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  • 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
  • C12N15/8257—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 for the production of primary gene products, e.g. pharmaceutical products, interferon
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  • 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
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
  • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)

Definitions

• the present invention in some embodiments thereof, relates to nucleic acid constructs, cells including plant cells, plants and methods of producing same for generating and/or increasing content of artemisinin in a cell including plant cells or a plant and, more particularly, but not exclusively, to artemisinin production in non-host cells including plant cells or plants for the preparation of therapeutic compositions.
• artemisinin was identified as the principle compound in A. annua extract with anti-malarial activity. Its structure was determined to be sesquiterpene lactone with an endoperoxide bridge. In addition to their anti-malarial properties, artemisinins have been recently shown to be cytotoxic for cancer cells [Efferth, T. (2006) Current Drug Targets 7: 407-421]. Artemisinin levels in A. annua are usually in the range of 0.01 to 1 % of total dry weight, which together with the fact that complete chemical synthesis of artemisinin is complex and inefficient at least partially accounts for the drug's high price.
• artemisinin is a product of the isoprene pathway, one of the main biosynthetic pathways in plants.
• the direct precursor of amorpha-4,11-diene, the first specific substrate in the biosynthesis of artemisinin is farnesyl diphosphate (FDP), which is produced by the condensation of two molecules of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP).
• FDP farnesyl diphosphate
• IPP isopentenyl diphosphate
• DMAPP isomer dimethylallyl diphosphate
• these two compounds can be derived from either the plastid- localized deoxyxylulose-5 -phosphate (DXP) pathway or the cytosolic mevalonate pathway (MVA).
• the former includes seven reactions (catalyzed by seven enzymes) starting with pyruvate and glyceraldehyde 3 -phosphate leading to the production of geranyl diphosphate (GDP).
• GDP geranyl diphosphate
• the MVA pathway starts with the condensation of acetyl-CoA and includes five steps for the production of IPP, following which DMAPP chain elongation leads to the formation of FDP, the universal precursor for the production of sesquiterpenes. These are generated by a cyclization reaction catalyzed by sesquiterpene synthases, such as amorphadiene synthase (ADS) forming amorpha-4,11-diene from FDP.
• ADS amorphadiene synthase
• HMG-CoA reductase in artemisinin production. Specifically, they showed that overexpression of the 3-hydroxy-3-methyl- glutaryl-coenzyme A reductase (HMGR) gene (hmgr) from Catharanthus roseus L. in Artemisia annua L. resulted in higher levels of artemisinin as compared to non- transgenic plants.
• HMGR 3-hydroxy-3-methyl- glutaryl-coenzyme A reductase
• US 2008261280 provides methods for engineering genetically transformed plants and microalgae for the mevalonate and isoprenoid biosynthetic pathways.
• US 2009136925 provides methods for identifying terpenoid regulatory region- regulatory protein associations and for modulating expression of sequences of interest (e.g. a sequence encoding an enzyme involved in artemisinin biosynthesis, e.g., an enzyme such as amorpha-4,11-diene synthase or CYP71AV1).
• sequences of interest e.g. a sequence encoding an enzyme involved in artemisinin biosynthesis, e.g., an enzyme such as amorpha-4,11-diene synthase or CYP71AV1.
• US 2011300546 provides methods of utilizing the PTS gene and R A interference of the ADS gene to increase patchouli alcohol content in Artemisia annua L. plants.
• EP 2204449 provides nucleotide sequences from Artemisia annua encoding enzymes in biosynthesis of dihydroartemisinic acid. Specifically, EP 2204449 discloses artemisinic/dihydroartemisinic aldehyde dehydrogenase, DBR1, used in processes to produce dihydroartemsinic aldehyde, dihydroartemisinic acid or artemisinic acid in a host cell.
• DBR1 artemisinic/dihydroartemisinic aldehyde dehydrogenase
• a method of generating and/or increasing content of artemisinin in a cell comprising exogenous ly expressing within the cell: (i) a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes formation of amorpha-4,11-diene from farnesyl diphosphate (FDP); and (ii) a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 11(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and (iii) a polynucleotide comprising a nucleic acid sequence encoding amorpha-4,11-diene monooxygenase (CYP71AV1) which catalyzes oxid
• a nucleic acid expression construct comprising: (i) a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes the formation of amorpha-4,11-diene from farnesyl diphosphate (FDP); and (ii) a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 11(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and (iii) a polynucleotide comprising a nucleic acid sequence encoding amorpha-4,11-diene monooxygenase (CYP71AV1) which catalyzes oxidation of amorpha-4,11-diene and/or artemisinic alcohol.
• ADS amorphadiene synthase
• a cell comprising a heterologous polynucleotide
• the heterologous polynucleotide comprises: (i) a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes the formation of amorpha-4,11-diene from farnesyl diphosphate (FDP); and (ii) a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta-11(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and (iii) a polynucleotide comprising a nucleic acid sequence encoding amorpha-4,11-diene monooxygenase (CYP71AV1) which catalyzes oxidation
• ADS amorphadiene synthase
• a plant comprising the plant cell of some embodiments of the invention.
• a method of producing artemisinin comprising: (a) generating and/or increasing content of the artemisinin in a cell according to the method of some embodiments of the invention, and (b) isolating the artemisinin from the cell, thereby producing the artemisinin.
• a method of producing artemisinin comprising: (a) providing the plant cell of some embodiments of the invention, and (b) isolating the artemisinin from the plant cell, thereby producing the artemisinin.
• a method of generating and/or increasing content of artemisinin in a cell comprising exogenously expressing within the cell a nucleic acid construct which comprises: (i) a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which is capable of forming amorpha-4,11-diene from farnesyl diphosphate (FDP); (ii) a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 11(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; (iii) a
• polynucleotide comprising a nucleic acid sequence encoding amorpha-4,11-diene monooxygenase (CYP71AV1) which catalyzes oxidation of amorpha-4,11-diene and/or artemisinic alcohol; (iv) a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450; and (v) a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase ⁇ tHMG), thereby generating and/or increasing content of artemisinin in the cell (e.g., in the plant cell).
• CYP71AV1 amorpha-4,11-diene monooxygenase
• CPR cytochrome P450 reductase
• an isolated artemisinin produced by the method of some embodiments of the invention.
• a polynucleotide system comprising: (i) a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes formation of amorpha-4,11-diene from farnesyl diphosphate (FDP); and (ii) a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 11(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and (iii) a polynucleotide comprising a nucleic acid sequence encoding amorpha-4,11-diene monooxygenase (CYP71AV1) which catalyzes oxidation of amorpha-4,11-diene and/or artemisinic alcohol.
• ADS amorphadiene synthase
• a nucleic acid construct system comprising: (i) a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes formation of amorpha-4,11-diene from farnesyl diphosphate (FDP); and (ii) a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta-11(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and (iii) a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding amorpha-4,11-diene monooxygenase (CYP71AV1) which catalyze
• kit comprising the system of any of some embodiments of the invention and instructions for use in transformation of a cell.
• the cell is a plant cell.
• the method further comprising subjecting the cell to light and/or to oxygen prior to the isolating of the artemisinin.
• the method further comprising exogenous ly expressing within the cell: (iv) a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• the method further comprising exogenously expressing within the cell: (v) a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl- coenzyme A reductase (tHMG).
• a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl- coenzyme A reductase (tHMG).
• the nucleic acid construct further comprises: (iv) a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• the nucleic acid construct further comprises: (v) a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG).
• tHMG 3-hydroxy-3-methylglutaryl-coenzyme A reductase
• the heterologous polynucleotide further comprises: (iv) a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• the heterologous polynucleotide further comprises: (v) a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG).
• tHMG 3-hydroxy-3-methylglutaryl-coenzyme A reductase
• at least two of the polynucleotides of (i)-(v) are comprised in a single nucleic acid construct.
• polynucleotides (i) and (ii) are comprised in a single nucleic acid construct.
• polynucleotides (i) and (iii) are comprised in a single nucleic acid construct.
• polynucleotides (ii) and (iii) are comprised in a single nucleic acid construct.
• polynucleotides (i)-(iii) are comprised in a single nucleic acid construct.
