apigenin and G, genistein) in a pH 9.2 Tris-HCl buffer.
  • A resveratrol
  • B piceatannol
  • C pinosylvin
  • D pterostilbene
  • E naringenin
  • Figure 31 shows biotransformation of arachidin-5 by protein fractions from elicited peanut hairy root culture.
  • HPLC chromatograms UV 320 nm
  • ethyl acetate extracts from the 60 min incubation mixtures contained 40 ⁇ arachidin-5 with (A) 30 ⁇ g heated microsomal fraction as control and (B) 30 ⁇ g microsomal fraction. All reactions were done in a.pH 9.2 Tris- HCl buffer.
  • Figure 32 shows substrate specificity of resveratrol prenyltransferase in microsomal fraction of elicited peanut hairy root.
  • A Chemical structures of oxyresveratrol and its prenylated product.
  • B HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 ⁇ oxyresveratrol, 300 ⁇ DMAPP, 10 mM MgCl 2 , 5 mM DTT and 30 ⁇ g microsomal fraction (above); heated denatured microsomal fraction (below) in a pH 9.2 Tris- HCl buffer for 60 min.
  • C HPLC-PDA-ESI-MS 3 analysis of oxyresveratrol, prenylated oxyresveratrol and its derivative.
  • Figure 33 shows enzymatic characterization of stilbenoid prenyltransferase genes (AhR4DT-9bl3, AhRPT-lOkl, AhRPT-4el, AhRPT-4elO, and AhRPT5m3) transiently expressed in Nicotiana benthamiana leaf.
  • Figure 34 shows resveratrol prenyltransferase activities in transgenic tobacco ⁇ Nicotiana tabacum) expressing AhRPT-lOkl gene cloned from peanut hairy roots.
  • HPLC chromatograms UV 320 nm of ethyl acetate extraction of 500 ⁇ reaction mixture of 200 ⁇ resveratrol, 300 ⁇ DMAPP, 10 mM MgCl 2 and 10 mM DTT incubated with 50 g crude protein of transgenic N.
  • Figure 35 shows the enzymatic characterization of recombinant stilbenoid
  • Figure 37 shows the phylogenetic relationship between peanut stilbenoid
  • prenyltransferases and related prenyltransferases accepting aromatic substrates are: Aa, Allium ampeloprasum; Ah, Arachis hypogaea; At, Arabidopsis thaliana; CI, Citrus limon; Cp, Cuphea avigera var pulcherrima; Cr, Chlamydomonas reinhardtii; Ct, Cudrania tricuspidata; Gm, Glycine max; HI, Humulus lupulus; Hv, Hordeum vulgare; La, Lupinus albus; Le, Lithospermum erythrorhizon; Ma, Morus alba; Os, Oryza sativa; Pc, Petroselinum crispum; Sf, Sophora flavescens; Ta, Triticum aestivum; Zm, Zea mays.
  • VTE2-ls Homogentisate phytyltransferases
  • HGGTs homogentisate geranylgeranyltransferases
  • VTE2-2s homogentisate solanesyltransferases
  • PPT geranyltransferase
  • PPTs -hydroxybenzoate polyprenyltransferases
  • Figure 38 shows the substrate specificity of AhR4DT-l and AhR3'DT-l.
  • A Chemical structures of prenyl acceptors used for substrate specificity analysis and their prenylated products: stilbenoids (a, resveratrol, b, piceatannol, c, oxyresveratrol, d, pinosylvin, e, piceid and f, pterostilbene), flavanone (g, naringenin), flavone (h, apigenin) and isoflavone (i, genistein).
  • stilbenoids a, resveratrol, b, piceatannol, c, oxyresveratrol, d, pinosylvin, e, piceid and f, pterostilbene
  • flavanone g, naringenin
  • flavone h, apigenin
  • isoflavone i, genistein
  • Figure 39 shows the microscopic analysis of subcellular localization of AhR4DT-l and AhR3'DT-l fused with GFP in onion epidermal cells upon particle bombardment.
  • A, D Plasmids containing CaMV35S-TEV-GFP and RS-TP-mCherry
  • B, E AhR4DT-l-GFP and RS- TP-mCherry
  • C, F AhR3 'DT-l-GFP and RS-TP-mCherry were mixed and introduced into onion epidermal cells by particle bombardment.
  • Al , B 1 , C 1 Bright field images of onion cells.
  • A2, B2, C2 Expression pattern of mCherry from construct CaMV35S-RS-TP-mCherry
  • A3 Expression pattern of GFP from construct CaMV35S-TEV-GFP
  • A4 Merge of A2 and A3
  • F2 Expression pattern of GFP from construct AhR3'DT-l-GFP
  • CaMV35S cauliflower mosaic virus 35S promoter
  • TEV tobacco etch virus translational enhancer
  • GFP green fluorescent protein
  • RS-TP rubisco small subunit transit peptide.
  • Figure 40 shows the enzyme activities and transcript coexpression during elicitation time course in peanut hairy root cultures.
  • A Prenyltransferase activities from crude cell-free extracts.
  • B Relative transcript accumulation of AhR4DT-l and
  • C AhR3 'DT-1 as determined by RT-qPCR.
  • D Uniquely mapped RNA-Seq reads coverage over reference A. hypogaea transcripts AhR4DT-l, (E) AhR3 'DT-1, (F) AhR3 'DT-2/3 and (G) AhR3 'DT-4 as described. E9 and E72, 9 h and 72 h MeJA + CD treatment; C9 and C72, 9 h and 72 h control treatments.
  • Figure 41 shows the proposed pathway of prenylated stilbenoids in peanut.
  • Stilbenoids identified from the medium of peanut hairy root culture upon elicitors treatment are bolded and their proposed pathway is highlighted in yellow.
  • Other prenylated stilbenoids identified in fungal-challenged peanut seeds are divided into three groups based on the prenyl unit and hydroxyl groups on their stilbene backbone.
  • Prenylation reactions catalyzed by AhR4DT-l and AhR3'DT-l identified in this study are labeled with red solid arrows.
  • Enzymatic reactions confirmed in peanut are marked with black solid arrow, while other proposed reactions are labeled in black arrows with dashed lines.
  • Pinosylvin, 3-methyl-2-butenyl-3'-resveratrol and the prenylation product of piceatannol by AhR3'DT-l have not been reported in peanut tissue.
  • Figure 42 shows the prenylation of resveratrol by AhR3'DT-l.
  • Figure 43 shows NMR spectra of the lmM 3 -methyl-2-butenyl-3' -resveratrol isolated from a large-scale enzymatic assay.
  • Figure 44 shows Prenylation of resveratrol by AhR4DT-l.
  • benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan-AhR4DT- 1 was confirmed by HPLC and LC-MS n analysis.
  • Figure 45 shows the Comparison of AhR4DT-l and AhRPT-9i2.
  • Figure 46 shows a comparison of AhR3'DT-l, AhRPT-10a4 and AhRPT-10d4.
  • Figure 47 shows a comparison of AhR3'DT-2 and AhR3'DT-3.
  • Figure 48 shows a structural analysis of AhR3'DT-4.
  • FIG. 49 shows a temperature dependency of AhR4DT-l and AhR3'DT-l activity.
  • AhR4DT-l (A) and AhR3'DT-l (B) activities were measured at various temperature (20, 25, 28, 30, 37, 40 and 50 °C) in 100 mM Tris-HCl buffer (pH 9.0) for 40 mins.
  • Figure 50 shows the incubation time and amounts of microsomal fraction dependency of AhR4DT-l and AhR3'DT-l activity.
  • Figure 51 shows a pH dependency of AhR4DT-l and AhR3'DT-l activity.
  • AhR4DT-l (A) and AhR3'DT-l (B) activities at various pH values were measured in three different buffers: 100 mM Tris-HCl buffer at pH 7.0, 8.0, 8.6 and 9.0; 100 mM Glycine-NaOH buffer at pH 8.6, 9.0, 9.4, 10.0 and 10.6; and 100 mM NaHC0 3 -Na 2 C0 3 buffer atpH 9.2, 9.7, 10.2 and 10.7. All the reactions were performed at 28 °C for 40 min.
  • Figure 52 shows a divalent cation dependency of AhR4DT-l and AhR3'DT-l activity.
  • AhR4DT-l (A) and AhR3'DT-l (B) activity with various divalent cation were measured with 10 mM MnCl 2 , FeCl 2 , CaCl 2 , CoCl 2 , ZnC12, NiCl 2 , or CuCl and the enzyme activity was compared with the reaction of 10 mM MgCl 2 .
  • Reactions without divalent cation and 10 mM EDTA instead of MgCl 2 were used as controls. All the reactions were performed in 100 mM Tris-HCl buffer (pH 9.0) at 28 °C for 40 min. (t.a., trace amount ( ⁇ 0.5%), n.d., Not detected.). Means and the standard deviation (error bars) were calculated from three replicates.
  • Figure 53 shows the kinetic values of AhR4DT-l and AhR3'DT-l.
  • Figure 54 shows the prenylation of piceatannol by AhR4DT-l .
  • Substrate specificity of AhR4DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan-AhR4DT-l was analyzed by HPLC and LC-MS.
  • Figure 55 shows the prenylation of pinosylvin by AhR4DT-l .
  • Substrate specificity of AhR4DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan- AhR4DT-l was analyzed by HPLC and LC-MS.
  • Figure 56 shows the prenylation of oxyresveratrol by AhR4DT-l.
  • Substrate specificity of AhR4DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan-AhR4DT-l was analyzed by HPLC and LC-MS n .
  • Figure 57 shows the prenylation of piceatannol by AhR3'DT-l.
  • Substrate specificity of AhR3'DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan- AhR3'DT-l was analyzed by HPLC and LC-MS.
  • Figure 58 shows the prenylation of oxyresveratrol by AhR3'DT-l.
