WO1997033974A2 - Dna encoding alpha-1(1,4)-glucan acetyl-transferase - Google Patents

Dna encoding alpha-1(1,4)-glucan acetyl-transferase Download PDF

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Publication number
WO1997033974A2
WO1997033974A2 PCT/EP1997/001117 EP9701117W WO9733974A2 WO 1997033974 A2 WO1997033974 A2 WO 1997033974A2 EP 9701117 W EP9701117 W EP 9701117W WO 9733974 A2 WO9733974 A2 WO 9733974A2
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Prior art keywords
enzyme
nucleotide sequence
mac
present
acetyl
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PCT/EP1997/001117
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French (fr)
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WO1997033974A3 (en
Inventor
Florence Dal Degan
Peter Poulsen
Jan Marcussen
Susanne OXENBøLL SøRENSEN
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Danisco A/S
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Application filed by Danisco A/S filed Critical Danisco A/S
Priority to NZ331426A priority Critical patent/NZ331426A/en
Priority to BR9708029-2A priority patent/BR9708029A/en
Priority to PL97328829A priority patent/PL328829A1/en
Priority to EP97908181A priority patent/EP0906413A2/en
Priority to JP9532251A priority patent/JP2000506023A/en
Priority to AU20243/97A priority patent/AU720991B2/en
Publication of WO1997033974A2 publication Critical patent/WO1997033974A2/en
Publication of WO1997033974A3 publication Critical patent/WO1997033974A3/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.)
    • C12N9/1025—Acyltransferases (2.3)
    • 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

Definitions

  • the present invention relates to an enzyme.
  • the present invention also relates to a nucleotide sequence coding for the enzyme.
  • Boos et al (I) observed the formation of acetyl-maltose and acetyl-oligomaltosides after accumulation of maltose or maltooligosides in E. coli. They also observed the formation of acetyl-maltose and acetyl-oligomaltosides in vitro when maltose or maltotriose, acetyl-coenzyme A and a cytosolic E. coli extract were mixed together Boos et al (2).
  • E. coli linkage map In addition, they cloned a 3.4 kb DNA fragment containing the gene in a high copy plasmid. Over-expressed maltose transacetylase was then purified to homogeneity from cell free extracts of an E. coli strain harbouring the above mentioned plasmid. The enzyme was shown to be a homodimer with two identical subunits of 20 kDa. The km (mM) and Vmax ( ⁇ mol/min x mg enzyme) values of this enzyme for the substrates glucose, maltose and acetyl-coenzyme A were
  • an enzyme having ⁇ (l ,4) glucan acetyl-transferase activity wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof.
  • a recombinant enzyme having ⁇ (l ,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof.
  • a recombinant enzyme having ⁇ (l,4) glucan acetyl-transferase activity wherein the enzyme has the amino acid sequence shown as SEQ ID No. 1.
  • a recombinant enzyme having ⁇ (l ,4) glucan acetyl-transferase activity, wherein the recombinant enzyme is immunologically reactive with an antibody raised against a purified recombinant enzyme according to the above-mentioned aspect of the present invention.
  • nucleotide sequence coding for the enzyme of the present invention or a sequence that is complementary thereto.
  • nucleotide sequence comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto.
  • nucleotide sequence or the enzyme of the present invention there is provided a construct comprising or expressing the nucleotide sequence or the enzyme of the present invention.
  • a vector comprising or expressing the construct or the nucleotide sequence or the enzyme according to the present invention.
  • a plasmid comprising or expressing the vector, the construct or the nucleotide sequence or the enzyme according to the present invention.
  • a transgenic organism comprising or expressing the plasmid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention.
  • a modified carbohydrate preferably starch prepared by a method comprising or expressing or using the present invention.
  • the enzyme of the present invention may be obtainable from any one of a bacterium, a fungus, an alga, a yeast, or a plant.
  • the enzyme is obtainable from E. coli.
  • the ⁇ (l,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac.
  • the gene coding for the ⁇ (l,4) glucan acetyl-transferase of the present invention is also sometimes referred to as the mac gene.
  • a seventh aspect of the present invention there is provided a nucleotide sequence having the sequence shown as SEQ ID No. 2.