• the polynucleotides wherein (i)-(iv) are comprised in a single nucleic acid construct are comprised in a single nucleic acid construct
• polynucleotides wherein (i)- (v) are comprised in a single nucleic acid construct are comprised in a single nucleic acid construct.
• the nucleic acid sequence further comprises a nucleic acid sequence encoding a mitochondrial signal peptide to thereby direct localization of the polypeptide into the mitochondria of the cell.
• the nucleic acid sequence comprises the nucleic acid sequence encoding an amorphadiene synthase (ADS).
• ADS amorphadiene synthase
• the nucleic acid construct further comprises: (vi) a polynucleotide comprising a nucleic acid sequence encoding a polypeptide which enables selection of a cell expressing the nucleic acid construct.
• the polypeptide which enables selection of a cell expressing the nucleic acid construct comprises a polypeptide which confers antibiotic resistance to a cell expressing the nucleic acid construct.
• the polypeptide which confers the antibiotic resistance isjieomycin phosphotransferase II (nptll).
• each of the polynucleotides further comprises a promoter sequence for directing expression of the nucleic acid sequence in the cell.
• at least two of the promoters are not identical.
• each of the polynucleotides further comprises a terminator sequence for controlling expression of the nucleic acid sequence in the cell.
• the plant is a tobacco plant.
• the plant is selected from the group consisting of tobacco, aspen, tomato, marguerite and lettuce.
• the artemisinin is characterized by liquid chromatography-mass spectrometry/mass spectrometry (LC- MS/MS) operated in multiple reaction monitoring (MRM) mode and monitoring MRM traces m/z 283.2 ⁇ 219 and 283.2 ⁇ 265.
• LC- MS/MS liquid chromatography-mass spectrometry/mass spectrometry
• the artemisinin is characterized by liquid chromatography-high resolution mass spectrometry (LC-HR- MS) m/z value of 283.1530 Da.
• the polynucleotide system of some embodiments of the invention further comprises: (iv) a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• the polynucleotide system of some embodiments of the invention further comprises: (v) a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl- coenzyme A reductase (tHMG).
• tHMG 3-hydroxy-3-methylglutaryl- coenzyme A reductase
• the polynucleotide system of some embodiments of the invention further comprises: (vi) a polynucleotide comprising a nucleic acid sequence encoding a polypeptide which enables selection of a cell expressing the nucleic acid construct.
• the nucleic acid construct system of some embodiments of the invention further comprises: (iv) a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• the nucleic acid construct system of some embodiments of the invention further comprises: (v) a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG).
• the nucleic acid construct system of some embodiments of the invention further comprises: (vi) a
• polynucleotide comprising a nucleic acid sequence encoding a polypeptide which enables selection of a cell expressing the nucleic acid construct.
• the polypeptide which enables selection of a cell expressing the nucleic acid construct comprises a polypeptide which confers antibiotic resistance to a cell expressing the nucleic acid construct.
• the polypeptide which confers the antibiotic resistance isjieomycin phosphotransferase II (nptll).
• nptll phosphotransferase II
• all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
• methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control.
• the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
• FIG. 1A is a schematic illustration of the mevalonate and proposed artemisinin pathways. Engineered genes are marked in red.
• FIG. IB is a schematic illustration of a mega vector comprising the gene constructs assembled to engineer artemisinin production. Arrows indicate genes, boxes indicate promoters and terminators, and the difference between the two constructs CYTART or MIT ART used to generate transgenic tobacco plants is in the use of ADS or mtADS.
• FIG. 2A-J are photographs illustrating subcellular localization of ADS and mtADS.
• the photographs illustrate transient expression of EGFP fused to ADS ( Figures 2A-E) or mtADS ( Figures 2F-J) in A. thaliana protoplasts.
• Transient expression of RFP (for cytosol labeling) and MitoTracker-stained mitochondria are illustrated in red
• EGFP fluorescence is illustrated in green
• plastid auto fluorescence is illustrated in blue.
• Micrographs of merged figures of RFP (Figure 2E) or MitoTracker (Figure 2J) with EGFP and Figures 2D and 21 illustrate bright-field.
• FIGs. 3A-B are photographs illustrating the expression of tHMG and artemisinin pathway genes in transgenic Nicotiana tabacum plants.
• Total RNA extracted from plants transformed with pRCS16F[kan][tHMG][CPR][ADS][CYP][DBR2] ( Figure 3A, transgenic tobacco plant line 6) and pRCS16F[kan][tHMG][CPR][mtADS][CYP][DBR2] ( Figure 3B, transgenic tobacco plant line 7) was analyzed for tHMG, ADS, mtADS, CYP71A V1, CPR and DBR2 by RT- PCR.
• FIG. 4A-C illustrate identification of artemisinin in extracts from metabolically engineered tobacco by LC-HR-MS.
• Ion current m/z values of 283.1530 and 286.1733 Da, recorded for artemisinin and artemisinin-3D respectively, are characteristic for these molecules.
• Ion current m/z value of 283.1530 is shown for control GFP and ADS-non-expressing transgenic tobacco plants ( Figure 3C).
• FIGs. 5A-D illustrate identification of artemisinin in engineered tobacco extracts by LC-MS/MS.
• Figure 4D shows pure artemisinin standard.
• MRM traces m/z 283.2 ⁇ 219 are shown in the upper chromatograms and 283.2 ⁇ 265 in lower chromatograms.
• FIGs. 6A-C illustrate identification of amorpha-4,11-diene in engineered tobacco cell suspension cultures by GC-MS.
• Figure 6A Single ion 189 m/z chromatograms of dodecane extract from metabolically engineered MITART
• Figure 6B GFP
• Figure 6C amorpha,4-l l,diene standard
• FIG. 7 is a pSAT2A.nosP.tHMG expression plasmid.
• FIG. 8 is a pSAT5.1.hspP.CYP expression plasmid.
• FIG. 9 is a pSAT4.1A.rbcP.CPR expression plasmid.
• FIG. 10 is a pSAT6A.supP.DBR2 expression plasmid.
• FIG. 11 is a pSAT5A.35SP.ADS expression plasmid.
• FIG. 12 is a pSAT5A.35SP.mtADS expression plasmid.
• FIG. 13 is a pSATlA.ocsAocsP.nptll.ocsT expression plasmid.
• the present invention in some embodiments thereof, relates to polynucleotide systems, nucleic acid constructs, nucleic acid constructs systems, cells such as plant cells, plants and methods of producing same for generating and/or increasing content of artemisinin in a cell including a plant cell or plant and, more particularly, but not exclusively, to artemisinin production in non-host cells including plant cells or plants for the preparation of therapeutic compositions.
• the present inventors have generated novel vectors for expression of the artemisinin pathway genes from A. annua in various types of plants and have transformed plant cells and plants using these novel vectors.
• the present inventors have further shown artemisinin production from transgenic plants, including tobacco plants. The aforementioned validates beyond any doubt the value of the present methods in producing artemisinin from plants.
• the present inventors have constructed vectors for expression of the artemisinin pathway genes from A. annua i.e. ADS, CYP71A V1 and DBR2, as well as CYP71A V1 reducing partner CPR (see Figure IB).
• ADS was cloned with (mtADS) or without (ADS) mitochondrial signal peptide to allow accumulation of the enzyme in the organelle or cytosol.
• HMG-CoA N' terminal truncated hydroxymethylglutaryl-CoA reductase (tHMG) from yeast was placed into these vectors (Figure IB).
• the vectors of some embodiments of the invention were used to develop transgenic plants (e.g.
• Nicotiana tabacum for production of artemisinin.
• Transgenic tobacco plants generated using the vectors of some embodiments of the invention expressed all artemisinin pathway genes ( Figures 3A-B) and authentic artemisinin accumulated in the engineered plants.
• the identity of artemisinin was confirmed by two different liquid chromatography-mass spectrometry (LC-MS) detection techniques, namely LC-high-resolution-MS (LC-HR-MS, Figures 4A-C) and LC-MS/MS ( Figures 5A-D) with multiple reactions monitoring (MRM) mode.
