Functional characterization of the first aromatic prenyltransferase which catalyzes the prenylation of umbelliferone to produce demethylsube

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A. tumefaciens LBA4404 (pAL4404)

A. tumefaciens strain LBA4404 and the binary vector method was generated by Dr. P.J.J Hooykaas at Leiden University in the Netherlands. This strain has a disarmed Ti plasmid pAL4404 plasmid, which only contains the T-DNA vir region (genes responsible for vir gene induction and T-DNA transfer), and is a widely used for plant transformation. This strain is resistant against rifampicin while plasmid pAL4404 provides resistance against streptomycin.

Saccharomyces cerevisiae WAT11 and WAT21

S. cerevisiae WAT11 and WAT21 were generated by Dr. Denis Pompon (Laboratoire d’Ingénierie des Protéines Membranaires (LIPM), Gif-sur-Yvette, France). They are derived from the W303 Strain. In this strains, the endogenous cytochrome P450 reductase gene (CPR) was replaced by the Arabidopsis thaliana reductases AtR1 or AtR2 (for WAT11 and WAT21 respectively) under the control of the GAL10-CYC1 promoter. This promoter is repressed by glucose and induced by galactose. These strains are ADE2 and therefore are unable to grow on a medium without adenine.

pCR®8/GW/TOPO® (Invitrogen)

pCR®8/GW/TOPO® (Invitrogen) is a kit developed by Invitrogen in order to allow efficient cloning of PCR product. This plasmid can serve as an entry vector for the Gateway® technology. This vector is provided linearized with single 3’-thymidine (T) overhangs coupled with a Topoisomerase. Taq DNA polymerases are enzymes that are known to add a single deoxyadenosine (A) to 3’ end of their PCR products. When these PCR products are mixed to the pCR®8/GW/TOPO®, the topoisomerase allows a quick and efficient insertion of the PCR product to generate a recombinant plasmid (Figure 45).

RNAs Extraction from plant material

Extraction of RNAs from plants is delicate due to the fact that a large quantity of RNase is present in the working environement. In addition RNAs are very sensitive to temperature and degrades very quickly at room temperature. Therefore special care is taken in account to solve these problems. A RNAse neutralizing solution (RNaseZap® – Ambion) was applied to the working bench and the user wear gloves for the whole extraction.
Extraction was performed using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s recommendations. Immediately after the extraction, samples were used for measurement of concentrations, aliquoted and freezed at -80 °C. The quality of samples was tested by migrating samples on agarose gel and quantified.

Amplification of DNA fragments by a Polymerase chain reaction (PCR) approach

PCR is a method for amplifying DNA in vitro. The PCR starts with a heat denaturation step at 95°C which leads to the separation of the two DNA strands. The second step allows the annealing of oligonucleotidic primers to the DNA matrix (generally 50 to 65 °C depending o3n the primer sequence). The melting temperature of the primers is based on the length and quantity of purine basis and is calculated as follow:
Tm= 69.3+0.41 x (%GC) – 650/N, where N is the number of nucleotides present in the primer. The annealing temperature must be fixed close to the melting temperature, it is generally fixed as Tm= -5 °C.
The third step is performed at 72 °C which is generally the optimal working temperature of the Taq DNA polymerase. This step allows the synthesis of the complementary strand of the DNA Matrix starting from the primer. The enzyme needs free dNTPs present in reaction mixture to synthesize this strand. Time duration for this elongation step depends on the length of the sequence to be amplified (generally 1 min/1000 base pairs). These three main cycles are repeated for specific numbers that permit to amplify specific fragments of DNA.

