EP1190069A2 - Surexpression dans des levures ou des plantes d'un gene codant pour la glycerol 3-phosphate acyltransferase - Google Patents

Surexpression dans des levures ou des plantes d'un gene codant pour la glycerol 3-phosphate acyltransferase

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Publication number
EP1190069A2
EP1190069A2 EP00941822A EP00941822A EP1190069A2 EP 1190069 A2 EP1190069 A2 EP 1190069A2 EP 00941822 A EP00941822 A EP 00941822A EP 00941822 A EP00941822 A EP 00941822A EP 1190069 A2 EP1190069 A2 EP 1190069A2
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Prior art keywords
seq
protein
dna
organism
sequence
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German (de)
English (en)
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Ravinder K. Jain
Samuel L. Mackenzie
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National Research Council of Canada
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National Research Council of Canada
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil

Definitions

  • the invention relates to the production of fats and oils for commercial and industrial uses. More particularly, the invention relates to a process by which natural oil or fat levels in organisms may be increased, fatty acid composition of triacylglycerides may be altered, and to nucleotide sequences which may be introduced into organisms to cause the increase, and plasmids, vectors, etc. useful in the process.
  • Non-animal-source oils are produced mainly for edible purposes but their use in non- edible applications is expected to increase due to declining fossil fuel supply '(Kinney, 1998). More than 65 million metric tons of vegetable oils are produced currently with a total value of $US 25 billion 2 ' 3 (Browse et al., 1998; Murphy, 1999). World demand for vegetable oils has increased by 300% since 1960. Further, the market share of animal-derived fats has declined from 39% of the total in 1960 to 26% in 1990. All of these factors have contributed to a demand by industry for higher vegetable oil content in plant seeds in order to be cost effective during production and processing 4 (Bright and Hawkes, 1998). Biotechnology offers avenues for meeting this demand through identification and manipulation of the biochemical pathways that lead to oil production.
  • Glycerol-3-phosphate acyltransferase catalyses the first reaction in triacylglyceride synthesis via the Kennedy pathway. It uses glycerol-3 -phosphate (G-3-P) and acyl-coenzyme A (acyl-CoA) thioesters to synthesise lysophosphatidic acid (LPA).
  • G-3-P glycerol-3 -phosphate
  • acyl-CoA acyl-coenzyme A
  • LPA acyl-CoA
  • the remaining reactions are catalysed by a /ysophosphatidic acid acyltransferase (LPAAT), phosphatidic acid phosphatase (PAPase) and diacylglycerol acyltransferase (DAGAT).
  • a plastidial GPAT gene has been used by others to change the fatty acid composition of membrane lipids and to improve chilling tolerance (Murata et al. 8 , 1992 and Nishizawa 9 , 1996).
  • the bacterial GPAT gene (plsB) has also been used to change the fatty acid composition of membrane lipids and to decrease chilling tolerance
  • Another object of the invention is to produce DNA clones, constructs and vectors suitable for modifying the genomes of organisms to increase the production of triacylglycerides (TAGs), relative to the wild type.
  • TAGs triacylglycerides
  • An additional object of the invention is to produce an organism having an altered fatty acid composition in its triacylglycerides, relative to the wild type.
  • Still a further object of the invention is to identify, isolate and clone a genetic element that may be used to modify the natural formation of triacylglycerols in plants in order to increase the yield of commercial plant oils, or to modify their composition to achieve specific commercial improvements of plants and plant products.
  • the invention provides a method for increasing the oil content of an organism by inserting in the organism a DNA encoding a protein having glycerol 3- phosphate acyltransferase activity.
  • the invention provides an organism transformed with a DNA, wherein the DNA encodes a protein having GPAT activity, and the organism, after transforming, has enhanced ability to produce triacylglycerides.
  • the invention provides a vector for genetically transforming an organism, wherein the vector comprises a DNA encoding a protein having GPAT activity, and the organism, after transforming, exhibits enhanced production of triacylglycerides.
  • the invention provides a method for modifying the fatty acid composition of triacylglycerides produced by an organism, wherein the organism is transformed with a DNA encoding a protein having GPAT activity.
  • the invention relates to a method for expressing in an organism at least one additional DNA sequence encoding a protein having GPAT activity.
  • the invention pertains to micro-organisms. In a particularly preferred embodiment, to yeasts and plants.
  • the method of the invention is particularly suited to the production of oil seed plants having enhanced TAG content, or having modified fatty acid composition in their TAGs.