• LC-MS liquid chromatography-mass spectrometry
• a method of generating and/or increasing content of artemisinin in a cell comprising exogenous ly expressing within the cell:
• a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes formation of amorpha-4,11-diene from farnesyl diphosphate (FDP); and
• a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 11(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and
• DBR2 artemisinic aldehyde delta- 11(13) reductase
• a polynucleotide comprising a nucleic acid sequence encoding amorpha- 4,11-diene monooxygenase (CYP71AV1) which catalyzes oxidation of amorpha-4,11- diene and/or artemisinic alcohol;
• CYP71AV1 amorpha- 4,11-diene monooxygenase
• artemisinin refers to the compound having a chemical formula of C15H22O5 and a chemical structure as shown in Formula I below, which is highly effective as an anti-malaria drug.
• the biosynthesis of artemisinin is depicted in Figure 1.
• increasing content of artemisinin refers to at least about 0.1 %, at least about 0.5 %, at least about 1 %, at least about 2 %, at least about 3 %, at least about 4 %, at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, increase in the content of artemisinin in the cell (e.g.
• a plant cell as compared to a native cell (i.e., a cell not modified with the polynucleotides of the invention, e.g., a non-transformed cell of the same species) which is grown (or cultured) under the same (e.g., identical) growth conditions.
• a native cell i.e., a cell not modified with the polynucleotides of the invention, e.g., a non-transformed cell of the same species
• the increase in the content of artemisinin in the cell is compared to (e.g., relative to) the content in native cell grown under the same (e.g., identical) growth conditions.
• generating artemisinin refers to at least upregulating the biosynthesis of artemisinin within a cell.
• generating artemisinin refers to producing artemisinin within a cell which does not produce artemisinin when non- transformed to express the exogenous polynucleotide of some embodiments of the invention.
• ADS amorphadiene synthase
• Non-limiting examples of coding sequences of amorphadiene synthase catalytic activity are provided in GenBank Accession NOs. Q9AR04.2 (SEQ ID NO: 11 for polypeptide) and GenBank Accession NO. HQ315833.1 (SEQ ID NO: 50 for polynucleotide) from Artemisia annua; GenBank Accession NOs. AEQ63683.1 (SEQ ID NO: 12 for polypeptide) and JF951730.1 (SEQ ID NO: 13 for polynucleotide) from a synthetic construct; and GenBank Accession NOs. AFA34434.1 (SEQ ID NO: 51 for polypeptide) and JQ319661.1 (SEQ ID NO: 52 for polynucleotide).
• the polynucleotide comprises a nucleic acid sequence encoding a polypeptide having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the polypeptide set forth in SEQ ID NO: 11 (GenBank Access No. Q9AR04.2), wherein the polypeptide catalyzes the formation of amorpha-4,11-diene from farnesyl diphosphate (FDP).
• FDP farnesyl diphosphate
• Homology e.g., percent homology, identity + similarity
• Homology comparison software including for example, the BlastP or TBLASTN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters, when starting from a polypeptide sequence; or the tBLASTX algorithm (available via the NCBI) such as by using default parameters, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
• NCBI National Center of Biotechnology Information
• default parameters for tBLASTX include: Max target sequences: 100; Expected threshold: 10; Word size: 3; Max matches in a query range: 0; Scoring parameters: Matrix - BLOSUM62; filters and masking: Filter - low complexity regions;
• the polynucleotide comprises a nucleic acid sequence having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence identity to the polynucleotide set forth in SEQ ID NO: 50 (GenBank Accession No. HQ315833.1), wherein the polynucleotide encodes a polypeptide which catalyzes the formation of amorpha-4,11-diene from farnesyl diphosphate (FDP).
• FDP farnesyl diphosphate
• the nucleic acid sequence further comprises a nucleic acid sequence encoding a mitochondrial signal peptide to thereby direct localization of the polypeptide into the mitochondria of the cell.
• Non-limiting examples of mitochondrial signal peptides which can be conjugated to the nucleic acid sequence of some embodiments of the invention (e.g., by recombinant techniques) include Nicotiana plubaginifolia atp2-l gene for mitochondrial ATP synthase: GenBank Access Nos. CAA26620.1/X02868.1 (SEQ ID NOs: 54 and 53), Mitochondrial import receptor subunit TOM20: GenBank Access Nos. NP_198909.1/NM_123458.4 (SEQ ID NOs: 56 and 55), Arabidopsis thaliana 2- oxoglutarate dehydrogenase subunit El : GenBank Access Nos.
• BAE99494.1/AK227494.1 (SEQ ID NOs: 58 and 57) and Saccharomyces cerevisiae COX4 mitochondrial targeting sequence (SEQ ID NO: 60 for the polypeptide; and SEQ ID NO: 59 for the polynucleotide).
• the mitochondria signal peptide which is conjugated to nucleic acid sequence of some embodiments of the invention is the Saccharomyces cerevisiae COX4 mitochondrial targeting sequence (SEQ ID NO: 60 for the polypeptide; and SEQ ID NO: 59 for the polynucleotide).
• the nucleic acid sequence encoding the amorphadiene synthase further comprises a nucleic acid sequence encoding a mitochondrial signal peptide to thereby direct localization of the amorphadiene synthase into the mitochondria of the cell.
• artemisinic aldehyde delta- 11(13) reductase refers to a polypeptide which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde, essentially as shown in Figure 1 and is described in Example 1 of the Examples section which follows.
• GenBank Accession NOs. ACH61780.1 SEQ ID NO: 14 for polypeptide
• EU704257 SEQ ID NO: 15 for polynucleotide
• the polynucleotide comprises a nucleic acid sequence encoding a polypeptide having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the polypeptide set forth in SEQ ID NO: 14 (GenBank Accession NO. ACH61780.1), wherein the polypeptide catalyzes the reduction of artemisinic aldehyde to dihydroartemisinic aldehyde.
• the polynucleotide comprises a nucleic acid sequence having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence identity to the polynucleotide set forth in SEQ ID NO: 15 (GenBank Accession NO. EU704257), wherein the polypeptide catalyzes the reduction of artemisinic aldehyde to dihydroartemisinic aldehyde.
• the nucleic acid sequence encoding the artemisinic aldehyde delta- 11(13) reductase (DBR2) further comprises a nucleic acid sequence encoding a mitochondrial signal peptide to thereby direct localization of the artemisinic aldehyde delta- 11(13) reductase (DBR2) into the mitochondria of the cell.
• amorpha-4,l l-diene monooxygenase CYP71AV1
• CYP71AV1 refers to a polypeptide which catalyzes oxidation of amorpha-4,11-diene and/or artemisinic alcohol, essentially as shown in Figure 1 and is described in Example 1 of the Examples section which follows.
• Non-limiting examples of coding sequences of amorpha-4,11-diene monooxygenase (CYP71AV1) catalytic activity are provided in GenBank Accession NOs. ABC41927.1 (SEQ ID NO: 16 for polypeptide) and DQ315671.1 (SEQ ID NO: 17 for polynucleotide) from Artemisia annua; GenBank Accession NO. Q1PS23.1 (SEQ ID NO: 18 for polypeptide) from Artemisia annua; GenBank Accession NOs. ADU25498.1 (SEQ ID NO: 19 for polypeptide) and HQ315834.1 (SEQ ID NO: 20 for polynucleotide) from Artemisia annua; GenBank Accession NOs.
• the polynucleotide comprises a nucleic acid sequence encoding a polypeptide having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the polypeptide set forth in SEQ ID NO: 16 (GenBank Accession NO. ABC41927.1), wherein the polypeptide catalyzes oxidation of amorpha-4,11-diene and/or artemisinic alcohol.
• the polynucleotide comprises a nucleic acid sequence having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the polynucleotide set forth in SEQ ID NO: 17 (GenBank Accession NO.
• the nucleic acid sequence encoding the amorpha-4,11-diene monooxygenase further comprises a nucleic acid sequence encoding a mitochondrial signal peptide to thereby direct localization of the amorpha-4,11-diene monooxygenase (CYP71AV1) into the mitochondria of the cell.