  • Substrate specificity of AhR3'DT-l in, microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration vAthAgrobacterium tumefaciens LBA4404 harboring pBIB-Kan- AhR3'DT-l was analyzed by HPLC and LC-MS.
  • Figure 59 shows the cloning strategy of binary vectors for peanut prenyltransferase genes screening.
  • Figure 60 shows the cloning strategy of binary vectors for subcellular localization of AhR4DT-l or AhR3'DT-l.
  • Figure 61 shows the cloning strategy of binary vectors containing GFP for subcellular localization control.
  • Figure 62 shows the primer efficiencies for real-time qPCR.
  • AhR4DT-l The efficiency of AhR4DT-l (A) was tested in 5x serial dilutions of peanut hairy root cDNA (range of 2 to 0.00032 ng).
  • Figure 63 shows a table of Prenyltransferase transcripts described in this application. Activities of expressed protein products and loci of best alignment in diploid Arachis reference genomes are shown.
  • Figure 64 shows a table of AhR4DT-l and AhR3'DT-l activity from N. benthamiana leaves fractions. The preparation of fractions and the resveratrol prenyltransferase assay are described in "Materials and Methods.” Values are the mean ⁇ standard deviation for three replicates.
  • Figure 65 shows a table of comparison of kinetic values of AhR4DT-l, AhR3'DT-l and prenyltransferase activity identified from peanut hairy root.
  • the apparent K m and V max values of AhR4DT-l and AhR3'DT-l for resveratrol, piceatannol and DMAPP were measured using the microsomal fraction of N. benthamiana leaves transiently expressing these two enzymes. Values are the mean ⁇ standard deviation of three replicates.
  • the apparent K m and F max values of prenyltransferase activity identified from the microsomal fraction of peanut hairy root for resveratrol and DMAPP were previously reported by Yang et al. (2016).
  • Figures 66A-66B show a list of primers used in this study. The restriction site on each primer is underlined.
  • Figure 67 shows substrates used for specificity assay and the prenylated products from reaction mixtures catalyzed by AhR4DT-l or AhR3'DT-l . Analysis was done by HPLC-PDA- electrospray ionization-MS3.
  • Figure 68 shows a list of plasmids used in this study. For details of the binary vectors, see Materials and Methods.
  • Figures 69A-69B show accession numbers of proteins used for phylogenetic analysis.
  • Figure 70 shows a primer design for analysis of transgenic plants expressing peanut stilbenoid-specific prenyltransferases (AhR4DT-l and AhR3'DT-l). Targeting position of primers, SubLoc-F-pBIBKan and SubLoc-R-pBIBKan on (A) pBIBKan, (B) pBIB-Kan- ' AhR4DT- 1 and (C) pBIB-Kan-AhR3'DT- 1.
  • Figure 71 shows a characterization of transgenic tobacco plants expressing peanut stilbenoid prenyltransferase AhR4DT-l gene.
  • Figure 72 shows a characterization of transgenic tobacco plants expressing peanut stilbenoid prenyltransferase AhR3 'DT-1 gene.
  • Figure 73 shows a phenotype of transgenic hairy roots of tobacco expressing peanut stilbenoid prenyltransferase AhR3 'DT-1.
  • hairy root lines developed from wild type of tobacco (WT), transgenic tobacco transformed with pBIB-Kan vector (pBIBKan - control), and AhR3 'DT-1 -expressing line 12 (Line 1 and Line 2). Hairy roots were developed via Agrobacterium rhizogenes- ediated transformation.
  • phytoalexins A substantial part of non-host defense responses in many plants is the pathogen-induced production of secondary metabolites, generally termed phytoalexins, that locally restrict disease progression due to bioactivities toxic to the pathogen (reviewed in Ahuja et al., 2012).
  • Peanut or groundnut (Arachis hypogaea) tissues mount a defense against infection by the soil fungus Aspergillus flavus and other pathogens by overproducing stilbene derivatives around sites of wounding and elicitor perception (Sobolev, 2013).
  • Resveratrol (3,5,4'-trihydroxy-stilbene) one of the most studied phytoalexin stilbenoids, has attracted great attention because of its bioactive properties shown through in vitro and in vivo assays to benefit human health.
  • Prenylated stilbenoids naturally produced as phytoalexins in the peanut plant possess one or two isoprenyl moieties bound to the aromatic ring of the stilbene molecule ( Figure 1).
  • these compounds exhibit similar or enhanced bioactivity in in vitro experiments.
  • arachidin-1 and resveratrol showed similar anti-inflammatory activity in lipid polysaccharide-treated RAW 264.7 macrophages and this correlated with the inhibition of prostaglandin E 2 production (Chang et al., 2006; Djoko et al., 2007).
  • Arachidin-1, arachidin-2 and arachidin-3 were more effective than resveratrol in inhibiting inducible nitric oxide production (Sobolev et al., 2011).
  • arachidin-1 inhibited lipid oxidation more effectively than resveratrol (Abbott et al., 2010), and arachidin-2 and arachidin-3 showed greater potency over resveratrol in inhibiting production of intracellular reactive oxygen species (Sobolev et al., 2011).
  • Aracbidin-1 further showed higher cytotoxicity than resveratrol to leukemia HL-60 cells (Huang et al., 2010) and other cancer cells (SK-MEL, KB, BT-549, and SK-OV-3) (Sobolev et al., 2011).
  • arachidin-1 and arachidin-3 were shown to bind to human cannabinoid receptors 2 (hCBR2s), while the affinity of their non-prenylated analogous, piceatannol and resveratrol for hCB2Rs was 5- to 10-fold lower.
  • hCBR2s cannabinoid receptors 2
  • stilbenoids arachidin-1, arachidin-2 and arachidin-3
  • more than 20 other prenylated stilbenoids have been described in peanut tissues (Sobolev et al., 2006; Wu et al., 2011; Sobolev, 2013; Sobolev et al, 2016).
  • the biosynthesis of stilbenoids derives from both the phenylpropanoid and acetate pathways. These merge to produce resveratrol by the action of resveratrol synthase which catalyzes the cyclization of 4-coumaroyl- CoA and malonyl-CoA (Schoppner and Kindl, 1984).
  • the prenylation step in which either of two prenyl patterns (3,3-dimethylallyl or 3-methyl-but-l-enyl) are introduced to various positions of the stilbene backbone ( Figure 1), along with the oxidation, methylation and cyclization steps plays a major role in the diversification of peanut prenylated stilbenoids.
  • the enzymes involved in resveratrol biosynthesis have been elucidated (Chong et al., 2009), the enzymes involved in the prenylation steps of resveratrol or any other stilbenoid have not been described.
  • prenyltransferase was found to be the critical activity for coupling the aromatic compound biosynthesis and terpenoid biosynthesis, the latter leading to the formation of the prenyl unit (Yazaki et al., 2009).
  • MVA mevalonic acid
  • MEP 2-C-methyl-D-erythritol-4-phosphate
  • DMAPP dimethylallyl pyrophosphate
  • mevastatin an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase involved in the MVA pathway was used in hairy root cultures of ginseng to study the biosynthesis of ginsenosides (Zhao et al., 2014); while clomazone, a herbicide that inhibits l-deoxy-D-xylulose-5-phosphate synthase (DXS) during early steps of DMAPP biosynthesis in the plastid was used to investigate the synthesis of monoterpenes in Catharanthus roseus (Han et al., 2013).
  • DXS l-deoxy-D-xylulose-5-phosphate synthase
  • Hairy roots of peanut have the capability to produce and secrete resveratrol (3,5,4'- trihydroxy-trara-stilbene), piceatannol (3,5,3',4'-tetrahydroxy-tra3 ⁇ 4y-stilbene) and their prenylated analogs, arachidin-3 and arachidin-1, in the medium upon co-treatment with MeJA and CD as elicitors (Yang et al., 2015a).
  • Mevastatin had no significant effect on the yields of resveratrol, piceatannol and prenylated stilbenoids with the exception of arachidin-5 and arachidin-1, which showed a 27% and 41% increase in yield, respectively, after a 72 -h treatment ( Figure 3).
  • non-inhibitor and mevastatin treated groups had significant increases in the yields of arachidin-5, arachidin-1, arachidin-2, and arachidin-3 indicating that mevastatin did not inhibit the accumulation of these prenylated stilbenoids (Figure 3).
  • micromolar increase of resveratrol was about 1.54 fold greater than the overall decrease in the accumulation of arachidin-5, arachidin-1, arachidin-2, and arachidin-3, suggesting that these prenylated stilbenoids may have been derived from resveratrol.