  • nucleotide sequence or the enzyme of the present invention there is provided a construct comprising or expressing the nucleotide sequence or the enzyme of the present invention.
  • a vector comprising or expressing the construct or the nucleotide sequence or the enzyme according to the present invention.
  • a plasmid comprising or expressing the vector, the construct or the nucleotide sequence or the enzyme according to the present invention.
  • a transgenic organism comprising or expressing the plasmid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention.
  • a modified carbohydrate preferably starch prepared by a method comprising or expressing or using the present invention.
  • the enzyme of the present invention may be obtainable from any one of a bacterium, a fungus, an alga, a yeast, or a plant.
  • the enzyme is obtainable from E.coli.
  • the ⁇ (1 ,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac.
  • the gene coding for the ⁇ (l,4) glucan acetyl-transferase of the present invention is also sometimes referred to as the mac gene.
  • the enzyme comprises the amino acid sequence shown as SEQ ID No 1 , or a variant, homologue or fragment thereof.
  • the enzyme has the amino acid sequence shown as SEQ ID No 1.
  • the enzyme is encoded by a nucleotide sequence comprising the nucleotide sequence shown as SEQ ID No 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto.
  • the enzyme is encoded by the nucleotide sequence shown as SEQ ID No 2.
  • the organism is a plant.
  • the nucleotide sequence is a DNA sequence.
  • the enzyme or nucleotide sequence(s) coding for same may be used in vitro or in vivo in combination with one or more other enzymes or nucleotide sequence(s) coding for same, which enzymes or nucleotide sequence(s) coding for same are preferably prepared by use of recombinant DNA techniques.
  • an in vivo enzymatic modification process can be followed by an in vitro enzymatic modification process.
  • the enzymes used need not necessarily be the same enzymes.
  • variant in relation to the enzyme include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has ⁇ (l,4) glucan acetyl-transferase activity, preferably having at least the same activity of the enzyme shown as SEQ ID No. 1.
  • homologue covers homology with respect to structure and/or function providing the resultant enzyme has ⁇ (l ,4) glucan acetyl-transferase activity.
  • sequence homology preferably there is at least 75 % . more preferably at least 85% , more preferably at least 90% homology to the sequence shown as SEQ ID No. 1. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 1.
  • variant in relation to the nucleotide sequence coding for the 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 codes for an enzyme having ⁇ (l ,4) glucan acetyl-transferase activity, preferably having at least the same activity of the enzyme shown as SEQ ID No. 1.
  • homologue covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for an enzyme having ⁇ (l,4) glucan acetyl-transferase activity.
  • sequence homology preferably there is at least 75 % , more preferably at least 85% , more preferably at least 90% homology to the sequence shown as SEQ ID No. 2. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 2.
  • the above terms are synonymous with allelic variations of the sequences.
  • nucleotide in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention.
  • the nucleotide sequence is not a native nucleotide sequence.
  • native nucleotide sequence means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment.
  • the enzyme of the present invention can be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
  • the enzyme of the present invention may be used in conjunction with other enzymes.
  • the enzyme is not a native enzyme.
  • native enzyme means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.
  • construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes the nucleotide sequence directly or indirectly attached or fused to a promoter.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl- intron or the ADH intron, intermediate the promoter and the nucleotide sequence.
  • the terms do not cover the natural combination of the gene coding for the enzyme ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
  • One highly preferred embodiment of the present invention therefore relates to the nucleotide sequence of the present invention operatively linked to a heterologous promoter.
  • the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant, such as potato, into which it has been transferred.
  • a marker which allows for the selection of the genetic construct in, for example, a plant, such as potato, into which it has been transferred.
  • markers which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
  • vector includes expression vectors and transformation vectors.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • transformation vector means a construct capable of being transferred from one species to another - such as from an E.coli plasmid to an Agrobacterium to a plant.
  • tissue includes tissue and organ, which tissue and organ can be isolated tissue and isolated organ, as well as tissue and organ when within an organism.
  • organ in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be e; ⁇ pressed when present in the organism.
  • the organism is a plant.
  • transgenic organism in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the enzyme according to the present invention and/or the products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism.
  • the nucleotide sequence is incorporated in the genome of the organism.