• the method further comprising exogenously expressing within the cell:
• a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• CPR cytochrome P450 reductase
• Cytochrome P450 reductase has been isolated from various organisms such as human, mouse, rat, dog, yeast, Drosophila melanogaster, flies, rabit, Bos taurus, boar, guinea pig (Cavia porcellus), sea urchin, Pan troglodytes, chimpanzee (Pan troglodytes), Trypanosoma brucei, fungus (Aspergillus nidulans), green alga (Chlamydomonas reinhardtii), Toxoplasma gondii ME49, and Artemisia annua, and the coding sequences of Cytochrome P450 reductase catalytic activity can be found in various databases including the National Center for Biotechnology Information (NCBI).
• NCBI National Center for Biotechnology Information
• Cytochrome P450 reductase from Homo sapiens is provided in GenBank Accession Nos. NM 000941.2 (SEQ ID NO: 29, for polynucleotide) and NP 000932.3 (SEQ ID NO: 30 for polypeptide); and the sequence of Cytochrome P450 reductase from Artemisia annua is provided in GenBank Accession NOs. ABI98819.1 (SEQ ID NO: 31 for polypeptide) and DQ984181 (SEQ ID NO: 32 for polynucleotide).
• the polynucleotide comprises a nucleic acid sequence encoding a polypeptide having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the polypeptide set forth in SEQ ID NO: 31 (GenBank Accession NO. ABI98819.1), wherein the polypeptide catalyzes the reduction of cytochrome p450.
• the polynucleotide comprises a nucleic acid sequence having at least 80 %, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, e.g., 100 % sequence homology or identity to the polynucleotide set forth in SEQ ID NO: 32 (GenBank Accession NO. DQ984181), wherein the polynucleotide encodes a polypeptide which catalyzes the reduction of cytochrome p450.
• the nucleic acid sequence encoding the cytochrome P450 reductase (CPR) further comprises a nucleic acid sequence encoding a mitochondrial signal peptide to thereby direct localization of the cytochrome P450 reductase (CPR) into the mitochondria of the cell.
• the method further comprising exogenously expressing within the cell: (v) a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3 -hydroxy-3 -methylglutaryl-coenzyme A reductase (tHMG).
• a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3 -hydroxy-3 -methylglutaryl-coenzyme A reductase (tHMG).
• HMGR 3 -hydroxy-3 - methylglutaryl-co-enzyme-A reductase catalyzes the conversion of HMG- CoA to mevalonate and is one of the early steps in the mevalonic acid pathway leading to production of isoprenoids. It is also considered as the rate-limiting enzyme in this pathway in eukaryotic cells.
• HMGR is an integral membrane protein localized in the endoplasmic reticulum; its N-terminal region consists of a membrane-spanning domain and its catalytically active domain is located in the C-terminal region.
• sequences of the wild type (non-mutated) form of 3 -hydroxy-3 - methylglutaryl-coenzyme A reductase are known from various organisms including plants (e.g., Artemisia annua), rat, mouse, human, zebrafish, Arabidopsis thaliana, Xenopus laevis, Nasonia vitripennis, Sus scrofa, Andida dubliniensis CD36, Drosophila melanogaster, Macaca mulatta, Salmo salar, Gallus gallus, Bos yaurus, Aedes aegypti, Uncinocarpus reesii 1704, Candida tropicalis MYA-3404, Pediculus humanus corporis, Culex quinquefasciatus, Danio rerio, and more (See via NCBI web site).
• plants e.g., Artemisia annua
• rat e.g., Artemis
• GenBank Accession NOs. Q43319 SEQ ID NO: 33 for polypeptide
• GenBank Accession NOs. AAB67527 SEQ ID NO: 34 for polypeptide
• U22382.1 SEQ ID NO: 35 for polynucleotide
• GenBank Accession NOs. AA09766.1 SEQ ID NO: 62 for the polypeptide
• BK006946.2 SEQ ID NO: 61 for the polynucleotide
• N-terminal truncation removes the membrane-binding region which includes a sterol-sensing domain that is required for feedback regulation and hence forms a soluble deregulated enzyme.
• a mutated form of yeast 3-hydroxy-3- methylglutaryl-coenzyme A reductase refers to an hyperactive form of 3- hydroxy-3-methylglutaryl-coenzyme A reductase, which comprises the catalytic portion of the enzyme but which is devoid of the domain causing feedback inhibition of the cytosolic mevalonate pathway (MVA) pathway.
• the membrane spanning domain of the HMG-CoA protein is removed (ca. 500-550 amino acids are removed from the N-terminal portion of the polypeptide), alternatively the sterol-sensing domain contained within this region can be mutated to be non-functional.
• An exemplary sequence of the N-terminal truncated 3- hydroxy-3-methylglutaryl-coenzyme A reductase is set forth in SEQ ID NOs: 63 and 64 for the polynucleotide and polypeptide sequences, respectively.
• any of the polynucleotides of (i)-(v) is comprised in a nucleic acid construct along with a promoter for directing transcription of the nucleic acid sequence in a host cell (e.g., in a plant cell).
• At least two of the promoters are not identical.
• each of the polynucleotides further comprises a terminator sequence for controlling expression of the nucleic acid sequence in the cell (e.g., the plant cell).
• At least two of the polynucleotides of (i)-(v) are comprised in a single nucleic acid construct.
• polynucleotides (i) and (ii) are comprised in a single nucleic acid construct.
• polynucleotides (i) and (iii) are comprised in a single nucleic acid construct. According to some embodiments of the invention polynucleotides (ii) and (iii) are comprised in a single nucleic acid construct.
• polynucleotides (i)-(iii) are comprised in a single nucleic acid construct.
• polynucleotides wherein (i)- (iv) are comprised in a single nucleic acid construct.
• polynucleotides wherein (i)-(v) are comprised in a single nucleic acid construct are comprised in a single nucleic acid construct.
• the nucleic acid construct further comprises: (vi) a polynucleotide comprising a nucleic acid sequence encoding a polypeptide which enables selection of a cell expressing the nucleic acid construct.
• any selection polypeptide may be used to distinguish cells transformed with the nucleic acid constructs from cells not transformed with the nucleic acid construct.
• Exemplary selection polypeptides include, but are not limited to, nptll, hpt, acc3, aadA (antibiotic selection); bar and pat (herbicide selection) or dhfr (antimetabolite selection).
• the polypeptide which enables selection of a cell expressing the nucleic acid construct comprises a polypeptide which confers antibiotic resistance to a cell expressing the nucleic acid construct.
• the polypeptide which confers the antibiotic resistance is neomycin phosphotransferase II (nptll).
• nptll neomycin phosphotransferase II
• SEQ ID NO: 38 and SEQ ID NO: 37 GenBank accession V01547).
• the nucleic acid construct may further comprise additional enzymes.
• Exemplary enzymes which may be incorporated into the nucleic acid constructs of the present invention include, but are not limited to, aldehyde dehydrogenase 1 and farnesyl diphosphate synthase.
• the method of generating and/or increasing content of artemisinin in a cell comprising exogenously expressing within the cell a nucleic acid construct which comprises:
• a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which is capable of forming amorpha-4, 1 1-diene from farnesyl diphosphate (FDP);
• ADS amorphadiene synthase
• a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 1 1(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde;
• DBR2 artemisinic aldehyde delta- 1 1(13) reductase
• CYP71AV1 4, 1 1-diene monooxygenase which catalyzes oxidation of amorpha-4, 1 1- diene and/or artemisinic alcohol
• a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450; and (v) a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG).
• CPR cytochrome P450 reductase
• tHMG yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase
• exogenously expressing within the cell refers to upregulating the expression level of an exogenous polynucleotide within the cell (e.g., a plant cell) by introducing the exogenous polynucleotide into the cell and expressing the polynucleotide by recombinant means.
• expressing refers to the expression at the ribonucleic acid (RNA) level and/or at the polypeptide level.
• RNA ribonucleic acid
• the expression of the polynucleotide in the cell can be in a stable or transient manner, so as to produce the desired RNA and polypeptide molecules within the cell.
• exogenous refers to a heterologous polynucleotide or to a polynucleotide which overexpression thereof is desired in a cell.
• the heterologous polynucleotide may not be naturally expressed within the cell, e.g., can be derived from another cell of the same species, from another organism or from another species.
