WO1998054335A1 - Modification in vivo de galactomannane de guar par expression d'arn antisens d'uridine diphosphogalactose epimerase - Google Patents

Modification in vivo de galactomannane de guar par expression d'arn antisens d'uridine diphosphogalactose epimerase Download PDF

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Publication number
WO1998054335A1
WO1998054335A1 PCT/IB1998/000891 IB9800891W WO9854335A1 WO 1998054335 A1 WO1998054335 A1 WO 1998054335A1 IB 9800891 W IB9800891 W IB 9800891W WO 9854335 A1 WO9854335 A1 WO 9854335A1
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Prior art keywords
galactose
mannose
containing compound
nucleotide sequence
udp
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PCT/IB1998/000891
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English (en)
Inventor
Morten JØRSBOE
Janne Brunstedt
Steen Guldager Pedersen
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Danisco A/S
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Application filed by Danisco A/S filed Critical Danisco A/S
Priority to KR1019997010932A priority Critical patent/KR20010012962A/ko
Priority to BR9809493-9A priority patent/BR9809493A/pt
Priority to NZ500951A priority patent/NZ500951A/en
Priority to EP98921695A priority patent/EP0983369A1/fr
Priority to CA002291449A priority patent/CA2291449A1/fr
Priority to JP50042399A priority patent/JP2002500515A/ja
Priority to AU74463/98A priority patent/AU733483B2/en
Priority to GB9927197A priority patent/GB2342920B/en
Publication of WO1998054335A1 publication Critical patent/WO1998054335A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—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
    • C12N9/90—Isomerases (5.)

Definitions

  • the present invention relates to a modification process.
  • the present invention relates to an in vivo modification process.
  • Galactomannans are a heterogenous group of cell wall polysaccharides consisting of a ⁇ -1- 4 linked mannan backbone with varying numbers of ⁇ -1-6 linked galactose side chains.
  • the galactomannans of most significant industrial use are obtained from the endosperms of the legumes guar (Cyamopsis tetragonolobus) and locust bean (Ceratonia siliqua). These galactomannans differ in their galactose content, with guar having a galactose to mannose ratio of approximately 1:1.6, whereas the ratio for locust bean gum (LBG) is approximately 1:3.4.
  • LBG low calorie products
  • guar gum with galactose contents of 10-34% was obtained (Bulpin et ai 1990).
  • Analysis of the gelation behaviour of me modified guar gum showed that a preparation with a galactose content of 24% formed mixed gels with carrageenan displaying similar rheological properties as LBG.
  • the galactose content of untreated guar gum was 38% and 23 % for LBG.
  • the yield of galactomannan is reduced because a 40% reduction in galactose content corresponds to approximately 15% less modified guar gum.
  • the released galactose may be undesirable in the final product and may have to be removed.
  • PCT/EP96/05581 relates to in vivo modification of a mannose/galactose containing compound - such as guar gum - in an organism (or part thereof) capable of synthesising that compound by a method that is not native to that organism - such as by a method that makes use of recombinant DNA techniques.
  • the modification may occur in relation to any one or more of the precursors of the compound (e.g. mannose and/or galactose) or in relation to the compound itself (i.e. modification of the mannose and/or galactose units of a compound comprising same).
  • PCT/EP96/05581 relates to an in vivo modification process that affects, preferably increases, the mannose-to-galactose ratio of either an organism (or part thereof) capable of producing a mannose/galactose containing compound or of a mannose/galactose containing compound thereof.
  • This in vivo modification process is not a naturally occurring process.
  • PCT/EP96/05581 it is possible to alter the internal in vivo ratio of mannose to galactose within an organism and/or the ratio of mannose to galactose of a mannose/galactose compound thereof.
  • an in vivo modification process that affects the mannose-to-galactose ratio of either an organism (or part thereof) capable of producing a mannose/galactose containing compound or of a mannose/galactose containing compound thereof
  • the in vivo modification process comprises expressing a nucleotide sequence that has an effect on: (a) the mannose-to-galactose ratio of mannose and galactose components of a mannose/galactose containing compound; and/or (b) the mannose-to-galactose ratio of mannose and galactose precursors for a mannose/galactose containing compound; and wherein the nucleotide sequence is antisense to at least a part of the gene for a UDP-galactose epimerase enzyme.