  • the transgenic organism is a plant.
  • the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present invention, piasmids according to the present invention, cells according to the present invention, tissues according to the present invention, or the products thereof.
  • the transgenic organism can also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a heterologous promoter.
  • the transgenic organism does not comprise the combination of a promoter and the nucleotide sequence coding for the enzyme according to the present invention, wherein both the promoter and the nucleotide sequence are native to that organism 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 5 ⁇ 7-intron or an ADH intron.
  • Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
  • TMV 5' signal sequence see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
  • 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, tuber, sprout, root and leaf tissues.
  • 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.
  • Ti plasmids from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizo genes 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 piasmids 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 appear 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 me 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 piasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these piasmids 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.
  • 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. M13 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.
  • 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.
  • infection of a plant may be done on a certain pan or tissue of the plant, i.e. on a part of a leaf, a tuber, 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.
  • the present invention relates to an enzyme having ⁇ (l,4) glucan acetyl- transferase activity and a nucleotide coding for same.
  • the present invention also provides a modified carbohydrate (preferably starch) obtainable from use of the same.
  • DH5 ⁇ -pM AC3 (which contains a 3.2 kb EcoP ⁇ -Pstl fragment from E. coli comprising the mac gene).
  • the deposit number is NCIMB 40789. This deposit concerns the plasmid pMAC3.
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • NF1830-pMAC5 (which contains the E.coli mac gene).
  • the deposit number is NCIMB 40790.
  • This deposit concerns the plasmid pMAC5.
  • a highly preferred aspect of the present invention therefore relates to an enzyme having ⁇ (l,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof; and wherein the enzyme is expressed by a nucleotide sequence obtainable from either deposit number NCIMB 40789 or deposit number NCIMB 40790.
  • nucleotide sequence comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto, and wherein the nucleotide sequence is obtainable from either deposit number NCIMB 40789 or deposit number NCIMB 40790.
  • the present invention also provides a modified carbohydrate (preferably starch) obtainable from use of this same plasmid.
  • Figure 1 which shows the nucleotide sequence corresponding to SEQ ID No. 2:
  • Figure 2 which shows the amino acid sequence corresponding to SEQ ID No. 1 ;
  • Figure 3 which shows a nucleotide sequence comprising the sequence
  • FIG. 4 which is a plasmid map of pMAC1
  • FIG. 5 which is a plasmid map of pMAC2
  • Figure 6 which is a plasmid map of pMAC3
  • Figure 7 which is a plasmid map of pMAC5 ;
  • FIG. 8 which is a plasmid map of pMAC8
  • Figure 9 which is a plasmid map of pMAC9.
  • Figure 10 which is a plasmid map of pMAClO. Some details on the Figures are as follows: Figure 1
  • the E coli mac gene was amplified with primers:
  • the 704 bp PCR product was digested with EcoRl and HindlU and inserted in pUH ⁇ 21-2 digested with the same restriction enzymes.
  • the PCR product was digested with BamHI and PstI and inserted in pBETP5 digested with the same enzymes.
  • the SBE TV-mac fusion was control sequenced with primer # C028
  • the 35S terminator-m ⁇ c fusion was sequenced with primer # B456 og # C027.
  • the mac gene was isolated from the 4.3 kb EcoRI fragment from ⁇ phage 8C4 (151) from the Kohara collection (4).
  • the fragment was inserted into the EcoRI site of plasmid pBluescript II SK (+) in both orientations yielding piasmids pMAC1 and pMAC2 ( Figures 4 and 5).
  • these piasmids gave rise to highly elevated maltose acetyltransferase levels indicating that the 4.3 kb EcoRI fragment contains the mac gene.
  • the nucleotide sequence of the 3.2 kb EcoRI-PstI insert in pMAC3 was then determined by automated sequencing on an A.L.F. sequencer.
  • the 3137 bp DNA sequence revealed a 372 bp region of the 3' end of the E. coli acrB gene and three open reading frames potentially encoding proteins of 124, 126, and 183 amino acids ( Figure 3).
  • the mac gene was inserted after an isopropylthiogalactosidase (IPTG) inducible phage T7-promoter Al in pUHE21-2 to give pMAC5 ( Figure 7).