• the heterologous polynucleotide comprises a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the cell, which is present and/or naturally expressed within the cell.
• the cell is a prokaryotic cell.
• the cell is a eukaryotic cell.
• the cell is a yeast cell, a microbial cell, a fungi cell, a mammalian cell, an animal cell, a frog cell, a human cell and the like.
• the cell is a plant cell.
• each of the polynucleotides of some embodiments of the invention further comprises a promoter sequence for directing expression of the nucleic acid sequence in the cell.
• promoter refers to a region of DNA which lies upstream of the transcriptional initiation site of a gene to which R A polymerase binds to initiate transcription of RNA.
• the promoter controls where (e.g., which tissue, e.g., which portion of a plant) and/or when (e.g., at which stage or condition in the lifetime of an organism) the gene is expressed.
• Any suitable promoter sequence can be used by the nucleic acid construct of the present invention, and exemplary promoters are described hereinunder.
• the nucleic acid construct (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
• a typical cloning vector may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
• such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
• the nucleic acid construct of some embodiments of the invention typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
• the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
• Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
• the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
• the other upstream promoter elements determine the rate at which transcription is initiated.
• the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed.
• cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al, (1987) Genes Dev. 1 :268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al, (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
• neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912- 916] or mammary gland- specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
• Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.
• CMV cytomegalovirus
• the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
• Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation of the exogenous polynucleotide.
• Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
• Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.
• the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
• a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
• the vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
• the expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
• IRS internal ribosome entry site
• mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
• Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
• SV40 vectors include pSVT7 and pMT2.
• Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205.
• exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
• viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
• viruses infect and propagate in specific cell types.
• the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
• the type of vector used by some embodiments of the invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
• bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) as described in Liang CY et al, 2004 (Arch Virol. 149: 51-60).
• HTLV-I human T cell leukemia virus type I
• AcMNPV Autographa californica nucleopolyhedrovirus
• Recombinant viral vectors are useful for in vivo expression of the exogenous polynucleotide since they offer advantages such as lateral infection and targeting specificity.
• Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
• Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
• nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
• nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
• viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
• Useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
• the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
• a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
• Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
• LTRs long terminal repeats
• such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
• the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
• the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
• a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
• Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
• the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide.
• a fusion protein or a cleavable fusion protein comprising the protein encoded by the exogenous polynucleotide of some embodiments of the invention (the "exogenous polypeptide" hereinafter) and a heterologous protein can be engineered.
• a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein.
• the exogenous polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al, (1990) J. Biol. Chem. 265: 15854-15859].
• prokaryotic or eukaryotic cells can be used as host-expression systems to express the exogenous polypeptide of some embodiments of the invention.
• host-expression systems include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence.
• Mammalian expression systems can also be used to express the polypeptides of some embodiments of the invention.
• bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).
• yeast a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447.
• vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.
• nucleic acid constructs and methods for expressing the exogenous polynucleotide in a plant cell Following is a non-limiting description of nucleic acid constructs and methods for expressing the exogenous polynucleotide in a plant cell.
• the expression of the coding sequence can be driven by a number of promoters.
• viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:51 1-514; Nilsson et al, Physiol. Plant 100:456-462, 1997]
• coat protein promoter to TMV can be used.
• plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J.
• the promoter which is used to express the polynucleotide of some embodiments of the invention within a plant cell is the octopine synthase gene (ocs) promoter (SEQ ID NO: 39); the nopalin synthase (nos) promoter (SEQ ID NO: 40); the hspl8.1 heat shock inducible promoter (HS) (SEQ ID NO: 43); the super promoter (sup) (SEQ ID NO: 44); rubisco promoter (rbc) (SEQ ID NO: 41); and/or the 35S CaMV promoter (SEQ ID NO: 42).
• ocs octopine synthase gene
• the terminator sequence which is used for controlled expression of the polynucleotide of some embodiments of the invention within a cell is the octopine synthase gene (ocs) terminator (SEQ ID NO: 45); the nopalin synthase (nos) terminator (SEQ ID NO: 46); the rubisco (rbc) terminator (SEQ ID NO: 47); and/or the 35S CaMV promoter (SEQ ID NO: 48) and the ags terminator (SEQ ID NO: 49).
• ocs octopine synthase gene
• nos nopalin synthase
• rbc rubisco
• the nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication.
• the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible with propagation in cells.
• the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
• the nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells.
• stable transformation the exogenous polynucleotide is integrated into the plant cell genome and as such it represents a stable and inherited trait.
• transient transformation the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
• the Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
• DNA transfer into plant cells There are various methods of direct DNA transfer into plant cells.
• electroporation the protoplasts are briefly exposed to a strong electric field.
• microinjection the DNA is mechanically injected directly into the cells using very small micropipettes.
• microparticle bombardment the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
• Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein.
• the new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant.
• Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant.
• the advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
• Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages.
• the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening.
• stage one initial tissue culturing
• stage two the initial tissue culture is established and certified contaminant-free.
• stage two the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals.
• stage three the tissue samples grown in stage two are divided and grown into individual plantlets.
• the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.
• the transgenic plants are generated by transient transformation of leaf cells, meristematic cells or the whole plant.
• Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
• Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al, Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants are described in WO 87/06261.
• the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting.
• a suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus.
• Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
• Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tatlor, Eds. "Plant Virology Protocols From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.
• a buffer solution e.g., phosphate buffer solution
• the virus When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
• a plant viral polynucleotide in which the native coat protein coding sequence has been deleted from a viral polynucleotide, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral polynucleotide, and ensuring a systemic infection of the host by the recombinant plant viral polynucleotide, has been inserted.
• the coat protein gene may be inactivated by insertion of the non-native polynucleotide sequence within it, such that a protein is produced.
• the recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters.
• Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters.
• Non-native (foreign) polynucleotide sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non- native plant viral subgenomic promoters if more than one polynucleotide sequence is included.
• the non-native polynucleotide sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
• a recombinant plant viral polynucleotide is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non- native coat protein coding sequence.
• a recombinant plant viral polynucleotide in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide.
• the inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters.
• Non-native polynucleotide sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
• a recombinant plant viral polynucleotide is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
• the viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus.
• the recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants.
• the recombinant plant viral polynucleotide is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.
• a technique for introducing exogenous polynucleotide sequences to the genome of the chlorop lasts involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous polynucleotide is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts.
• the exogenous polynucleotides selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast.
• the exogenous polynucleotide includes, in addition to a gene of interest, at least one polynucleotide stretch which is derived from the chloroplast's genome.
• the exogenous polynucleotide includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous polynucleotide. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference.
• a polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
• the exogenous polynucleotides of some embodiments of the invention are introduced into a single host plant by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell.
• the transformed cell can then be regenerated into a mature plant using the methods described hereinabove.
• the regenerated transformed plants can then be cross-bred and resultant progeny selected using conventional plant breeding techniques for increased content of artemisinin.
• expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing into a single plant-cell a single nucleic-acid construct including a plurality of different exogenous polynucleotides.
• a construct can be designed with a single promoter sequence which can transcribe a polycistronic messenger R A including all the different exogenous polynucleotide sequences.
• the polynucleotide sequences can be inter-linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence.
• IRES internal ribosome entry site
• a transcribed polycistronic RNA molecule encoding the different polypeptides described above will be translated from both the capped 5' end and the two internal IRES sequences of the polycistronic RNA molecule to thereby produce in the cell all different polypeptides.
• the construct can include several promoter sequences each linked to a different exogenous polynucleotide sequence.
• artemisinin can be produced in tobacco suspension cultures.
• tobacco suspension cultures can be initiated e.g. from young leave explants of transgenic plants (e.g. transformed with the vectors of the present teachings).
• Explants can be placed on solid growth media (e.g. Murashige and Skoog (MS) growth media) supplemented with various supplements (e.g.
• solid growth media e.g. Murashige and Skoog (MS) growth media
• RNA-in situ hybridization Methods of determining the level in the cell or the plant of the RNA transcribed from the exogenous polynucleotide are well known in the art and include, for example, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-in situ hybridization.