  • Divalent cations were absolutely required for resveratrol prenyltransferase activity and Mg was the most effective these (100%), followed by Mn 2+ (71.8%), Fe z+ (14.6%) and Ca + (0.9%) (Figure 27).
  • No prenyltransferase activity was detected when a divalent cation was absent from the reaction ( Figure 28).
  • Isoprenoid precursor isopentenyl diphosphate (IPP) was tested as a prenyl donor for the prenyltransferase with resveratrol as the prenyl acceptor and no activity was detected in the assay (Figure 28).
  • the apparent k m values for resveratrol and DMAPP were calculated as 111.1 ⁇ 40.44 ⁇ and 91.89 ⁇ 7.032 ⁇ , respectively ( Figure 29).
  • stilbenoids (piceatannol, pinosylvin, pterostilbene and oxyresveratrol), flavanone (naringenin), flavone (apigenin) and isoflavone (genistein) were incubated with a microsomal fraction using DMAPP as a prenyl donor.
  • DMAPP DMAPP
  • AhR4DT a membrane-bound prenyltransferase specific for stilbenoids from peanut hairy root
  • flavonoid prenyltansferases As the limiting enzyme involved in the biosynthesis of prenylated flavonoids, flavonoid prenyltansferases have been a focus of previous research particularly in plants of the Leguminosae family known to accumulate these type of specialized metabolites.
  • SfG6DT and SfiLDT which prenylate genistein to produce wighteone (6-dimethylallylgenistein) and isoliquiritigenin to dimethyllylisoliquiritigenin respectively, were identified in the same species (Sasaki et al., 2011).
  • This enzyme catalyzes the dimethylallylation of glycinol at position 4 to produce the precursor of the phytoalexin, glyceollin I (Akashi et al., 2008). More recently, GuA6DT, a flavone prenyltransferase was identified in another legume species, liquorice (Glycyrrhiza uralensis) (Li et al., 2014).
  • AhR4DT catalyzes the dimethylallylation of resveratrol at C-4 and is derived from the microsomal fraction of elicited peanut hairy roots.
  • AhR4DT shows several common features with other prenyltransferases, such as those described above. For instance, all prenylation activities mentioned above and demonstrated here require a divalent cation as cofactor and a basic buffer for optimal reaction rate (except for GmG4DT which reaction rate optimum is pH 7.5 buffer for the reaction, (Akashi et al., 2008).
  • both resveratrol synthase and chalcone synthase use 4-coumaroyl-CoA as a substrate and perform three condensation reactions with malonyl-CoA to form a linear tetraketide, which is later folded into new ring structures.
  • These two enzymes are distinguished by a special property of stilbene synthases, which looses the terminal carboxyl group as C0 , resulting in release of 4 C0 2 .
  • each reaction catalyzed by chalcone synthase releases 3 C0 2 molecules. It has been suggested that stilbene synthase may have developed from chalcone synthase via gene duplication and mutation rendering new and improved functions (Tropf et al., 1994).
  • abiotic elicitors to induce the biosynthesis of stilbenoid in hairy root cultures provides for an axenic sustainable and controlled production platform for these specialized metabolites which can be leveraged to study their biosynthetic pathway.
  • the main prenylated stilbenoids produced by peanut hairy root can be categorized into two groups according to the structure of their prenyl side chains.
  • One group includes arachidin-2 and arachidin-5 (identified in this study) having a 3,3-dimethylallyl moiety.
  • This is the most common type of prenylation found in prenylated stilbenoids from various plant families. For instance, longistylines C, longistylines D and chiricanine A found in Lonchocarpus chiricanus (Leguminosae) (Ioset et al., 2001) also have the same dimethylallyl moiety.
  • Artoindonesianin N with one dimethylallyl moiety and 4-dimethylallyl-oxystilbene were reported in Artocarpus integer (Moraceae) (Boonlaksiri et al., 2000) and A. gomezianus (Moraceae) (Hakim et al., 2002) respectively.
  • Mappain found in Macaranga mappa (Euphorbiaceae) (Van Der Kaaden et al., 2001) has one dimethylallyl moiety and one geranyl moiety, while schweinfurthin C with two geranyl moieties was isolated from M. alnifolia ⁇ Euphorbiaceae) (Yoder et al., 2007).
  • the dimethylallyl moiety is also the most common prenylation pattern present in prenylated flavonoids, which are mostly found in the following families: Cannabaceae, Guttiferae, Leguminosae, Moraceae, Rutaceae, and Umbelliferae.
  • prenyltransferases are responsible for these dimethylallylation and geranylation attaching DMAPP and GPP, respectively, to different positions of the stilbenoid and flavonoid skeletons.
  • One major group of prenylated stilbenoids in peanut hairy roots is represented by arachidin-1 and arachidin-3 which harbor a 3-methyl-but-l-enyl moiety.
  • this unique prenylated form has been reported in peanut and very few other species.
  • 3-methyl-but-l-enyl-oxystilbene isolated from A. integer (Moraceae) (Boonlaksiri et al, 2000) is the only prenylated stilbenoid with this moiety reported in a species other than peanut. Due to the difference in the position of the olefinic bond on the prenylated moieties, 3-methyl-but-l-enyl stilbenoids such arachidin-3 and arachidin-1 have higher lipophilicity as evident from a later retention time in reverse-phase HPLC chromatograms when compared with their 3,3-dimethylallyl analogs, arachidin-2 and arachidin-5 respectively.
  • arachidin-1, arachidin-2, arachidin-3, and arachidin- 5 are the major prenylated stilbenoids secreted and accumulated in the spent culture together with resveratrol.
  • CD is not added in the hairy root cultures, only limited amounts of resveratrol and stilbenoids are detected in the medium.
  • Authentic standards of resveratrol and piceatannol were obtained from Biophysica and Axxora, respectively; arachidin-1, arachidin-2, arachidin-3, and arachidin-5 standards were purified from elicited peanut hairy root as described below.
  • Pinosylvin, oxyresveratrol, pterostilbene, naringenin, apigenin, and genistein used in this study were purchased from Sigma- Aldrich.
  • Chiricanine-A was purified from fungal-challenged peanut seeds as described below.
  • DMAPP was obtained from Isoprenoids, LC. Stock solutions of inhibitors, mevastatin (100 mM, Sigma- Aldrich) and clomazone (100 mM, Sigma- Aldrich), were prepared in ethanol and stored at 4 °C.
  • the trays were evenly sprayed with the fungal spores of Aspergillus caelatus NRRL 25528 (lOVmL), placed into autoclave bags, and incubated at 30 °C for 96 h. The bags were opened every 24 h to allow fresh air to the peanuts and growing fungus.
  • the column was subsequently eluted with 0.5 L of CHC1 3 , 1.0 L of EtOAc, 1.0 L of acetone, and 1.0 L of MeOH (all solvents were purchased from Fisher). Eight fractions were collected from the column and analyzed by , HPLC. Fractions containing chiricanine A were combined, evaporated to dryness with a rotary evaporator, and subjected to further purification on a similar silica gel column. The column was subsequently eluted with 0.4 L of CHC1 3 , 1.5 L of CHCl 3 EtOAc (1 : 1), 1.3 L of EtOAc, and 1.0 L of acetone. Twenty four fractions were collected.
  • Hairy root cultures of peanut (Arachis hypogaea) cv. Hull line 3 established by the Medina-Bolivar laboratory (Condori et al., 2010) were maintained in 50 mL modified Murashige and Skoog medium (MSV) (Condori et al., 2010) with 3% sucrose in 250 ml flasks.
  • MSV Murashige and Skoog medium
  • the spent medium of nine-day-old hairy root cultures was discarded and replaced with 50 mL fresh MSV medium containing 3% sucrose with 100 ⁇ methyl jasmonate (MeJA) and 9 g/L methyl-P-cyclodextrin (CD; CavasolTM) as elicitors and incubated in the dark at 28 °C for an additional 72 hours as previously described (Yang et al., 2015a). After elicitation. period, one milliliter spent medium was partitioned with ethyl acetate twice. The combined organic phase was evaporated under nitrogen gas and dissolved in methanol for subsequent HPLC and mass spectrometry analyses.
  • MSV medium containing 3% sucrose with 100 ⁇ methyl jasmonate (MeJA) and 9 g/L methyl-P-cyclodextrin (CD; CavasolTM) as elicitors and incubated in the dark at 28 °C for an additional 72 hours as previously described (Yang et al.