Polymerase chain reaction for Phire plant direct PCR kit

This PCR differs from a classical reaction only for the primers annealing temperature, which were measured using the Tm calculator and instructions available at Thermo Scientific Phire Hot Start II DNA Polymerase has an engineered DNA-binding domain that enhances the polymerase activity. Additionaly, it exhibits extremely high resistance to many PCR inhibitors found in plants. Reaction mix for PCR contained, 0.5 µM of each primer, 0.4 µl of Phire Hot Start II DNA Polymerase, 10 µl of 2x Phire Plant PCR Buffer and 0.5 µl of Plant tissue (dilution protocol) for a 20 µL of total reaction volume.
The PCR starts with an initial denaturation step at 98°C for 5 min, and then 40 cycles of amplification (5 sec at 98°C, 5 sec at 62 to 78°C, and then 20-40 sec at 72°C). A final elongation step was carried at 72°C for 1 min. Two different primer pairs (nptII dir and nptII rev (i) and 35S dir and x-transgene rev (ii)) were utilized for checking the presence of two genes (nptII (i) and transgene along with the part of 35S promoter: 35S:: transgene (ii)) in different potentially transformed palnts of R. graveolens. Primer pair utilized for the amplification of the nptII gene along with the temperature of hybridization is listed in atable (Table 12), while the second pair of primers will be discussed latterely in the respective part.

Analysis of DNA and RNA by agarose gel electrophoresis

Agarose gel electrophoresis is a technic that is used to separate DNA or RNA on the basis of their sizes. Concentration of the gel can be adjusted depending on the type of molecule to be analyzed. A gel with a concentration of 1% will be used for analysis of a wide range of molecules. This gel is prepared by dissolving 1g agarose in 100 ml TAE 1X buffer (Tris acetate 40 mM, EDTA 1 mM). Samples are supplemented with 6X loading buffer (Tris 50 mM pH 7.5; EDTA 100 mM pH 7.5; 10% glycerol; 0.05% bromophenol blue) and loaded on the gel. Separation of the sample is realized at 100 V (10 V/cm) for 20 to 45 min depending on type of sample. In addition to samples, a colored marker of size (Fermentas massruler ready -to-use # SM0403) is loaded on the gel to help to determine the approximate sizes of the bands appearing on the gel after staining with Ethidium Bromide (EtBr) or Sybr Safe. EtBr, is an intercalating agent and commonly used as fluorescent tag in molecular biology. Once intercalated into DNA it fluoresces when exposed to Ultraviolet light (λ=254 nm). EtBr is highly toxic. Syber® Safe DNA Gel stain (Invitrogen) is an alternative to EtBr. It has been classified as less toxic than EtBr for users as well as for environment. It is also an intercalating agent and is available in solution 10.000 time concentrated (Invitrogen) in Dimethyl sulfoxide (DMSO). This solution is finally diluted in a ratio of 1:10000. DNA stained with Syber® Safe DNA Gel stain can be visualized by blue light transilluminator or a standard UV transilluminator. However it is advised to avoid the exposure of DNA stained with Syber® Safe to UV if it is going to be used in cloning process.

Extraction of DNA from agarose gel

Once migration and visualization of samples on agarose gel is finished, DNA can be extracted from the gel. For this purpose, the band is cut out from the gel using a clean scalpel and is transferred in a 2 ml microtube. The DNA is than extracted from the gel using the QIAquick Gel Extraction Kit (Annex). Once extracted, the DNA can be used for subsequent experiments such as cloning, sequencing or re-amplification by PCR.

Digestion of DNA fragment by restriction enzymes

All the restrictions have been done with the FastDigest® Restriction Enzymes commercialized by Fermentas (Thermo scientific). These enzymes allow a short time incubation (reducing the time of incubation by a factor of 10 as compared to classical enzymes) and provide to do most of the digestion in a single and common buffer. Digestion reaction is generally carried out in a final volume of 20 µl containing 2 µl of 10X buffer, 1 µl of restriction enzyme and 1 µg of DNA. Reaction mixture is incubated at 37 °C for 5 min to 1 h.