• RT-PCR reverse transcription polymerase chain reaction
• Methods of determining the level in the cell or the plant of the polypeptide encoded by the exogenous polynucleotide are well known in the art and include, for example, Western blot analysis, activity assay, immunostaining, immunohistochemistry, immunofiuoerescence and the like.
• the plant cell forms part of a plant.
• the increase in the content of artemisinin in the plant is compared to the content in native plant grown under the same (e.g., identical) growth conditions.
• a plant comprising the plant cell of some embodiments of the invention.
• plant encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), flowers, and plant cells, tissues and organs.
• the plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.
• Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Cichorium spp., Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia
• the plant is a tobacco plant.
• the plant is Artemisia annua plant.
• the plant is an aspen, a tomato, a marguerite or a lettuce.
• a method of producing artemisinin comprising: (a) generating and/or increasing content of the artemisinin in a cell or plant according to the method of some embodiments of the invention, and (b) isolating the artemisinin from the cell or plant, thereby producing the artemisinin.
• the method of producing artemisinin is effected by: (a) providing the cell of some embodiments of the invention, and (b) isolating the artemisinin from the cell, thereby producing the artemisinin.
• the method further comprises providing and/or maintaining conditions suitable for artemisinin production within the cell.
• the method of producing artemisinin further comprises subjecting the cell or the plant to light and/or to oxygen prior to the isolating of the artemisinin.
• the cell or plant may be subjected to intense light or to daylight, for e.g. several minutes, several hours or several days, in order to increase artemisinin content in the cell or plant.
• artemisinin can be also isolated from a culture medium of the cells (e.g. plant cells), which produce artemisinin (e.g., from the natural artemisinin producing Artemisia annua (sweet wormwood) plant cells or from any cell (e.g., a plant cell) engineered to produce artemisinin by recombinant techniques according to the methods described herein).
• artemisinin or its precursor amorphadiene can than be purified from the cell liquid culture by, for example, in-situ product removal approach as previously described [Farhi, M. et al. (2011) Metab. Eng. 13: 474-481].
• a two-phase partitioning culture may be employed by adding a volume of a biocompatible solvent, e.g. 10 %-20 % (v/v) n-dodecane, methyl oleate or isopropyl myristate, as the organic phase or a solid adsorbent e.g. Amberlite resin, Diaion HP-20 or activated charcoal.
• a biocompatible solvent e.g. 10 %-20 % (v/v) n-dodecane, methyl oleate or isopropyl myristate
• a solid adsorbent e.g. Amberlite resin, Diaion HP-20 or activated charcoal.
• isolated refers to at least partially separated from the cell producing same. In a specific embodiment, isolated refers to free of pathogenic contaminants.
• artemisinin can be determined within the plant, in a cell culture medium (e.g., a plant cell culture medium), or in a plant extract, essentially as described in the general materials and experimental procedures section of the Examples section which follows.
• plant tissues are dried and ground, mixed with deuterium labeled artemisinin (Toronto Research Chemicals) and extracted by sonication (e.g., for 15 minutes) with 2 ml hexane. After partitioning into methanol (e.g., 1 ml) phases are separated and the methanolic layer is concentrated under a nitrogen stream to about 100 ⁇ .
• deuterium labeled artemisinin Toronto Research Chemicals
• Liquid chromatography-mass spectrometry (LC-MS) analysis can be performed using Agilent 1200 series rapid resolution liquid chromatography system coupled to Agilent 6410 triple quadrupole mass spectrometer.
• Agilent 1200 series rapid resolution liquid chromatography system coupled to Agilent 6410 triple quadrupole mass spectrometer.
• two Zorbax Eclipse XDB-C18 (100x2.1 mm, 1.8 ⁇ , Agilent Technologies) columns are connected in sequence followed by Synergy Fusion-RP (100x2 mm, 2.5 ⁇ , Phenomenex). Columns temperature is maintained at 40 °C, with an injection volume of about 10 ⁇ .
• Chromatographic analysis can be performed using a binary gradient (See e.g., Table 2 in the Examples section which follows).
• the mass spectrometer can be equipped with electrospray ionization ion source which is operated in positive mode upon the following parameters: capillary voltage 4000V, nebulizer pressure 241 kPa, drying gas 10 1/min, gas temperature 350 °C, 99.5 % nitrogen is used as nebulizer and drying gas and 99.999 % nitrogen is used as a collision gas.
• artemisinin can be detected by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) operated in multiple reaction monitoring (MRM) mode by monitoring transitions, e.g. 283 [M H + ] ⁇ 247, 283 ⁇ 265, 283 ⁇ 219, 283.2 ⁇ 151.1 and/or 265 ⁇ 247 under the following optimized parameters: fragmentor voltage 80V and CID energy 4eV.
• MRM multiple reaction monitoring
• LC-HRMS liquid chromatography-high resolution mass spectrometry
• an Accela LC system coupled with an LTQ Orbitrap Discovery hybrid FT mass spectrometer (Thermo Fisher Scientific Inc.) can be used. Chromatographic analysis is performed using two LC columns as described for LC-MS/MS and following a binary gradient program (See e.g., Table 3 in the Examples section which follows). Columns temperature is maintained at 40 °C, flow rate was 250 ⁇ /min and injection volume was 10 ⁇ .
• the mass spectrometer is equipped with an Atmospheric-pressure chemical ionization (APCI) ion source operated in positive ionization mode.
• APCI Atmospheric-pressure chemical ionization
• the ion source parameters can be as follows: corona discharge needle current 5 ⁇ , capillary temperature 250 °C, sheath gas rate 50 (arb), auxiliary gas rate 10 (arb), vaporizer temperature 400 °C. 99.5 % nitrogen is used as sheath and auxiliary gas.
• Ion transfer optic parameters are optimized for protonated artemisinin using the automatic tune option. Mass spectra are acquired in m/z 200-800 Da range, resolution was 30000. The LC-MS system is controlled and data can be analyzed using Xcalibur software (Thermo Fisher Scientific Inc.).
• artemisinin can be detected by liquid chromatography-high resolution mass spectrometry (LC-HR-MS) wit accurate mass ions in the range of m/z 283.1000-283.4000.
• LC-HR-MS liquid chromatography-high resolution mass spectrometry
• the artemisinin is detected by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) operated in multiple reaction monitoring (MRM) mode and monitoring MRM traces m/z 283.2
• LC-MS/MS liquid chromatography-mass spectrometry/mass spectrometry
• MRM multiple reaction monitoring
• the artemisinin produced by the method of some embodiments of the invention, from the cell (e.g., from the plant cell) or plant of some embodiments of the invention has a pharmaceutical grade purity of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, e.g., 100% purity.
• an isolated artemisinin produced by the method of some embodiments of the invention.
• any of the polynucleotides described hereinabove can be included in a system (e.g., a cocktail of polynucleotides).
• isolated polynucleotide refers to at least partially separated form the natural environment, e.g., from the plant cell naturally expressing same.
• polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes formation of amorpha-4, 1 1-diene from farnesyl diphosphate (FDP); and (ii) a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 1 1(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and (iii) a polynucleotide comprising a nucleic acid sequence encoding amorpha-4, 1 1-diene monooxygenase (CYP71AV1) which catalyzes oxidation of amorpha-4, 1 1-diene and/or artemisinic alcohol;
• ADS amorphadiene synthase
• DBR2 artemisinic aldehyde delta- 1
• system further comprises:
• a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• system further comprises:
• a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG).
• any of the polynucleotides comprised in the system can be ligated in a nucleic acid construct suitable for expression in a host cell.
• a system of isolated nucleic acid constructs comprising: (i) a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes formation of amorpha-4, 1 1-diene from farnesyl diphosphate (FDP); and (ii) a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 1 1(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and (iii) a nucleic acid construct comprising
• system further comprises:
• nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• system further comprises:
• nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG).
• system further comprises:
• nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding a polypeptide which enables selection of a cell expressing the nucleic acid construct.
• kit comprising the system of any of some embodiments of the invention and instructions for use in transformation of a cell.