  • a two-phase HPCCC solvent system (hexane: ethyl acetate: methanol: water (4:5:3:3, v/v/v/v) was equilibrated at room temperature in a 2 L separatory funnel.
  • the upper phase of this solvent mixture was used as stationary phase and the lower one was used as mobile phase for preparative HPCCC (Dynamic Extractions) system.
  • the multilayer coil was filled with the stationary phase at a flow rate of 8 mL/min while spinning at 1600 rpm. Then the hydrodynamic equilibrium was established by pumping 6 mL/min of mobile phase into the column until a clear mobile phase was eluted at the outlet.
  • Crude extract (300 mg) was dissolved in 5 mL of the two-phase solvent and manually injected. The effluent was monitored at UV 340 nm. The fractions were collected every 30 s, dried in a SpeedVac, and analyzed by HPLC as described above. According to the HPLC profiles, the fractions collected between 27 to 32 min contained arachidin-1 and arachidin-5, whereas those collected between 53 to 62 min contained arachidin-2 and arachidin-3. Fractions containing arachidin-1 and arachidin-5 were combined as one sample, whereas those containing arachidin-2 and arachidin-3 were combined as a separate sample. Then, the organic solvent in the samples was removed using rotavapor.
  • aqueous mixtures containing the target compounds were extracted with ethyl acetate (1:1, v/v), evaporated nearly to dryness with a rotavapor and redissolved in methanol. Then these combined fractions were applied to TLC plates (TLC silica gel 60 RP-18 F 2 5 4 s, Millipore) and separated using as developing solvent a solvent system composed of methanol: water: acetic acid (15:45:40, v/v/v). After separation, the purified prenylated stilbenoids on the adsorbent were scraped off, re-dissolved in methanol and dried under nitrogen gas for subsequent 1H and 13 C NMR spectra analysis. In summary, 4.6 mg of arachidin-5, 20.3 mg of arachidn-1, 5.2 mg of arachidin-2 and 17.8 mg of arachidin-3 were purified from the peanut hairy root culture medium using the HPCCC and preparative TLC method.
  • Enzyme solutions from peanut hairy roots for prenyltransferase assay were prepared using nine-day-old roots elicited with 100 ⁇ MeJA and 9 g/L CD for 48 h.
  • Ten grams (fresh weight) of elicited hairy root tissues were grinded and homogenized in 20 mL of extraction buffer composed of 100 mM Tris-HCL buffer (pH 7.6), 10 mM dithiothreitol (DTT) and 2.5% (w/v) polyvinylpyrrolidone (average mol wt 40,000; PVP-40, Sigma) using a mortar and pestle.
  • the homogenate was centrifuged at 12,000xg for 15 min at 4 °C to remove the cell debris.
  • Crude cell -free extracts were obtained by passing 2.5 mL of the 12,000 xg supernatant through a PD-10 desalting column (GE Healthcare), using 100 mM Tris-HCL (pH 9.2) containing 10 mM DTT as equilibration buffer. About 13.5 mL of the remaining 12,000 xg supernatant were subsequently centrifuged at 156,000 xg for 45 min at 4 °C. The 156,000 xg supernatant was collected and cleaned up through a PD-10 desalting column (GE Healthcare) equilibrated with 100 mM Tris- HC1 buffer (pH 9.2) containing 10 mM DTT.
  • microsomal pellet was resuspended in 100 mM Tris-HCl buffer (pH 9.2) containing 10 mM DTT, re-centrifugated (156,000xg for 45 min), and finally resuspended in 1 mL of the same buffer for subsequent enzyme reaction.
  • crude cell-free extract was prepared from 12-day-old non-elicited peanut hairy roots following a similar procedure to that described for elicited roots above except that no DTT was included in the extraction and equilibration buffers.
  • Protein quantification Protein contents in the various enzyme solutions were determined using the Coomassie protein assay (Thermo Scientific) using bovine serum albumin as standard.
  • the standard assay condition contained 1 mM resveratrol with 50 ⁇ g crude cell-free extract from 12-day-old non-elicited peanut hairy root in 500 ⁇ , of 100 mM Tris-HCl buffer (pH 7.6). To study the effect of reducing agent on the degradation of resveratrol, additional 5 mM DTT was added to the standard assay mixture. After 30 min incubation at 28 °C, the reaction mixture was extracted with 500 ⁇ , ethyl acetate and the amount of resveratrol remaining in the mixture was analyzed by HPLC. i the control group, crude cell-free extract was heated at 99 °C for 20 min.
  • the standard assay was performed in a total volume of 500 ⁇ , containing 100 ⁇ resveratrol, 300 ⁇ DMAPP as a prenyl donor, 10 mM MgCl 2 , 5 mM DTT and 30 ⁇ g microsomal fractions in 100 mM Tris-HCl buffer (pH 9.2). After 60 min incubation at 28 °C, the reaction mixture was terminated by adding 20 ⁇ , of 6 M HCl and extracted with 500 ⁇ , of ethyl acetate. The extracts were dried under nitrogen gas, dissolved in methanol and the reaction product was quantified by HPLC analysis. The prenylation activity for each reaction was quantified by the molar concentration of generated prenylated product per second with one milligram microsomal fractions (kat/mg).
  • prenyltransferase activities under varying incubation times (30, 60, 90 and 120 min) with 30 ⁇ g microsomal fraction and varying mount of microsomal faction (30, 60, 90 and 120 ⁇ g) incubated for 60 min were measured.
  • activities of prenyltransferase were measured using 100 mM Tris-HCl buffer at pH 7.0, 8.0, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, and 10.0.
  • varying concentration (10, 20, 40, 80, 160, 320, and 640 ⁇ ) of resveratrol with a fixed concentration of DMAPP (640 ⁇ ) and varying concentration (10, 20, 40, 80, 160, 320, and 640 ⁇ ) of DMAPP with a fixed concentration of resveratrol (640 ⁇ ) were incubated with microsomal fractions of peanut hairy root in a total volume of 250 ⁇ ⁇ at 28 °C for 60 min. These reactions were used to calculate V m and K m values using nonlinear regression analysis of Michaelis-Menten equation using Graphpad Prism 6 software.
  • the first flavonoid-specific prenyltransferase, SfN8DT-l was cloned from a cDNA (EST) library of Sophora flavescens cell cultures and its enzymatic activity was characterized using the microsomal fraction of recombinant yeast (Sasaki et al., 2008). Sequence homology to SfN8DT-l was the basis for discovery of several other flavonoid prenyltransferases, such as SfiLDT and SfG6DT in S. flavescens (Sasaki et al., 2011) and LaPTl in Lupinus albus (Shen et al., 2012).
  • transcripts from our set of 2,591,753 transcript assemblies encoded full-length protein sequences of 101 to 432 amino acid residues that aligned to the set of flavonoid prenyltransferase sequences over a length of at least 100, with >80% sequence identity.
  • the cultivated peanut genome is allotetraploid thought to have been formed as the result of a single hybridization between two closely-related diploid species, Arachis duranensis and Arachis ipaensis. Reference sequence assemblies of the latter diploid genomes were reported recently (Bertioli et al., 2016), and these provided a draft proxy reference we used to evaluate and potentially reduce our transcriptome to unique genie loci.
  • microsomal fraction of yeast expressing a truncated form of LaPTl showed 6-fold higher activity than that of the full-length protein (Shen et al., 2012).
  • Reasons for these observations may be low tolerance of plant transit peptides in yeast, correlated with incorrect folding and decreased stability of the prenyltransferases, resulting in low enzymatic activity. Rather than removing N-terminal putative transit peptide sequences and reexamining the yeast expression system as in the flavonoid prenyltransferase studies mentioned, we shifted to a heterologous plant expression system.
  • benthamiana leaves was incubated with DMAPP and resveratrol to test prenyltransferase activities.
  • One of the cDNA products (amplified with primers PT-lO-FW-Notl/PT-k-RV-iQwI, Figure 66) showed resveratrol dimethylallyltransferase activity (Figure 35). No activity was observed in the crude cell extract of N. benthamiana control leaves that were infiltrated with Agrobacterium harboring an empty binary vector (Figure 35).
  • NMR predict tool was used to generate multiple 1H and 13 C spectra for with various combinations of prenyl positions, with reference to the 4-hydroxyphenyl ring (Banfi and Patiny, 2008; Castillo et al., 2011). Two possible prenyl positions appear to be of highest probability and matched with the experimentally obtained 1H and C NMR spectra ( Figures 43 A and 43 B).
