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WO1993018158A1 - Enzyme de plante recombinee - Google Patents

Enzyme de plante recombinee Download PDF

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Publication number
WO1993018158A1
WO1993018158A1 PCT/GB1993/000432 GB9300432W WO9318158A1 WO 1993018158 A1 WO1993018158 A1 WO 1993018158A1 GB 9300432 W GB9300432 W GB 9300432W WO 9318158 A1 WO9318158 A1 WO 9318158A1
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WIPO (PCT)
Prior art keywords
sequence
enzyme
plant
acp
acyl
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PCT/GB1993/000432
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English (en)
Inventor
Susan Amanda Hellyer
Richard Safford
Neil Martin Loader
Antoni Ryszard Slabas
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Unilever Plc
Unilever N.V.
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Application filed by Unilever Plc, Unilever N.V. filed Critical Unilever Plc
Priority to EP93904281A priority Critical patent/EP0629243A1/fr
Priority to AU35731/93A priority patent/AU3573193A/en
Publication of WO1993018158A1 publication Critical patent/WO1993018158A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—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 involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • This invention relates to the novel nucleotide sequence of a gene encoding a plant enzyme, functional equivalents thereof, vectors containing the novel nucleotide sequence, a method of producing the enzyme, a method of altering the characteristics of a plant and plants having been so altered.
  • oils or lipids consist of fatty acid residues (relatively long hydrocarbon chains) joined by ester links to propan -1, 2, 3 - triol (otherwise known as glycerol).
  • acyl-ACP A carboxylase and fatty acid synthetase.
  • the end product of de novo fatty acid biosynthesis is palmitoyl-ACP (Acyl Carrier Protein) which is then rapidly elongated to stearoyl-ACP.
  • a highly specific stearoyl-ACP desaturase ensures that the only unsaturated fatty acid in high abundance is oleoyl-ACP.
  • Another enzyme, acyl-ACP Another enzyme, acyl-ACP
  • thioesterase is thought to play an important role in the chain termination of fatty acid biosynthesis by catalysing the hydrolysis of acyl-ACP to free fatty acid and ACP. It is believed that subsequently the free fatty acid is incorporated into complex lipids.
  • Acyl-ACP thioesterases may be characterised by their preferred substrates.
  • acyl-ACP thioesterases showing preferential enzymatic activity for long chain fatty acids eg C18 compounds
  • long chain fatty acids eg C18 compounds
  • acyl-ACP thioesterases from plants have been identified and purified. These include the enzymes from Avocado and Safflower (Ohlrogge et al., [1978]
  • the invention provides a nucleotide
  • sequence encoding an enzyme precursor having an acyl-ACP thioesterase activity, comprising the sequence of
  • nucleotides 169-1269 shown in Figure 1 (Seq. ID No. 1) or functional equivalents thereof.
  • nucleotide sequence of the invention include, for example: those nucleotide sequences which encode the same polypeptide with the same activity (i.e. which cleave acyl-ACP molecules) but which, by virtue of the degeneracy of the genetic code, possess a different nucleotide sequence; sequences which encode substantially the same polypeptide but wherein there may be one or more conserved amino acid substitutions (i.e.
  • sequences which encode substantially the same polypeptide which preferably share at least 50% amino acid homology and more preferably at least 60% homology) but wherein there may be one or more minor deletions or truncations; and sequences which hybridize under standard conditions to the complement of nucleotides 169-1269.
  • Such functional equivalents will have at least 75% nucleotide sequence homology and preferably at least 85% homology.
  • An example of a functional equivalent is the sequence pNL3 (Seq. ID No. 3) shown in Figure 2.
  • a particular example of a functional equivalent is the sequence comprising the antisense equivalent to the sequence of the invention. Whilst antisense sequences are not generally understood to be functional equivalents, use of the term functional equivalent is intended for the purposes of the present application to encompass such sequences.
  • Another particular example of functional equivalents are those sequences which code for the mature enzyme rather than the enzyme precursor.
  • the precursor includes an N-terminal transit peptide of about 4kDa.
  • the N-terminal 27-37 amino acids preferably the N terminal 29-35 amino acids
  • the transit peptide could be exchanged for the transit peptide of another protein (e.g. that of ACP ) .