  • nucleotide sequence to affect in vivo the mannose-to-galactose ratio of either an organism (or part thereof) capable of producing a mannose/galactose containing compound or of a mannose/galactose containing compound thereof, wherein the nucleotide sequence is antisense to at least a part of the gene for a UDP-galactose epimerase enzyme, and wherein the nucleotide sequence has an effect on: (a) the mannose-to-galactose ratio of mannose and galactose components of a mannose/galactose containing compound; and/or (b) the mannose-to-galactose ratio of mannose and galactose precursors for a mannose/galactose containing compound.
  • the nucleotide sequence is antisense to at least a part of the coding sequence for a UDP-galactose epimerase enzyme.
  • mannose/galactose containing compound means a compound comprising at least one mannose group and at least one galactose group.
  • mannose/galactose containing compound is galactomannan.
  • mannose/galactose containing compound is guar gum.
  • the organism capable of producing a mannose/galactose containing compound is a guar plant and the mannose/galactose containing compound thereof is galactomannan.
  • other galactomannan producing plants are encompassed such as fenugreek and lucerne. Plants that are considered not to produce appropriate quantities of galactomannan belong to the family Solanacea and the species Nicotiana tabacum.
  • organism (or part thereof) capable of producing a mannose/galactose containing compound also includes any suitable organism - in particular a plant - capable of producing a mannose/galactose containing compound, such that the internal in vivo ratio of mannose to galactose of that organism is altered.
  • the term also includes any part of an organism that is capable of producing a mannose/galactose containing compound, such that the ratio of mannose to galactose of that part is altered.
  • the term also includes a part when within an organism or in a live culture medium. Preferably, the part is when within an organism per se. An example of a part is seed.
  • mannose and galactose precursors includes mannose per se or derivatives thereof and/or galactose per se or derivatives thereof as precursors for the biosynthesis of a mannose/galactose containing compound, preferably galactomannan.
  • mannose and galactose precursors includes mannose per se or derivatives thereof and/or galactose per se or derivatives thereof as precursors for the biosynthesis of a mannose/galactose containing compound, preferably galactomannan.
  • the term means mannose per se or derivatives thereof (such as mannose-6-phosphate or GDP- mannose) and/or galactose per se or derivatives thereof as precursors for the biosynthesis of galactomannan, preferably guar galactomannan.
  • the in vivo mannose-to-galactose ratio of the organism (or part thereof) or mannose/galactose containing compound thereof is higher than that of the guar plant or the galactomannan thereof.
  • the in vivo mannose-to-galactose ratio of the organism (or part thereof) or mannose/galactose containing compound thereof is substantially similar to that of the locust bean or the galactomannan thereof.
  • the organism (or part thereof) or mannose/galactose containing compound thereof is a guar plant or the gum thereof.
  • the present invention also covers a mannose/galactose containing compound when prepared by the process of the present invention.
  • This mannose/galactose containing compound will be referred to as a mannose/galactose containing compound according to the present invention.
  • the present invention also covers a foodstuff comprising a mannose/galactose containing compound according to the present invention.
  • the present invention also covers a composition - such as a foodstuff - comprising a mannose/galactose containing compound according to the present invention admixed with another polysaccharide.
  • a composition - such as a foodstuff - comprising a mannose/galactose containing compound according to the present invention admixed with another polysaccharide.
  • other saccharide is any one or more of xanthan, carrageenan and agarose.
  • the present invention covers methods for preparing compositions or foodstuffs according to the present invention comprising mixing the mannose/galactose containing compound according to the present invention with another suitable ingredient.
  • the broad aspects of the present invention can be achieved by the use of a nucleotide sequence that is antisense to at least a part of the gene for a UDP-galactose epimerase enzyme.
  • UDP-galactose epimerase enzyme converts UDP-glucose to UDP-galactose which is incorporated into the galactomannan.
  • expression of antisense UDP-galactose epimerase reduces the epimerase activity and thereby increases the in vivo mannose-to-galactose ratio of mannose and galactose precursors for the biosynthesis of a mannose containing compound, such as galactomannan.
  • a preferred aspect of the present invention relates to a construct comprising or expressing the nucleotide sequence of the present invention.
  • Another preferred aspect of the present invention relates to a vector comprising or expressing the construct or nucleotide sequence of the present invention.
  • Another preferred aspect of the present invention relates to a plasmid comprising or expressing the vector, construct or nucleotide sequence of the present invention.
  • Another preferred aspect of the present invention relates to a transgenic organism comprising or expressing the plasmid, vector, construct or nucleotide sequence of the present invention.