• nucleic acid construct comprising:
• a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes the formation of amorpha-4,1 1-diene from farnesyl diphosphate (FDP); and (ii) a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 1 1(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and
• ADS amorphadiene synthase
• DBR2 artemisinic aldehyde delta- 1 1(13) reductase
• a polynucleotide comprising a nucleic acid sequence encoding amorpha- 4,1 1-diene monooxygenase (CYP71AV1) which catalyzes oxidation of amorpha-4, 1 1- diene and/or artemisinic alcohol.
• CYP71AV1 amorpha- 4,1 1-diene monooxygenase
• the nucleic acid construct further comprises:
• a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• the nucleic acid construct further comprises:
• a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG).
• a polynucleotide comprising a nucleic acid sequence encoding a polypeptide which enables selection of a cell expressing the nucleic acid construct as described hereinabove, e.g., a polypeptide which confers antibiotic resistance to a cell expressing the nucleic acid construct, such as the_neomycin phosphotransferase II.
• a cell e.g., a plant cell
• a heterologous polynucleotide comprising:
• a polynucleotide comprising a nucleic acid sequence encoding amorphadiene synthase (ADS) which catalyzes the formation of amorpha-4,1 1-diene from farnesyl diphosphate (FDP); and
• ADS amorphadiene synthase
• a polynucleotide comprising a nucleic acid sequence encoding artemisinic aldehyde delta- 1 1(13) reductase (DBR2) which catalyzes reduction of artemisinic aldehyde to dihydroartemisinic aldehyde; and
• DBR2 artemisinic aldehyde delta- 1 1(13) reductase
• the heterologous polynucleotide further comprises:
• a polynucleotide comprising a nucleic acid sequence encoding cytochrome P450 reductase (CPR) which catalyzes reduction of cytochrome p450.
• CPR cytochrome P450 reductase
• the heterologous polynucleotide further comprises:
• a polynucleotide comprising a nucleic acid sequence encoding a mutated form of yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMG).
• the heterologous polynucleotide further comprises: (vi) a polynucleotide comprising a nucleic acid sequence encoding a polypeptide which enables selection of a cell expressing the nucleic acid construct as described hereinabove, e.g., a polypeptide which confers antibiotic resistance to a cell expressing the nucleic acid construct, such as the_neomycin phosphotransferase II.
• the artemisinin produced from the cell or plant according to the teachings of the invention is a highly effective therapeutic agent.
• the artemisinin produced according to the teachings of the present invention may be used for treatment of malaria, cancer (e.g. hepatoma cancer), parasites (e.g. helminth parasites, neospora caninum), and towards any effective therapeutic utility.
• the artemisinin produced in accordance with the present teachings may be used for treatment is any subject in need thereof, e.g. in mammals (e.g., humans and animals).
• artemisinin is typically derivatized. Because artemisinin itself has physical properties such as poor bioavailability that limit its effectiveness, semisynthetic derivatives of artemisinin have been developed. These include: Artesunate (water-soluble: for oral, rectal, intramuscular, or intravenous use), Artemether (lipid- soluble: for oral, rectal or intramuscular use), Dihydroartemisinin, Artelinic acid, Artenimol and Artemotil.
• the artemisinin produced from the plant according to the teachings of the invention and derivatives of same is a highly effective therapeutic drug, e.g., an anti- malaria, anti cancer and anti-parasites infection drug, which can be used per se, and/or can form part of a pharmaceutical composition where it is mixed with suitable carriers or excipients.
• a highly effective therapeutic drug e.g., an anti- malaria, anti cancer and anti-parasites infection drug
• a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• active ingredient refers to artemisinin accountable for the biological effect.
• physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
• An adjuvant is included under these phrases.
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
• excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
• CNS central nervous system
• neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
• pharmacological strategies designed to increase the lipid solubility of an agent e.g., conjugation of water- soluble agents to lipid or cholesterol carriers
• the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
• each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
• compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
• Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuos infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
• the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen- free water based solution, before use.
• a suitable vehicle e.g., sterile, pyrogen- free water based solution
• compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (i.e. artemisinin or its derivative(s), e.g., a synthetic derivative) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., malaria, cancer or parasites infection) or prolong the survival of the subject being treated.
• active ingredients i.e. artemisinin or its derivative(s), e.g., a synthetic derivative
• the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
• a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
• the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
• Dosage amount and interval may be adjusted individually to provide tissue levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
• MEC minimum effective concentration
• the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
• dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
• compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may, for example, comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
• Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
• compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
• a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
• Tobacco var. Samsun
• aspen Populus tremula
• marguerite Argyranthemum frutescens
• lettuce Lactuca sativa
• mtADS mitochondrial targeted amorphadiene synthase
• CDS ADS coding sequence
• tHMG yeast 3-hydroxy-3-methylglutaryl- coenzyme A reductase
• tHMG was cloned as EcoRl/BamHl fragment into pSAT2A.nosP, producing pSAT2.nosP.tHMG, in which the tHMG ORF was expressed under the control of the nopaline synthase promoter (nos) (as shown in Figure 7);
• CYP71A V1 was cloned as Xhol/Kpnl fragment into pSAT5.1A.hspP, producing pSAT5.1.hspP.CYP, in which the CYP71A V1 ORF was expressed under the hps 18.1 heat shock inducible promoter (HS) (as shown in Figure 8);
• DBR2 was cloned as EcoRllKpnl fragment into pSAT6A.supP, producing pSAT6A.supP.DBR2, in which the DBR2 ORF was expressed under the control of the super promote
• the ADS or mtADS ORFs were cloned as EcoRllBamHl fragments into pSAT5A.35SP, producing pSAT5A.35SP.ADS and pSAT5A.35SP.mtADS, respectively, in which the mitochondria-targeted or free ADS ORF were expressed under the control of tandem constitutive 35 S CaMV promoter (as shown in Figures 11 and 12, respectively).
• the tHMG, CYP71A VI and DBR2 expression cassettes from pSAT2A.nosP.tHMG, pSAT5.1.hspP.CYP, pSAT6A.supP.DBR2 were successively cloned as Ascl/I-Ppol, I-Ceul/PI-Pspl, PI- Pspl/PI-Pspl, respectively, into their corresponding sites into pRCS16F [as previously described in Tovkach, A. et al. (2009) Plant J. 57: 747-757], producing pRCS 16F[tHMG] [AaCYP] [AaDBR2] .
• the constitutive kanamycin resistance expression cassette was cloned, as Ascl/Ascl fragment from pSATlA.ocsAocsP.nptll.ocsT [as previously described in Tzfira, T. et al. (2005) supra; Chung, S. M. et al. (2005) Trends Plant Sci. 10: 357-361] into the same site of pRCS 16F[tHMG] [AaCYP] [AaDBR2], producing pRCS16F[kan][tHMG][CYP][DBR2].
• ADS and mtADS were cloned in frame upstream of EGFP in pSAT6-EGFP-Cl vector as previously described [Chung S.M. (2005), supra].
• the resulting plasmids were used for transformation of protoplasts isolated from Arabidopsis thaliana leaf mesophyll using the TAPE-Arabidopsis Sandwich protoplast isolation method previously described [Wu FH et al. (2009) Plant Methods 24: 16] and cellular localization of EGFP signal was analyzed as reported previously [Spitzer-Rimon B. et al. (2010) Plant Cell 22: 1961-1976].
• Plant transformation vectors were mobilized into Agrobacterium tumefaciens strain AGL0 by electroporation. Nicotiana tabacum 'Samsun' was transformed using standard leaf disc transformation method as previously described [Marton, I. et al. (2010) Plant Physiology 154: 1079-1087].
• Nicotiana tabacum 'Samsun' was transformed using standard leaf disc transformation method as previously described [Marton, I. et al. (2010) Plant Physiology 154: 1079-1087].
• To generate control GFP-transgenic tobacco plants Agrobacterium tumefaciens strain AGL0 carrying pRCS2-EYFP-CHS/DsRed- P/ECFP was used as previously described [Tzfira, T. et al. (2005) supra].
• Tobacco suspension culture was initiated from young leaves explants of TO MIT ART transgenic plants producing artemisinin or control GFP-transgenic tobacco plants.