  • prenyltransferase(s) from the peanut hairy root transcriptome other primer pairs were designed based on the alignment of the putative prenyltransferase transcripts ( Figure 66).
  • additional PCR amplicons were amplified from the cDNA of 9-hour-elicited peanut hairy roots and five were subsequently subcloned into binary vectors and transiently expressed in N. benthamiana leaves for prenyltransferase activity assays using DMAPP and resveratrol as substrates.
  • Four additional cDNAs were identified as resveratrol prenyltransferase genes.
  • the other three cDNA clones (two amplified with primers and one amplified with primers VTS-FW-NotlfPT-m-KV-Kpnl, Figure 66) exhibited the same catalytic activities as AhR3'DT-l, converting resveratrol into 3 -methyl-2-butenyl-3' -resveratrol using DMAPP as prenyl donor.
  • AhR3'DT-2, AhR3'DT-3 and AhR3'DT-4 we named these AhR3'DT-2, AhR3'DT-3 and AhR3'DT-4.
  • AhR3'DT-l exhibited the highest activity.
  • AhR3'DT-2 and AhR3'DT-3 exhibited activity levels that were 18% and 17% of AhR3'DT-l respectively, while AhR3'DT-4 reached only 5% that of AhR3'DT-l ( Figure 35).
  • peanut prenyltransferases The gene structure of peanut prenyltransferases was estimated by aligning the transcripts . to available Arachis diploid progenitor genome sequence references (Bertioli et al., 2016). Four loci in each became apparent as candidate origins. Three of these were on pseudochromosome 8 in each genome, contained within a span containing notable gaps and described by Bertioli et al. (2016) as effected by a genomic reduction during polyp loidization. Only AhR3'DT-4 aligned completely to pseudochromosome 1 ( Figure 63). Eliminating poor alignments in either progenitor, we estimated two loci in A. duranensis and two in A.
  • AhRPT-9i2 likely reduces its encoded protein structure to eight transmembrane spans (Figure 45), suggesting that the integrity of the nine transmembrane domains in AhR4DT-l are essential for its activity.
  • Two inactive transcripts of AhR3'DT-l encode a C-terminal extension that does not appear to have transmembrane properties (Figure 46).
  • this sequence is highly variable in these three less active forms, suggesting this uncharacterized region may play a role in influencing the
  • HG homogentisate
  • PHB p- hydroxybenzoate
  • AhR3'DT-l the enzyme with the highest activity among its group, along with AhR4DT- 1 were selected for further biochemical characterization. Similar to the previous resveratrol prenyltransferase activity characterized from peanut hairy roots (Yang et al., 2016), the specific activity in the microsomal fraction of N. benthamiana leaves expressing AhR4DT-l or
  • AhR3'DT-l were 8.9-fold and 13.9-fold higher than that in the crude cell-free extracts, respectively ( Figure 64). Therefore, we used the microsomal fraction enriched with AhR4DT-l or AhR3'DT-l for subsequent enzymatic assays. Reactions were incubated with resveratrol, DMAPP and Mg 2+ as cofactor. Although the optimum activities of AhR4DT-l and AhR3'DT-l were observed at 37 °C and 30 °C, respectively, in the microsomal fraction of N.
  • Mg 2+ was the most effective (100%) for AhR4DT-l, followed by Mn 2+ (83.9%) and Fe 2+ (6.4%) (Figure 52A).
  • Mn 2+ 210.3%
  • Mg 2+ forms a bidentate complex with DMAPP which gets stabilized for an efficient transferase reaction.
  • AhR3'DT-l each exhibited a higher V max /K m value for resveratrol than piceatannol, suggesting that both of these prenyltransferases prefer resveratrol over piceatannol as substrate.
  • AhR3'DT-l The results ( Figure 38; Figures 54 to 58; Figure 67) showed that AhR4DT-l can selectively catalyze piceatannol, pinosylvin and oxyresveratrol into arachidin-5, chiricanine A and prenylated oxyresveratrol (the position of the prenyl moiety on the prenylated oxyresveratrol remains undetermined), respectively.
  • Pterostilbene which has two methoxy groups at the C-3 and C-5 positions along with piceid, a resveratrol glucoside with a glycosidic group at C-3 position, were not prenylated by either AhR4DT-l or AhR3'DT-l ( Figure 38), suggesting that either or both hydroxyl groups on the C- 3 and C-5 of stilbene backbone might be crucial for substrate recognition by AhR4DT-l and AhR3'DT-l.
  • prenyl donor specificity of AhR4DT-l and AhR3'DT-l in addition to DMAPP, other prenyl diphosphates, including isopentenyl pyrophosphate (IPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) were examined with resveratrol as a prenyl acceptor.
  • IPP isopentenyl pyrophosphate
  • GPP geranyl pyrophosphate
  • FPP farnesyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • the iPSORT program predicted a chloroplast transit peptide (cTP) in AhR4DT-l and a mitochondrial targeting peptide (mTP) in AhR3'DT-l.
  • cTP chloroplast transit peptide
  • mTP mitochondrial targeting peptide
  • ChloroP and TargetP predictions suggested AhR4DT-l contained neither cTP nor mTP, while AhR3'DT-l contained an N-terminal cTP.
  • AhR4DT-l-GFP and AhR3'DT-l-GFP gene fusion constructs driven by the CaMV35S-TEV promoter were expressed transiently in onion epidermal cells via particle bombardment.
  • AhR4DT-l and AhR3'DT-l in peanut are localized to plastids, similar to flavonoid prenyltransferases such as SfN8DT-l, GmG4DT and LaPTl characterized in other plant species (Yazaki et al., 2009; Shen et al., 2012; Sasaki et al., 2008).
  • Figures 6D-G represent sample-normalized counts of reads that mapped unambiguously to these transcript references.
  • mock treatments assayed at 9 h and 72 h
  • AhR4DT-l mRNA stood out, however, as accumulating to 2-3x over control levels early in response to addition of MeJA + CD (Figure 40D).
  • AhR3 'DT-1 on the other hand, among treatments assayed, reached its highest levels only 72 h post-elicitation (Figure 40E).
  • AhR3 'DT-2/3 and -4 transcripts were clearly detectable by RNA-Seq across the time course, but did not appear to change in response to elicitation ( Figure 40F-G). These results indicate the activation of AhR4DT-l and AhR3 'DT-1 genes correlates with stress elicitation in peanut hairy root tissue, and that the accumulation of mRNA encoding these two enzymes correlates temporally with their prenyltransferase activities observed and with their catalyzed product accumulation.
  • niRNA of similar enzymes we show to exhibit activities that are not relevant, or less relevant, to arachidin-2 and 3 -methyl-2-butenyl-3' -resveratrol production in peanut are not noticeably transcriptionally responsive to the elicitation.
  • products attributable to AhR4DT-l and AhR3'DT-l activities do not accumulate in peanut hairy root cultures in response to the control treatments used here (Yang et al., 2015), protein expression and/or localization are likely to be controlled by mechanisms beyond the transcript accumulation observed.
  • Prenylation of aromatic compounds plays an important role in the diversification of plant secondary metabolites and contributes to the enhancement of the biological activity of these polyphenolic compounds (Yazaki et al., 2009).
  • flavonoid prenyltransferase genes include SJN8DT-1, SfiLDT, S/G6DT, SfFPT, GmG4DT, GuA6DT and LaPTl cloned from legume species (Sasaki et al., 2008, 2011; Chen et al., 2013; Yazaki et al., 2009; Li et al., 2014; Shen et al., 2012), along with MalDT and CtlDT&om non-legume species Morus alba and Cudrania tricuspidata, respectively (Wang et al., 2014).
  • each of these enzymes is a membrane-bound protein containing several putative transmembrane a-helices.
  • the subcellular localization of the two stilbenoid prenyltransferases described here are primarily or exclusively in the plastid, as are the five flavonoid prenyltransferases previously characterized.
  • each enzyme contains two conserved aspartate-rich motifs.
  • stilbenoid prenyltransferases are monophyletic to other plant prenyltransferases accepting aromatic substrates (Figure 40).
  • AhR4DT-l specifically transfers a 3,3-dimethylallyl group to the A-ring at the C-4 position of resveratrol, piceatannol and pinosylvin.