  • inventions include methods of expressing or allowing expression or transforming any one of the nucleotide sequence, the construct, the plasmid, the vector, the cell, the tissue, the organ or the organism, as well as the products thereof.
  • Further preferred aspects of the present invention include uses of the antisense nucleotide sequence according to the present invention for preparing or treating foodstuffs, including animal feed.
  • One of the key advantages of the present invention is that by using the anti-sense sequence it is possible to increase the mannose-to-galactose ratio of organisms or mannose containing compounds thereof, in particular in vivo modified guar gum.
  • the antisense nucleotide sequence may be used in vivo in combination with one or more other nucleotide sequences, which nucleotide sequences are preferably prepared by use of recombinant DNA techniques.
  • the nucleotide sequence of the present invention may be used in combination with one or more gene products to further affect the mannose-to-galactose ratio, which gene products are preferably prepared by use of recombinant DNA techniques.
  • the gene products can be any one or more of peptides, polypeptides, proteins, enzymes and RNA.
  • at least one of the gene products is an enzyme that is expressed by a nucleotide sequence that is not a natural nucleotide sequence.
  • a natural nucleotide sequence means an entire nucleotide sequence that is in its natural environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its natural environment.
  • the antisense nucleotide sequence according to the present invention is used in conjunction with one or more genes encoding enzymes that are required for the biosynthesis of GDP-mannose - namely the enzyme phosphomannose isomerase (PMI) and/or the enzyme phosphomannose mutase and/or the enzyme GDP-mannose pyrophosphorylase - and/or with one or more genes encoding an ⁇ - galactosidase.
  • PMI phosphomannose isomerase
  • phosphomannose mutase and/or the enzyme GDP-mannose pyrophosphorylase - and/or with one or more genes encoding an ⁇ - galactosidase.
  • nucleotide sequence according to the present invention is used in conjunction with a gene encoding the enzyme phosphomannose isomerase (PMI) and/or with a gene encoding an ⁇ -galactosidase.
  • PMI phosphomannose isomerase
  • ⁇ -galactosidase a preferred PMI and ⁇ -galactosidase are disclosed and discussed in PCT/EP96/05581 (the contents of which are incorporated herein by reference).
  • the present invention also encompasses the use of variants, homologues or fragments of the nucleotide sequence according to the present invention.
  • variants, homologues or fragments of the nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence is similar to a nucleotide sequence that is antisense to at least a part of the gene for a UDP-galactose epimerase enzyme and wherein the resultant nucleotide sequence is capable of affecting the mannose-to-galactose ratio as defined above.
  • allelic variations of the sequences are synonymous with allelic variations of the sequences.
  • the UDP-galactose epimerase enzyme - the gene sequence of which the nucleotide sequence of the present invention is antisense to - may be any suitable UDP-galactose epimerase enzyme.
  • the UDP-galactose epimerase enzyme - the gene sequence of which the nucleotide sequence of the present invention is antisense to - is an endogenous UDP-galactose epimerase enzyme or an enzyme having a sequence similar thereto (such as at least 85% sequence similarity, preferably at least 90% sequence similarity, more preferably at least 95% sequence similarity, more preferably at least 98% sequence similarity).
  • the UDP-galactose epimerase enzyme - the gene sequence of which the nucleotide sequence of the present invention is antisense to - may be or may comprise the arnino acid sequence as presented in SEQ ID No. 1 or SEQ ID No. 2, or a variant, homologue or fragment thereof.
  • the UDP- galactose epimerase enzyme - the gene sequence of which the nucleotide sequence of the present invention is antisense to - may be or may comprise the amino acid sequence as presented in SEQ ID No. 1 or SEQ ID No. 2.
  • nucleotide sequence of the present invention is antisense to the UDP-galactose epimerase enzyme coding sequence that is presented in SEQ ID No. 1 or SEQ ID No. 2, or a variant, homologue or fragment thereof.
  • nucleotide sequence of the present invention is antisense to the UDP-galactose epimerase enzyme coding sequence that is presented in SEQ ID No. 1 or SEQ ID No. 2.
  • the terms “variant”, “homologue” or “fragment” in relation to the arnino acid sequence for the preferred UDP-galactose epimerase enzyme vis-a-vis the antisense nucleotide sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) arnino acid from or to the sequence providing the resultant enzyme has UDP-galactose epimerase activity, preferably having at least the same activity of the enzyme comprising the sequence shown as SEQ ID No. 1 or SEQ ID No. 2.