• Explants were placed on solid Murashige and Skoog (MS) growth media supplemented with 2 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/1 kinetin and grown for ca. 4 weeks until calli were formed. Calli were then transferred to a solid MS media supplemented with benzylaminopurine (BA) 0.5 mg/1 and 0.5 mg/1 1-naphthaleneacetic acid (NAA) and subcultured every 3 weeks.
• BA benzylaminopurine
• NAA 1-naphthaleneacetic acid
• Suspension cultures were initiated by placing callus fragments in flasks with liquid media based on basal MS containing 200 mg/1 KH 2 PO 4 , 1 mg/1 thiamine, 100 mg/1 myo-inositol, 0.4 mg/1 2,4-D and 3% sucrose and grown with continuous shaking for up to 4 weeks.
• stem explants were transformed as previously described [Tzfira, T. et al. (1997) Plant Mol. Biol. Rep. 15: 219-235] with A. tumefaciens strain AGL0 carrying MITART vector.
• Transgenic M82 and Micro-Tom tomatoes were regenerated after transformation with A. tumefaciens strain AGL0 carrying the MITART construct as previously described in Vishnevetsky et al. [Vishnevetsky, M. et al. (1999) The Plant Journal 20: 423-431].
• Marguerite and lettuce transgenic plants were generated by direct adventitious shoot regeneration obtained from in-situ grown leaves as previously described [Glassner, H., (2006) M.Sc, Thesis, The Hebrew University of Jerusalem] and using A. tumefaciens carrying the MITART construct.
• the primers used for RT-PCR analysis are listed in Table 1 , below.
• cDNA amplification was conducted with an initial denaturation step of 94 °C for 3 min, followed by 30 cycles of 94 °C for 10 s, 58 °C for 10 s, 72 °C for 40 s, and a final elongation step of 72 °C for 10 min.
• PCR amplification was also conducted with samples generated without RT.
• Dried and ground leaf samples tobacco, A. annua, aspen, tomato or marguerite
• 100 mg were supplemented with 10 ng deuterium labeled artemisinin (Toronto Research Chemicals) and extracted by sonication for 15 min with 2 ml hexane.
• phases were separated and the methanolic layer was concentrated to approximately 100 ⁇ under a nitrogen stream.
• the cultures were freeze-dried in a lyophilizer for approximately 24 hours before grinding and extraction as with the leaves samples.
• LC-MS/MS analysis was performed using Agilent 1200 series rapid resolution liquid chromatography system coupled to Agilent 6410 triple quadrupole mass spectrometer.
• Agilent 1200 series rapid resolution liquid chromatography system coupled to Agilent 6410 triple quadrupole mass spectrometer.
• two Zorbax Eclipse XDB-C18 100 x 2.1 mm, 1.8 ⁇ , Agilent Technologies
• Synergy Fusion-RP 100 x 2 mm, 2.5 ⁇ , Phenomenex
• Column temperature was maintained at 40 °C, injection volume was 10 ⁇ .
• Chromatographic analysis was performed using a binary gradient as follows:
• Table 2 The binary gradient used for LC-MS/MS analysis
• the mass spectrometer was equipped with electrospray ionization ion source which was operated in positive mode upon the following parameters: capillary voltage 4000 V, nebulizer pressure 241 kPa, drying gas 10 1/min, gas temperature 350 °C, 99.5 % nitrogen was used as nebulizer and drying gas and 99.999 % nitrogen was used as a collision gas.
• Artemisinin was detected in MRM mode by monitoring three transitions (283 [MH + ] ⁇ 247, 283 ⁇ 265 and 283 ⁇ 219) under following optimized parameters: fragmentor voltage 80 V and CID energy 4eV.
• the LC-MS system was controlled and data was analyzed using MassHunter software (Agilent Technologies Inc.).
• Table 3 The binary gradient used for LC-HR-MS analysis
• the mass spectrometer was equipped with an APCI ion source operated in positive ionization mode.
• the ion source parameters were as follows: corona discharge needle current 5 ⁇ , capillary temperature 250 °C, sheath gas rate 50 (arb), auxiliary gas rate 10 (arb), vaporizer temperature 400 °C. 99.5 % nitrogen was used as sheath and auxiliary gas.
• Ion transfer optic parameters were optimized for protonated artemisinin using the automatic tune option. Mass spectra were acquired in m/z 200-800 Da range, resolution was 30000. The LC-MS system was controlled and data were analyzed using Xcalibur software (Thermo Fisher Scientific Inc.).
• Leaf samples of tobacco, A. annua, aspen, tomato or marguerite 500 mg from each independent line were ground in liquid nitrogen and extracted twice by sonication for 30 min with 2 ml hexane and 600 ng of the sesquiterpene valencene as internal standard.
• the extract was partially purified on a silica gel column (100 mesh) and washed with hexane. The eluate was concentrated under nitrogen stream before analyzing a 1 ⁇ aliquot by gas chromatography-mass spectrometry (GC-MS).
• amorpha-4,11-diene production by tobacco suspension cultures a two-phase partitioning culture was employed by adding 10 %-15 % (v/v) n-dodecane as the organic phase. Cultures were grown for 29 days and the organic layer was then sampled for GC-MS as previously described [Farhi, M. et al. (2011) supra].
• the GC-MS system was composed of a TRACE GC 2000 gas chromatograph and a TRACE DSQ quadrupole mass spectrometer (ThermoFinnigan). GC was run in a 30 m Rtx-5Sil MS column with 0.25- ⁇ film thickness (Restek). The injection temperature was set at 250 °C, with an initial oven temperature of 100 °C for 1 min, followed by a 5 °C/min ramp to 270 °C. MS was operated in EI mode (70 eV) in both scanning mode (40-325 m/z) and selected ion monitoring of the molecular and fragment ions (204 and 119, 161 and 189 m/z). Amorpha-4,11-diene was identified and quantified using the selected ions' responses compared to that of the valencene internal standard as described previously [Farhi, M. et al. (2011) supra].
• HMG-R is the main rate-limiting step in the MVA pathway, its activity is regulated by feedback inhibition and its overexpression in plants enhances the accumulation of terpenoids.
• a mutated HMG-R enzyme ⁇ tHMG was generated to overcome the negative regulation and tHMG was cloned into the plant transformation vectors.
• artemisinin pathway genes ADS, CYP71A V1 and DBR2 were cloned from A. annua.
• each gene was placed between different promoters and terminators ( Figure IB).
• the two plant binary transformation vectors CYTART and MITART were used to generate transgenic Nicotiana tabacum plants. 7 lines generated with the CYTART vector carrying ADS and 8 lines generated with the MITART vector carrying mtADS, all from independent transformation events, were rooted and grown in a greenhouse. No phenotypic differences were observed between artemisinin-producing ADS and mtADS plants and control lines. Expression of all five genes was confirmed by RT-PCR analysis in these transgenic lines ( Figures 3A-B).
• MITART containing construct achieved better than CYTART derived transgenes, and up to eight-fold higher levels of artemisinin were generated by mt ⁇ DS-expressing line as compared to ⁇ DS-expressing lines (e.g. transgenic tobacco lines with cytosolic ADS produced about 0.75, 0.88, 0.94 and 0.48 ⁇ g artemisinin/g dry weight, whereas tobacco lines with mtADS generated about 5.0, 5.9, 6.3 and 6.8 ⁇ g artemisinin/g dry weight).
• the terpenoid was identified by comparison of the retention time and MS to those obtained from amorpha-4,11-diene extracted from A. annua and yeast producing amorpha-4,11-diene. Additionally, MS analysis indicated that the engineered tobacco cell suspension culture was also able to biosynthesis oxidized derivatives of amorpha-4,11-diene. No amorpha-4,11-diene, or its derivatives, was accumulate in GFP control lines. Extracts from suspension cultures, grown with or without a dodecane overlay, are monitored for artemisinin production by LC-HR-MS.
• the MIT ART and CYTART vectors were used to genetically transform several, systematically unrelated, plant species. Several transgenic lines, arising from the different transformation events, were obtained and regenerated from tomato, lettuce, aspen and marguerite. Plantlets were transferred to the greenhouse for growth and are being analyzed using LC-HR-MS and GC-MS analysis for production of artemisinin and its precursors in different plant tissues and under different growth conditions and treatments.

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