  • This enzyme exhibits biochemical properties that match well with the prenyltransferase activity identified from elicited peanut hairy roots (Yang et al., 2016), including Km values of resveratrol/DMAPP and identical preferences for prenyl acceptors and divalent cations.
  • the second stilbenoid specific prenyltransferase characterized here was AhR3'DT-l, that recognizes 3,5,4'-trihydroxystilbene and adds a 3,3-dimethylallyl group to C-3' of the B-ring.
  • AhR3'DT-l none of the prenylation products of resveratrol and piceatannol catalyzed by AhR3'DT- 1 were detected in peanut hairy root culture or peanut hairy root tissue.
  • AhR3'DT-l showed a lower Km for resveratrol and piceatannol, indicating a higher affinity for these prenyl acceptor substrates.
  • its affinity for DMAPP was much lower than that of AhR4DT-l.
  • arachidin-l and arachidin-3 Differing from arachidin-5, arachidin-2, and most other prenylated flavonoids which harbor a 3,3- dimethylallyl moiety, arachidin-l and arachidin-3 have a unique 3-methyl-but-l-enyl moiety ( Figure 41).
  • the biosynthesis pathway(s) of arachidin-1 and arachdin-3 have not been fully elucidated, however several biosynthetic routes leading to their production could be proposed when considering results from our previous and current studies.
  • AhR4DT-l identified here initiates the first step in the biosynthesis of prenylated stilbenoids in peanut by catalyzing the prenylation of resveratrol and piceatannol to form arachidin-2 and arachidin-5, respectively.
  • arachidin-5 is also formed via hydroxylation of arachidin-2 by a stilbenoid monooxygenase (P450).
  • Flavonoid 3'- monooxygenases for example, are known to catalyze 3 '-hydroxylation of the flavonoid backbone (Tanaka and Brugliera, 2013).
  • arachidin-3 might be hydroxylated by a monooxygenase to produce arachidin-1 ( Figure 41). Further enzyme discovery and testing are needed to explore these possibilities.
  • arachidin-2 and arachidin-5 might be directly converted to arachidin-3 and arachidin-l by an isomerase which could shift the olefmic bond position on their prenylated moieties ( Figure 41).
  • arachidin-2 or arachidin-5 might be converted into an intermediate product which is further modified into arachidin-3 or arachidin-l through multiple enzymatic steps ( Figure 41). It is still unclear whether arachidin-2 derivative and arachidin-5 derivative found in the peanut hairy root culture were one of these intermediates involved in the biosynthesis of arachidin-3 and arachidin-l, which are the predominant compounds in the culture ( Figure 41).
  • arachidin-3 and arachidin-l were directly synthesized from resveratrol and piceatannol catalyzed by AhR4DT-l or another specific prenyltransferase utilizing 3-methyl-but- 1-enyl pyrophosphate as prenyl donor. Although, as far as we know, this kind pyrophosphate has never been described in plants.
  • Hairy root of peanut cv. Hull line 3 was was previously established by transforming peanut cotyledonary leaves with Agrobacterium rhizogenes strain ATCC 15834 and maintained in modified Murashige and Skoog (MSV) medium under continuous darkness at 28 °C as described before (Condori et al., 2010). The procedure of elicitation for stilbenoids production in peanut hairy root culture was performed according to Yang et al., (2015).
  • Authentic standards of resveratrol and piceatannol were obtained from Biophysica and Axxoram, respectively.
  • Arachidin-1, arachidin-2, arachidin-3 and arachidin-5 standards were purified from elicited peanut hairy root cultures as described previously (Yang et al., 2016).
  • Pinosylvin, oxyresveratrol, pterostilbene, naringenin, apigenin, genistein, and IPP, GPP, FPP and GGPP were purchased from Sigma- Aldrich.
  • DMAPP used in this study was obtained from Isoprenoids.
  • RNA was extracted from elicited root tissue at 0.5, 3, 9, 18, 24 and 72 hours using TRIzol reagent (Life Technologies), according to the manufacturer's instructions. For controls, RNA was likewise extracted from cultures prior to treatment (t 0) and from non-elicited cultures, collected 9 and 72 hours after mock treatment, being refreshed with fresh MSV medium.
  • transcript assemblies were aligned independently to the Arachis duranensis and Arachis ipaensis genomes available in PeanutBase (www.peanutbase.org and Bertioli et al., 2016), which confirmed clustering of highly similar forms and allowed us to approximate a reduced ⁇ , hypogaea sequence reference against which we could quantify reads coverage attributable to the enzyme transcripts under study. Reads mapped to the resulting four genomic loci were then isolated using Samtools 1.3.1 (Li et al., 2009b) and re-mapped to A. hypogaea transcript references, determined here.
  • RNA- Seq reads were performed using Tophat2, v 2.0.7 (Kim et al., 2013) using the genome guided option. Uniquely mapped read counts from each sample were assessed using HTSeq v 0.6. lpl (Anders et al., 2015).
  • RNA of 9-hour-elicited peanut hairy roots was prepareded by TRIzol reagent and the cDNA was synthesized using iScriptTM Select cDNA Synthesis kit (Bio- Rad) using oligo (dT) primer.
  • N-terminal primer and three C-terminal primers were synthesized with flanking Notl and Kpnl restriction sites, respectively ( Figure 66) and PCR using these primers was performed with ExTaq DNA Polymerase (Takara) following the program below: initial denaturation (3 min, 94°C); 30 cycles (30s, 94°C; 30s, 52°C; 1 min 30s, 72 °C); and a final extension step (10 min, 72 °C).
  • Three amplicons (including AhR3 'DT-1) were subcloned into a pGEM-T vector (Promega) and multiple clones from each amplicon were sequenced at University of Chicago Comprehensive Cancer Center (UCCCC).
  • Protein sequences were aligned using MUSCLE (Edgar 2004), and a neighbor-joining phylogenetic tree computed with PhyML (Guindon and Gascuel 2003) using the Dayhoff substitution model and 100 bootstrapped replicates.
  • the cloning strategy of binary vectors is showed in Figure 59.
  • the sequence of the double enhanced cauliflower mosaic virus 35S promoter (CaMV35S) fused to the translational enhancer from tobacco etch virus (TEV) was amplified from plasmid pR8-2 (constructed by Medina-Bolivar and Cramer, 2004) and subcloned into pGEM-T vector using Ca35S-FW-SM-l/TEV-RW-iV primers ( Figure 66) with SaWNotl flanking restriction sites.
  • the pGEM-CaMV35S-TEV and pGEM-T vectors containing putative prenyltransferase gene were digested with SalllNotl and NotllKpnl, respectively.
  • a high copy number vector, pBC KS(-) digested with KpnVSall was used as a transition vector.
  • Two fragments of full-length cDNA and CaMV35S-TEV promoter were ligated into the transition vector in a 16 °C overnight reaction with T4 ligase (NEB).
  • the engineered A. tumefaciens was grown in 5 mL of YEP medium, containing 50 mg/L of kanamycin (Sigma- Aldrich) and 30 mg/L of streptomycin (Sigma- Aldrich) for antibiotic selection, at 28°C on an orbital shaker at 200 rpm. After cultivation for 2 days, 5 mL of bacterial suspension was inoculated into 50 mL of fresh YEP medium containing the antibiotics and allowed to grow for one additional day under the same conditions.
  • the "middle tier" of JV. benthamiana leaves were harvested for prenylation activity screening.
  • Five grams of leaf tissue (fresh weight) were grounded and homogenized using a mortar with pestle in 10 mL of extraction buffer containing 100 mM Tris-HCl (pH 7.6) and 10 mM dithiothreitol (DTT).
  • the crude cell-free extract was obtained by passing the 12,000 ⁇ g supernatant through a PD-10 desalting column (GE Healthcare) equilibrated with 100 mM Tris-HCl (pH 9.0) containing 10 mM DTT.
  • the total protein concentration was determined by coomassie protein assay (Thermo Scientific) using bovine serum albumin as standard.
  • resveratrol 100 ⁇
  • DMAPP 300 ⁇
  • MgCl 2 10 mM
  • DTT 5 mM
  • Tris-HCl buffer 100 mM, pH 9.0
  • the enzyme reaction was terminated by addition of HC1 (6 M, 20 ⁇ ,) and then the reaction mixture was extracted with ethyl acetate (1 mL).
  • the ethyl acetate extract was dried under nitrogen gas and dissolved in 300 ⁇ , of methanol.
  • the reaction product was identified and quantified using HPLC ESI-MS" analysis as previously described (Yang et al., 2016).
  • the reaction of crude cell-free extract of N. benthamiana leaves infiltrated with A. tumefaciens harboring the empty pBIB-Kan vector was used as control.