  • the term “homologue” covers homology with respect to structure and/or function.
  • sequence homology preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to an enzyme comprising the sequence shown as SEQ ID No. 1 or SEQ ID No. 2. More preferably there is at least 95% , more preferably at least 98% , homology to an enzyme comprising the sequence shown as SEQ ID No. 1 or SEQ ID No. 2.
  • variants in relation to the nucleotide sequence coding for the UDP-galactose epimerase enzyme vis-a-vis the antisense nucleotide sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for or is capable of coding for an enzyme having UDP-galactose epimerase activity, preferably having at least the same activity of the enzyme comprising the sequence shown as SEQ ID No. 1 or SEQ ID No. 2.
  • homologue covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for or is capable of coding for an enzyme having UDP-galactose epimerase activity.
  • sequence homology preferably there is at least 75%, more preferably at least 85% , more preferably at least 90% homology to a sequence comprising the sequence shown as SEQ ID No. 1 or SEQ ID No. 2. More preferably there is at least 95%, more preferably at least 98%, homology to a sequence that comprises the sequence shown as SEQ ID No. 1 or SEQ ID No. 2.
  • variants in relation to the antisense nucleotide sequence according to the present invention - namely a sequence that is antisense to that coding for the UDP-galactose epimerase enzyme - include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence is antisense to a sequence that codes for or is capable of coding for an enzyme having UDP-galactose epimerase activity, preferably having at least the same activity of the enzyme comprising the sequence shown as SEQ ID No. 1 or SEQ ID No. 2.
  • homologue covers homology with respect to structure and/or function providing the resultant nucleotide sequence is antisense to a sequence that codes for or is capable of coding for an enzyme having UDP-galactose epimerase activity.
  • sequence homology preferably there is at least 75% , more preferably at least 85%, more preferably at least 90% homology to a sequence comprising a sequence that is antisense to the sequence shown as SEQ ID No. 1 or SEQ ID No. 2. More preferably there is at least 95%, more preferably at least 98% , homology to a sequence comprising a sequence that is antisense to the sequence shown as SEQ ID No. 1 or SEQ ID No. 2.
  • the transgenic organism of the present invention includes an organism comprising any one or more of the nucleotide sequences according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention, or the products thereof, including combinations thereof.
  • the transgenic organism can also comprise any one or more of the nucleotide sequences of the present invention under the control of one or more heterologous promoters .
  • the transgenic organism does not comprise the combination of a promoter and the nucleotide sequence according to the present invention, wherein both the promoter and the nucleotide sequence are native to that organism (or part thereof) and are in their natural environment.
  • promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Mond theory of gene expression.
  • the promoter could additionally include one or more features to ensure or to increase expression in a suitable host.
  • the features can be conserved regions such as a Pribnow Box or a TATA box.
  • the promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention.
  • suitable other sequences include the S/zi-intron or an ADH intron.
  • Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
  • the nucleotide sequence according to the present invention is under the control of a promoter that allows expression of the nucleotide sequence.
  • the promoter may be a cell or tissue specific promoter. If, for example, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, stem, sprout, root and leaf tissues.
  • the promoter for the nucleotide sequence of the present invention can be the ⁇ -Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the ⁇ -Amy 637 promoter) as described in PCT/EP95/02195.
  • the promoter for the nucleotide sequence of the present invention can be the ⁇ -Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the ⁇ -Amy 351 promoter) as described in PCT/EP95/02196.
  • the Amy 351 promoter it is possible to inactivate a part of it so that the partially inactivated promoter expresses the nucleotide sequence in a more specific manner such as in just one specific tissue type or organ.
  • the term "inactivated” means partial inactivation in the sense that the expression pattern of the promoter is modified but wherein the partially inactivated promoter still functions as a promoter.
  • the modified promoter is capable of expressing the nucleotide sequence in at least one (but not all) specific tissue of the original promoter.
  • examples of other partial inactivation of a promoter sequence include altering the folding pattern of the promoter sequence, or binding species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognised by, for example, a specific RNA polymerase.
  • Another, and preferable, way of partially inactivating the Amy 351 promoter is to truncate it to form fragments thereof. Another way would be to mutate at least a part of the sequence so that the RNA polymerase can not bind to that part or another part.