Table of contents :

1 Phenylpropanoids
1.1 Biosynthetic pathway of phenylpropanoids
1.2 Coumarins and furanocoumarins
1.2.1 Coumarins
1.2.2 Biosynthetic pathway of coumarins
1.3 Furanocoumarins
1.3.1 Distribution of furanocoumarins in the plant Kingdom
1.3.2 Role of furanocoumarins
1.3.3 Localization of furanocoumarins in plants
1.3.4 Storage of furanocoumarins in plant cell
1.3.5 Biosynthetic pathway of furanocoumarins
1.3.6 Biological properties of furanocoumarins Photolysis and photo-oxydation of furanocoumarins Photocylcloaddition and photo-dimerization Reaction of furanocoumarins with nucleic acid Reaction of furanocoumarins with lipids Reaction of furanocoumarins with proteins
1.3.7 Furanocoumarins in the pharmaceutical industry Photochemotherapy (PUVA) Other implications of furanocoumarins as pharmaceutics
1.3.8 Toxicity caused by furanocoumarins Phytophotodermatitis Melanoma Interaction of furanocoumarins with drugs
2 Prenyltransferases
2.1 Role of prenyltransferases in plants
2.1.1 Implication of prenyltransferases in the biosynthesis of primary metabolites Biosynthesis of isoprenoids Isopentenyl diphosphate isomerase (IPPI) Reaction mechanism of GPP synthase Subcellular Localization of the biosynthetic pathway of isoprenoids Diversity of molecules derived from the isoprenoid biosynthetic pathway Biosynthesis of isoprenoid quinones DHNA phytyltransferases HGA Prenyltransferases p-hydroxybenzoic acid prenyltransferase Prenyltransferases involved in the biosynthesis of other plant essential products Cytokinins (Zeatin) Biosynthesis of photosynthetic pigments Post translational modification of proteins
2.1.2 Prenyltransferases in biosynthesis of secondary metabolites General introduction Biosynthesis of plant prenylated polyphenols Polyphenols Biological activities of prenylated polyphenols Biochemical studies of polyphenol prenyltransferases of plants Biochemical studies for soluble-type aromatic prenyltransferases Biochemical studies for membrane bound aromatic prenyltransferases. Isolation of aromatic prenyltransferases of plant polyphenols Shikonin biosynthesis Flavonoid and isoflavonoid biosynthesis Structure activity relationship of Prenyltransferases Three dimensional modeling of aromatic prenyltransferase Reaction mechanism of aromatic prenyltransferases of plant polyphenols Evolution of aromatic prenyltransferases of plants Heterologous expression systems for the characterization of plants membrane-bound aromatic prenyltransferases
3 Objective of the project Material and Methods
1 Material
1.1 Plant material
1.1.1 P. crispum
1.1.2 N. benthamiana
1.1.3 R. graveolens
1.2 Bacterial strains
1.2.1 E. coli Top 10
1.2.2 E. coli GeneHogs® (Invitrogen)
1.2.3 E. coli M15
1.2.4 E. coli BL21 (DE3) (Novagen)
1.2.5 E. coli ccdb survival
1.2.6 E. coli HB101 (pRK2013)
1.2.7 E. coli MC1061
1.3 Agrobacterial strains
1.3.1 A. tumefaciens C58C1RifR (pGV2260)
1.3.2 A. tumefaciens LBA4404 (pAL4404)
1.4 Yeast strains
1.4.1 Saccharomyces cerevisiae WAT11 and WAT21
1.5 Recombinant plasmids and vectors
1.5.1 pYeDP60
1.5.2 pQE30 (Qiagen)
1.5.3 pGEX-KG (Amersham Biosciences)
1.5.4 pCR®8/GW/TOPO® (Invitrogen)
1.5.5 Binary vector systems pBin-GW pGWB2
1.6 Culture media
1.6.1 For bacteria Liquid LB Solid LB YEB
1.6.2 Yeast medium YPGA SGI YPGE YPL
1.6.3 Culture media and conditions used for plants culture Murashige and Skoog (MandS) In vitro culture of R. graveolens
1.6.4 Growth of plants in soil
1.7 Antibiotics
2 Molecular biology
2.1 DNA Extraction from plant
2.2 RNAs Extraction from plant material
2.3 Amplification of DNA fragments by a Polymerase chain reaction (PCR) approach
2.3.1 Classic Polymerase chain reaction
2.3.2 High Fidelity PCR
2.3.3 Reverse transcription
2.3.4 Verification of the stably transformed plants using the Phire plant direct PCR k Preparation of samples Polymerase chain reaction for Phire plant direct PCR kit
2.4 Analysis of DNA and RNA by agarose gel electrophoresis
2.5 Extraction of DNA from agarose gel