  • IPTG isopropylthiogalactosidase
  • pMAC5 Figure 7
  • Cultures of E coli strain NF1830 (MC1000. recAl . F' lacIqlZ: :tm5, a gift from Niels Fiil, University of Copenhagen) harbouring pMAC5 was found to have highly elevated levels of maltose acetyltransferase, when expression of the mac gene is induced by addition of IPTG to the growth medium.
  • a 1 L LB culture of NF1830-pMAC5 supplemented with ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml) was grown at 37°C with vigorous shaking until the A600 reached 0.7.
  • IPTG was added to a final concentration of 2mM and growth was continued for four hours.
  • the cells were harvested by centrifugation (10 min. at 4 000 x g) and washed by resuspension in 200 ml 0.9% NaCl. The cell pellet was then resuspended in 250 ml 20 mM potassium phosphate pH 7.5 containing 0.4 mM PMSF, 0.4 mg/ml pepstatin and 1.6 mM EDTA.
  • the suspension was sonicated 5 x 1 min. using a Vibra Cell VC 600 with a 19 mm High Gain Horn and extender (all from Sonics and Materials Inc. , USA).
  • the homogenate was clarified by centrifugation for 60 min. at 90 000 x g at 4°C and subsequent filtration through a 0.22 ⁇ m filter. Purification of Recombinant Mac
  • the resulting crude extract was applied to a Q-Sepharose 26/10 column (Pharmacia Biotech) equilibrated with 20 mM potassium phosphate pH 7.5 (hereinafter called "buffer A") at a flow rate of 2 ml/min.
  • buffer A 20 mM potassium phosphate pH 7.5
  • the column was washed with 300 ml of buffer A and the bound protein was eluted by applying a 0 to 0.3 M NaCl linear gradient in buffer A (300 ml).
  • the fractions containing enzyme activity were pooled and applied to a 8 ml Affi-Gel Blue (Biorad) column (16 mm x 26 cm) equilibrated with buffer A at a flow rate of 1 ml/min.
  • the column was washed with 50 ml of the same buffer containing 0.4 M NaCl. The enzyme was then eluted with the same buffer containing 2 M NaCl. The active pool was dialysed overnight against buffer A and subsequently concentrated to approximately 3 ml in a Centriprep-30 (Amicon). This fraction was applied to a 6 ml Acetyl-coA-Minileak column equilibrated with buffer A at a flow rate of 0.3 ml/min.
  • This affinity resin was made by coupling 200 mg of Acetyl-coA to 5 g (dry weight) of Minileak High (Kem-En-Tek, Denmark) in 10 ml of 1 M NaCO 3 pH 11 for 20h at room temperature.
  • the column was washed with 20 ml of buffer A. It was then turned upside down and the pure enzyme was eluted in less than 20 ml with buffer A containing 0.5 M NaCl.
  • the purification of the maltose acetyltransferase to homogeneity was achieved after three chromatographic steps. From 11 culture we were able to get 5.8 mg pure Mac. The yield was 29% and the enzyme was purified 80-fold. The purity of the enzyme was assessed both by SDS-PAGE and mass spectrometry. The latter revealed a molecular mass of 19,982 Da.
  • the concentration of pure Mac solutions was estimated spectrophotometrically at 280 nm using an extinction coefficient of 0.66 as determined from the amino acid composition of Mac according to (5).
  • the acetyl-transferase activity of Mac was assayed spectrophotometrically according to a modified Alpers' assay (6).
  • a Perkin Elmer Lambda 18 spectrophotometer was used.
  • the assay mixture of a total volume of 1 ml contained a 50 mM potassium phosphate, 2 mM EDTA buffer at pH 7.5, 100 ⁇ l of maltose 1M, 100 ⁇ l of Acetyl-coA 0.4 mM, 10 ⁇ l 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 40 mM dissolved in methanol and 10 ⁇ l enzyme.
  • the reaction was started by the addition of enzyme or maltose and was monitored at 412 nm at 25°C.
  • One activity unit was defined as the amount of enzyme that produced an increase in absorbance of 1 per minute at 25°C.
  • An extinction coefficient of 13 600 M -1 x cm -1 was used for DTNB in order to calculate the consumption of acetyl coenzyme A.