  • Another modification is to mutate the binding sites for regulatory proteins for example the CreA protein known from filamentous fungi to exert carbon catabolite repression, and thus abolish the catabolite repression of the native promoter.
  • the present invention therefore concerns affecting mannose-to-galactose ratios by the use of recombinant DNA techniques.
  • the present invention also concerns affecting mannose-to-galactose ratios of or within a plant - such as by preparing a transgenic plant. Even though the enzyme and the nucleotide sequence of the present invention are not disclosed in EP-B-0470145 and CA- A-2006454, those two documents do provide some useful background commentary on the types of techniques that may be employed to prepare transgenic plants according to the present invention. An adaption of some of these background teachings is now included in the following commentary.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • the present invention relates to a vector system which carries the nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant.
  • the vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system.
  • Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3 , 1-19.
  • One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
  • Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above.
  • the nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appears to be essential for insertion of modified T- DNA into the plant genome.
  • the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct.
  • the vector system is preferably an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
  • the nucleotide sequence or construct of the present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant.
  • An example of a useful microorganism is E. coli, but other microorganisms having the above properties may be used.
  • a vector of a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.
  • the Ti-plasmid harbouring the nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
  • cloning vectors which contain a replication system in E. coli and a marker which allows a selection of the transformed cells.
  • the vectors contain for example pBR 322, pUC series, Ml 3 mp series, pACYC 184 etc.
  • the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector.
  • the contained plasmid is used for the transformation in E.coli.
  • the E.coli cells are cultivated in a suitable nutrient medium and then harvested and lysed.
  • the plasmid is then recovered.
  • sequence analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
  • the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected.
  • T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A-120516; Hoekema. in: The Binary Plant Vector System Offset-drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4: 1-46; and An et al., EMBO J. (1985) 4:277-284.
  • infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
  • a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive.
  • the wound is then inoculated with the Agrobacterium.
  • the inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
  • tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
  • Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
  • transgenic guar plants according to the present invention can be achieved by transformation of guar cotyledonary explants by the method according to Joersbo and Okkels (PCT/DK95/00221) using Agrobacterium tumefaciens LBA4404.
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • E.coli DH5 ⁇ pGEPI42 The deposit number is NCEVIB 40881.
  • NCIMB 40881 contains clone GEPI42 - which comprises SEQ ID No. 1.
  • NCIMB 40882 contains clone GEPI48 - which comprises SEQ ID No. 2.
  • the nucleotide sequence of the present invention is antisense to the UDP-galactose epimerase enzyme coding sequence that is obtainable from deposit number NCIMB 40881 or NCIMB 40882, or is a variant, homologue or fragment thereof. According to a more preferred embodiment the nucleotide sequence of the present invention is antisense to the UDP-galactose epimerase enzyme coding sequence that is obtainable from deposit number NCIMB 40881 or NCIMB 40882.
  • a cDNA expression library representing mRNA from immature guar seeds was constructed in the plasmid pcDNAII (Invitrogen Corporation) and transformed into the E. coli strain ToplOF' .
  • the quality of the cDNA library was controlled by purification of plasmids from a number of separate ToplOF' colonies, picked at random. Restriction enzyme analysis revealed that all examined plasmids were recombinant.
  • the E. coli strain PL-2 is not able to metabolise galactose due to a defective galE- gene while the two other genes of the gal-operon, galK and galT, are intact (Buttin, J Mol Biol, 7, 164-182 (1963); Wu and Kalckar, Proc Nat Acad Sci, USA 55, 622-629 (1966). Thus, insertion of an active UDP-galactose epimerase gene in PL-2 would allow this strain to grow on galactose. Transformation of PL-2 and selection
  • PL-2 cells were made competent by the method of Hanahan (Techniques for transformation of E. coli. IRL Press, Oxford (ISBN 0-947946-18-17), 109-135 (1985). A titer of 5 x 10 6 transformed cells/ ⁇ g library plasmid was obtained.
  • the selection medium was essentially a minimal medium added galactose, consisting of M9 salts (Maniatis et al, Molecular cloning, a laboratory manual, Cold Spring Harbor, New York (ISBN 0-87969-136-0) (1982) and added 0.05 g/1 threonine, 0.05 g/1 leucine, 0.05 g/1 methionine, 1.0 g/1 of thiamin-HCl, 50 mg/1 ampicillin, 0.8 g/1 fructose, 0.9 g/1 agarose and 6 or 8 g/1 galactose.