2.6 Digestion of DNA fragment by restriction enzymes
2.7 Ligation
2.7.1 pCR8®/GW/ TOPO®
2.7.2 Ligation in other vectors
2.8 Construction of recombinant binary plasmid for genetic transformation of plant cells using the Gateway® Technology
2.8.1 Recombination reaction via Gateway® technology
2.9 Preparation of competent E. coli bacteria
2.9.1 Preparation of electro-competent bacteria
2.9.2 Preparation of chemically competent bacteria
2.10 Transformation of competent E. coli bacteria
2.10.1 Electroporation
2.10.2 Heat shock
2.11 Transformation of A. tumefaciens
2.11.1 Heat shock Preparation of chemically-competent Agrobacteria Heat shock transformation of Agrobacteria Transformation of agrobacteria using the triparental mating method Technical approach
2.12 Extraction of plasmidic DNA
2.13 Sequencing
2.14 Induction of the expression of genes by UV
3 Heterologous expression system
3.1 Prokaryote expression system
3.1.1 Expression
3.1.2 Purification of protein
3.2 Eukaryotes expression system
3.2.1 Yeast Expression system Preparation of competent yeast Transformation of yeast Protein expression in yeast Conditions of culture Preparation of yeast culture Microsomes preparation
3.3 Heterologous Expression in plants
3.3.1 Transient expression in N. benthamiana
3.3.2 Inoculum Preparation
3.3.3 Agro-infiltration of leaves
3.4 Stable transformation of R. graveolens
3.4.1 Germination of R. graveolens seeds
3.4.2 Preparation of bacterial inoculum
3.4.3 Transformation of hypocotyls of R. graveolens
4 Methods of biochemical analysis
4.1 Quantification of proteins
4.2 Quantification of P450 by CO spectrum
4.3 Measurement of enzymatic parameters
4.3.1 For Cytochrome P450 Determination of kinetic constants
4.3.2 For prenyltransferases Determination of enzyme kinetics Measurement of the inhibition of the Pt activity by furanocoumarins and determination of the inhibition constant
4.4 HPLC Analysis
4.4.1 Metabolisation of substrate
4.4.2 Analysis of extracts of phenylpropanoids
4.5 Analysis by mass spectrometry (MS)
4.5.1 Preparation of samples for analysis for quantification Detection and quantification of hydroxylated products Detection and quantification of prenylated products
4.6 Synthesis of substrats
4.6.1 Synthesis of CoA esters Production of CoA esters Kinetics of chemical reactions Synthesis of shikimic and quinic acid esters Synthesis of shikimic acid and quinic acid derivatives
5 Methods of analytical analysis
5.1 Preparation of phenylpropanoids extracts
5.2 Quantification of expression of gene by real time PCR
5.2.1 Preparation of material
5.2.2 Preparation of the reaction mix
6 Statistical analysis of data
Results and discussion
Chapter I: New putative aromatic prenyltransferases
1 Identification of candidate genes encoding for enzymes belonging to the aromatic prenyltransferases family
1.1 Identification and isolation of candidate genes
1.1.1 In silico data mining A. gigas homogentisic acid phytyltransferases Identification of a gene encoding for a putative Pt of P. sativa
1.1.2 PCR approach using degenerated primers
1.1 In silico analysis of the putative prenyltransferase sequences
1.1.1 Consensus sequences
1.1.2 Protein typology
1.1.3 Subcellular localization
1.1.4 A new group of prenyltransferases on the basis of phylogenetic analysis
1.2 Conclusion
Chapter II: Development of a heterologous transient expression system for membranous proteins using Nicotiana benthamiana
2 Development of heterologous expression system of N. benthamiana
2.1 Introduction
2.2 Validation of a transient expression system in N. benthamiana using the Green Fluorescent Protein (GFP)
2.2.1 Recombinant plasmid: pBIN-m-gfp5-ER
2.2.2 Preparation of Agrobacterium inoculum and inoculation of N. benthamiana leaves….
2.2.3 Determination of the best concentration of agrobacteria to be used for infiltrating N. benthamiana leaves
2.2.4 Improvement of the expression of GFP Co-expression of GFP with p19
