USRE46642E1 - Method of production of recombinant sucrose synthase, use thereof in the manufacture of kits for determination of sucrose, production of ADPglucose and production of transgenic plants whose leaves and storage organs accumulate high contents of ADPglucose and starch - Google Patents

Method of production of recombinant sucrose synthase, use thereof in the manufacture of kits for determination of sucrose, production of ADPglucose and production of transgenic plants whose leaves and storage organs accumulate high contents of ADPglucose and starch Download PDF

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USRE46642E1
USRE46642E1 US14/267,538 US200514267538A USRE46642E US RE46642 E1 USRE46642 E1 US RE46642E1 US 200514267538 A US200514267538 A US 200514267538A US RE46642 E USRE46642 E US RE46642E
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plant
sucrose
adpg
starch
production
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Miren Edurne Baroja Fernandez
Francisco José Muñoz Perez
Francisco Javier Pozueta Romero
Maria Teresa Moran Zorzano
Nora Alonso Casajus
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Publica de Navarra
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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.)
    • 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.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1062—Sucrose synthase (2.4.1.13)
    • 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)
    • 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
    • C12N15/8245—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 carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91097—Hexosyltransferases (general) (2.4.1)
    • G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)

Definitions

  • the invention relates to optimization of the production of recombinant sucrose synthase (SS) in soluble, active form employing an appropriate strain of Escherichia coli, the use of SS for making kits for determination of sucrose, design of optimized forms of SS for the synthesis of ADPglucose (ADPG), and the production of transgenic plants whose leaves and storage tissues accumulate high levels of ADPG and amylose-enriched starch as a result of overproduction of cytosolic ADPG in plants which overexpress SS.
  • SS sucrose synthase
  • ADPG ADPglucose
  • Starch is the main storage form of carbohydrates in plants. It accumulates in large amounts in organs such as seeds (wheat, barley, maize, pea, etc.) and tubers (potato and yam among others) and is a fundamental constituent of the human diet. Furthermore, starch is widely used in the paper, cosmetic, pharmaceutical and food industries, and is also used as an essential component for the manufacture of biodegradable plastics and environment-friendly paints. Since it is made up of covalently bound glucose molecules, investigation of the processes involved in the synthesis of this polysaccharide is a top priority in various areas of industrial production.
  • ADPG is the universal precursor of starch biosynthesis in plants, both in heterotrophic organs ( FIG. 1A ) and in leaves ( FIG. 2A ), and it is widely assumed that its production is controlled exclusively by the enzyme ADPG pyrophosphorylase (AGPase) or ADPG synthase (EC 2.7.7.27) (Okita, T. W. (1992) Is there an alternative pathway for starch synthesis? Plant Physiol. 100, 560-56; Müller-Röber, B., Sonnewald, U. Willmitzer, L. (1992) Inhibition of the ADPglucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J.
  • This ratio of amylose and amylopectin depends on, among other things, the concentration of ADPG in the plant cell (Clarke, B. R., Denyer, K., Jenner, C. F., Smith, A. M. (1999) The relationship between the rate of starch synthesis, the adenosine 5′-diphosphoglucose concentration and the amylose content of starch in developing pea embryos. Planta 209, 324-329).
  • SS (EC 2.4.1.13, SS) (UDP-glucose:D-fructose-2-glucosyl transferase) is a reversible enzyme that catalyses the production of UDPG and fructose from sucrose and UDP.
  • SS has classically been regarded as having the role of producing UDPG, metabolic processing of which eventually gives rise to the production of starch in heterotrophic tissues such as endosperm and tubers (Zrenner, R., Salanoubat, M., Willmitzer, L., Sonnewald, U. (1995) Evidence for the crucial role of sucrose synthase for sink strength using transgenic potato plants. Plant J.
  • Sugar nucleotides such as UDPG and ADPG are produced commercially from pyrophosphorylase reactions catalysed by enzymes such as UDPG pyrophosphorylase (UGPase) and AGPase, respectively, based on the use of an expensive substance called glucose-1-phosphate (G1P).