  • sequence also comprises a suitable 5' untranslated region, including a promoter, to enable expression in appropriate host cells.
  • a suitable 5' untranslated region including a promoter
  • the sequence also comprises a suitable 3' untranslated region.
  • this 3' untranslated region can comprise other signals, such as a polyadenylation signal.
  • the invention provides an enzyme precursor having an acyl-ACP thioesterase activity, comprising the amino acid sequence shown in Figure 1 (Seq. ID No. 1) or functional equivalents thereof.
  • functional equivalents can include mature enzymes lacking the N terminal transit peptide (which is thought to be cleaved between Ala33 and Val 34, or in the vicinity thereof).
  • Other functional equivalents include chimaeric polypeptides comprising the transit peptides of other proteins (such as that from ACP).
  • acyl-ACP thioesterase from Brassica napus (oil seed rape), the gene for which has been cloned and sequenced by the inventors.
  • Figures 1 and 2 A number of sequence variations can be seen (detailed in Figure 3).
  • Some of these nucleotide sequence differences result in different deduced amino acid sequences, as shown in Figure 4. It is thought that all these sequence variations are manifestations of the same phenomenon: it is believed that the B. napus acyl-ACP thioesterase enzyme is encoded by a multi-gene family (i.e.
  • the invention provides a nucleotide sequence encoding an enzyme precursor having acyl-ACP thioesterase activity and comprising the amino acid sequence shown in Figure 1, or functional equivalents thereof.
  • the invention provides a vector
  • a further aspect of the invention comprises a cell
  • Such a transformed cell may be of bacterial, fungal, plant or animal origin.
  • the invention provides a method of producing an enzyme precursor having acyl-ACP thioesterase activity comprising the steps of: inserting a sequence encoding the enzyme precursor or a functional equivalent into a suitable expression vector; transforming a suitable host cell with said vector; growing said transformed host cell in suitable culture conditions; and obtaining the enzyme precursor from the host cells and/or from the culture medium.
  • a sequence is employed which encodes a
  • the enzyme precursor may be advantageous to obtain the enzyme precursor free from contamination by other plant proteins or products, thus in a preferred embodiment the enzyme precursor is expressed in an animal or bacterial cell. Most preferably a microorganism is used to express the enzyme precursor. Conveniently the precursor is secreted.
  • the invention has a number of applications. It might prove possible, by under- or over-expressing this enzyme precursor (or mature enzyme), to alter the properties of the storage lipid molecules of the plant in which the enzyme precursor is expressed. For instance, by over-expressing the enzyme precursor it might be possible to bring about premature termination of the hydrocarbon chain elongation during fatty acid biosynthesis, thus resulting in lipids with shorter hydrocarbon chain fatty acid components. Such "medium chain” lipids are useful as "hardening stock" in the production of margarines and detergents. Alternatively, by use of an anti-sense construct, it might be possible to reduce the levels of active enzyme in the plant, resulting in the increased production of longer chain fatty acids. Equally, because the enzyme catalyses the last stage in fatty acid
  • plants to be altered could include rape, sunflower, safflower, soybean, peanut, cotton, oil palm or corn.
  • the invention provides a method of altering the characteristics of a plant, comprising introducing into the plant the sequence of the invention or a functional equivalent thereof, so as to alter the level of acyl-ACP thioesterase activity.
  • the enzyme precursor or function equivalent is selectively expressed in the plant seeds. This can be achieved by expressing the precursor or functional
  • a seed specific promoter e.g. those for the ACP, napin and cruciferin genes.
  • acyl-ACP thioesterase activity are those influenced by levels of the enzyme precursor or mature enzyme, such as fatty acid yield and composition.
  • the characteristic altered is the storage oil yield or composition of the seeds of the plant.
  • the acyl-ACP thioesterase from B. napus exhibits a strong substrate specificity, with oleoyl-ACP (18:1-ACP) being the preferred substrate.
  • the product hydrocarbon should have chain lengths shorter than 18 carbon atoms.
  • One plant which contains storage lipids with fatty acid components of the preferred length is Cuphea.
  • Figure 1 shows the DNA sequence of cDNA clone pNL2 which encodes the B. napus acyl-ACP thioesterase gene and the deduced amino acid sequence of the enzyme.