  • N-terminal sequencing of pure Mac was performed using an Applied Biosystems 476A protein sequencer. One nanomole of protein was desalted by RP-HPLC on a C2 column (4.6/30) prior to loading onto the sequencer. The N-terminal sequence of Mac was determined up to residue was determined up to residue 48 and was in complete concordance with the nucleotide sequence of the mac gene ( Figure 1). Furthermore, the N-terminal methionine residue was not present on the mature protein ( Figure 2).
  • Mac may be a trimer.
  • the isoelectric point of Mac was determined by isoelectric focusing on a PhastGel IEF 4-6.5 (Pharmacia) and was found to be 5.7.
  • the pH profile of Mac was investigated between pH 5 and 8.5 at a 100 mM maltose concentration in 50 mM buffers containing 100 mM NaCl. Under these conditions, the pH optimum was 7.7.
  • Mac The pH stability of Mac was examined at 25 °C between pH 3.0 and 10.0. Mac was instantaneously inactivated at pH 3.0 but was stable between pH 4.0 and 10.0 for at least six hours.
  • thermostability of Mac was investigated at pH 7.5 between 40 and 70°C. After incubation for four hours at 40°C and 50°C, the remaining activity of Mac was 100% and 75%, respectively. Its half-life was 70 min, and 22 min at 60°C and 70°C, respectively.
  • the substrate preference of Mac towards the carbohydrate acetyl-acceptor substrate was investigated by measuring the initial rate of the acetylation of various carbohydrates (used at 50 and 100 mM concentrations) following the procedure described in "Determination of enzyme concentration and activity". The results are presented in Tables 1 , 2 and 3. Among the monosaccharides tested, glucose was the best substrate and among the disaccharides tested, maltose and isomaltose were the best substrates. Table 3. Comparison of the relative activity of Mac towards various maltooligosaccharides as acetyl-acceptors.
  • the SBE-Mac fusion was inserted in the E. coli expression vector pAL781 (Invitrogene, San Diego. USA) in order to over-express the fusion enzyme in E. coli and analyse the activity.
  • pAL781 Invitrogene, San Diego. USA
  • a comparison of the highly over-expressed SBE-Mac fusion and the purified wild type Mac enzyme on SDS gels showed that the fusion migrated slightly slower due to the 27 amino acid extension.
  • the fusion retained the ability to use maltose as a substrate for acetylation.
  • the fusion enzyme appears to be intact and is fully active in E. coli. Therefore, it may be assumed, that the SBE-Mac fusion enzyme will be active in potatoes.
  • the E coli mac gene was amplified with primers: 5'-CGG GAT CCG AGC ACA GAA AAA GAA AAG ATG-3' (upper primer with BamHI site) and 5' -AAC TGC AGA TTT TGC ATA ACA GTT GC-3' (lower primer with PstI site) and pMAC3 as template.
  • the PCR product was digested with BamHI and PstI and inserted in pBETP5 (see PCT patent application No. WO 94/24292, the contents of which are incorporated herein by reference) digested with the same enzymes yielding pMAC8.
  • the mac gene is inserted in an expression cassette that provides tuber specific expression from a patatin promoter and transcription termination at a CaMV 35S terminator.
  • the Mac enzyme is fused to 102 amino acids of the N-terminus of the potato starch branching enzyme including a 75 amino acid transit peptide that directs the mac gene product to the potato tuber amyloplasts.
  • a segment containing the nodium - i.e. a segment taken from 2 mm above and 5 mm below the nodium - was cut from in vitro grown potato plants or mannose selected shoots (for mannose selection see our earlier patent applications WO 93/05163 and/or WO 94/20627).
  • the leaf was removed from the nodium segment, and the segment was placed vertically on agar plates with MS medium (Sigma) supplemented with 60 g sucrose/1 and 2 mg 6-benzyl-aminopurine/l.
  • the nodium segments were grown for 7 days at 20°C with a 16 hour light period and an 8 hour dark period. Subsequently, the plates were wrapped in alu-foil and placed in the dark at 20°C.
  • the minitubers were harvested after about 28 days and applied for western analysis in order to detect Mac expression.