  • UDPase UDPG pyrophosphorylase
  • AGPase AGPase
  • G1P glucose-1-phosphate
  • An alternative to this practice for production of sugar nucleotides is based on the use of SS, development of which has largely been hampered by the limitations of Escherichia coli for expressing and efficiently processing a large number of eukaryotic proteins.
  • SS intended for the production of sugar nucleotides has had to be purified by expensive processes of purification of proteins from plant extracts (patent DE4221595 (1993), Purified sucrose synthase enzyme useful for production of nucleotide-activated sugars or oligosaccharides).
  • This SS obtained from plant extracts has the disadvantage that it has a predilection for UDP and very low affinity for ADP (Pressey R (1969) Potato sucrose synthase: purification, properties, and changes in activity associated with maturation. Plant Physiol. 44, 759-764; Nguyen-Quock, B., Krivitzky, M., Huber, S.
  • the present invention describes the development of a system based on the use of an appropriate strain of E. coli and on the use of a suitable expression vector that permits the large-scale production and fast and easy purification of different variants of recombinant SS in its active form. Some of these variants have greater affinity for ADP than those obtained from plant extracts and can be used both for the production of UDPG and ADPG from inexpensive substances such as sucrose, UDP and ADP.
  • Chromatographic techniques constitute a powerful tool for determining the sucrose content of complex samples such as plant extracts, sera, urine, fruit juice, wines, fruit and foodstuffs (D'Aoust, M-A., Yelle, S, Nguyen-Quock, B. (1999) Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit. Plant Cell 11, 2407-2418; Tang, G-Q., Sturm, A. (1999) Antisense repression of sucrose synthase in carrot affects growth rather than sucrose partitioning. Plant Mol. Biol. 41, 465-479; Frias, J., Price, K. R., Fenwich, G. R., Hedley, C.
  • sucrose determination has been developed, based on determination of the glucose-1-phosphate released by the action of sucrose phosphorylase of bacterial origin (Vinet, B., Panzini, B., Boucher, M., Massicotte, J. (1998) Automated enzymatic assay for the determination of sucrose in serum and urine and its use as a marker of gastric damage. Clin. Chem. 44, 2369-2371).
  • the present invention describes the development of a simple, reliable and inexpensive alternative method for the determination of sucrose in a sample based on the use of SS and coupling enzymes which hydrolyse ADPG or UDPG.
  • SS might be involved in the direct synthesis of ADPG necessary for the biosynthesis of starch (Baroja-Fernández, E., Mu ⁇ oz, F. J., Saikusa, T., Rodr ⁇ guez-López, M., Akazawa, T., Pozueta-R ⁇ umlaut over (m) ⁇ omero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant Cell Physiol. 44, 500-509).
  • Patent WO02067662 Robert, F., Danny, L., Yong-Ling, R. “Modification of sucrose synthase gene expression in plant tissue and uses therefor” Patent WO0245485; Christopher, B., William, B., Robert, H. “Bacterial sucrose synthase compositions and methods of use” Patent WO9803637).
  • the invention relates firstly to the development and optimization of a method of production of large amounts of recombinant SS that is soluble, can be purified easily and has high specific activity, based on the use of a suitable strain of E. coil and on the use of an expression vector that makes it possible to obtain SS with a histidine tail.
  • the invention further relates to the procedure followed for making kits for determination of sucrose based on the use of the enzyme product with SS activity coupled to enzymes that metabolize ADPG or UDPG. It further relates to optimization of the production of sugar nucleotides such as ADPG or UDPG starting from variants of SS specially designed for this purpose.
  • details are given of the design of transgenic plants with high content of sucrose, ADPG and starch and a high amylose/amylopectin ratio following overexpression of SS.
  • pSS was digested with the NcoI and NotI restriction enzymes.
  • the fragment released (which contains the cDNA encoding SS, SSX) was cloned on the same restriction sites of the pET-28a(+) expression plasmid (Novagen) ( FIG. 3B ) which possesses a nucleotide sequence in the polylinker region that encodes a histidine-rich sequence, which becomes fused with the recombinant protein.
  • the resulting plasmid (designated pET-SS, FIG. 3C ) was inserted by electroporation in various strains of E. coli.
  • coli strain BLR(DE3) (Novagen) transformed with pET-SS was deposited in the Spanish Type Culture Collection on 29 Oct. 2003, located in the Research Building of Valencia University, Burjassot Campus, Burjassot 46100 (Valencia, Spain) with the deposition number CECT:5850. The bacteria were incubated at 20° C. in LB medium. Overexpression of SSX was effected by addition of 1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) in 100 ml of cell culture grown at 20° C.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the bacteria were collected and resuspended in 4 ml of binding buffer (Novagen, His-bind purification kits), then sonicated and centrifuged at 40,000 g for 20 minutes.
  • the supernatant which contains the recombinant SS with an amino acid sequence rich in histidine residues at the N-terminal end, was passed through an affinity column of the His-bind protein purification kit from Novagen. Following the instructions with the kit, SS was eluted with 6 ml of the recommended elution buffer, which contained 200 mM of imidazole instead of 1 mol. After elution, the protein was quickly submitted to dialysis to remove any trace of imidazole, which inactivates SS irreversibly.
  • the mutated variant SS5 was designed, giving rise to the construction pSS5. This was done using the QuikChange Site-Directed Mutagenesis kit (Stratagene). pSS5 was digested with NcoI and NotI. The fragment released (which contains SS5) was cloned on the same restriction sites of the pET-28a(+) expression plasmid giving rise to pET-SS5, which was inserted by electroporation in E. coli BLR(DE3). The E. coli strain XL1 Blue transformed with pSS5 was deposited in the Spanish Type Culture Collection on 29 Oct. 2003, located in the Research Building of Valencia University, Burjassot Campus, Burjassot 46100 (Valencia, Spain) with the deposition number CECT:5849.
  • SS was overexpressed (a) constitutively, (b) specifically in leaves and (c) specifically in storage organs such as tubers.
  • p35S-SS-NOS was digested successively with the enzymes NotI, T4 DNA polymerase and HindIII and was cloned within the binary plasmid pBIN20 ( FIG. 4A ) (Hennegan, K. P., Danna, K. J. (1998) pBIN20: An improved binary vector for Agrobacterium-mediated transformation. Plant Mol. Biol. Rep. 16, 129-131) which had previously been digested successively with the enzymes EcoRI, T4 DNA polymerase and HindIII. The plasmid thus obtained was designated pBIN35S-SS-NOS ( FIG. 4C ).
  • PCR was used for amplifying the promoter region (designated RBCS) of the gene that encodes the small subunit of RUBISCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) of tobacco (Barnes, S. A., Knight, J. S., Gray, J. C. (1994) Alteration of the amount of the chloroplast phosphate translocator in transgenic tobacco affects the distribution of assimilate between starch and sugar. Plant Physiol. 106, 1123-1129).
  • RBCS promoter region
  • RUBISCO ribulose-1,5-bisphosphate carboxylase/oxygenase
  • This nucleotide sequence (which confers specific expression in photosynthetically active cells) was inserted in the pGEMT-easy vector (Promega), giving rise to pGEMT-RBCSprom ( FIG. 5A ).
  • This construction was digested with HindIII and NcoI and the fragment released was cloned in the corresponding restriction sites of p35S-SS-NOS, giving rise to pRBCS-SS-NOS ( FIG. 5B ).
  • This construction was digested successively with HindIII, T4 DNA polymerase and NotI.
  • the fragment released was cloned in pBIN20 digested successively with HindIII, T4 DNA polymerase and EcoRI.
  • the resulting construction was designated pBINRBCS-SS-NOS ( FIG. 5C ).
  • both pBIN35S-SS-NOS and pBINRBCS-SS-NOS were inserted in A. tumefaciens C58:GV2260 (Debleare, R., Rytebier, B., de Greve, H., Debroeck, F., Schell, J., van Montagu, M., Leemans, J. (1985) “Efficient octopine Ti plasmid-derived vectors of Agrobacterium mediated gene transfer to plants” Nucl. Acids Res.
  • kits designed for the determination of sucrose shown in the following Scheme I of enzymatic reactions involved in the kit for spectrophotometric/fluorimetric determination of sucrose based on the conversion of sucrose to a sugar nucleotide and then conversion of this to glucose-1-phosphate, glucose-6-phosphate and NAD(P)H.
  • the kit is based on the action of SS on the sucrose molecule in the presence of a nucleotide diphosphate (e.g. UDP or ADP), releasing equimolar amounts of fructose and the corresponding sugar nucleotide.
  • a nucleotide diphosphate e.g. UDP or ADP
  • UDPG UDPG pyrophosphatase of the Nudix type (EC 3.6.1.45)
  • the G1P released by the action of these hydrolytic enzymes is transformed by the action of phosphoglucomutase (PGM), yielding glucose-6-phosphate (G6P), which in its turn can be made to undergo a coupling reaction with NAD(P)+ by the action of the enzyme G6P dehydrogenase (G6PDH), producing 6-phosphogluconate and NAD(P)H, which can easily be determined by fluorimetry and by spectrophotometry at 340 nm.
  • the NAD(P)H released can be coupled to the action of FMN-oxidoreductase/luciferase, yielding light, which is quantified spectrophotometrically.
  • the UDPG produced can be coupled with UDPG dehydrogenase (EC 1.1.1.22) which, in the presence of NAD, gives rise to equimolar amounts of UDP-glucuronate and NADH, which can be determined by fluorimetry or by spectrophotometry at 340 nm.
  • the NADH released can be coupled to the action of FMN-oxidoreductase/luciferase, yielding light, which is quantified spectrophotometrically.
  • ADPG bacterial ADPG pyrophosphatase
  • bacterial ADPG pyrophosphatase EC 3.6.1.21
  • bacterial ADPG pyrophosphatase EC 3.6.1.21
  • Zandueta-Criado A.
  • Rodr ⁇ guez-López M.
  • Lasa I., Akazawa, T., Pozueta-Romero, J. (2001)
  • Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli.
  • G6P glucose-6-phosphate
  • NAD(P)+ by the action of the enzyme G6P dehydrogenase, producing 6-phosphogluconate and NAD(P) H, which can easily be determined by fluorimetry or spectrophotometry at 340 nm.
  • Examples are described below, which show in detail the procedure for cloning a cDNA that encodes an isoform of SS of potato in a suitable expression vector and in a strain of E. coil optimized for the production and accumulation of the enzyme in its active form.
  • Other examples describe the use of the recombinant SS for making assay kits for the determination of sucrose in plant samples, serum, urine, fruit juices, sweetened fruit drinks, refreshing drinks, etc.
  • Another example describes the use of variants of SS optimized for the large-scale production of sugar nucleotides such as UDPG and ADPG.
  • Another example describes the production of plants with high content of sucrose, ADPG and starch and a high amylose/amylopectin ratio as a result of the high ADPG-producing activity in plants that overexpress SS.
  • the amino acid sequence deducted from SEQ ID NO: 3 is slightly different from SS4 and is therefore designated SSX.
  • the amino acid sequence deducted after expression of SEQ ID NO: 3 in the pET-28a(+) plasmid is SEQ ID NO: 4, which includes a histidine-rich sequence of 38 amino acids fused with the amino-terminal end of the amino acid sequence deducted from SEQ ID NO: 3.
  • recombinant SSX in the total protein pool of CECT:5850 is approximately 20%, compared to the very low productivity of recombinant SS (30 micrograms per gram of bacteria) described in the literature (Nakai, T., Tonouchi, N., Tsuchida, T., Mori, H., Sakai, F., Hayashi, T. (1997) “Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia Coli” Biosci. Biotech. Biochem. 61, 1500-1503; Li, C. R., Zhang, X. B., Hew, C. S.
  • the unit is defined as the amount of enzyme that catalyses the production of one micromol of UDPG per minute.
  • UDPG Three grams of UDPG of high purity was produced efficiently and economically after incubation for 12 hours at 37° C. of 100 milliliters of a solution containing 1 M sucrose, 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl 2 /15 mM KCl/100 mM UDP and 30 units of recombinant SS from potato obtained after expression of PET-SS in BLR(DE3) and subsequent purification. Reaction came to an end after heating the solution at 100° C. for 90 seconds and then centrifugation at 10,000 g for 10 minutes.
  • This isoform designated SS5, was obtained by point mutagenesis of SSX using the QuikChange Site-Directed Mutagenesis kit (Stratagene) and successive use of the following pairs of primers whose sequences are [SEQ ID NO 5, SEQ ID NO: 6], [SEQ ID NO: 7, SEQ ID NO: 8] and [SEQ ID NO: 9, SEQ ID NO: 10].
  • the nucleotide sequence obtained, designated SS5, is SEQ ID NO: 11.
  • the changes in the amino acid sequence of SS5 (Susy 5) relative to SS4-Susy 4-(present in databases) are shown shaded in Table I.
  • SEQ ID NO: 12 The amino acid sequence deducted after expression of SEQ ID NO: 11 in the pET-28a(+) plasmid is SEQ ID NO: 12, which includes a histidine-rich sequence of 38 amino acids fused with the amino-terminal end of the amino acid sequence deducted from SEQ Ill NO: 11.
  • Table I includes said histidine-rich sequence of 38 amino acids fused to the amino-terminal portion of SS5.
  • the recombinant SS5 obtained after expression of pET-SS5 had a Vmax of 80 units/mg of protein and 65 units/mg of protein in the presence of UDP and ADP, respectively.
  • ADPG Three grams of ADPG of high purity was produced efficiently and economically after incubation for 12 hours at 37° C. of 100 milliliters of a solution containing 1 M sucrose, 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl 2 /15 mM KCl/100 mM ADP and 30 units of recombinant SS from potato obtained after expression of pET-SS5 in BLR(DE3) and subsequent purification in a His-bind column. Reaction came to an end after heating the solution at 100° C. for 90 seconds and then centrifugation at 10,000 g for 10 minutes. The supernatant was applied to a preparative-scale HPLC chromatograph (Waters Associates) for purification of the ADPG.
  • a preparative-scale HPLC chromatograph Waters Associates
  • Determination of the amount of sucrose present in the test sample is based on fluorimetric determination or spectrophotometric determination (at 340 mn) of the NAD(P)H produced according to the coupled reactions shown in schemes I and II.
  • Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant Cell Physiol. 44, 500-509).
  • FIGS. 8-10 present the results obtained in leaves of potato plants that overexpress SS both constitutively (35S-SS-NOS), and specifically (RBCS-SS-NOS).
  • the SS activity in the leaves of any of these plants is 2-10 times higher than in the same organ of a wild-type plant (WT). These leaves had the following characteristics:
  • FIGS. 12-14 show the results obtained in potato tubers that overexpress SS constitutively (35S-SS-NOS). These results are essentially identical to those observed in tubers that overexpress SS under the control of a specific tuber promoter (promoter of the patatina gene).
  • the SS activity in the tubers of any of these plants is ??? times greater than in the same organ of a wild-type plant.
  • These tubers had the following characteristics:
  • AGPase is the only source of ADPG.
  • ADPG levels have never been investigated in AGPase-deficient plants.
  • FIG. 15A the levels of starch in AGPase-deficient TL25 Arabidopsis plants (Lin, T. P., Caspar, T., Somerville, C. R., Preiss, J. (1988) Isolation and characterization of a starchless mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity.
  • FIG. 1 Mechanisms of starch biosynthesis in heterotrophic organs.
  • A “Classical” mechanism according to which SS is involved in the production of UDPG, which is eventually converted to starch after the combined action of UDPG pyrophosphorylase (UGPase), cytosolic phosphoglucomutase (PGM), plastidial phosphoglucomutase, ADPG pyrophosphorylase (AGPase) and starch synthase.
  • UDPase UDPG pyrophosphorylase
  • PGM cytosolic phosphoglucomutase
  • AGPase ADPG pyrophosphorylase
  • starch synthase “Alternative” mechanism according to which SS is involved in the direct production of ADPG in the cytosol. The ADPG is then transported to the amyloplast by the action of a translocator. Once inside the amyloplast, the starch synthase utilizes the ADPG for producing starch.
  • FIG. 2 Mechanisms of biosynthesis of starch in leaves.
  • A “Classical” mechanism according to which the entire process of starch biosynthesis takes place inside the chloroplast. According to this view, starch metabolism and sucrose are not connected. Moreover, SS does not take part in the gluconeogenic process.
  • B “Alternative” mechanism of starch biosynthesis according to which SS is involved in the direct synthesis of ADPG in the cytosol. The ADPG is then transported to the interior of the plastid where the starch synthase utilizes it as substrate for the reaction of starch synthesis.
  • FIG. 3 Stages in construction of the pET-SS expression plasmid from pET-28a(+) and pSS.
  • FIG. 4 Stages in construction of the pBIN35S-SS-NOS expression plasmid from pBIN20 and p35S-SS-NOS.
  • FIG. 5 Stages in construction of the pRBCS-SS-NOS expression plasmid from pGEMT-RBCSprom, p35S-SS-NOS and pBIN20.
  • FIG. 6 Expression of pET-SS in different strains of Escherichia coli.
  • A SS activity (in milliunits (mU) per milligram of bacterial protein) in bacterial extracts transformed with pET or with pET-SS. The reaction took place in the direction of degradation of sucrose and production of ADPG.
  • the reaction cocktail contained 50 mM HEPES (pH 7.0), 1 mM EDTA, 20% polyethylene glycol, 1 mM MgCl 2 , 15 mM of KCl and 2 mM of ADP. Reaction took place for 10 minutes at 37° C.
  • B SDS-PAGE of protein extracts from the various strains of E. coli transformed with pET and with pET-SS. The position of the recombinant SSX is indicated with an asterisk.
  • FIG. 7 Determination of sucrose at different stages of development of barley endosperm using the kit based on the coupled reactions of SS, ADPG (UDPG) pyrophosphatase, PGM and G6PDH.
  • the results were identical to those obtained in parallel by (a) use of a kit based on the coupled reactions of SS and UDPG dehydrogenase and (b) use of high-performance chromatography (HPLC) with amperometric detection in a DX-500 Dionex system connected to a Carbo-Pac PAI column.
  • HPLC high-performance chromatography
  • FIG. 8 SS activity in leaves of wild-type (WT) potato plants and potato plants that overexpress SSX following integration of the constructions 35S-SS-NOS (by the action of the strain of Agrobacterium tumefaciens CECT:5851) or RBCS-SS-NOS in their genome.
  • Activity is expressed in milliunits (mU) per gram of fresh weight. The unit is defined as the amount of SS required for producing one micromol of ADPG per minute.
  • FIG. 9 Content of starch in leaves of wild-type (WT) potato plants and potato plants that overexpress SSX fallowing integration of the constructions 35S-SS-NOS (by the action of the strain of Agrobacterium tumefaciens CECT:5851) or RBCS-SS-NOS in their genome.
  • FIG. 10 Content of ADPG in leaves of wild-type (WT) potato plants and potato plants that overexpress SSX following integration of the constructions 35S-SS-NOS (by the action of the strain of Agrobacterium tumefaciens CECT:5851) or RBCS-SS-NOS in their genome.
  • FIG. 11 Transitory accumulation of (A) starch and (B) ADPG during a photoperiod of 8 hours of light and 16 hours of darkness in leaves of WT plants ( ⁇ ), 35S-SS-NOS ( ⁇ ) and RBCS-SS-NOS ( ⁇ ).
  • FIG. 12 SS activity (referred to fresh weight, FW) in tubers of wild-type potato plants (WT), regeneration controls (RG) and potato plants that overexpress SSX (lines 4, 5, 6 and 12) after integration of the construction 35S-SS-NOS in their genome (by the action of the strain of Agrobacterium tumefaciens CECT:5851).
  • the activity is expressed in milliunits (mU) per gram of fresh weight.
  • the unit is defined as the amount of SS required for producing one micromol of ADPG per minute.
  • FIG. 13 Content of starch (referred to fresh weight, FW) in tubers of wild-type potato plants (WT), regeneration controls (RG) and potato plants that overexpress SSX (lines 4, 5, 6 and 12) after integration of the construction 35S-SS-NOS in their genome (by the action of the strain of Agrobacterium tumefaciens CECT:5851).
  • FW fresh weight
  • WT wild-type potato plants
  • RG regeneration controls
  • SSX lines 4, 5, 6 and 12
  • FIG. 14 Content of ADPG (referred to fresh weight, FW) in tubers of wild-type potato plants (WT) and potato plants that overexpress SSX after integration of the construction 35S-SS-NOS in their genome (by the action of the strain of Agrobacterium tumefaciens CECT:5851).
  • FW fresh weight
  • FIG. 15 Content of (A) starch and (B) ADPG in leaves of AGPase-deficient Arabidopsis thaliana TL25.
  • FIG. 16 Content of (A) starch and (B) ADPG in leaves of AGPase-deficient potato AGP62 and AGP85.

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ES200400257A ES2245867B1 (es) 2004-02-05 2004-02-05 Procedimiento produccion sacarosa sintasa recombinante a partir de escherichia coli y uso en fabricacion de kits de determinacion de sacarosa, produccion de azucares-nucleotidos y obtencion de plantas transgenicas con alto contenido de almidon y alto balance amilosa/amilopectina.
US14/267,538 USRE46642E1 (en) 2004-02-05 2005-01-27 Method of production of recombinant sucrose synthase, use thereof in the manufacture of kits for determination of sucrose, production of ADPglucose and production of transgenic plants whose leaves and storage organs accumulate high contents of ADPglucose and starch
PCT/ES2005/070010 WO2005075649A1 (fr) 2004-02-05 2005-01-27 Procede de production de saccharose synthase recombinante, son utilisation dans la fabrication de kits de determination de saccharose, production de adpglucose et obtention de plantes transgeniques dont les feuilles et les organes de reserve accumulent une forte teneur en adpglucose et amidon
US10/587,372 US8168856B2 (en) 2004-02-05 2005-01-27 Method of production of recombinant sucrose synthase, use thereof in the manufacture of kits for determination of sucrose, production of adpglucose and production of transgenic plants whose leaves and storage organs accumulate high contents of adpglucose and starch

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ES2339094B1 (es) * 2008-11-13 2011-03-18 Iden Carbohydrate Biotechnology, S.L. Procedimiento para la produccion de plantas transgenicas que presentan alto contenido en compuestos antioxidantes, alta capacidad antioxidante y resistencia al empardecimiento.
CN106834343B (zh) * 2017-02-21 2019-09-13 中国农业大学 蔗糖合成酶在调控植物果实发育中的应用
KR101985668B1 (ko) * 2017-10-30 2019-06-04 명지대학교산학협력단 벼 유래 OsSUS4 유전자를 이용한 도열병 저항성이 조절된 형질전환 식물체의 제조방법 및 그에 따른 식물체
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DE102020111560A1 (de) 2020-04-28 2021-10-28 Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen Enzymkaskaden auf Basis von Saccharose Synthase und Pyrophosphorylase zur Umsetzung von ADP zu ATP
CN111793641B (zh) * 2020-07-20 2022-07-19 中国农业科学院郑州果树研究所 甜樱桃PavSS或PavSPS基因在调控果实着色或果实成熟软化中的用途
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