  • AhR4D-l were harvested 24, 48, 72 and 96 hours post-infiltration.
  • the AhR4D-l activity in the crude cell-free extract of N. benthamiana leaves increased with the post- infiltration period from 24 to 72 hours, while the activity in the leaves harvested from 96 hours post-infiltration was similar to 72 hours (data not shown).
  • both AhR4DT-l and AhR3'DT-l were predicted as membrane bound proteins by TMHMM 2.0, the microsomal fraction of N. benthamiana leaves at 72 hours post-infiltration was used to study the biochemical properties of AhR4DT-l or AhR3'DT-l .
  • the basic reaction and measurement for AhR4DT-l and AhR3'DT-l activity were the same as that for prenylation activity screening with exception of using 30 ⁇ g of microsomal fraction of N. benthamiana leaves as enzyme instead of crude cell-free extract of N. benthamiana leaves in a 500 of reaction.
  • the enzymatic reactions were performed in Tris-HCl buffer (100 mM, pH 7.0 to 9.0), glycine-NaOH buffer (100 mM pH 8.6 to 10.6) andNaHC0 3 -Na 2 C0 3 buffer (100 mM, pH 9.2 to 10.7).
  • the optimal reaction temperatures for AhR4DT-l and AhR3'DT-l were tested at 20, 25, 28, 30, 37, 40, and 50 °C in Tris-HCl buffer (100 mM, pH 9.0).
  • 10 mM MnCl 2 , FeCl 2 , CaCl 2 , CoCl 2 , ZnCl 2 , NiCl 2 , or CuCl 2 was added to the reaction mixture instead of MgCl 2 , and the enzyme activity was compared with the reaction containing MgCl 2 .
  • the reactions without divalent cation and 10 mM EDTA instead of MgCl 2 were used as controls.
  • varying concentrations (10, 20, 40, 80, 160, 320, and 640 ⁇ ) of resveratrol or piceatannol with a fixed concentration of DMAPP (640 ⁇ ) and varying concentrations (10, 20, 40, 80, 160, 320, and 640 ⁇ ) of DMAPP with a fixed concentration of resveratrol (640 ⁇ ) were incubated with 30 ⁇ g of microsomal fractions of N. benthamiana leaves expressing AhR4DT-l or AhR3'DT-l to calculate the apparent K m and V max values by nonlinear regression analysis of the Michaelis-Menten equation using GraphPad Prism 6 software.
  • the prenyl acceptor specificity of AhR4DT-l and AhR3'DT-l were tested using 100 ⁇ of each stilbenoid (resveratrol, piceatannol, oxyresveratrol, pinosylvin, pterostilbene and piceid), flavanone (naringenin), flavone (apigenin), and isoflavone (genistein) with 300 ⁇ DMAPP as a prenyl donor, while the prenyl donor specificities of these two enzymes were tested using 300 ⁇ prenyl diphosphates (DMAPP, IPP, GPP, FPP, GGPP) with 100 ⁇ resveratrol as a prenyl acceptor. All these reactions were performed in a total volume of 500 ⁇ , with 100 mM Tris-HCl buffer (pH 9.0) at 28°C for 40 min.
  • mGFP5 modified green fluorescence protein
  • PT-9M3-RV- BamHI or PT-lOkl-RV-BamHI reverse primer with Ca35S-FW-5'fl/I-2 forward primer were used to amplify the full-length of AhR4DT-l or AhR3'DT-l with CaMV35S-TEV promoter region from pBC-CaMV35S-TEV-9bl3 and pBC-CaMV35S-TEV-10kl vector, which were created during the construction of the binary vector ( Figure 60; Figure 68).
  • the PCR products were then cloned into pGEM-T vector for sequencing validation.
  • CaMV35S- TEV-AhR4DT-l and CaMV35S-TEV-AhR3 'DT-1 fragments were isolated and inserted into pGEM-mGFP5-l to yield pGEM-CaMV35S-TEV-AhR4DT-l-GFP and pGEM-CaMV35S- ,TEV-AhR3 'DT-l-GFP, respectively.
  • mGFP5 gene was amplified from pR8-2 using primers mgfp5-FW-NotI/mgfp5-RW-Xp «I and cloned into pGEM-T vector to give pGEM- mGFP5-2.
  • Two fragments CaMV35S-TEV digested from pGEM-CaMV35S-TEV by SaWNotl and mGFP5 digested from pGEM-mGFP5-2 by NotVKpnl were inserted into pBC KS(-) vector to form pBC-CaMV35S-TEV-GFP.
  • the fragment of CaMV35 S-TEV-GFP was eventually subcloned into binary vector pBIB-Kan to form pBIB-Kan-GFP ( Figure 61).
  • target plasmid and 5 g of pt-rk plasmid were together coated on 50 of 60 mg/mL tungsten particles (Ml 7, 1 ⁇ ; Bio-Rad) in the presence of 1 M CaCl 2 and 15 mM spermidine.
  • tungsten particles Ml 7, 1 ⁇ ; Bio-Rad
  • plasmid-coated particles were dried on plastic discs and accelerated with a helium burst at 1100 psi in a bombardment chamber. Bombarded onion epidermal peels were kept on plates containing MS medium for 60 hours in the dark.
  • the localization of the expressed proteins in the transformed cell was visualized with a Nikon Eclipse E800 microscope with a 20x/0.5W Fluor water immersion objective.
  • Confocal fluorescence images were obtained by using Nikon digital eclipse CI microscope system with 488 nm laser illumination and 525/50 nm filter for GFP fluorescence and 543 nm laser with 595/50 nm filter for RFP fluorescence.
  • Primers for AhR4DT-l and AhR3 'DT-1 were designed using Allele ID (PREMIER Biosoft). Two reference genes, ACT7 (encoding actin 7) and EFal (encoding elongation factor al) were selected previously (Condori et al., 2011) and used to normalize the expression of AhR4DT-l and AhR3 'DT-1 in peanut hairy roots.
  • AhR3'DT-4 have been deposited in the GenBankTM database under the accession numbers
  • explants were placed in regeneration medium (modified MS' medium containing 1 mg/L BAL and 0.1 mg/L NAA), 600 mg/L cefotaxime and 200 mg/L kanamycin for selection of transgenic plants.
  • regeneration medium modified MS' medium containing 1 mg/L BAL and 0.1 mg/L NAA
  • 600 mg/L cefotaxime and 200 mg/L kanamycin for selection of transgenic plants.
  • transgenic callus that developed at the inoculation site were harvested and transferred to medium containing 600 mg/1 cefotaxime and 200 mg/L kanamycin, and maintained at 24 °C for another 2 weeks for shoot development. Newly developed shoots were transferred to antibiotic-free medium for roots of whole plants ( Figure 11).
  • genomic DNA was isolated from transgenic lines using DNeasy Plant Mini Kit (QIAGEN). Primer pairs that targeted the outside of prenyltransferase gene on pBIB-Kan vectors were used for molecular characterization of transgenic lines.
  • the transgenic plants transformed with empty pBIB-Kan vector showed an amplicon of 523 bp, while the AhR4DT-l and AhR3'DT-l transgenic lines gave amplicons of 2673 bp and 2619 bp, respectively.
  • the AhR4DT-l or AhR3'DT-l activity in these transgenic lines was further confirmed via enzymatic assay ( Figures 71C and 72C). In summary, five AhR4DT-l -expressing tobacco plants and nine AhR3'DT-l -expressing tobacco plants of N. tabacum were established ( Figures 71 and 72).
  • ChloroP a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8:
  • TopHat2 accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.
  • peanut (Arachis hypogaea) seeds challenged by an Aspergillus flavus strain. J. Agric. Food Chem. 64: 579-584.
  • GMAP A genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21 : 1859-1875.
  • a stilbenoid-specific prenyltransferase utilizes dimethylallyl pyrophosphate from the plastidic terpenoid pathway. Plant Physiol. 171: 2483-98.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Nutrition Science (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Le procédé et le système de la présente invention concernent l'identification de gènes de prényltransférase provenant de chevelu racinaire d'arachide traité par un éliciteur. L'une des prényltransférases, AhR4DT-1, catalyse une réaction clé impliquée dans la biosynthèse des stilbénoïdes prénylés, dans laquelle le resvératrol est prénylé à sa position C-4 pour former de l'arachidine-2, tandis qu'une autre, AhR3'DT-1, a été capable d'ajouter le groupe prényle à la position C-3'du resvératrol. Chacune de ces prényltransférases présente une spécificité élevée pour les substrats stilbénoïdes, et leur localisation subcellulaire dans le plaste a été confirmée par microscopie à fluorescence. L'analyse de structure des stilbénoïdes prénylés suggèrent que ces deux activités de prényltransférase représentent les premières étapes déterminées dans la biosynthèse d'un grand nombre de stilbénoïdes prénylés et de leurs dérivés dans l'arachide.
EP17814218.8A 2016-06-16 2017-06-16 Prényltransférases stilbénoïdes provenant de plantes Pending EP3472309A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662351117P 2016-06-16 2016-06-16
PCT/US2017/037995 WO2017218967A2 (fr) 2016-06-16 2017-06-16 Prényltransférases stilbénoïdes provenant de plantes