  • the media are hereafter called M9- ⁇ S6 (containing 6 g/1 galactose) or M9-ES8 (containing 8 g/1 galactose).
  • Competent PL-2 cells were transformed with the guar cDNA library and cells were plated onto the selective substrate M9-ES6 or M9-ES8. After two days at 37°C, colonies appeared (approx 0.1 % of the total number of transformants). A total of 48 colonies were selected.
  • Plasmids from colony 42 and colony 48 were purified and retransformed into competent PL-2 cells and plated on M9-ES6 or M9-ES8. In both retransformation experiments, a large number of colonies appeared after two days at 37°C. About ten independent colonies from each were analysed for UDP-galactose epimerase activity and all extracts contained similar high levels of UDP-galactose activity as found in the original colonies 42 and 48. This experiment demonstrates that the UDP-galactose epimerase activity detected in the PL-2 derived colonies 42 and 48 is encoded by the cDNA inserts.
  • Sequence ID No. 1 and No. 2 show partial nucleotide sequences of the inserts in colonies 42 and 48, respectively, along with the deduced arnino acid sequences.
  • Antisense nucleotide sequences to the above-mentioned clones of interest are prepared and are used to transform guar gum by following the above-mentioned techniques.
  • the present invention is based on the surprising finding that it is possible to increase the mannose-to- galactose ratio of guar gum by the insertion of a nucleotide sequence that is an antisense nucleotide sequence.

Abstract

L'invention porte sur un procédé de modification in vivo, lequel procédé permet d'influer sur le rapport mannose - galactose d'un organisme (ou d'une partie d'organisme) capable de produire un composé contenant un mannose/galactose ou d'un composé de cet organisme contenant un mannose/galactose. Le procédé de modification in vivo consiste à exprimer une séquence nucléotidique influant sur, (a), le rapport mannose - galactose de composants mannose et galactose d'un composé contenant un mannose/galactose et/ou, (b), le rapport mannose - galactose de précurseurs de mannose et de galactose d'un composé contenant un mannose/galactose. La séquence nucléotidique précitée est antisens à l'égard d'au moins une partie du gène pour l'uridine diphosphogalactose épimérase.
PCT/IB1998/000891 1997-05-28 1998-05-27 Modification in vivo de galactomannane de guar par expression d'arn antisens d'uridine diphosphogalactose epimerase WO1998054335A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
KR1019997010932A KR20010012962A (ko) 1997-05-28 1998-05-27 유디피-갈락토스 에피머라제 안티센스 알엔에이의 발현에의한 구아 내의 갈락토만난 생체내 변형
BR9809493-9A BR9809493A (pt) 1997-05-28 1998-05-27 Modificação in vivo de galactomananas em guar por expressão de arn de anti-sentido de udp-galactose epimerase
NZ500951A NZ500951A (en) 1997-05-28 1998-05-27 In vivo modification of galactomannans in guar by expression of udp-galactose epimerase antisense rna
EP98921695A EP0983369A1 (fr) 1997-05-28 1998-05-27 MODIFICATION $i(IN VIVO) DE GALACTOMANNANE DE GUAR PAR EXPRESSION D'ARN ANTISENS D'URIDINE DIPHOSPHOGALACTOSE EPIMERASE
CA002291449A CA2291449A1 (fr) 1997-05-28 1998-05-27 Modification in vivo de galactomannane de guar par expression d'arn antisens d'uridine diphosphogalactose epimerase
JP50042399A JP2002500515A (ja) 1997-05-28 1998-05-27 Ｕｄｐ−ガラクトースエピメラーゼアンチセンスｒｎａの発現によるグアールにおけるガラクトマンナンのインビボ改変
AU74463/98A AU733483B2 (en) 1997-05-28 1998-05-27 In vivo modification of galactomannans in guar by expression of UDP-galactose epimerase antisense RNA
GB9927197A GB2342920B (en) 1997-05-28 1998-05-27 In vivo modification of galactomannans in guar by expression of udp-galactose epimerase antisense rna

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GBGB9710990.4A GB9710990D0 (en) 1997-05-28 1997-05-28 Modification process
GB9710990.4 1997-05-28