2.3 Development of a N. benthamiana -based transient expression system for the expression and the functional characterization of a membranous enzyme (CYP
2.4 Development of a N. benthamiana heterologous expression system in order to do the functional characterization of aromatic prenyltransferases
2.4.1 Presence and stability of potential substrates for Prenyltransferases involved in the biosynthesis of furanocoumarins
2.4.2 Validation of the N. benthamiana transient expression system for the expression of SfN8DT-1, a flavonoid prenyltransferase isolated from S. flavesc Recombinant plasmid: pGWB5-SfN8DT-1 Preparation of inoculum and infiltration of plants Development of N. benthamiana transient expression system for in vitro functional characterization Preparation of plant microsomes and test for the enzyme activity N. benthamiana transient expression system for in vivo characterization of SfN8DT-1..
2.4.3 Discussion and conclusions
Chapter III: Functional characterization of the first aromatic prenyltransferase which catalyzes the prenylation of umbelliferone to produce demethylsube
3 Functional characterization of the umbelliferone prenyltransferase
3.1 Introduction
3.2 Cloning of the PcPt coding sequence into the pBIN-GW plasmid
3.3 Transient expression of PcPT in N. benthamiana
3.3.1 In vivo characterization of PcPt
3.3.2 Biochemical (in vitro) characterization of PcPt Enzymatic characterization Screening for other potential substrates Biochemical and kinetic characterization of PcPt Optimal incubation time Optimal pH Saturated concentration of DMAPP Measurement of the kinetic constants of PcPt Measurement of uncompetitive inhibition
3.4 Stable expression of PcPt
3.4.1 Construction of transgenic R. graveolens plants using PcPt as a transgene
3.4.2 Molecular characterization of transgenic plants
3.4.3 Relative Expression level of PcPt assessed by real time PCR
3.4.4 Quantification of coumarins and furanocoumarins Is there a relationship between a tissue specific expression pattern of PcPt and the coumarin/furanocoumarin content in Parsley (P. crispum)? Analysis of the coumarin and furanocoumarin composition in P. crispum plants Quantification of the PcPt expression within various organs of P. crispum
3.4.5 Metabolic engineering of the biosynthetic pathway of furanocoumarin in N. benthamiana Co-infiltration of tobacco leaves with Agro-pBIN-p19, Agro-pBIN-C2’H and Agro-pBIN-PcPt Analyses of samples by mass spectrometry
3.4.6 Conclusion and Discussion
Chapter IV: Functional characterization of other putative aromatic prenyltransferases
4 Functional characterization of other putative aromatic prenyltransferases
4.1 Cloning of the six aromatic prenyltransferase coding sequence into a plant expression vector (either pBIN-GW or pGWB2)
4.2 Functional characterization of PsPt
4.2.1 General presentation
4.2.2 Umb, a substrate for PsPt ?
4.3 Functional characterization of CliPt
4.3.1 Transient expression of prenyltransferase of CliPt in N. benthamiana In vivo characterization of CliPt In vitro characterization of CliPt
4.3.2 Construction of transgenic R. graveolens plants overexpressing CliPt Molecular characterization of CliPt transgenic plants Overexpression of CliPt: detection through RT-qPCR. Metabolic profiles of transformed R. graveolens plants: Quantification of geranylated Umb and geranylated esculetin
4.3.3 Attempt for functional characterization for other aromatic prenyltransferases Transient expression system of N. benthamiana Stable expression in R. graveolens
4.4 Discussion and conclusions
5 General conclusion
6 Perspectives
7 Résumé en français pour validation par le conseil scientifique
7.1 Chapitre 1 : Identification et clonage de nouveaux gènes codants pour des Prenyltransferases
7.2 Chapitre 2 : Mise au point d’un système d’expression adapté pour l’expression de protéines membranaires
7.3 Chapitre 3: Caractérisation fonctionnelle de nouvelles prenyltransferases impliquées dans la synthèse de furocoumarines.

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