  • Figure 2 shows the nucleotide sequence of cDNA clone pNL3 which encodes an allelic variant of B. napus-acyl-ACP thioesterase, and the deduced amino acid sequence.
  • Figure 3 shows the variations in nucleotide sequence between pNL2 and pNL3,
  • Figure 4 shows the variations in the deduced amino acid sequence of the proteins encoded by pNL2 and pNL3
  • Figure 5 shows the amino acid sequences obtained by direct sequencing of peptides generated by enzymatic digestion of B. napus acyl-ACP thioesterase, with boxes indicating residues which show variation from the amino acid sequence expected from the cDNA sequences, and
  • Figure 6 shows a B. napus genomic Southern blot probed with an oilseed rape thioesterase probe.
  • Figures 1 and 2 show the nucleotide sequence of pNL2 and pNL3 respectively, full length cDNA clones of the B.
  • napus acyl-ACP thioesterase gene together with the deduced amino acid sequences of the respective proteins encoded.
  • Several differences are observed between the amino acid sequence obtained directly from the protein and that deduced from the cDNA clones (see Figure 5).
  • Clones pNL2 and pNL3 were obtained in the following manner.
  • RNA was isolated from 25-30 days after flowering (D.A.F.) B. napus embryos, as previously described [Hall et al. (1978), Proceedings of the National Academy of Sciences 75 , 3196-3200], and used to construct a lambda gt10 cDNA.
  • the cDNA was made essentially according to the manufacturer's instructions (Pharmacia) and then ligated into the vector and packaged in vitro.
  • double stranded oligo dT-primed cDNA was synthesised from poly A + RNA according to Gubler and Hoffman [Gene (1983) 25:203-269]; EcoRI linkers were ligated to the ends, and the resulting material cloned into a bacteriophage vector, lambda gt10 (Amersham International).
  • oligonucleotide probe (RAAT 3AS - see below, Seq. ID No. 5), derived from the rape thioesterase peptide VMMNQDT.
  • RAAT 3AS oligonucleotide probe
  • pTZ 18R U . S . B .
  • overlapping fragments of pNL1 were sequenced in both directions .
  • ORF open reading frame
  • a second B. napus cDNA embryo library was constructed in lambda ZAPII (Stratagene), according to the manufacturer's protocol. 100,000 plaques were screened with a 730bp Bglll pNL1 fragment and eight positive clones were
  • the eight clones were purified to homogeneity and in vivo excised, according to the Sratagene protocol, to give rescued plasmids containing the cDNA insert within the polylinker. Restriction analysis revealed that two plasmids, pNL2 and pNL3, contained inserts of 1560bp and 1460bp respectively. pNL2 and pNL3 were subcloned into M13 and sequenced. Translation of the resulting DNA sequences showed both pNL2 and pNL3 to contain ORFs of 363 amino acids, but differing in the length of their 3' non-coding sequences. Neither pNL2 nor pNL3 contained an initiating methionine and, in order to obtain this, the technique of 5' RACE (Rapid amplification of cDNA ends) was employed,
  • the mixture was heated to 95°C for 7 min then held at 72°C. 10ul 10 ⁇ PCR buffer (BRL), 200uM dNTPs and 2.5 units Taq polymerase were added, heated at 45°C for 2 min then 72°C for 40 min. To PCR amplify the products, the mixture was heated to 94°C for 45 min to denature cDNA, primer annealed at 50° for 25 min and extended at 72°C for 3 min. This cycle was repeated 34 times, followed by final extension at 72°C for 10 mins.
  • BBL PCR buffer
  • 200uM dNTPs 200uM dNTPs
  • Taq polymerase 2.5 units
  • RACE 6 was identical in sequence to pNL2 and RACE 1 was identical to pNL3.
  • the complete nucleotide and deduced amino acid sequences of pNL2 and pNL3 are shown in Figure 1 and Figure 2 respectively.
  • the 2 composite clones pNL2 and pNL3 represent entire copies of the structural genes of rape acyl-ACP thioesterase. Excluding poly(A) tails, pNL2 contains 1642bp and pNL3 1523bp. Both pNL2 and pNL3 have putative initiation methionines at nucleotide 169, which show good homology to the plant consensus translation initiation sequence, and to encode open reading frames (ORFs) of 366 amino acids.
  • ORFs open reading frames
  • the two clones differ in the length of their 3' non-coding sequences.
  • the open reading frames of pNL2 and pNL3 encode polypeptides of 41,960 Da and 41,983 Da respectively and these represent the
  • Figure 3 shows the nucleotide sequence variation between cDNA clones pNL2 and pNL3.
  • Step 1 Preparation of cell free extract
  • Rape seed (100g), which had been harvested between 40-60 days post anthesis (after flowering), and stored at -70°C, was homogenised in 250ml 20mM potassium phosphate, pH 7.3, containing 2mM dithiothreitol and 0.2mM
  • the filtrate was brought to 30% saturation by the addition of solid ammonium sulphate and the suspension was stirred for 30 minutes prior to centrifugation at 20,000g for 30 minutes.
  • the supernatant was refiltered through glass wool, and brought to 50% saturation with solid ammonium sulphate. After stirring for 45 minutes the extract was centrifuged for 20 minutes at 30,000g. The pellets were stored at -20oC until required.
  • Ammonium sulphate pellets were resuspended in Buffer A (20mM bis-Tris HCl, pH 6.5, containing 2mM dithiothreitol and 0.2mM PMSF) and centrifuged at 30 000g for 10 minutes to remove particulate matter.
  • Step 3 Fast Flow Q-Sepharose chromatography
  • the supernatant was diluted with Buffer A until the conductivity was less than 20 ⁇ 10 -4 ohms (final volume 200ml) and loaded onto a Fast Flow Q-Sepharose column (2.5cm ⁇ 12cm) equilibrated in 20mM bis-Tris HCl, pH 6.5. After loading, the column was washed with equilibration buffer until absorbance at 280nm was less than 0.15.
  • Enzyme activity was eluted with a 300ml gradient from 0-0.5M NaCl in Buffer A. Fractions (5ml) were collected and assayed for activity. The flow rate throughout was 1.3ml minute -1 . The unbound sample was assayed for activity and typically less than 10% of the activity loaded did not bind. Thioesterase activity eluted between 150 and 300mM NaCl. Fractions containing activity were pooled (115-120ml) and stored overnight at -70°C.
  • Step 4 ACP-Sepharose chromatography
  • the Q-Sepharose pooled fractions were rapidly thawed and applied to an ACP-Sepharose column (1cm ⁇ 13cm)
  • Step 5 F.P.L.C Mono P chromatography
  • the ACP-Sepharose pool was dialysed against 2.51 25mM bis-Tris, iminodiacetic (pH 7.1) acid for 4.5 hours with one change of buffer after two hours.
  • the conductivity of the dialysed sample was checked and, if necessary, the sample was diluted with 25mM bis Tris pH 7.1 until the conductivity was less than 10 ⁇ 10 -4 ohms.
  • the sample was loaded via a 50ml superloop onto a Mono P column (HR 5/20) equilibrated in 25mM bis-Tris iminodiacetic acid pH 7.1 at a flow rate of 1 ml minute -1 at room temperature.
  • the column was washed with equilibration buffer and activity was eluted with 50ml 10% polybuffer 74 (v/v)/iminodiacetic acid, pH 4.0, at a flow rate of 1 ml minute -1 .
  • Fractions (1ml) were collected into tubes which contained 50ul 1M bis-Tris pH 9.9 to immediately neutralise the elutant since thioesterase activity appeared to be unstable at low pH.
  • the column was calibrated with a blank run prior to loading the enzyme. Under these chromatographic conditions, all activity bound to the column and typically 40-50% of the activity loaded was recovered in five fractions.
  • Concentrated sample (final volume 1ml) was brought to 0.2M NaCl and stored at -20°C overnight. Samples stored at -20°C lost almost no activity while those stored at 4°C lost typically 60% activity.
  • Step 6 F.P.L.C. Superose 12 gel filtration
  • the Mono P concentrated pool was centrifuged on an MSE centaur for 5 minutes prior to loading onto two Superose 12 gel filtration columns HR10/30 (1 ⁇ 30cm) in series. To obtain maximum purification, care was taken not to overload the column and therefore 200ul aliquots of the supernatant were loaded for each run.
  • the column running buffer was 50mM sodium phosphate, pH 7.2,
  • the enzyme was further characterised.
  • the intact enzyme was found to have a molecular weight of about 70 kDa, being composed of a dimer of approximately 38kDa sub-units.
  • a degraded form of the enzyme (33kDa) was found to co-purify with the intact 38kDa subunits.
  • the pH optimum was 9.5.
  • the preferred substrate of the B. napus enzyme was found to be oleoyl-ACP (18:1-ACP).
  • the partial amino acid sequence of the purified protein was determined as described below.
  • Buffer B to elute isocratically a major absorbance peak which contained no protein and was probably a contaminant from 4-vinyl-pyridine. Protein peaks were collected manually into Eppendorf tubes, fractions containing acyl-ACP thioesterase polypeptides (38 and 33kDa) were pooled.
  • the pooled purified enzyme preparation was brought to near dryness on a Univap concentrator.
  • the appropriate digestion buffer detailed below, was added and the pH was measured using indicator paper and adjusted with 1M NaOH. During the concentration step, a heavy precipitate formed which disappeared on neutralisation.
  • the final volume of samples prepared for digestion was 100-150ul.
  • Preparations containing 3-7ug (80-180 pmoles) of purified thioesterase polypeptides (38 and 33kDa) were digested with 0.15ug Endoproteinase Lys-C (Boehringer Mannheim). To check for auto-digestion a control sample was prepared with 0.15ug protease in 150ul 100mM Tris/HCl (pH 8.5), 2mM EDTA (digestion buffer). Incubations were left at 37°C for 10-16 hours. Additional Endoproteinase Lys-C (0.15ug) was added to the thioesterase and control incubations.
  • TPCK-typsin (Cooper Biomedical) overnight at 37 ⁇ C.
  • a control sample was prepared with 0.18ug TPCK-trypsin in 100ul 0.2M ammonium carbonate/1mM calcium chloride (digestion buffer) and incubated as for the protein sample.
  • Fresh TPCK-trypsin was prepared and a further 0.18ug was added to the protein and control incubations. Both samples were left at 37°C for 8 hours. Samples were stored at -20°C prior to reversed-phase chromatography.
  • Reversed-phase chromatography was performed at room temperature on Gilson HPLC equipment adapted for microbore column chromatography. Samples were loaded, via a 100ul loop, onto an Aquapore RP 300-C8 microbore column (250 ⁇ 1mm id; 7u) pre-equilibrated in 0.1% TFA. Flow rate was 0.1ml/minute. Peptides were eluted with a gradient of increasing buffer B (90% acetonitrile; 0.085% TFA) 0-70% over 100 minutes. Absorbance was monitored at 214nm and peaks were collected manually into Eppendorf tubes. Prior to loading sample, acetonitrile gradients were performed until a reproducible low baseline was obtained.
  • N-terminal Protein Sequencing was performed on an Applied Biosystems model 475 protein sequencer as described previously (Cottingham et al., [1988] Biochim. Biophys. Acta 954, 201-207). A number of peptide sequences were obtained. These are shown in Figure 5 and are Seq. ID Nos 12 (peptide 26), 13 (peptide 15), 14 (peptide 30), 15 (peptide 34) and 16 (peptide 39). The residues boxed in the tryptic peptides are different to those expected from translation of the cDNA sequences. These differences are thought to be due to the allelic nature of the gene.
  • Figure 5 shows a genomic Southern blot probed with thioesterase - specific probe. Molecular standard markers (obtained by a PstI digest of lambda DNA) are shown on the right hand side with their size shown in kilobases. The middle four lanes represent controls having a known copy number (1, 2, 5 or 10) whilst Hpa, Sac and Bam are restriction digests of rape genomic DNA resulting in 12, 17 and 16 copies respectively per haploid genome. The probe used was a 764bp fragment generated by PCR, using primers PTB1 AS and PTB2AS (Seq. ID Nos 17 and 18 respectively), shown below:
  • This example describes the construction of a plant
  • thioesterase fragment linked to a seed-specific acyl carrier protein (ACP) promoter for use in altering fatty acid composition and oil yield in transgenic oil seeds.
  • the 764 bp fragment of pNL2 was produced by PCR amplification using the primers PTB AS1.
  • PTB AS1 and PTB AS2 incorporated Sad and Kpn1 restriction sites respectively into the fragment.
  • a 100ul PCR reaction contained 10ul 10x reaction buffer (Stratagene), 200uM dNTPs, 50 pmole PTB AS1, 50 pmole PTB AS2, 100pg pNL2, 2.5 units Taq (Stratagene). Reaction conditions were 92° for 45 sec, 55°C for 45 sec, 72°C for 90 sec for 25 cycles followed by 72°C for 10 min.
  • the 764 bp fragment was recovered via DEAE paper from agarose gels, digested with Sad and Kpn I, re-run on agarose gels and recovered from DEAE paper.
  • a 1.4kb BamHI-BglII fragment of pTZ5BS [de Silva et al., (1992) Plant Molecular Biol. 18:1163-1172], representing a seed specific ACP promoter sequence, was ligated into BamHI-restricted pTZl8R [Mead et al., (1986) Prot. Engin. 1: 64-74] to produce pTZAP1.
  • pTZAP1 was restriction analysed to confirm correct orientation of ACP promoter fragment.
  • pTZAP1 was restricted with Sad and Kpn1 and recovered from agarose gel via DEAE paper. The recovered pTZAP1 was ligated with the 764 bp NL2 fragment from above to form pNL4.
  • pNL2 and the plant transformation vector pBI 101 were digested with BamH1 and Sad.
  • the 2.15kb fragment of pNL2 and the pBI 101 vector fragment were recovered from DEAE cellulose and ligated to form pETR1.
  • pETR1 was subjected to restriction analysis to confirm correct insertion of gene fragments.
  • the vector ⁇ ETR1 was transferred into Agrobacterium tumefaciens pGV3850 (Zambryski et al., (1983) EMBO J.
  • Brassica napus (cv. Westar) stem segments were transformed with the above binary Agrobacterium strain using the procedure of Fry et al. [(1987) Plant Cell Reports 6: 321-325] with the following modifications: kanamycin
  • pGV3850 containing pAPIGUS [de Silva et al. (1992), Plant Molecular Biology, 18: 1163-1172], a plasmid in which the ACP promoter controls the expression of the beta-glucuronidase (GUS) reporter gene.
  • APIGUS transformed plants were analysed for oil content and for fatty acid composition.
  • Table 1 shows the percentage total fatty acid (% TFA) content and the fatty acid composition of seeds from the transgenic plants.
  • the fat content of the control APIGUS seeds (shown as AP1-TAK in Table 1) is seen to range from 38.1%-27.3%.
  • the fat content of the pETR1 antisense thioesterase seeds ranged from 43.2%-14.7%.
  • the reduced oil content of the pETR1 seeds could result from reduced levels of thioesterase activity.
  • Acyl-ACP thioesterase serves as the chain terminating enzyme of de novo fatty acid biosynthesis, cleaving the synthesized acyl-ACP to free fatty acid and ACP. As such, decreasing the level of this terminating enzyme, via antisense technology, could have limited the overall rate of oil synthesis in the seed.
  • polyunsaturated fatty acids such as those from plants 19 and 9 is highly desirable for use as cooking oils since they are less susceptible to oxidation.
  • transformation vectors containing sense rape thioesterase genes linked to the seed specific ACP promoter for use in altering fatty acid yield and composition in transgenic oil seeds.
  • thioesterase cDNA which contained a plastid targetting sequence
  • a chimaeric thioesterase gene was constructed in which a rape ACP plastid transit sequence was fused to a partial thioesterase cDNA which encoded for the mature enzyme.
  • the chimaeric thioesterase gene was linked to the seed specific rape ACP promoter.
  • PCR reaction 100ul contained 10ul 10x reaction buffer (Stratagene), 200uM dNTPs, 50 pmole PTB 26, 50 pmole PTB 33, 100ng pNL2, 2.5 units Taq (Stratagene). Mixture heated 92°C for 30 sec, 55°C for 30 sec, 72°C for 60 sec for 25 cycles, followed by 72°C for 10 min for final extension.
  • the PTB 26 primer introduced a Xbal
  • pNL6 was constructed by restricting pNL2 with EcoRI and BglII (partial) recovering the 1.2kb fragment, via DEAE paper; and ligating into EcoRI/BamH1 restricted PTZ19.
  • a 166 bp ACP transit sequence was obtained from rape ACP cDNA clone 29C08 [Safford et al. (1988), Eur. J. Biochem.
  • PTB 31 introduced a Sail site into the fragment and PTB 32 introduced Sad and Pst1 sites.
  • the PCR product was recovered from agarose gel, via DEAE paper, restricted with Sail and Sad and cloned into M13 to confirm correct
  • thioesterase sequence pNL11 was restricted with Pst1 and a 1.1kb fragment recovered and ligated into Pst1 restricted, phosphatased pNL12. This produced pNL13.
  • the 1.4kb ACP promoter was released from pTZ5BS by restricting with BamH1 and Sal1 and was ligated into
  • pNL13 and pNL14 were both restricted with Sal1.
  • a 1.2kbb fragment was recovered from pNL13 and ligated into the linearised, phosphatased pNL14 vector to yield pNL15.
  • pNL15 was restricted with BamH1 and Sad to release a 2.8kb fragment containing the ACP promoter, the ACP transit sequence and the mature thioesterase sequence. This fragment was ligated into the plant transformation vector pBI101 which had been
  • A. tumefaciens pGV3850 via the previously described direct DNA uptake method.
  • the resulting A. tumefaciens strain was used to transform B. napus stem segments as described in Example 3 above. From the resulting transgenic plants, seeds will be analysed for altered thioesterase levels and for altered oil content and fatty acid composition.
  • a plant transformation vector was constructed containing this cDNA linked to the ACP promoter sequence.
  • Primer PTB 34 added an additional 6 bp of thioesterase sequence onto the RACE 6 product previously described and also a Sail restriction site.
  • PTB 35 introduced a HindIII site into the fragment.
  • the 180bp PCR fragment was recovered from DEAE paper restricted with Sail and HindIII and ligated into Sal1/HindIII restricted pBluescript SK + to give pNL17. DNA sequence analysis was carried out to confirm correct sequence.
  • a 440bp fragment containing the transit peptide plus part of the mature thioesterase coding sequence was obtained by
  • the PTB 36 primer introduced a HindIII site 3' to the thioesterase initiating ATG and PTB 37 introduced an EcoRI site 3' to the Bgl II site.
  • the 440bp PCR fragment was recoved via DEAE paper, restricted with HindIII and EcoRI and ligated into HindIII/EcoR1 restricted M13 to yield
  • a 1.4kb Sal1-Sad fragment was released from pNL19 and ligated into Sal1-Sad restricted pNL14 to yield pNL20.
  • pNL20 was restricted with BamH1 and Sad to yield a 2.8kb fragment containing the ACP promoter linked to the thioesterase cDNA.
  • This 2.8kb fragment was ligated into the plant transformation vector pBI101, in which the BamH1-Sad fragment encoding the b-glucuronidase (GUS) gene had been removed.
  • the final vector, pRATE was propagated in E. coli and transferred into A. tumefaciens pGV3850 as previously described.
  • the resulting A. tumefaciens strain was used to transform B. napus stem segments as described in Example 3. From the resulting transgenic plants, seeds will be analysed for altered thioesterase levels and for altered oil content and fatty acid composition.

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  • Microbiology (AREA)
  • Nutrition Science (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne une séquence nucléotidique, codant une enzyme possédant une activité acyl-ACP thioestérase, qui comprend des nucléotides de 169 à 1269 de la séquence en figure 1 ou équivalents fonctionnels. L'invention concerne également un polypeptide possédant une activité enzymatique, des vecteurs, des cellules hôtes, et des plantes transgéniques comprenant la nouvelle séquence nucléotidique avec un procédé de modification des caractéristiques d'une plante et un procédé de production de l'enzyme.
PCT/GB1993/000432 1992-03-03 1993-03-03 Enzyme de plante recombinee WO1993018158A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP93904281A EP0629243A1 (fr) 1992-03-03 1993-03-03 Enzyme de plante recombinee
AU35731/93A AU3573193A (en) 1992-03-03 1993-03-03 Recombinant plant enzyme

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB929204583A GB9204583D0 (en) 1992-03-03 1992-03-03 Recombinant plant enzyme
GB9204583.0 1992-03-03

Publications (1)

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WO1993018158A1 true WO1993018158A1 (fr) 1993-09-16

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EP (1) EP0629243A1 (fr)
GB (1) GB9204583D0 (fr)
WO (1) WO1993018158A1 (fr)

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994010288A3 (fr) * 1992-10-30 1995-01-05 Calgene Inc Thioesterases a chaine moyenne provenant de plantes
WO1995013390A3 (fr) * 1993-11-10 1995-07-06 Calgene Inc Sequences de thioesterases vegetales actives sur les substrats acyl-(proteine vectrice d'acyle)
US5654495A (en) * 1992-10-30 1997-08-05 Calgene, Inc. Production of myristate in plant cells
WO1998006862A1 (fr) * 1996-08-09 1998-02-19 Calgene Llc Procedes de fabrication de composes carotenoides et d'huiles speciales a partir de graines de plantes
EP0795020A4 (fr) * 1995-09-29 1999-02-24 Calgene Inc Sequences de stearoyl-(proteine vectrice d'acyle)-thioesterase vegetales et procedes pour augmenter la teneur en stearate des huiles de graines vegetales
US6331664B1 (en) 1997-05-05 2001-12-18 Dow Agrosciences Llc Acyl-ACP thioesterase nucleic acids from maize and methods of altering palmitic acid levels in transgenic plants therewith
US6429356B1 (en) 1996-08-09 2002-08-06 Calgene Llc Methods for producing carotenoid compounds, and specialty oils in plant seeds
US6653530B1 (en) 1998-02-13 2003-11-25 Calgene Llc Methods for producing carotenoid compounds, tocopherol compounds, and specialty oils in plant seeds
US6841717B2 (en) 2000-08-07 2005-01-11 Monsanto Technology, L.L.C. Methyl-D-erythritol phosphate pathway genes
US6872815B1 (en) 2000-10-14 2005-03-29 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US6956155B1 (en) 1999-06-04 2005-10-18 Consejo Superior De Investigaciones Cientificas High oleic high stearic plants seeds and oils
US7067647B2 (en) 1999-04-15 2006-06-27 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
EP1650304A3 (fr) * 1993-03-11 2006-07-12 The Rockefeller University Clonage et production par voie recombinante de phospholipase de vespides et thérapies immunologiques s'y rapportant
US7112717B2 (en) 2002-03-19 2006-09-26 Monsanto Technology Llc Homogentisate prenyl transferase gene (HPT2) from arabidopsis and uses thereof
US7141267B2 (en) 1999-06-04 2006-11-28 Consejo Superior De Investigaciones Cientificas (Csic) High oleic/high stearic sunflower oils
US7161061B2 (en) 2001-05-09 2007-01-09 Monsanto Technology Llc Metabolite transporters
US7230165B2 (en) 2002-08-05 2007-06-12 Monsanto Technology Llc Tocopherol biosynthesis related genes and uses thereof
US7238855B2 (en) 2001-05-09 2007-07-03 Monsanto Technology Llc TyrA genes and uses thereof
US7244877B2 (en) 2001-08-17 2007-07-17 Monsanto Technology Llc Methyltransferase from cotton and uses thereof
US7262339B2 (en) 2001-10-25 2007-08-28 Monsanto Technology Llc Tocopherol methyltransferase tMT2 and uses thereof
US7592015B2 (en) 1999-06-04 2009-09-22 Consejo Superior De Investigaciones Cientificas Use of high oleic high stearic oils
US20130031678A1 (en) * 2009-12-18 2013-01-31 Honggang Zheng Brassica plants yielding oils with a low total saturated fatty acid content
US9185861B2 (en) 2010-05-25 2015-11-17 Cargill, Incorporated Brassica plants yielding oils with a low alpha linolenic acid content
US9695434B2 (en) 2010-05-25 2017-07-04 Cargill, Incorporated Brassica plants yielding oils with a low alpha linolenic acid content
WO2024020359A1 (fr) * 2022-07-22 2024-01-25 Pioneer Hi-Bred International, Inc. Plantes brassica ayant des allèles de fata2-14 mutants produisant des huiles ayant une faible teneur totale en acides gras saturés

Citations (3)

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WO1991016421A1 (fr) * 1990-04-26 1991-10-31 Calgene, Inc. Thioesterases vegetales
WO1992011373A1 (fr) * 1990-12-20 1992-07-09 E.I. Du Pont De Nemours And Company Sequences de nucleotides de genes d'acyle-acp thioesterase de soja
WO1992020236A1 (fr) * 1991-05-21 1992-11-26 Calgene, Inc. Thioesterases a chaine moyenne d'origine vegetale

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1991016421A1 (fr) * 1990-04-26 1991-10-31 Calgene, Inc. Thioesterases vegetales
WO1992011373A1 (fr) * 1990-12-20 1992-07-09 E.I. Du Pont De Nemours And Company Sequences de nucleotides de genes d'acyle-acp thioesterase de soja
WO1992020236A1 (fr) * 1991-05-21 1992-11-26 Calgene, Inc. Thioesterases a chaine moyenne d'origine vegetale

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BIOTECHNOLOGY vol. 10, no. 1, January 1992, NEW YORK US page 59 'Advances in gene technology: Feeding the world in the 21st century' *
PLANT LIPID BIOCHEMISTRY, STRUCTURE AND UTILIZATION; NINTH INTERNATIONAL SYMPOSIUM ON PLANT LIPIDS, KENT, ENGLAND, UK, JULY 8-13, 1990. 1990, PORTLAND PRESS LTD., LONDON, ENGLAND pages 157 - 159 HELLYER, A., ET AL. 'Acyl-acyl-carrier protein thioesterase from oil seed rape purifiaction and characterization' *