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BR (1) BR9809493A (fr)
CA (1) CA2291449A1 (fr)
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WO2000047755A2 (fr) * 1999-02-10 2000-08-17 E.I. Du Pont De Nemours And Company Genes modificateurs d'udp-glucose
AU733483B2 (en) * 1997-05-28 2001-05-17 Dupont Nutrition Biosciences Aps In vivo modification of galactomannans in guar by expression of UDP-galactose epimerase antisense RNA
US6992236B1 (en) 1999-02-10 2006-01-31 E. I. Du Pont De Nemours And Company Plant UDP-glucose epimerases
US7053268B1 (en) 1999-06-17 2006-05-30 Danisco A/S Promoter
US7265265B2 (en) 2002-11-14 2007-09-04 Pioneer Hi-Bred International, Inc. Genes for galactomannan production in plants and methods of use
US7585818B2 (en) 2005-05-10 2009-09-08 Halliburton Energy Services, Inc. Nonnatural galactomannans and methods of use
US7863256B2 (en) 2005-03-02 2011-01-04 Fidia Farmaceutici S.P.A. Amide derivatives of hyaluronic acid in osteoarthrosis
US7884087B1 (en) * 1998-07-06 2011-02-08 Fidia Farmaceutici S.P.A. Amides of hyaluronic acid the derivatives thereof and a process for their preparation
US8080418B2 (en) 2007-03-09 2011-12-20 Corning Incorporated Method of making a three dimensional cell culture matrix

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WO1997020937A2 (fr) * 1995-12-04 1997-06-12 Danisco A/S Procede de modification

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DÖRMANN, P. ET AL.: "Functional expression of Uridine 5'diphospho-glucose 4-epimerase (EC 5.1.3.2) from Arabidopsis thaliana in Saccharomyces cerevisiae and Escherichia coli.", ARCHIEVES IN BIOCHEMISTRY AND BIOPHYSICS, vol. 327, no. 1, 1 March 1996 (1996-03-01), XP002076759 *
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU733483B2 (en) * 1997-05-28 2001-05-17 Dupont Nutrition Biosciences Aps In vivo modification of galactomannans in guar by expression of UDP-galactose epimerase antisense RNA
US7884087B1 (en) * 1998-07-06 2011-02-08 Fidia Farmaceutici S.P.A. Amides of hyaluronic acid the derivatives thereof and a process for their preparation
US8575129B2 (en) 1998-07-06 2013-11-05 Fidia Farmaceutici S.P.A. Amides of hyaluronic acid and the derivatives thereof and a process for their preparation
US7294762B2 (en) 1999-02-10 2007-11-13 E. I. Du Pont De Nemours And Company Plant UDP-galactose epimerases
WO2000047755A2 (fr) * 1999-02-10 2000-08-17 E.I. Du Pont De Nemours And Company Genes modificateurs d'udp-glucose
US7741535B2 (en) 1999-02-10 2010-06-22 E.I. Du Pont De Nemours And Company Plant UDP-galactose epimerases
US6992236B1 (en) 1999-02-10 2006-01-31 E. I. Du Pont De Nemours And Company Plant UDP-glucose epimerases
US7968766B2 (en) 1999-02-10 2011-06-28 E.I. Du Pont De Nemours And Company Plant UDP-galatose epimerases
WO2000047755A3 (fr) * 1999-02-10 2001-03-08 Du Pont Genes modificateurs d'udp-glucose
US7053268B1 (en) 1999-06-17 2006-05-30 Danisco A/S Promoter
US7265265B2 (en) 2002-11-14 2007-09-04 Pioneer Hi-Bred International, Inc. Genes for galactomannan production in plants and methods of use
US7863256B2 (en) 2005-03-02 2011-01-04 Fidia Farmaceutici S.P.A. Amide derivatives of hyaluronic acid in osteoarthrosis
US7585818B2 (en) 2005-05-10 2009-09-08 Halliburton Energy Services, Inc. Nonnatural galactomannans and methods of use
US8080418B2 (en) 2007-03-09 2011-12-20 Corning Incorporated Method of making a three dimensional cell culture matrix

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KR20010012962A (ko) 2001-02-26
GB2342920A (en) 2000-04-26
AU7446398A (en) 1998-12-30
AU733483B2 (en) 2001-05-17
CN1264428A (zh) 2000-08-23
EP0983369A1 (fr) 2000-03-08
GB9710990D0 (en) 1997-07-23
NZ500951A (en) 2001-09-28
GB2342920B (en) 2001-08-15
GB9927197D0 (en) 2000-01-12
JP2002500515A (ja) 2002-01-08
BR9809493A (pt) 2000-06-20
CA2291449A1 (fr) 1998-12-03

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