  • seed oil plant and “oil seed crop” are meant to encompass any plant or crop from which the oil may be isolated in marketable quantity.
  • Some plants or crops having TAGs with particularly interesting fatty acid composition are grown for the production of TAGs, even though the lipid content is low (e.g. less than 1 wt%).
  • the method of the invention may be used in such plants to increase the content of TAG.
  • Preferred plants or crops are those having a seed lipid content of at least 1 wt% (in the wildtype).
  • Borago o ⁇ cinalis Brassica species, for example mustards, canola, rape, B. campestris, B. napus, B. rapa; Cannabis sativa (Hemp, widely uses as a vegetable oil in Asia); Carthamus tinctorius (Safflower); Cocos nucifera (Coconut); Crambe abyssinica (Crambe); Cuphea species (Cuphea produce medium chain fatty acids of industrial interest); Elaeis guinensis (African oil palm); Elaeis oleifera (American oil palm); Glycine max (Soybean); Gossypium hiristum (Cotton - American); Gossypium barbadense (Cotton - Egyptian); Gossypium herbaceum (Cotton - Asiatic); Helianthus annus (Sunflower); Linum usitatissimum (Linseed or flax); Oenethera bien
  • GPAT Three types of plant GPAT have been reported: plastidial (P), mitochondrial (M), and cytosolic (ER). They exhibit different specificities towards acyl-ACP and acyl- CoA derivatives of fatty acids "(Frentzen, 1993).
  • the P-GPAT is mainly concerned with phospholipid biosynthesis in chloroplasts and has been shown to use both acyl- ACPs and acyl-CoAs as substrates, although the latter with a lower efficiency 12 (Wilkinson and Bell, 1997).
  • the ER form of GPAT is the most important for TAG biosynthesis but a plant gene has not yet been cloned.
  • the ER-GPAT uses acyl- CoAs.
  • the GPAT from the enteric bacterium Escherischia coli can use both acyl- ACP and acyl-CoA equally well 13 (Wilkinson and Bell, 1997).
  • the invention also relates to substantially homologous DNA sequences from plants encoding proteins with deduced amino acid sequences of 25% or greater identity, and 40% or greater similarity, isolated and/or characterized and/or designed by known methods using the sequence information of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO:4 or SEQ ID NO:5, and to parts of reduced length that are still able to function as inhibitors of gene expression by use in an anti-sense, co-suppression (TranSwitch; 14 Jorgensen and Napoli 1994) or other gene silencing technologies.
  • results from averages of statistically-significant numbers of plants or seeds according to the invention are best compared with results from averages of statistically-significant numbers of untransformed (wild-type) plants or seeds of the same genotype grown under identical conditions at the same time. This allows for the variability of individual plants of the same genotype, particularly when such plants are grown under different conditions.
  • the actual number of plants or seeds used to form the required average may vary, but should be enough to provide a generally constant average whenever such number is selected. Generally, the number should be at least 10, and is more preferably at least 20, 30, 50 or 100.
  • oil contents or compositions may be compared with plants of the same species transformed with an open vector (the same vector as that used for the introduction of the DNA of the invention, but with such DNA omitted), grown under identical conditions at the same time. Again, an average of results from a number of such plants, as well as plants transformed according to the present invention, is preferred (the numbers being the same as those indicated above).
  • the GPAT of the current invention is useful in manipulating GPAT activity, and triacylglycerol bioassembly in plants.
  • a construct containing the GPAT gene in a sense orientation possibly under the control of a tissue-specific promoter
  • the expression of GPAT and accumulation of seed oil can be enhanced or the acyl composition of the seed oil altered.
  • Yet another example would be to express the GPAT cDNA under the control of a constitutive promoter (e.g. 35S ; 15 Datla et al., 1993) to increase the TAG content of vegetative tissues (leaves, roots, stems). This may have particular advantages for altering the starch/oil ratio in root crops.
  • GPAT expression can be silenced to some degree by anti-sense or co- suppression (Transwitch) phenomena ( 16 De Lange et al., 1995; 17 Mol et al., 1990; 18 Jorgensen and Napoli, 1994; 19 Kinney, 1995; 20 Vaucheret et al, 1998; 21 Taylor, 1998).
  • Transwitch anti-sense or co- suppression
  • silencing GPAT in a seed specific manner may result in a reduction in TAG accumulation. This could have applications, for example, in reducing the oil content in seed barley to enhance stability during storage.
  • seed-specific silencing may lead to a relatively high accumulation of DAG or an increase in the DAG/TAG ratio in the developing or mature seed.
  • Some of the manipulations and deliverables which are possible using the GPAT gene or a part thereof, include, but are not limited to, the following: seeds with increased or decreased oil content; seeds containing oils with an enhanced diacylglycerol content, seed oils with an altered acyl composition; plants producing larger or heavier seeds; plants exhibiting an enhanced or altered capacity to accumulate storage compounds in other storage organs (e.g. tubers, roots).
  • seeds with increased or decreased oil content seeds containing oils with an enhanced diacylglycerol content, seed oils with an altered acyl composition
  • plants producing larger or heavier seeds plants exhibiting an enhanced or altered capacity to accumulate storage compounds in other storage organs (e.g. tubers, roots).
  • Figure 1 shows the Seed oil content of wild type (Wt) Arabidopsis thaliana, and thaliana transformed with: vector only (pHS737); the unmodified GPAT encoding DNA from safflower (ctpGPA); the GPAT encoding DNA from safflower from which the transit peptide has been deleted (ctpGPA-TP); the GPAT encoding DNA from safflower from which the transit peptide has been deleted and the ER retention signal has been added (ctpGPA+ERRS); the GPAT encoding DNA from Escherichia coli (plsB); and the GPAT encoding DNA from Escherichia coli to which the ER retention signal has been added (plsB+ERRS).
  • vector only pHS737
  • ctpGPA the unmodified GPAT encoding DNA from safflower
  • ctpGPA-TP the GPAT encoding DNA from safflower from which
  • the inventors chose to use the well-accepted model plant system Arabidopsis thaliana for the cloning of GPAT, as a host system for genetic engineering to alter GPAT expression, and to study the effects of altering GPAT expression on seed triacylglycerol bioassembly.
  • Arabidopsis thaliana a typical flowering plant, has gained increasing popularity as a model system for the study of plant biology.
  • Arabidopsis has come to be widely used as a model organism in plant molecular genetics, development, physiology and biochemistry ( 22 Meyerowitz and Chang, 1985; 23 Meyerowitz, 1987; 24 Goodman et al., 1995).
  • This model dicotyledonous plant is also closely related to Brassica crop species and it is increasingly apparent that information concerning the genetic control of basic biological processes in Arabidopsis will be transferable to other species ( 25 Lagercrantz et al., 1996).
  • an anthocyanin pathway-specific transcriptional activator from the monocot maize designated as R (the myc transcription factor involved in activation of biosynthetic genes for anthocyanin production in the aleurone cells of maize kernels), was expressed in the dicot
  • hypocotyl DeBlock et al., 1989
  • cotyledonary petiole 37 Moloney et al, 1989
  • wound infection a wound infection
  • particle bombardment/biolistic methods 38 Sanford et al., 1987; 39 Nehra et al, 1994; 40 Becker et al., 1994
  • polyethylene glycol-assisted protoplast transformation 41 Rhodes et al., 1988;
  • plant promoters to direct any intended up- or down-regulation of transgene expression using constitutive promoters (e.g. those based on CaMV35S), or by using promoters which can target gene expression to particular cells, tissues (e.g. napin promoter for expression of transgenes in developing seed cotyledons), organs (e.g. roots), to a particular developmental stage, or in response to a particular external stimulus (e.g. heat shock).
  • constitutive promoters e.g. those based on CaMV35S
  • Particularly preferred plants for modification according to the present invention include Arabidopsis thaliana, borage (Borago spp.), Canola, castor (Ricinus communis), cocoa bean (Theobroma cacao), corn (Zea mays), cotton (Gossypium spp), Crambe spp., Cuphea spp., flax (Linum spp.), Lesquerella and Limnanthes spp., Linola, nasturtium (Tropaeolum spp.), Oenothera spp., olive (Olea spp.), palm
  • Oilseed crops are plant species that are capable of generating edible or industrially useful oils in commercially significant yields, and include many of the plant species listed above. Such oilseed crops are well known to persons skilled in the art.
  • transgenic oilseed plant Once a transgenic oilseed plant has been produced according to the present invention, it can be grown and harvested in conventional ways. Oil may be extracted from harvested seed by collecting and crushing the seed, and/or by methods of solvent extraction, in which the crushed seeds are contacted with a solvent for the oil and the resulting solution is filtered off or decanted and the solvent removed. Other conventional and traditional methods of oil extraction may be used, if desired.
  • the method of the invention encompasses the transformation of any organism with a DNA encoding a protein having GPAT activity.
  • the inventors have demonstrated the role of GPAT in regulating the amount of TAG by expressing, in yeast and in the plant Arabidopsis thaliana, a P-GPAT gene (ctpgpat) from safflower 45 (Bhella and MacKenzie, 1994) and the GPAT gene (plsB) from E. coli.
  • plastidial proteins encoded by nuclear genes are targeted to plastids by a transit peptide (tp). Removal of the tp will confine such proteins to the cytosol. Since the enzymes of TAG biosynthesis are present in the endoplasmic reticulum ( ⁇ R) and TAGs are synthesised at the ⁇ R, an ⁇ R retention signal 46 (errs; Jackson et al., 1990) which has been shown to target many heterologous proteins including E.coli LPAAT to the ⁇ R (Weier et al. 47 , 1998) was used to target P-GPAT without a tp. The plsB gene was used as such and also with an added errs sequence. It is generally accepted in the art that proteins having 60% or greater sequence homology will have identical functionalities. Nonetheless, many cases are known in which far lower sequence homologies (e.g. 25 to 30%) exist and yet the proteins have identical functionalities.
  • heterologous genes may be particularly advantageous, because the encoded proteins may not be subject to regulation (such as feedback inhibition, or inhibition by native inhibitors) in the same way as the native GPAT.
  • the inventors used the vector pYES2 (Invitrogen) for transformation of yeast, and the vector pHS737, for transformation of Arabidopsis thaliana.
  • Examples of other vectors are:
  • pYeDP60 (Urban P, Cullin C, Pompon D 1991. Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells. Biochimie 72: 463-472); pCGS109 (Botstein D, David RW, Fink GR, Taunton-Rigby A, Knowlton RG, Mao J-I, Moir DT, Goff CG 1987. GAL 1 yeast promoter linked to non galactokinase gene. US patent No. 4661454); pYEUra3 (Clontech).
  • pHS737 and pHS738 (Selvaraj and Hirji; unpublished); pRD400 (Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W. 1992. Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 122:383- 384.); pBinl9 (Frisch DA, Harris-Haller LW, Yokubaitis NT, Thomas TL, Hardin SH, Hall TC. 1995. Complete sequence of the binary vector Bin 19.
  • An open reading frame (orf, -1.1 kb) without tp was amplified by PCR from the ctpgpat cDNA.
  • Another chimeric gene containing an errs at its 3' end was also PCR amplified.
  • the orf of the E.coliplsB gene (-2.5 kb) was PCR amplified from bacterial DNA without modification or with an errs at its 3 ' end.
  • the blunt-end PCR fragments generated using Pfu DNA polymerase were cloned into pSK II (Strategene) cloning vector and were sequenced to confirm the nucleotide sequence as well as incorporation of restriction sites and errs sequences into the chimeric genes.
  • the modified genes were labelled as ctpgpat-tp, ctpgpat-tp+errs, plsB and plsB+errs.
  • the intact ctpgpat cDNA was also used (Bhella and MacKenzie 48 , 1994).
  • the sequences of the modified and unmodified genes are shown in SEQ. ID. NOS. 1 to 5.
  • the ctpgpat or plsB chimeric genes were retrieved as BamHl or Bgl ⁇ segments, respectively, and were cloned into the BamHl site of the yeast expression vector, pYES2 (Invitrogen), under the transcriptional control of a galactose inducible promoter (GAL1).
  • GAL1 galactose inducible promoter
  • the LNVScl strain (Invitrogen) of yeast was transformed with the above recombinant constructs by the heat shock method (Elble 49 , 1992) to assess the functionality of the genes and the derived proteins.
  • Yeast cells containing chimeric GPAT genes were grown in SC-Ura (Bio 101) containing glucose. A 2.5 mL culture was initiated and grown for 18 hr at 28°C. A fresh 10 mL culture was grown by adding equal number of yeast cells to this culture and grown for another 24 hr.
  • GPAT gene expression was induced by transferring cells to growth medium containing galactose as follows : the cells were then transferred to 10 mL SC-Ura and galactose and the GPAT gene expression was induced for 24 hr. The yeast cells were in stationary phase by then. Cells were either used for protein extraction for enzyme assay or for lipid analysis.
  • GPAT genes produced functionally active protein when expressed by a galactose inducible promoter in yeast cells. Extracted GPATs were assayed for activity in vitro, by looking at the production of lysophosphatidic acid, and the results are listed in Table 1.
  • glucose and galactose indicate cells grown on glucose medium, and those grown on medium containing galactose (to induce the promoter), respectively. N.B. comparison in Table 4 can only be made within a construct, as the cells in different constructs may be at a different growth stage.
  • lipid content is increased by enhancing GPAT activity. No manipulation of growth medium, growth conditions or substrates is required to achieve a higher lipid content.
  • chimeric genes were cloned into the BamHl site of the plant transformation vector, pHS737, under the control of a tandem 35S CaMV promoter with AMV translational enhancer and 35S polyA for constitutive expression.
  • the recombinants were transferred into Agrobacterium tumefaciens GV 3101 fox Arabidopsis thaliana transformation.
  • Arabidopsis plants were transformed by the floral dip method (Clough and Bent 53 , 1998). Seeds (Tj) from these plants were collected and selected on a growth medium containing kanamycin. Transgenic plants were grown to maturity and seeds (T ) from 10 individual plants were collected and used for lipid analysis. Wild type and plants transformed with vector alone were grown as controls along with the transformed plants.
  • the fatty acid composition of seeds was determined by GC analysis following extraction of the oil and conversion of the triglycerides to fatty acid methyl esters. A known amount of C15 triglyceride was added to the seed sample as a tracer before oil extraction. Total seed lipid content was estimated on the basis of the recovery of C15 fatty acid methyl ester. C17:0 methyl ester was used as an internal standard for the chromatography. Fatty acid methyl esters were analysed using an HP 5850 gas chromatograph equipped with a DB-23 column (30m X 0.25mm; J & W Scientific, Folsom, CA). The GC conditions were: injector temperature and flame ionisation detector temperature, 250°C. After an initial hold at 180°C for 1 min, the oven temperature was programmed to 240°C at 4°C/min and held at this temperature for 10 min.
  • Seeds of plants transformed using only the pHS737 vector were indistinguishable in oil content from wild type control plants grown under the same conditions. All other gene constructs produced higher seed oil content.
  • the unmodified ctpgpat which would be expected to be expressed in the plastid, produced oil increases ranging from 10 to 21%. This suggests that LPA is released from the plastids and subsequently converted to TAGs. On average the greatest increase in oil was observed in seeds of transformants carrying the ctpgpat-tp gene (average +22%).
  • the plsB gene increased seed oil content by an average of 15%.
  • the addition of an ER targeting sequence resulted in an average seed oil increase of 18%.
  • Seeds of plants transformed with the vector only did not differ significantly in average weight from wild type plants. Seeds of individual plants from each construct were significantly heavier than wild type and the pHS737 control; e.g. 315-2, 301-2, 302-6, 303-3 and 304-15. However, increased seed oil content was not always positively correlated with increased seed weight; e.g. 303-7 and 304-1.
  • Phenotypes presenting increased seed oil content and weight would result in increased yield from oilseed crops. Those presenting an increase in seed oil content without an increase in weight would provide more oil per tonne of seed, representing an additional advantage to oil seed producers.
  • Seed oils from plants transformed with the vector alone were not significantly different from the wild type.
  • the Kennedy pathway is common to all organisms. Transformation of yeast or plants with DNA encoding GPAT activity can be used both to enhance oil content, and to alter the fatty acid composition of TAGs.
  • the use of GPATs with different acyl-CoA or acyl-ACP specificities can be used to tailor the fatty acid composition of the TAGs produced by the micro-organism or plant.
  • the method of the invention can manipulate oil synthesis in other organisms such as yeast, other fungae and algae for producing commodity and speciality oils. Increasing the oil content of feed quality grains would reduce the need for adding exogenous fats in the diets of animals and birds 54 (Kishore and Shewmaker, 1999).
  • Safflower plastidial GPAT the GPAT used in the examples, prefers unsaturated acyl-CoA or acyl-ACP
  • E. coli GPAT also used in the examples, prefers saturated acyl-CoA or acyl-ACP.
  • These genes can be used to modify the type of fatty acid at the sn- ⁇ position of TAGs. This enables the production of structured TAGs, in which the fatty acids occupying each position may be controlled. It is believed that fatty acid absorption and physiological effect are related to TAG structure, and not just gross composition. This also has implications for manipulating fat content in humans and other animals.
  • a plasmid library containing a mixture of plasmids (pYES2:ct/?gp ⁇ t-t/?, pYES2:ctpgpat-tp+errs, pYES2:plsB and pYES2 plsB+errs) was deposited, according to the Budapest Treaty, on May 24, 2000, at the International Depository Authority of Canada (Winnipeg, Manitoba, Canada), under accession number ID AC 240500-2 and reference pYEASTOIL.
  • a plasmid library containing a mixture of plasmids (pHS131:ctpgpat, pHS131:ctpgpat-tp, pHS131:ctpgpat-tp+errs, pHS131:plsB and pHS737: fcS+err.s') was deposited according to the Budapest Treaty, on May 24, 2000, at the International Depository Authority of Canada (Winnipeg, Manitoba, Canada), under accession number ID AC 240500-1 and reference pPLANTOIL.
  • SEQ ID NO: 1 is the DNA ctpgpat (encoding intact safflower plastidial GPAT)
  • SEQ ID NO: 2 is the DNA ctpgpat-tp (encoding safflower plastidial GPAT minus transit peptide)
  • SEQ ID NO: 3 is the DNA ctpgpat-tp+errs (encoding safflower plastidial GPAT minus transit peptide plus ER retention sequence)
  • SEQ ID NO: 4 is the ONAplsB (encoding E. coli GPAT)
  • SEQ ID NO: 5 is plsB+errs (encoding E. coli GPAT plus ER retention sequence)
  • SEQ ID NO: 6 is the protein encoded by ctpgpat (SEQ ID NO: 1; intact safflower plastidial GPAT)
  • SEQ ID NO: 7 is the protein encoded by ctpgpat-tp (SEQ ID NO: 2; safflower plastidial GPAT minus transit peptide)
  • SEQ ID NO: 8 is the protein encoded by ctpgpat-tp+errs (SEQ ID NO: 3; safflower plastidial GPAT minus transit peptide plus ER retention sequence)
  • SEQ ID NO: 9 is the protein encoded by plsB (SEQ ID NO: 4; E. coli GPAT)
  • SEQ ID NO: 10 is the protein encoded by plsB+errs (SEQ ID NO: 5; E. coli GPAT plus ER retention sequence)
  • Arabidopsis fad2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis.
  • the Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19: 645-653
  • Floral dip a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743

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Abstract

L'invention concerne un procédé permettant d'augmenter la concentration de triacylglycéride dans un organisme, et/ou de modifier la composition des acides gras de la triacylglycéride par expression, dans un organisme, d'un ADN codant pour une protéine présentant une activité de glycérol-3-phosphate acyltransférase (GPAT). Des exemples préférés de l'invention sont illustrés au moyen de l'Arabidopsis thaliana et de levures, à l'aide de constructions préparées à partir de gènes GPAT plastidiaux issus du carthame et du E.Coli.
EP00941822A 1999-06-21 2000-06-20 Surexpression dans des levures ou des plantes d'un gene codant pour la glycerol 3-phosphate acyltransferase Withdrawn EP1190069A2 (fr)

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PCT/CA2000/000738 WO2000078974A2 (fr) 1999-06-21 2000-06-20 Surexpression dans des levures ou des plantes d'un gene codant pour la glycerol 3-phosphate acyltransferase

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US7033790B2 (en) 2001-04-03 2006-04-25 Curagen Corporation Proteins and nucleic acids encoding same
US7192762B2 (en) 2004-11-04 2007-03-20 E. I. Du Pont De Nemours And Company Mortierella alpina glycerol-3-phosphate o-acyltransferase for alteration of polyunsaturated fatty acids and oil content in oleaginous organisms
JP5890687B2 (ja) * 2010-02-03 2016-03-22 サントリーホールディングス株式会社 グリセロール−３−リン酸アシル基転移酵素ホモログとその利用
CN111394399B (zh) * 2019-01-03 2022-06-28 上海凯赛生物技术股份有限公司 一种降低长链二元酸中酰基甘油酯杂质含量的方法
CN112980876B (zh) * 2021-03-12 2023-01-24 中国农业科学院棉花研究所 GhGPAT12蛋白和GhGPAT25蛋白在调控棉花雄性生殖发育中的应用

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JP3007119B2 (ja) * 1990-01-12 2000-02-07 紀夫 村田 グリセロール―３―リン酸アシルトランスフェラーゼをコードするｄｎａ鎖
DK0567648T3 (da) * 1991-01-16 1996-03-04 Kirin Brewery Afkølingsresistente planter og produktion deraf
AU1034595A (en) * 1993-11-19 1995-06-06 Kirin Beer Kabushiki Kaisha Dna chain coding for glycero-3-phosphate acyltransferase and use thereof
GB9510927D0 (en) * 1995-05-31 1995-07-26 Ca Nat Research Council Plant and seed oil modification
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