Publications (2)

Publication Number Publication Date
EP3472309A2 true EP3472309A2 (fr) 2019-04-24
EP3472309A4 EP3472309A4 (fr) 2020-01-15

Family

ID=65758304

Family Applications (1)

Application Number Title Priority Date Filing Date
EP17814218.8A Pending EP3472309A4 (fr) 2016-06-16 2017-06-16 Prényltransférases stilbénoïdes provenant de plantes

Country Status (1)

Country Link
EP (1) EP3472309A4 (fr)

Also Published As

Publication number Publication date
EP3472309A4 (fr) 2020-01-15

Similar Documents

Publication Publication Date Title
US11535856B2 (en) Stilbenoid prenyltransferases from plants
Brückner et al. Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes
Hammerbacher et al. Biosynthesis of the major tetrahydroxystilbenes in spruce, astringin and isorhapontin, proceeds via resveratrol and is enhanced by fungal infection
Wang et al. Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua
Gülck et al. Synthetic biology of cannabinoids and cannabinoid glucosides in Nicotiana benthamiana and Saccharomyces cerevisiae
Lam et al. Completion of tricin biosynthesis pathway in rice: cytochrome P450 75B4 is a unique chrysoeriol 5′-hydroxylase
Akashi et al. Molecular cloning and characterization of a cDNA for pterocarpan 4-dimethylallyltransferase catalyzing the key prenylation step in the biosynthesis of glyceollin, a soybean phytoalexin
Qu et al. Differential accumulation of phenolic compounds and expression of related genes in black-and yellow-seeded Brassica napus
Gholami et al. Natural product biosynthesis in Medicago species
Shen et al. Characterization of an isoflavonoid-specific prenyltransferase from Lupinus albus
Sasaki et al. Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens
Dobritsa et al. LAP5 and LAP6 encode anther-specific proteins with similarity to chalcone synthase essential for pollen exine development in Arabidopsis
Munkert et al. Iridoid synthase activity is common among the plant progesterone 5β-reductase family
Lam et al. Cytochrome P450 93G1 is a flavone synthase II that channels flavanones to the biosynthesis of tricin O-linked conjugates in rice
Pang et al. Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula
Yang et al. Stilbenoid prenyltransferases define key steps in the diversification of peanut phytoalexins
CA2939981C (fr) Facteurs de transcription de bhlh25 pour ameliorer la production de metabolite secondaire dans les plantes
Lui et al. Convergent recruitment of 5′‐hydroxylase activities by CYP75B flavonoid B‐ring hydroxylases for tricin biosynthesis in Medicago legumes
Sugiyama et al. Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases
Xu et al. Biosynthesis and regulation of active compounds in medicinal model plant Salvia miltiorrhiza
Sharma et al. Present status of Catharanthus roseus monoterpenoid indole alkaloids engineering in homo-and hetero-logous systems
Jiang et al. Gene mining and identification of a flavone synthase II involved in flavones biosynthesis by transcriptomic analysis and targeted flavonoid profiling in Chrysanthemum indicum L.
EP2041284B1 (fr) Production de stilbènes et de leurs dérivés dans des cultures de racines végétales chevelues
Dhar et al. Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis
Yang et al. A stilbenoid-specific prenyltransferase utilizes dimethylallyl pyrophosphate from the plastidic terpenoid pathway

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20181218

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
RIC1 Information provided on ipc code assigned before grant

Ipc: C12P 7/22 20060101ALI20191204BHEP

Ipc: C12N 9/10 20060101AFI20191204BHEP

Ipc: C12P 7/42 20060101ALI20191204BHEP

A4 Supplementary search report drawn up and despatched

Effective date: 20191213

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20201102

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS