WO1998044780A1 - Amidons vegetaux et methodes permettant de produire lesdits amidons dans des hotes - Google Patents

Amidons vegetaux et methodes permettant de produire lesdits amidons dans des hotes Download PDF

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Publication number
WO1998044780A1
WO1998044780A1 PCT/US1998/006660 US9806660W WO9844780A1 WO 1998044780 A1 WO1998044780 A1 WO 1998044780A1 US 9806660 W US9806660 W US 9806660W WO 9844780 A1 WO9844780 A1 WO 9844780A1
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Prior art keywords
host
gene
starch
genes
maize
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PCT/US1998/006660
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English (en)
Inventor
Hanping Guan
Peter L. Keeling
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Exseed Genetics, Llc
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Priority to AU68828/98A priority Critical patent/AU6882898A/en
Priority to JP54294098A priority patent/JP2001519664A/ja
Priority to CA002283719A priority patent/CA2283719A1/fr
Priority to EP98914483A priority patent/EP1017270A4/fr
Publication of WO1998044780A1 publication Critical patent/WO1998044780A1/fr
Priority to US10/336,753 priority patent/US20030226176A1/en
Priority to US11/330,822 priority patent/US7285703B2/en

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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.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • C12N15/8245—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
    • 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)
    • C12N9/107—1,4-Alpha-glucan branching enzyme (2.4.1.18)
    • 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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Definitions

  • This invention relates to hosts containing constructs with genes from the starch pathway. More specifically the present invention relates to bacterial hosts that form plant like starches. Additionally the present invention relates to plant hosts that have genes from the starch pathway. The invention further relates to the starches produced by said hosts.
  • starch using industry includes diverse industries such as candy makers, makers of adhesives and paints, gravy makers, paper producers, etc. Since the demand for starch, (which is formed of amylose and amylopectin), has been dramatically increasing for specialized food and industrial uses, efforts have been undertaken to tailor the quantity and quality of starch for specific food and industrial uses.
  • Starch is the major form in which carbohydrates are stored in biological systems. Plant starch in chloroplasts is transitory and storage starch accumulates in storage organs of many plant. Starch can be found in all organs of most higher plants including leaves stems and roots and fruits and embryo and endosperm. In addition to higher plants starch, similar polysacchande (glycogen) has been found in bacteria. Many bacteria produce a reserve polysaccharide similar to the glycogen found in animals.
  • Storage polysaccharide has been classified as being in two groups, group one has storage in the cytsol of the cell and the second group within the plastid.
  • Escherichia coli produces a polysaccharide within the cytsol.
  • Starch producing plants typically store starch in the plastids. Typical starch bearing plants include cassava, potato, corn, peas, rice, wheat, barley. The main starch storing tissue of corn, rice wheat and barley and oats, the cereal grains, is the endosperm.
  • Starches are also classified by the plant source, for example cereal starches are from cereal grains such as maize, rice, wheat, barley, oats and sorghum; tuber and root starch are from potatoes and yams and cassava .
  • starch from plants consists of two major components: amylose and amylopectin. These intertwine in the starch granule of the plants.
  • Amylose is a linear polymer of alpha 1 -4 bonded anhydroglucose units while amylopectin is a branched polymer comprised of linear chains alpha 1-4 linked anhydroglucose units with branches resulting from alpha 1-6 linkages between the linear chains. It has been known for sometime that mutant genes in starch bearing plants can be expressed and that the properties of the starch can be altered. The proportion of the two components and their structures in the mutant primarily determine the physical-chemical properties of the starch.
  • the SSSIII gene from potato was transformed into E.coli deficient in glgA gene.
  • the effect of glgC and branching enzyme I and II in combination in a mutant E.coli has also been studied and glycogen like product was reported.
  • the starch industry that is commercial does not have a particular interest in the production of glycogen which is the polysaccharide produced by bacteria and animals (the health care industry may have some such interest).
  • the industry has thus not yet been able to generate tailored starches at reasonable prices through plant gene transformation. There remains a need for the industry to find new starches that are useful due to their changed characteristics such as lower viscosity and new starches that are useful because of their higher viscosity and new methods of producing such starches.
  • ADPGlc pyrophosphorylase plays a pivotal role in regulating the amount of starch synthesized, while starch synthase and starch branching enzyme primarily determine the starch structure.
  • SBE and SS Multiple forms of SBE and SS have been identified in many plants including maize, rice, pea and potato.
  • GBSS granule bound starch synthase
  • Maize is the only cereal crop from which the genes coding for the five forms of SS have been isolated. Clearly a number of these sequences are published and known to those of skill in the art. Genes coding for maize SBE have also been cloned and characterized. Previous reports have demonstrated that maize SBEI has a higher rate of branching amylose than SBEII and preferentially transfers longer chains, while SBEII shows a higher rate of branching amylopectin and preferentially transfers shorter chains. In comparison with SBE, less is known about the specificities and functions of multiple forms of SS. In Waxy maize, which lacks GBSS, only amylopectin is synthesized and amylose is missing.
  • GBSS encoded by waxy gene
  • GBSS is primarily responsible for the synthesis of amylose.
  • Study of waxy mutation in Chlamedomonas reinhardtii has suggested that GBSS is also involved in amylopectin synthesis.
  • Chlamedomonas reinhardtii SSII controls the synthesis of intermediate size glucans of amylopectin in Chlamedomonas
  • direct evidence for the functions of SS in higher plants is still missing.
  • Antisense technology has been used to study the functions of SS in potato, however, the results are inconclusive.
  • the mutation to the bacteria was the reduction of the activity of glycogen BE in the AC71(glgB-) so that the mutant was essentially free of BE activity.
  • the paper analyzed the debranched alpha-glucan isolated from the four different transformants. The first host was E. coli containing glgB and the second host was the AC71 without any transformed genes then AC71 transformed with maize BEII, and then AC71 with maize BEI , then AC71 with maize BEI and BEII.
  • the resultant polysaccharide products were analyzed by HPLC, by chain length and relative peak area and by mole distribution of chains.
  • E coli GS glycogen synthases
  • the industry still needs the option of producing plant like starches in a fermentation process from bacteria and thus without the necessity of breeding and growing environment sensitive plants; and, the option of producing plants that generate the specific tailored starch through a plant host. And the industry needs altered and new starches that are cereal like starches or root and tuber like starches in large quantities and inexpensively thus avoiding having to use chemical modification of starch.
  • Another object of the present invention is the synthesis of polysaccharides including amylose, amylopectin in E. coli, and/or fungal and yeast by plant starch synthesizing enzymes including SS, SBE, bacterial branching enzyme, glycogen synthase and other enzymes in other living organism.
  • Yet another object of my invention is using each or combination of these enzymes or modified enzymes studied in this patent to produce or to improve polysaccharides in any living organism including starch synthesis in plants.
  • the invention provides DNA constructs in a host that include most of the genes in the starch pathway of a plant such that the host produces a plant like polysaccharide. And in one embodiment produces maize starch including slightly different embodiments that make specific maize mutant like starch in a non plant host.
  • This invention encompasses a bacterial host containing a combination of two or more of such genes SSI, SSSIIa, SSIIb, SSSIII, GBSS, BEI and BEII when the combination does not form glycogen like material.
  • This invention encompasses a plant host transformed with any of the following maize genes or a plant host having a combination of two or more of the following maize genes SSI, SSIIa. SSIIb, SSSIII, GBSS, BEI and BEII in a hybrid or an inbred rice plant.
  • the present invention includes new' polysaccharide produced by a transformed host.
  • the host having a wildtype, which does not produce the new polysaccharide, the transformed host expressing at least two exogenous starch synthesis genes, the genes are selected from a group consisting of starch synthesis genes such as SSI SSIIa, SSIIb, SSIII, GBSS and optionally including at least one of the BEI and BEII genes wherein the transformed host is capable of producing such new polysaccharide.
  • the invention also covers a new polysaccharide wherein the host also expresses the exogenous genes selected from the following group consisting of bacterial glycogen inducing genes are selected from the group glgA, glgB, glgC and any mutants thereof. Or wherein the host also expresses the exogenous genes selected from the following group consisting of plant granule bound enzymes. And the new polysaccharide wherein the starch synthesis genes are selected from the group consisting of BEI and BEII.
  • the present invention broadly encompasses a host containing a transformed Gig C gene and at least one of the starch branching enzymes genes in a host in combination with at least one other transformed starch gene wherein the host produces a polysaccharide product. And a host containing transformed bacterial gene and at least one of the non starch branching enzymes selected from the group consisting of debranching enzymes and soluble starch synthase
  • a method of producing polysaccharides which are non glycogen like in a host comprising transforming a host capable of being used in a fermentation process, with genes selected from the group which produce starch synthesizing enzymes, or glycogen synthesizing enzymes such that the host produces nonglycogen like starch, and employing the host in a fermentation process that produces polysaccharides.
  • the host is bacteria, or a fungal or a yeast. Additionally the method of this invention includes the use of bacterial genes also such as the glycogen synthesizing genes including the glgC, glgA, glgB genes.
  • a method wherein the genes which produce starch synthesizing enzymes include genes encoding for starch soluble synthases I, Ila, lib and SS III (dull).
  • a method wherein the genes which produce starch synthesizing enzymes include genes encoding for starch debranching enzyme and branching enzymes.
  • the invention covers the modified starch synthesizing enzymes including the N-terminally truncated SS.
  • the invention covers a host transformed to carry a gene active in glycogen production, and at least one nonstarch branching gene active in the production of at least one of the following polysaccharides amylopectin and amylose in its original host.
  • the host can be a monocot or a dicot plant.
  • the host can be a cereal bearing plant. Or the host can be a bacteria.
  • the invention includes a host wherein at least one nonstarch genes active in the production of at least one of the following polysaccharides, amylopectin and amylose in its original plant, is selected from the group consisting of starch soluble starch synthase I, Iia, lib, III genes and debranching enzyme gene (sul), GBSS gene, sh2 gene and bt2 gene.
  • a host including at least one of the starch branching enzyme genes such as BEI gene, BEII gene.
  • the present invention can also be described as a host transformed to carry a gene active in ADPG production, and at least one starch gene active in the production of at least one of the following polysaccharides amylopectin and amylose in its original host wherein the host produces polysaccharides that are plant like starch and not glycogen like.
  • the host can be transformed to carry a pyrophosphorylase gene, and glycogen synthase gene.
  • the scope of the present invention includes a host deficient in alpha 1,4 glucan synthesizing ability and alpha 1,4-1,6 branching enzyme capability transformed to express at least one a plant starch soluble synthesis gene.
  • the host can also include being transformed to express at least one gene encoding for debranching enzyme, and/or a gene encoding for starch soluble synthase I, starch soluble synthase enzyme Iia, lib, starch soluble synthase enzyme III.
  • This host can including being transformed to express at least one gene encoding for starch branching enzyme.
  • This invention also includes the production of a glycogen like material in plants. Attached hereto are a number of plasmids described by the figures and by table one, that are part of the present invention and are claimed herein.
  • One such example is the plasmid wherein the plasmid is in a carrier host and the plasmid contains the SSIIa gene with the n terminus GENVMNVIVV and wherein the gene is approximately 1561 base pairs in length.
  • the invention includes mutant hosts such as mutant plants like waxy rice and potatoes and corn as example and wherein the host is a mutant E. Coli, or fungus.
  • FIG. 1 shows a graph which gives the relative peak area in percent and the chain length of glycogen and starch soluble synthase I (SSI), starch soluble synthase II (SSIIa), starch soluble synthase lib (SSIIb).
  • SSI starch soluble synthase I
  • SSIIa starch soluble synthase II
  • SSIIb starch soluble synthase lib
  • FIG. 2 shows plasmid pEXSC-MBEI with 7661 base pairs and promoter T7 and a Kanamycin gene and glgC and the maize starch branching enzyme I (MB El).
  • FIG. 3 shows plasmid pEXSC3C with 7461 base pairs and promoter T7 and ampicillin gene and the maize starch soluble synthase gene Iia.
  • pEXS3c is the 1082 bp Nde I-EcoRI fragment containing the N-terminus of MS SSIIa (from MSSIIa in pBSK) subcloned into the Nde I-EcoRI sites of pEXS3a, replacing the N-terminus of IIA-2 with the longer Iia N- terminus.
  • MSSIIa is the mature maize SSIIa and is 2090 bp long. The following sites are not contained in the MSSIIc insert: Apa I, Bglll, Eco V, Not, Spe I, and Xba I.
  • the N-terminus of this plasmid is AEAEAGGKD.
  • FIG. 4 shows plasmid pEXSC-MBEI-MBEII with 9971 base pairs and promoter T7 and a Kanamycin gene and glgC and the maize starch branching enzyme I (MBEI) and the maize starch branching enzyme II (MBEII).
  • FIG. 5 shows plasmid pEXSC-MBEII with 7521 base pairs and promoter T7 and a Kanamycin gene and glgC and the maize starch branching enzyme II (MBEII).
  • FIG. 6 shows plasmid pEXSC-3a with 7990 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the maize N-terminally truncated starch synthase gene Iia (MSSIIa-2).
  • the N-terminal sequence is GENVMNVI.
  • FIG. 7 shows plasmid pEXSC-8 with 7079 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the maize starch soluble synthase gene I and version I- 2.(MSSI-2), An N-terminally truncated SSI.
  • FIG. 8 shows plasmid pEXSC-9 with 7551 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the maize starch soluble synthase gene lib (SSIIb).
  • the N-terminal sequence is AAAPAGEE.
  • FIG. 9 shows plasmid pEXSC-10 with 7211 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the maize starch soluble synthase gene I, the full length SSI.
  • the N-terminal sequence is CVAELSREGPA
  • FIG. 10 shows plasmid pEXSCA with 6738 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the glgA gene.
  • FIG. 11 shows plasmid pEXSC9a with 7240 base pairs and promoter T7 and ampicillin gene and the maize starch soluble synthase gene IIb-2 (Maize SS IIb-2), an N-terminally- truncated SSIIb.
  • the N-terminal sequence is MNVVVVASECAP.
  • FIG. 12 shows plasmid pEXSWX with 6968 base pairs and promoter T7 and an ampicillin gene and the N-terminally-truncated maize WX (maize granular bound starch synthase).
  • the N-terminal sequence for wx is ASAGMNNVFVGAEMA.
  • FIG. 13 shows plasmid pEXSWX2 with 6980 base pairs and promoter T7 and an ampicillin gene and the ⁇ -terminally-truncated maize WX termed as wx2.
  • the ⁇ -terminus of wx2 is M ⁇ NNFVGAEMA.
  • FIG. 14 shows plasmid pEXSC9 with 7780 base pairs and promoter T7 and ampicillin gene and E. coli glgC gene and the maize starch soluble synthase genellb (Maize SS lib).
  • FIG. 15 shows plasmid pEXSClOd with 7112 base pairs and promoter T7 and ampicillin gene, E. coli glgC gene and the ⁇ -terminally-truncated maize starch soluble synthase gene I termed as Maize SSI-3).
  • the ⁇ -terminus of maize SSI-3 is MSIVFTGEASPYA.
  • FIG. 16 shows plasmid pEXSlO with 5300 base pairs and promoter T7 and ampicillin gene and the full length maize starch soluble synthase gene I termed as Maize SS I.
  • FIG. 17 shows plasmid pEXS8 with 7259 base pairs and promoter T7 and ampicillin gene and the ⁇ -terminally-truncated maize starch synthase gene I termed as SSI-2.
  • the ⁇ - terminal sequence is CVAELSRDLGLEPEG.
  • FIG. 18 shows plasmid pEXSCAl with 5128 base pairs and promoter T7 and ampicillin gene and the glgA.
  • pESCAl is a 1551 bp Spel-Sac I fragment containing glgA (from glgA in pBSK) subcloned into the Xba I- Sac I sites of p ET-23d which is commercially available from ⁇ ovagen in Madision Wisconsin under catalog number 69748-1 and called ET- 23d(+) D ⁇ A.
  • FIG. 20 shows the spectrum of the iodine glucan complex of the product produced by the host transformed with plasmids containing the glgC, the BEI, the BEII genes and glgA; glgC, the BEI, the BEII genes and maize SSI, SSI-2 and glgC, the BEI, the BEII genes and maize SSIIb, and glgC, the BEI, the BEII genes and maize SSIIa-2, and glgC, the BEI, the BEII genes, the X-axis is listing nm and the Y axis is reading absorbance.
  • FIG. 22 shows pExs-trc has 4178 base pairs with the trc promoter and the ampicillin gene.
  • PEXS trc is pTrc99A-Ndel which has been mutagenixed. (Nco I site in multiple cloning site of p Trc99A-Ndel is mutagenixed to Nde I using primers EXS63 AND EXS64.)
  • pEXS-trc contains only one Nde I site and no Nco I sites. The following sites are not contained in pEXS-trc; Bgl II, Cla I, Nco I, Not I, Sac II, SnaB I, Spe I, and Xho I.
  • FIG. 23 shows pEXS-trc3 has 4129 base pairs with the trc promoter and the ampicillin gene in partial and the Kanamycin gene.
  • the pEXS-trc3 is pEXS-trcl cut with Bgll (filled in)- Sca I and religated, deleting most of the Amp gene (304 nt from the 5" end remain).
  • the following sites are Not contained in p EXS-trc3: Apa I, Bgl II, Eco V, Nco I, Not I, SnaB I, and Spe I.
  • FIG. 24 shows the plasmid pEXS 102 having 7190 base pairs, adapted for plant transformation containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the Waxy 2 gene and the nos terminator and the ampicillin gene.
  • FIG. 25 shows the plasmid pEXS 103 having 6607 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide and the Waxy 2 gene and the nos terminator and the ampicillin gene.
  • FIG. 26 shows the plasmid pEXS 101 having 6979 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide and the gig B gene and the nos terminator and the ampicillin gene.
  • FIG. 27 shows the plasmid pEXS 100 having 7557 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the gig B gene and the nos terminator and the ampicillin gene.
  • FIG. 28 shows the plasmid pEXS 101 having 6273 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the gig A gene and the nos terminator and the ampicillin gene.
  • FIG. 29 shows the plasmid pEXS 66 having 6001 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the gig C 3 gene and the nos terminator and the ampicillin gene.
  • FIG. 30 shows the plasmid pEXS 65 having 6373 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the maize waxy gene and the nos terminator and the ampicillin gene.
  • FIG. 31 shows the plasmid pEXS 64 having 7073 base pairs, adapted for plant use containing the maize 10KD zein promoter,the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase Iia gene and the nos terminator and the ampicillin gene.
  • FIG. 32 shows the plasmid pEXS 63 having 6473 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase Iia gene and the nos terminator and the ampicillin gene.
  • FIG. 33 shows the plasmid pEXS 62 having 6773 base pairs, adapted for plant use containing the maize 10KD zein promoter,the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase 1-2 gene and the nos terminator and the ampicillin gene
  • FIG. 34 shows the plasmid pEXS 61 having 7013 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase lib gene and the nos terminator and the ampicillin gene.
  • FIG. 35 shows the plasmid pEXS 59 having 6858 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the E.coli glgA gene and the nos terminator and the ampicillin gene
  • FIG. 36 shows the plasmid pEXS 58 having 7658 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase Iia gene and the nos terminator and the ampicillin gene.
  • FIG. 37 shows the plasmid pEXS 56 having 6586 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the gig C 3 gene and the nos terminator and the ampicillin gene.
  • FIG. 38 shows the plasmid pEXS 54 having 7658 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the Maize SS Iia gene and the nos terminator and the ampicillin gene.
  • FIG. 39 shows the plasmid pEXS 53 having 7058 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the maize starch soluble synthase IIa-2 gene and the nos terminator and the ampicillin gene.
  • FIG. 40 shows the plasmid pEXS 52 having 7358 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and maize starch soluble synthase 1-2 gene and the nos terminator and the ampicillin gene.
  • FIG. 41 shows the plasmid pEXS 51 having 7398 base pairs, adapted for plant use containing the maize lOKD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and maize starch soluble synthase lib gene and the nos terminator and the ampicillin gene.
  • FIG. 42 shows photograph of eleven products of altered starch produced with the present invention.
  • C-I- II the glgC gene and the BEI and the BEII and EXS-10 plasmid that contains the gene SSI, having the N-terminus shown in Table 1.
  • FIG. 43 shows the DNA sequence and the protein sequence for glgA having 1488 base pairs.
  • FIG. 44 shows the DNA sequence and the protein sequence for glgB having 2361 base pairs.
  • FIG. 45a shows the DNA sequence for Zea mays 10-kDa zein gene having 2562 base pairs .
  • FIG. 45b shows the DNA sequence for Zea mays 10-kDa zein portion of the gene used as the promoter in a number of the plasmids discussed herein.
  • FIG. 46 shows the DNA sequence and the protein sequence for glgC3 (glgC 3 ) having 1328 base pairs containing two mutations P295D, K296E. This is a mutant of the wild type glgC gene.
  • FIG. 47 shows the DNA sequence and the protein sequence for glgC (glgC) having 1328 base pairs.
  • FIG. 48 shows the DNA sequence and the protein sequence for glgCwt (glgCwt) having 1328 base pairs. This is the glgC gene that is found in nature.
  • FIG. 49 shows the DNA sequence and the protein sequence for the maize waxy gene denoted wx herein.
  • FIG. 50 shows the DNA sequence and the protein sequence for the maize starch soluble synthase lib encoding gene having 2423 base pairs.
  • FIG. 51 shows the DNA sequence and the protein sequence for the maize starch soluble synthase Iia.
  • FIG. 52 shows the DNA sequence and the protein sequence for the maize starch soluble synthase 1-2 having 1749 base pairs.
  • FIG. 53 shows the DNA sequence and the protein sequence for the maize branching enzyme II.
  • FIG. 54 shows the DNA sequence and the protein sequence for the maize branching enzyme I.
  • FIG. 55 shows the DNA sequence and the protein sequence (153) for the transit peptide portion of the maize starch soluble synthase I.
  • FIG. 56 PCR analysis of transgenic rice plants.
  • the genomic DNA isolated from rice plants were PCR amplified using specific primers for the inserted gene.
  • the specific bands were identified on 1% agarose gel compared with non-transgenic rice plant.
  • FIG. 57 Activity staining of starch synthase on renaturing SDS-PAGE gel with Iodine solution.
  • FIG. 58 SSI-1, SSI-2, and SSI-3 construct diagram.
  • Three forms of SSI were constructed in the pET expression system (see Methods).
  • pExslOa encodes SSI-1, the full length maize SSI (583 amino acids).
  • pExs ⁇ encodes a truncated SSI, SSI-2, with amino acids #8-52 deleted from the N-terminus of SSI-1.
  • pExsld encodes the most truncated form of SSI, SSI-3, with the first 93 amino acids deleted from SSI-1.
  • a depiction of the waxy gene, encoding GBSS, is also included for comparison.
  • the amino acid motif KS/TGGL is indicated by the triangles.
  • the KS/TGGL motif is located 18 amino acids from the N-terminus in GBSS, while the motif is 106 amino acids from the N- terminus in maize SSI. Drawing not to scale.
  • FIG. 59 SSIIa-] and SSIIa-2 construct diagram.
  • Two forms of SSIIa were constructed in the pET expression system.
  • pExs3c encodes SSIIb-1, the putative full length maize SSIIb.
  • N-terminal sequencing of SSIIa- 1 revealed that the polypeptide chain started at amino acid #1, so the length of SSIIa-1 is 669 amino acids.
  • pExs3a encodes a truncated form of SSIIa.
  • SSIIa- 2 with the first 176 N-terminal amino acids deleted from SSIIa (493 amino acids total).
  • a depiction of the waxy gene, encoding GBSS, is also included for comparison.
  • the amino acid motif KTGGL is indicated by the triangles.
  • the KTGGL motif is located 18 amino acids from the N-terminus in GBSS, while the motif is 194 amino acids from the N-terminus in maize SSIIa.
  • FIG. 60 SSIIb-1 and SSIIb- 2 construct diagram. Two forms of SSIIb were constructed in the pET expression system (see Methods).
  • pExs9 encodes SSIIb-1, the putative full length maize SSIIb. N-terminal sequencing of SSIIb-1 revealed that the polypeptide chain started at amino acid #1, so the length of SSIIb-1 is 637 amino acids.
  • pExs9a encodes a truncated form of SSIIb, SSIIb-2, with the first 144 N-terminal amino acids deleted from SSIIb (492 amino acids total). A depiction of the waxy gene, encoding GBSS, is also included for comparison.
  • the amino acid motif KTGGL is indicated by the triangles.
  • the KTGGL motif is located 18 amino acids from the N-terminus in GBSS, while the motif is 158 amino acids from the N-terminus in maize SSIIb.
  • FIG. 61 Temperature Curves for SSI enzymes. All assay components, except enzyme and [U- 14 C]-ADPGlc, were mixed and then preincubated at each temperature for 3 minutes before addition of enzyme and ADPGlc. For all assays, the final concentration of [U- 14 C]- ADPGlc was 3 mM, while amylopectin was 6 mg/ml. Each point is an average of three separate determinations.
  • FIG.62 Temperature Optima of SSIIa- 1 and SSIIa-2. All assay components, except enzyme and [U- 14 C]-ADPGlc, were mixed and then preincubated at each temperature for 3 minutes before addition of enzyme and ADPGlc. For assays in the presence of 0.5 M citrate, 5 mg/ml amylopectin was used as primer. For assays without citrate, 10 mg/ml amylopectin was used. For all assays, the concentration of [U- 14 C]-ADPGlc was 3 mM. Each point is an average of three separate determinations.
  • FIG. 63 Temperature Optima of SSIIb-1 and SSIIb-2. All assay components, except enzyme and [U- I4 C] ADPGlc, were mixed and then preincubated at each temperature for 3 minutes before addition of enzyme and ADPGlc. For all assays, the concentration of [U- I4 C]ADPGlc was 3 mM and the concentration of glycogen was 40 mg/ml. Each point is an average of three separate determinations. PREFERRED EMBODIMENT - DESCRIPTION
  • Gene shall mean the entire gene sequence or any mutations or varieties of the codon that produce the desired activity in the host or alternatively the section or sections of the gene sequence necessary to produce the desired activity in the host.
  • glgC gene shall mean glgC 16 , glgC 3 and other mutants that produce the desired activity in the host.
  • Starch synthase gene shall mean full length SS, N-terminally-truncated SS or mutated SS with starch synthase activity.
  • Glycogen like-shall mean polysaccharide material such as those produced as the main starch product by E.coli in its native state and by the hosts as taught in the above described paper by Hanping Guan.
  • Non Glycogen like- shall mean polysaccharide material which is plant like and is not produced as the main starch product by E.coli in its native state and by the hosts as taught in the above described paper by Hanping Guan.
  • Plant like starch- is non glycogen like.
  • Transformed gene-shall mean a gene that was somewhere in the lineage of the plant or bacteria introduced into the plant by means other than nature. Thus the progeny of a transformed host would continue to contain a transformed gene.
  • Transformed host- shall mean any organism containing one or more of the novel plasmids and/or a novel combination of starch synthetic genes discussed herein.
  • MSS# maize soluble starch synthase
  • SS# will likewise mean starch synthase though not necessarily maize.
  • STS# will also designate soluble starch synthase.
  • GBSS granule bound starch synthase.
  • SBE# starch branching enzyme
  • MBE maize starch branching enzyme
  • MSBE# maize starch branching enzyme
  • BE# starch branching enzyme.
  • the present invention broadly encompasses transforming hosts such as bacteria or plants with plant starch synthetic genes that produce a non glycogen like material (a bacteria containing BEI and BEII from maize produces a glycogen like material).
  • Starch bearing plants and organisms hereinafter are referred to as the host.
  • One of the primary aspects of this invention is the generation of plant like starch from a bacterial host and the production of altered starch in a plant host.
  • the present invention has been exemplified in both bacteria and in transformed rice plants.
  • the host can contain though it is not a limitation, an unlimited supply of ADPG from the addition of the glgC gene (the bacterial gene) to the plant.
  • the present invention encompasses plasmids that contain the maize genes and/or the bacterial genes in a construct adapted for use in a bacteria and constructs adapted for use in a plant.
  • the plasmids in the plant construct preferably containing an active promoter recognized by the plant, a transit peptide, and the cleavage site that permits the protein to cleave from the transit peptide when crossing into the amyloplast in the plant.
  • the plasmids used in the rice transformation specifically encompassed the maize 10 kd zein promoter, and the transit peptide from the maize SSI gene in the constructs adapted for plant use.
  • the present invention also encompasses the plant producing the altered starch in the starch storage section of the plant or within the host cell and the altered starch itself. Additionally the present invention encompasses the combination of a number of starch genes in combination being active in a host such that the host produces differing non glycogen polysaccharides. Still further the present invention encompasses a method of making plant like starch in a bacterial host and the method of making altered plant like starch (altered in relationship to the type or amount of starch that the host makes without the constructs containing the genes), in a plant. Yet another object of the present invention is the addition of a gene that encodes for the substrate ADPG used to form starch.
  • the present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in a plant and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase I or its mutant form.
  • the present invention also encompasses the combination of a promoter adapted for use in a plant and optionally a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase I or its mutant form, and at least one gene encoding for branching enzyme transformed into a plant host.
  • the present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in a plant and a gene encoding for ADPGlc pyrophosphorylase, preferably a bacterial gene, and a gene encoding for starch synthase Iia or its mutant form.
  • the present invention also encompasses the combination of a promoter adapted for use in a plant and optionally a gene encoding for ADPGlc pyrophosphorylase, and a gene encoding for starch synthase Iia or its mutant form, and at least one gene encoding for branching enzyme transformed in to a plant host.
  • the present invention encompasses a plasmid having a promoter adapted for use in a plant and a gene encoding for ADPGlc pyrophosphorylase, preferably a bacterial gene, and a gene encoding for starch synthase lib and its mutant form.
  • the present invention also encompasses the combination for a promoter adapted of use in a plant and an optional gene encoding for ADPGlc pyrophosphorylase, and a gene encoding for starch synthase lib or its mutant form and at least one gene encoding for branching enzyme transformed in to a plant host.
  • the present invention encompasses a plasmid having a promoter adapted for use in a plant and a gene encoding for Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase Iia, starch synthase lib, DU1.
  • the present invention also encompasses the combination of a promoter adapted for use in a plant and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase Iia, starch synthase lib and DU1, , and at least one gene encoding for branching enzyme transformed in to a plant host.
  • the present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in a plant and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase Iia, lib and starch synthase III (DU1).
  • the present invention also encompasses the combination of a promoter adapted for use in a plant and an optional gene encoding for ADPGlc pyrophosphorylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase Iia, lib starch synthase III(DUl), and at least one gene encoding for branching enzyme, and at least one gene encoding for the debranching enzyme transformed in to a plant host.
  • ADPGlc pyrophosphorylase preferably a bacterial gene
  • the present invention encompasses a plasmid or combination of plasmids in the host having a promoter adapted for use in a bacteria or yeast and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase I.
  • the present invention also encompasses the combination of a promoter adapted for use in a bacteria or yeast and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase I, and at least one gene encoding for branching enzyme transformed in to a bacteria or yeast host.
  • the present invention encompasses a plasmid or combination of plasmids in the host having a promoter adapted for use in a bacteria or yeast and a gene encoding for Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase Iia.
  • the present invention also encompasses the combination of a promoter adapted for use in a bacteria or yeast and optionally a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase Iia, and at least one gene encoding for branching enzyme transformed in to a bacteria or yeast host.
  • the present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in a bacteria or yeast, and a maize gene encoding for starch synthase III(DUl).
  • the present invention also encompasses the combination of a promoter adapted for use in a bacteria or yeast and an optional gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase III, and at least one gene encoding for branching enzyme transformed in to a bacteria or yeast host.
  • the present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in bacteria or in yeast and a gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa,IIb, starch synthase III(DUl).
  • the present invention also encompasses the combination of a promoter adapted for use in bacteria or in yeast and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa,IIb, starch synthase III, and at least one gene encoding for branching enzyme transformed in to bacteria or into yeast hosts.
  • the present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in bacteria or in yeast and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa,IIb, starch synthase III
  • the present invention also encompasses the combination of a promoter adapted for use in bacteria or in yeast and a gene encoding for Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa,IIb, starch synthase III(DUl), and at least one gene encoding for branching enzyme, and at least one gene encoding for the debranching enzyme transformed in to a bacteria or into a yeast host.
  • the present invention encompasses the truncated versions of the SSI and the SSII and the SSIII genes that still provide protein that is sufficient to make the polysaccharide.
  • Starch biosynthesis in higher plants and glycogen biosynthesis in E. coli have similar reactions which use adenosine diphosphate glucose (ADPGlc) as a substrate.
  • ADPGlc adenosine diphosphate glucose
  • This similarity allows us to use plant starch synthase (SS) and starch branching enzyme (SBE) to complement the functions of glycogen synthase (GS) and glycogen branching enzyme (GBE) in E. coli G6MD3, which is deficient in GS and GBE.
  • Transformation of E.coli glgC gene and maize starch synthase gene in E.coli G6MD3 produced linear a 1,4 glucan similar to amylose.
  • coexpression of the glgC, maize starch synthase and maize branching enzyme produced branched polysaccharides.
  • coli G6MD3 resulted in the synthesis of different sizes of polysaccharide with DP 500-4000.
  • These polysaccharides synthesized in E .coli by maize SS have different physical-chemical properties than polysaccharides synthesized in natural organisms including starch from plant sources and glycogen from animals.
  • the polysaccharide can be used in food and nonfood industries to replace and/ or complement starch functionalities. A large amount of these polysaccharide can be produced by fermentation technology.
  • the following plasmids have been transformed into rice plants Transgenic 1, MSTSIA(pExs52) and glgC 3 (pExs66), MSTSIIa and glgC 3 (pExs53 and pExs56).
  • the second group of rice transformatns contain MSTSIIc and glgC 3 (pExs54 and pExs56).
  • the third group of transformation transgenic 5 MSTSIII and glgC 3 ( pExs 61 and pExs 66); transgenic 6 Mwx glgC 3 p Exs65 and pExs66).
  • figures 25-41 for plasmid maps and figure 43-55 for sequences used in the plasmid.
  • glgA and glgB and glgC were combined and transformed into rice. This is combining the rice plants starch pathway with the gene encoding for ADPG and the genes encoding for at least one of the following enzymes, SSI, SSII, SSIII, Debranching enzymes, BEI, BEII, GBSS (wx).
  • These plasmids could have been transformed into other cereals such as corn, wheat, barley, oats, sorghum, milo in substantially the plasmid that is shown in the figures for the plant host.
  • the promoter could be the waxy gene which is published, other additional zein promoters are known and could be used as the promoter.
  • the promoter used herein is described in Figures 45aand 45b.
  • plasmid with little additional work could be transformed into dicots such as potatoes, sweet potato, taro, yam, lotus cassava, peanuts, peas, soybean, beans, chickpeas.
  • the promoter could be selected to target the starch storage area of the particular dicots (some are roots some are tubers).
  • Various method of transforming monocots and dicots are known in the industry and the method of transforming the genes is not critical to the present invention.
  • the plasmid can be introduced into Agrobacterium tumefaciens by the freeze-thaw method of An et al.(1988) Binary vectors. In Plant Molecular Biology Manual A3, S.B. Gelvin and R.A.
  • a number of monocots are also starch bearing plants but until about a decade ago monocots were difficult to develop transformants.
  • the most prominent methods of transformation presently used in monocots is the gunning of micro projectiles into the plants or using Agrobacterium and subsequent regeneration of the plants from the transformed materials.
  • Various tissues and cells can now be transformed with plasmids into monocot hosts. In fact there are teaching from at least five ago on methods of transforming not only callus but also cotyledons. The methods of transforming plants and selecting for the transformants with either selectable or screen able markers are also well known.
  • EXAMPLE 1 Construction of the E. coli expression vector.
  • the expression vector pExs2 was derived from pET-23d (Novagen) and pGPl-2 (15).
  • the expression vectors pExs-trc and pExs-trc3 were derived from pTrc99a (Pharmacia) and pGPl-2.
  • the Bglll/Pstl fragment (2192 bp) containing the pBR322 origin of replication was deleted from pET-23d and replaced with the BamHI/Pstl fragment (3 kb) containing origin pl5A and kanamycin resistance gene from pGPl-2. This process generated plasmid pEXSl containing both ampicillin and kanamycin resistance genes.
  • the ampicillin resistance gene was inactivated by deletion of the Seal / Bgll fragment (360 bp, Bgll end was filled in and blunt- end ligated with Seal end). Inactivation of the ampicillin resistance gene in pEXS 1 generated the expression plasmid pEXS2, containing the T7 promoter, T7 terminator, kanamycin resistance gene and pl5A origin of replication. Plasmid pTrc99a was digested with Ndel, filled in with kelnow fragment and blunt-end ligated to remove Ndel site. A Ndel site was introduced at the Ncol site by mutagenesis to generate plasmid pExs-trc.
  • the Bgll and PvuII fragment(2.48 kb) in pExs-trc containing the pBR322 origin of replication was replaced by Bgll/BamHI (filled in with Klenow fragment) fragment (3 kb) containing origin pi 5 A and kanamycin resistance gene from pGPl-2 to generate pExs-trc2.
  • the ampicillin resistance gene was inactivated by deletion of the Seal / Bgll fragment (360 bp, Bgll end was filled in and blunt-end ligated with Seal end). Inactivation of the ampicillin resistance gene in pExs-trc2 generated the expression plasmid pExs-trc3.
  • primer Exs4 5'-CAAGAATGCTGCGGGAGTC-3'
  • primer Exs23 5'- AAGTCGACATATGTGCGTCGCGGAGCTGAGCAG-3'
  • primer Exs 57 5'- GGGCCCCATATGAGCATTGTCTTTGTAACCGG-3 *
  • primer Exsl 5'- CTCGGGCCCATATGGGGGAGAATGTTATGAA-3'
  • primer Exs2 5'-
  • Primer Exs 17 paired individually with primer Exs 16 and Exs55 was to modify the N-terminus of maize SSSIII to generate pExs-9 and pExs-9a.
  • EXAMPLE 2 Construction of expression plasmids for E. coli ADPGlc pyrophosphorylase, BE and maize SBE.
  • E. coli glgB gene was excised from plasmid pOP12 (16).
  • the BstXl (filled in) / Malawi fragment containing the glgB ribosome binding site and the full length glgB gene was cloned at the Smal site of pBluescriptSK- (Stratagene).
  • the glgB gene in pBluscriptSK- was subsequently cloned into pEXS2 at the Xbal / Sail sites to generate plasmid pEXSB.
  • Primer G (5'- GAAGATCTGGCAGGGACCTGCACAC-3')
  • primer H 5'-
  • GGACTAGTGCATTATCGCTCCTGTTTAT-3' were used to PCR the E. coli glgC gene coding for ADPGlc pyrophosphorylase from plasmid pOP12.
  • the glgC gene including its own ribosome binding site was subcloned into expression plasmid pEXS2 at the Xbal (filled in with Klenow fragment) and Notl site to generate plasmid pEXSc.
  • the genes coding for mature maize SBEI and SBEII along with a ribosome binding site were subcloned from plasmids pET- 23d-SBEI and pET-23d-SBEII into the plasmid pEXSc at the Spel site to form the plasmids pEXSc-SBEI and pEXSc-SBEII.
  • the gene coding for mature maize SBEII including a ribosome binding site was cloned into pEXSc-SBEI at the Xbal/Notl sites to form plasmid pEXSc-SBEI-SBEII.
  • E.coli glgc gene and genes encoding maize SBEI and SBEII were also cloned in plasmid pExs-trc and pExs-trc3 respectively and together as described for pExs2.
  • Homologous recombination was used for the strain construction. This was done according to the method described by Hamilton et al ( Journal of Bacteriology, 1989, 171 :4617-4622.) A temperature-sensitive pSClOl replicon was used to facilitate the selection. The gene coding for spectinomycin adenyltransferase was inserted at PvuII sites in plasmid pOP12 to form plasmid HPG9 which has spectinomycin resistance and has C-terminus of glgB gene and N-terminus of glgA gene deleted.
  • the transformed cell was cultured in 3 mL LB with 100 mg/mL Spectinomycin at room temperature overnight, the cells were plated on LB agar plate containing 100 mg/mL spectinomycin and incubated at 44°C overnight. Single colonies were inoculated on LB agar plate containing 100 mg/mL spectinomycin and 0.2% glucose and incubated at 44°C and at 37°C overnight. The colonies at 37°C were stained with iodine. The colony with negative staining was selected and grown in 100 mL LB at 37°C overnight. The cells were harvested and homogenized in an extraction buffer for assaying glycogen synthase and branching enzyme activities.
  • the 1DE3 lysogenization kit fromNovagen was used for site specific integration of 1DE3 prophage into E. coli HPG204 to form E. coli HPG204(DE3)[ was DThe lysate was prepared with Plvir and its transduction into E.
  • Plasmid pExs-2 and pExs-trc3 has kanamycin resistance and pl5A origin of replication. It is compatible with plasmid pET21a, pExs-trc, pTrc99A containing pBR322 origin. Expression plasmids pExs-2 and pET-21a were used to express SS and SBE in E. coli HPG204(DE3). Expression plasmids pExs-trc and pExs-trc3 were used for expression in E. coli G6MD3. This made it possible to transform different combinations of maize SS and SBE in E. coli HPG204(DE3), or G6MD3 which is deficient in GS and GBE activity.
  • An overnight culture of cells transformed with maize SS and SBE was diluted 1 :20 (v/v) in fresh LB containing 0.2 % glucose, 100 mg/mL ampicillin and 50 mg/mL kanamycin.
  • EXAMPLE 5 Isolation of highly branched a-glucan from E. coli.
  • Cell pellet (30 g) was resuspended and lysed by sonication in 150 mL 50 mM tris- acetate buffer (pH 7.5) containing 10 mM EDTA and 5 mM DTT. After a fraction of the homogenate was saved for assaying the STS and SBE activities, the homogenate was centrifuged at 20, OOOg for 50 min at 4°C. After collecting the supernatant, the pellet was resuspended in 150 mL water and boiled for 15 min with occasional stirring. The resuspension was centrifuged at 20, OOOg for 30 at room temperature. After collecting the supernatant, the pellet was washed again with 100 mL water as above.
  • TCA Tri chloric acid
  • the pellet is redissolved in 90 mL hot water by heating in boiling water bath. Insoluble materials are immediately removed by centrifugation at room temperature. Add 10 mL butanol to the supernatant, stay at 0 °C for 1 hr and centrifuge at 12000 rpm for 30 min at 4 °C. Repeat the step once.
  • the amylose precipitate is redissolved in 90 mL hot water by heating, and 10 mL butanol are added to the solution. After storing at 40 °C on ice for one hour, it is centrifuged at 4 °C for 30 min. Repeat the step once. The pellet is redissolved in 100 mL 10% butanol by heating.
  • the reaction mixture for STS contained 100 mM Bicine buffer, 10 mg/mL glycogen, 0.5 mg/mL BSA, 0.5 M sodium citrate, 25 mM potassium acetate, 10 mM GSH, 3 mM [14C]ADPGlc (500 dpm/nmol) and enzyme in a final volume of 0.1 mL.
  • the reaction was carried out at 25°C for 15 min and terminated by boiling for 2 min.
  • the unincorporated [14C] ADPGlc was separated with Dowex anion exchange column (200-400 mesh, Sigma Chemical Co.).
  • One unit of activity is defined as 1 nmol Glc incorporated into the a-glucan per min at 25°C. SBE activity was determined by phosphorylase stimulation assay. One unit of activity is defined as 1 mmol Glc incorporated into the a-glucan per min at 30°C.
  • the cell pellet was resuspended in sonication buffer (50 mM Tris-acetate, pH 7.5, 10 mM EDTA, and 5 mM DTT; 7 ml buffer per gram of cell mass), and cells were lysed using a Fisher 550 Sonic Dismembrator with 5 x 1 min. bursts with 30 sec. intervals. The homogenate was centrifuged at 9600g for 30 minutes. SSI in the supernatant was then precipitated by slowly adding neutralized saturated ammonium sulfate to 40% saturation. After stirring on ice for an additional 50 minutes, proteins were collected by centrifugation at 12700g for 45 minutes.
  • sonication buffer 50 mM Tris-acetate, pH 7.5, 10 mM EDTA, and 5 mM DTT; 7 ml buffer per gram of cell mass
  • the protein pellet was then redissolved in buffer A (50 mM Tris, pH 7.5, 1 mM EDTA, and 5 mM DTT) containing 0.1 M KCl and dialyzed against the same buffer, with one change of buffer. After dialysis, the sample was centrifuged at 13000g for 20 minutes to remove insoluble materials. The resulting supernatant was loaded onto an amylose affinity column pre-equilibrated with dialysis buffer, and the flow through was collected. The column was washed with 10 column volumes of buffer A containing 0.1 M KCl, and then with buffer A containing 0.5 M KCl and 0.5 M maltose, collecting fractions during both washes.
  • buffer A 50 mM Tris, pH 7.5, 1 mM EDTA, and 5 mM DTT
  • the active fractions were pooled and dialyzed overnight against buffer A, with one change of buffer. The next day, the amylose column sample was filtered and applied to a mono Q 5/5 FPLC column (Pharmacia). After washing with buffer A, a 20 ml 0-0.4 M KCl gradient was employed. The active fractions were electrophoresed on an 8% SDS-PAGE gel (31) to determine the purity of SSI in those fractions; the fractions which were apparently homogeneous were pooled and concentrated using a Centricon-30 spin column (Amicon).
  • One unit activity is defined as one ⁇ mol glucose incorporated into a-1 ,4 glucan per minute at 25 C using 5 mg/mL glycogen as primer.
  • Plasmid Protein STS activity BE activity Imax DP CL Yield (Mg/mL) (u/mg protein) (u/mg protein) (nm) (mg dry wt/g wet cell) pExsCA 580 700 10.6 3.3
  • Plasmid Protein STS activity BE activity Imax DP CL Yield (Mg/mL) (u/mg protein) (u/mg protein) (nm) ( mg dry wt/g wet cell)
  • ADPGlc Kinetics for STSI enzymes Assays and data evaluation are as in Table H. K-» are expressed as mM ADPGlc and N m are in ⁇ mo min/mg protein. 5 mg ml amylopectin was used as primer for all assays.
  • Amylose column 20 9.3 0.991 9.3 45.9 monoQ column 0.9 0.94 4.81 4.5 222
  • V ma _ (with amylopectin) 26.3 22.5 15.4 41.1 84.5 67.9
  • e ⁇ -te ⁇ ninally truncated form of SS, while any SS with the designation SS-1 is the full length version of the SS.
  • Table 15 The starch synthase activities of the chimerical enzymes. Generation of chimerical enzymes of maize starch synthase: the recombination of N-terminal extensions with C-terminal catalytic domains of starch synthase
  • the gene coding for N-terminal extensions of STSI, STSIia and STSIIb were inserted, in the same (+) or reverse (-) orientation of original N-terminal DNA sequence, in front of the C-terminal catalytic domains of WX2, STSIia and STSIIb, respectively.
  • the chimerical enzymes were expressed in E.coli, and the activities were assayed.
  • Corn starch is a milky , slightly thickened gel which is slightly if at all flowable.
  • Rice starch forms two levels the upper level is a thickened syrup like consistency more flowable then corn starch (less thick then corn starch) opaque milky color (more translucent then corn starch in this level) and a lower level which is a very white glob not transmitting much light through this bottom level of material. This lower level is formed in a very thick mass and does not appear flowable.
  • Corn amylopectin is slightly less white then the top level of rice starch and is a very slightly opaque milky color (more translucent then corn starch) slightly less flowable then the rice top level.
  • Potato dextrin is the most transparent almost appearing clear but is still opaque white and it is very flowable appearing only slightly less flowable then water.
  • Waxy Maize starch will flow very slowly and has the consistency of honey. The color is very opaque transmitting little light and the color is only slightly less light then corn starch.
  • SSI starch made from plasmid pExs-8 has two distinct levels. The top level appears clear and slightly thicker then the flowability of water. The bottom level appears as a precipitate. This sample resembles the ornaments that contain little figures and plastic flakes resembling snowflakes. Like those ornaments when turned upside down the sample appears to be falling snow. However the flakes in this sample appear to be slightly gummy and appear in the first moments of level mixing to form a opaque white liquid.
  • SSI starch made from a host containing the following two plasmids pExsC BEI BEII and pExs ⁇ is not as clear as the top level of pExs-8 and appears slightly less thick then pExs-8. It has even more flowability then does Potato Dextrin.
  • SSIIb starch made from a host containing the following two plasmids pExsC BEI BEII and pExs-9 is not as clear as the top level of pExs-8 and appears slightly less thick then pExs-8. It has even more flowability then does Potato Dextrin.
  • WAXY starch made from a host containing the following two plasmids pExsC BEI BEII and pExs-wx is not as clear as the top level of pExs-8 but seems to have a few tiny thread like chains that settle to the bottom and when mixed give the material a slightly more white color and appears slightly less thick then pExs-8. It has even more flowability then does Potato Dextrin.
  • SSII starch made from a host containing the following two plasmids pExsC BEI BEII and pExs-3a is the color of corn starch and maybe slightly whiter but not as white as the bottom level of pExs ⁇ and definitely transmitting more light through and has the flowability characteristic of pExs-8 when mixed.
  • glgA starch appears to have a very slight precipitate and is comparable in color to corn amylose pectin and ExsC BEI BEII and pExs-wx. And the flowability is between corn amylose and pExsC BEI BEII pExs-wx.
  • the samples of polysaccharides listed above form groups generally according to color as follows: waxy maize starch and corn starch and pExsC BEI BEII pExs3a and pExsc ⁇ are the whitest group.
  • the flowability characteristics of this group are fairly diverse. With corn starch a lump and Waxy maize starch only slightly flowable and pExsC BEI BEII and pExs- 3a and pExsC-8 more like water then syrup.
  • the second group contains corn amylopectin and pExsC BEI BEII pExs-wx and pExsC BEI BEII and pExs-Al which are less white and clearer.
  • the flowability of corn amylopectin is less then the other two members of this group but it is still similar.
  • the last group is the least white and thus the clearest.
  • This group includes pExsC BEI BEII and pExs-8, potato dextrin, pExsC BEI BEII and pExs-10, pExsC BEI BEII and pExs-9.
  • the flowability of this group is also similar to each other. Plant Hosts
  • the following plasmids have been transformed into rice plants.
  • the sequence for the mutant glgC gene is shown in Figure 46.
  • the plasmids are made substantially in a similar manner as described above for the production of bacterial plasmid.
  • the following combinations of plasmids have been transformed into rice plants.
  • plasmids including the combination that includes all of the maize genes SSI, SSII, SSII, BEI, BEII, and GBSS in one host or alternatively in two host that are then crossed to form a hybrid having the entire complement of up regulated starch genes are being developed.
  • sequences in the antisense positions could have placed the sequences in the antisense positions to down regulate these genes to the extent that maize genes will down regulate the partial homologous rice genes.
  • the first group of transgenic are group 1, including rice transformants (transformed by microprojectile bombardment) containing MSTSI-2 (pExs52) and glgC 3 (pExs66), MSTSIIa-2 and glgC 3 (pExs53 and pExs56).
  • the second group of rice transformants contains MSTSIIa and glgC 3 (pExs54 and pExs56).
  • the third group of transformation contain: transgenic 5 MSTSIIb and glgC 3 ( pExs 61 and pExs 66); transgenic 6 Maize wx and glgC 3 pExs65 and pExs ⁇ ).
  • glgA and glgB and glgC are combined and transformed into rice.
  • This last transformant is combining the rice plants starch pathway with the gene encoding for ADPG pyrophosphorylase and the bacterial genes.
  • the combination of the plasmids encoding for at least one of the following enzymes, SSI, SSIIa, SSIIb, SSIII, Debranching enzymes, BEI, BEII, GBSS (wx)and some or all of the bacterial starch genes is also useful.
  • There ae presently over 300 transformants in the greenhouse.
  • the TI transgenic rice plants have been screened and characterized ( Figure 56, 57). 12 plants have successfully expressed maize SSI-2 in rice seeds. 21 plants have successfully expressed maize SSIIb in rice seeds.
  • N-terminal truncation decreased the enzyme affinity for amylopectin, with the K., for amylopectin of the truncated SSI-3 being about 60%-90% higher than that of the full length SSI-1.
  • the effects of N-terminal truncation of SSIIa depend upon the assay conditions used. For both SSIIa-1 and SSIIa-2, the V max of each enzyme increased 2-fold upon raising assay temperature from 27 N C to 37 N C (Tables II and III). However, the effect of temperature on ADPGlc affinity was different for SSIIa-1 and SSIIa-2.
  • the K-, for ADPGlc was not affected by raising temperature.
  • the K_, of ADPGlc for the putative full length SSIIa-1 increased 2 fold upon raising the assay temperature from 27 N C to 37 N C (Table III).
  • the truncated SSIIa-2 exhibited a lower K_, for ADPGlc than SSIIa-1 did in all assay conditions used in this study except that they showed similar K_, values for ADPGlc when glycogen was used as a primer at 27EC.
  • N-terminal truncation of SSIIa appears to lower the K-, for ADPGlc under most assay conditions, it also must be noted that the maximal velocity of the truncated SSIIa-2 is decreased by about 2-4 fold when compared to SSIIa-1.
  • the truncated SSIIb-2 was found to be more temperature stable than the longer SSIIb- 1 in the presence of citrate, while little difference was observed in their pH activity profiles.
  • starch genes can produce new and altered starch in either host, plant or bacteria. Additionally, polysaccahrides very similar to corn starch can be produced in a bacterial host.

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Abstract

La présente invention concerne des hôtes qui contiennent des constructions présentant des gènes issus de la voie de synthèse de l'amidon. Plus spécifiquement, l'invention concerne des hôtes bactériennes qui forment des amidons végétaux. Elle concerne en outre des hôtes végétaux contenant des gènes issus de la voie de synthèse de l'amidon. Elle concerne enfin les amidons produits par lesdits hôtes.
PCT/US1998/006660 1997-04-04 1998-04-03 Amidons vegetaux et methodes permettant de produire lesdits amidons dans des hotes WO1998044780A1 (fr)

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Application Number Priority Date Filing Date Title
AU68828/98A AU6882898A (en) 1997-04-04 1998-04-03 Plant like starches and the method of making them in hosts
JP54294098A JP2001519664A (ja) 1997-04-04 1998-04-03 植物様デンプンおよび宿主においてそれらを作成する方法
CA002283719A CA2283719A1 (fr) 1997-04-04 1998-04-03 Amidons vegetaux et methodes permettant de produire lesdits amidons dans des hotes
EP98914483A EP1017270A4 (fr) 1997-04-04 1998-04-03 Amidons vegetaux et methodes permettant de produire lesdits amidons dans des hotes
US10/336,753 US20030226176A1 (en) 1998-04-03 2003-01-06 Plant like starches and the method of making them in hosts
US11/330,822 US7285703B2 (en) 1998-04-03 2006-01-11 Plant like starches and the method of making them in hosts

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WO2000008184A1 (fr) * 1998-07-31 2000-02-17 Aventis Cropscience Gmbh Vegetaux synthetisant un amidon modifie, leurs procedes de preparation, leur utilisation, ainsi que l'amidon modifie
WO2000022140A1 (fr) * 1998-10-09 2000-04-20 Planttec Biotechnologie Gmbh Forschung & Entwicklung MOLECULES D'ACIDE NUCLEIQUE CODANT UNE ENZYME DE RAMIFICATION DE BACTERIES DU GENRE NEISSERIA ET PROCEDE POUR PRODUIRE DES α-1,4 GLUCANES RAMIFIEES EN α-1,6
WO2000031282A1 (fr) * 1998-11-19 2000-06-02 Advanced Technologies (Cambridge) Limited Vegetaux genetiquement modifies presentant une modification de l'amidon
WO2000031274A1 (fr) 1998-11-19 2000-06-02 Advanced Technologies (Cambridge) Limited Plantes genetiquement transformees par modification de l'amidon
WO2000006755A3 (fr) * 1998-07-28 2000-09-08 Du Pont Modification de l'expression du gene de l'enzyme de biosynthese de l'amidon en vue de la production d'amidon dans des cultures cerealieres
WO2000073422A1 (fr) * 1999-05-27 2000-12-07 Planttec Biotechnologie Gmbh Cellules de plantes genetiquement modifiees et plantes dont l'activite d'une proteine amylosucrase et celle d'une enzyme ramifiante sont plus intenses
WO2000078984A2 (fr) * 1999-06-21 2000-12-28 Pioneer Hi-Bred International, Inc. Amelioration de la resistance de puits florale et augmentation de la stabilite de grenaison des plantes
WO2001019975A2 (fr) * 1999-09-15 2001-03-22 National Starch And Chemical Investment Holding Corporation Plantes dont une ou plusieurs enzymes de modification de l'amidon presentent une activite reduite
WO2002018606A1 (fr) * 2000-08-28 2002-03-07 E. I. Du Pont De Nemours And Company Nouveaux amidons produits par expression des genes heterologues de synthase d'amidon lie aux granules
US6495740B1 (en) 1996-06-27 2002-12-17 The Austrailian National University Manipulation of cellulose and/or β-1,4-Glucan
WO2005003312A3 (fr) * 2003-06-27 2005-10-13 Monsanto Technology Llc Elevation des niveaux d'huile dans les plantes
WO2005104822A1 (fr) * 2004-04-21 2005-11-10 Basf Plant Science Gmbh Plants de mais transgenique presentant des valeurs nutritives ameliorees
US7285703B2 (en) 1998-04-03 2007-10-23 Basf Plant Science Gmbh Plant like starches and the method of making them in hosts

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JP5750635B2 (ja) * 2010-07-15 2015-07-22 公立大学法人秋田県立大学 イネ変異体、澱粉の製造方法、澱粉、及びイネ変異体の製造方法

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US7154026B2 (en) 1996-06-27 2006-12-26 The Australian National University Manipulation of cellulose and/or β-1,4,-glucan
US6495740B1 (en) 1996-06-27 2002-12-17 The Austrailian National University Manipulation of cellulose and/or β-1,4-Glucan
US7285703B2 (en) 1998-04-03 2007-10-23 Basf Plant Science Gmbh Plant like starches and the method of making them in hosts
US6392120B1 (en) 1998-07-28 2002-05-21 E. I. Du Pont De Nemours And Company Modification of starch biosynthetic enzyme gene expression to produce starches in grain crops
US6570008B1 (en) 1998-07-28 2003-05-27 E. I. Du Pont De Nemours And Company Modification of starch biosynthetic enzyme gene expression to produce starches in grain crops
WO2000006755A3 (fr) * 1998-07-28 2000-09-08 Du Pont Modification de l'expression du gene de l'enzyme de biosynthese de l'amidon en vue de la production d'amidon dans des cultures cerealieres
US7385104B2 (en) 1998-07-31 2008-06-10 Bayer Cropscience Ag Plants synthesizing a modified starch, a process for the generation of the plants, their use, and the modified starch
US6596928B1 (en) 1998-07-31 2003-07-22 Bayer Cropscience Gmbh Plants synthesizing a modified starch, the generation of the plants, their use, and the modified starch
WO2000008184A1 (fr) * 1998-07-31 2000-02-17 Aventis Cropscience Gmbh Vegetaux synthetisant un amidon modifie, leurs procedes de preparation, leur utilisation, ainsi que l'amidon modifie
US7247769B2 (en) 1998-07-31 2007-07-24 Bayer Cropscience Gmbh Plants synthesizing a modified starch, a process for the generation of the plants, their use, and the modified starch
JP2002527068A (ja) * 1998-10-09 2002-08-27 プランテック バイオテクノロジー ゲーエムベーハー フォーシュング アンド エンテゥウィックラング ナイセリア（Ｎｅｉｓｓｅｒｉａ）属細菌からの分枝酵素をコードする核酸分子ならびにα−１，６−分枝α−１，４−グルカンの製造方法
US8716463B2 (en) 1998-10-09 2014-05-06 Bayer Bioscience Gmbh Method for the producing alpha-1, 6-branched alpha-1, 4-glucans from sucrose
US7732164B2 (en) 1998-10-09 2010-06-08 Bayer Bioscience Gmbh Method for producing α-1, 6-branched α-1, 4-glucans from sucrose
US7638311B2 (en) 1998-10-09 2009-12-29 Bayer Bioscience Gmbh Method for producing α-1, 6-branched α-1, 4-glucans from sucrose
US7833751B2 (en) 1998-10-09 2010-11-16 Bayer Bioscience Gmbh Method for producing alpha-1, 6-branched alpha-1, 4-glucans from sucrose
WO2000022140A1 (fr) * 1998-10-09 2000-04-20 Planttec Biotechnologie Gmbh Forschung & Entwicklung MOLECULES D'ACIDE NUCLEIQUE CODANT UNE ENZYME DE RAMIFICATION DE BACTERIES DU GENRE NEISSERIA ET PROCEDE POUR PRODUIRE DES α-1,4 GLUCANES RAMIFIEES EN α-1,6
US8771424B2 (en) 1998-10-09 2014-07-08 Bayer Bioscience Gmbh Method for the producing alpha-1, 6-branched alpha-1, 4-glucans from sucrose
US6699694B1 (en) 1998-10-09 2004-03-02 Planttec Biotechnologie Gmbh Method for producing α-1,6-branched α-1,4-glucans from sucrose
WO2000031274A1 (fr) 1998-11-19 2000-06-02 Advanced Technologies (Cambridge) Limited Plantes genetiquement transformees par modification de l'amidon
WO2000031282A1 (fr) * 1998-11-19 2000-06-02 Advanced Technologies (Cambridge) Limited Vegetaux genetiquement modifies presentant une modification de l'amidon
US6566585B1 (en) 1999-05-27 2003-05-20 Planttec Biotechnologie Gmbh Genetically modified plant cells and plants with an increased activity of an amylosucrase protein and a branching enzyme
WO2000073422A1 (fr) * 1999-05-27 2000-12-07 Planttec Biotechnologie Gmbh Cellules de plantes genetiquement modifiees et plantes dont l'activite d'une proteine amylosucrase et celle d'une enzyme ramifiante sont plus intenses
US8026411B2 (en) 1999-06-21 2011-09-27 Pioneer Hi-Bred International, Inc. Enhanced floral sink strength and increased stability of seed set in plants
US7193130B2 (en) 1999-06-21 2007-03-20 Pioneer Hi-Bred International, Inc. Expressing acid invertase or ADP-glucose pyrophosphorylase in floral tissue for enhanced floral sink strength and increased stability of seed in plants
WO2000078984A2 (fr) * 1999-06-21 2000-12-28 Pioneer Hi-Bred International, Inc. Amelioration de la resistance de puits florale et augmentation de la stabilite de grenaison des plantes
WO2000078984A3 (fr) * 1999-06-21 2001-02-15 Pioneer Hi Bred Int Amelioration de la resistance de puits florale et augmentation de la stabilite de grenaison des plantes
WO2001019975A3 (fr) * 1999-09-15 2001-09-27 Nat Starch Chem Invest Plantes dont une ou plusieurs enzymes de modification de l'amidon presentent une activite reduite
WO2001019975A2 (fr) * 1999-09-15 2001-03-22 National Starch And Chemical Investment Holding Corporation Plantes dont une ou plusieurs enzymes de modification de l'amidon presentent une activite reduite
US7619134B2 (en) 2000-08-28 2009-11-17 E.I. Du Pont De Nemours And Company Starches produced by the expression of heterologous granule bound starch synthase genes
WO2002018606A1 (fr) * 2000-08-28 2002-03-07 E. I. Du Pont De Nemours And Company Nouveaux amidons produits par expression des genes heterologues de synthase d'amidon lie aux granules
AU2004254373B2 (en) * 2003-06-27 2009-11-19 Monsanto Technology, Llc Elevation of oil levels in plants
WO2005003312A3 (fr) * 2003-06-27 2005-10-13 Monsanto Technology Llc Elevation des niveaux d'huile dans les plantes
WO2005104822A1 (fr) * 2004-04-21 2005-11-10 Basf Plant Science Gmbh Plants de mais transgenique presentant des valeurs nutritives ameliorees
US8030540B2 (en) 2004-04-21 2011-10-04 Basf Plant Science Gmbh Transgenic corn having enhanced nutritional qualities

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