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5654495A (en) * 1992-10-30 1997-08-05 Calgene, Inc. Production of myristate in plant cells
WO1994010288A3 (fr) * 1992-10-30 1995-01-05 Calgene Inc Thioesterases a chaine moyenne provenant de plantes
EP1650304A3 (fr) * 1993-03-11 2006-07-12 The Rockefeller University Clonage et production par voie recombinante de phospholipase de vespides et thérapies immunologiques s'y rapportant
WO1995013390A3 (fr) * 1993-11-10 1995-07-06 Calgene Inc Sequences de thioesterases vegetales actives sur les substrats acyl-(proteine vectrice d'acyle)
US5723761A (en) * 1993-11-10 1998-03-03 Calgene, Inc. Plant acyl-ACP thioesterase sequences
EP0795020A4 (fr) * 1995-09-29 1999-02-24 Calgene Inc Sequences de stearoyl-(proteine vectrice d'acyle)-thioesterase vegetales et procedes pour augmenter la teneur en stearate des huiles de graines vegetales
US6972351B2 (en) 1996-08-09 2005-12-06 Calgene Llc Methods for producing carotenoid compounds and specialty oils in plant seeds
WO1998006862A1 (fr) * 1996-08-09 1998-02-19 Calgene Llc Procedes de fabrication de composes carotenoides et d'huiles speciales a partir de graines de plantes
US6429356B1 (en) 1996-08-09 2002-08-06 Calgene Llc Methods for producing carotenoid compounds, and specialty oils in plant seeds
US6331664B1 (en) 1997-05-05 2001-12-18 Dow Agrosciences Llc Acyl-ACP thioesterase nucleic acids from maize and methods of altering palmitic acid levels in transgenic plants therewith
US6653530B1 (en) 1998-02-13 2003-11-25 Calgene Llc Methods for producing carotenoid compounds, tocopherol compounds, and specialty oils in plant seeds
US7141718B2 (en) 1999-04-15 2006-11-28 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US7067647B2 (en) 1999-04-15 2006-06-27 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
US7335815B2 (en) 1999-04-15 2008-02-26 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
US7265207B2 (en) 1999-04-15 2007-09-04 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US6956155B1 (en) 1999-06-04 2005-10-18 Consejo Superior De Investigaciones Cientificas High oleic high stearic plants seeds and oils
US7141267B2 (en) 1999-06-04 2006-11-28 Consejo Superior De Investigaciones Cientificas (Csic) High oleic/high stearic sunflower oils
US7592015B2 (en) 1999-06-04 2009-09-22 Consejo Superior De Investigaciones Cientificas Use of high oleic high stearic oils
US7435839B2 (en) 1999-06-04 2008-10-14 Consejo Superior De Investigaciones Cientificas High oleic high stearic plants, seads and oils
US6841717B2 (en) 2000-08-07 2005-01-11 Monsanto Technology, L.L.C. Methyl-D-erythritol phosphate pathway genes
US7405343B2 (en) 2000-08-07 2008-07-29 Monsanto Technology Llc Methyl-D-erythritol phosphate pathway genes
US8362324B2 (en) 2000-10-14 2013-01-29 Monsanto Technology Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US6872815B1 (en) 2000-10-14 2005-03-29 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US7420101B2 (en) 2000-10-14 2008-09-02 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
US7238855B2 (en) 2001-05-09 2007-07-03 Monsanto Technology Llc TyrA genes and uses thereof
US7161061B2 (en) 2001-05-09 2007-01-09 Monsanto Technology Llc Metabolite transporters
US7605244B2 (en) 2001-08-17 2009-10-20 Monsanto Technology Llc Gamma tocopherol methyltransferase coding sequence from Brassica and uses thereof
US7553952B2 (en) 2001-08-17 2009-06-30 Monsanto Technology Llc Gamma tocopherol methyltransferase coding sequence identified in Cuphea and uses thereof
US7244877B2 (en) 2001-08-17 2007-07-17 Monsanto Technology Llc Methyltransferase from cotton and uses thereof
US7595382B2 (en) 2001-08-17 2009-09-29 Monsanto Technology Llc Gamma tocopherol methyltransferase coding sequences from Brassica and uses thereof
US7262339B2 (en) 2001-10-25 2007-08-28 Monsanto Technology Llc Tocopherol methyltransferase tMT2 and uses thereof
US7112717B2 (en) 2002-03-19 2006-09-26 Monsanto Technology Llc Homogentisate prenyl transferase gene (HPT2) from arabidopsis and uses thereof
US7230165B2 (en) 2002-08-05 2007-06-12 Monsanto Technology Llc Tocopherol biosynthesis related genes and uses thereof
US20130031678A1 (en) * 2009-12-18 2013-01-31 Honggang Zheng Brassica plants yielding oils with a low total saturated fatty acid content
CN102984935A (zh) * 2009-12-18 2013-03-20 嘉吉公司 产生具有低总饱和脂肪酸含量的油的芸苔属植物
US9334483B2 (en) 2009-12-18 2016-05-10 Cargill, Incorporated Brassica plants with mutant FATA2 alleles yielding oils with a low total saturated fatty acid content
US10709079B2 (en) 2009-12-18 2020-07-14 Cargill, Incorporated Brassica plants yielding oils with a low total saturated fatty acid content
US9185861B2 (en) 2010-05-25 2015-11-17 Cargill, Incorporated Brassica plants yielding oils with a low alpha linolenic acid content
US9695434B2 (en) 2010-05-25 2017-07-04 Cargill, Incorporated Brassica plants yielding oils with a low alpha linolenic acid content
WO2024020359A1 (fr) * 2022-07-22 2024-01-25 Pioneer Hi-Bred International, Inc. Plantes brassica ayant des allèles de fata2-14 mutants produisant des huiles ayant une faible teneur totale en acides gras saturés

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GB9204583D0 (en) 1992-04-15

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