  • Potato minitubers transformed with the pMAC9 or pMAC10 constructs were examined by Western analysis for expression of the E. coli mac gene with antibodies raised towards the E. coli maltose acetyltransferase.
  • the analysis clearly demonstrated that 3 out of 5 MAC9 minitubers and 5 out of 7 MAC 10 minitubers gave a distinct expression of the E. coli maltose acetyltransferase.
  • These results indicate that the 75 amino acid SBE transit peptide, that was originally fused to the 209 amino acid SBE-Mac fusion, has been removed from the SBE-fusion. Furthermore, this implies that the transit peptide was correctly processed by the signal peptidase in the amyloplast membrane, and that the SBE-Mac fusion has been directed to the amyloplast. Immunoblots on potato tuber extracts
  • Potato tubers of comparable sizes were chosen and cut into pieces and homogenised in extraction buffer and Dowex (1 % , w/vol) using a mortar and pestle or an electric blender.
  • 5 ml extraction buffer 50 mM potassium phosphate pH 7.5, 2 mM EDTA, 0.5 mM PMSF
  • the mixture was allowed to stand on ice for 30 min and the insoluble material was removed by centrifugation. Protein concentration was measured using the BCA reagent (Pierce).
  • Mac activity was measured in duplicates or triplicates as follows: 0, 50, 100 or 200 ⁇ l potato extract, 10 ⁇ l of 1 mM acetyl-coenzyme A, 25 ⁇ l of 1 M glucose and assay buffer (50 mM potassium phosphate, 2 mM EDTA, pH 7.5) were mixed per microtiter plate well to give a total volume of 250 ⁇ l. The reaction was started by the addition of acetyl-coenzyme A. After 10 min. reaction at room temperature, 25 ⁇ l of freshly made 4 mM DTNB was added and A 405 was measured immediately. Two wells were prepared for each single assay, one with glucose and one without.

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Abstract

An enzyme is described. The enzyme has α(1,4) glucan acetyl-transferase activity.

Description

AN ENZYME
The present invention relates to an enzyme. The present invention also relates to a nucleotide sequence coding for the enzyme.
Boos and co workers in 1981 and 1982 (1, 2) presented evidence for the existence of an enzyme capable of acetylating maltose via transfer of the acetyl group from Acetyl-coenzyme A to maltose in E. coli. In particular, Boos et al (I) observed the formation of acetyl-maltose and acetyl-oligomaltosides after accumulation of maltose or maltooligosides in E. coli. They also observed the formation of acetyl-maltose and acetyl-oligomaltosides in vitro when maltose or maltotriose, acetyl-coenzyme A and a cytosolic E. coli extract were mixed together Boos et al (2).
Boos et al in 1981 stated that the activity responsible for maltose and maltodextrin acetylation was unknown. However, in their further studies of 1982 (2), Feundlieb and Boos named the unknown enzyme "maltose transacetylase" but then said that the function of maltose transacetylase in E. coli was unclear.
Later Brand and Boos (3) isolated an E. coli mutant lacking the gene encoding maltose transacetylase. This mutant enabled them to map the gene at 10.4 min on the
E. coli linkage map. In addition, they cloned a 3.4 kb DNA fragment containing the gene in a high copy plasmid. Over-expressed maltose transacetylase was then purified to homogeneity from cell free extracts of an E. coli strain harbouring the above mentioned plasmid. The enzyme was shown to be a homodimer with two identical subunits of 20 kDa. The km (mM) and Vmax (μmol/min x mg enzyme) values of this enzyme for the substrates glucose, maltose and acetyl-coenzyme A were
62 and 200, 90 and 110, and 0.018 and 166 respectively. Maltotriose and other oligosaccharides were found to be acetylated with a rate of 2% of the rate determined for glucose. In addition, Brand and Boos presented the following relative iransacetylation rates: glucose 1, maltose 0.55, mannose 0.2, fructose 0.07, galactose
0.04, maltotriose and other malto-oligosaccharides 0.02. Oligosaccharides are saccharides having less than ten sugar units. Despite of these findings Brand and Boos did not sequence the enzyme or the nucleotide sequence coding for the maltose transacetylase enzyme.
According to a first aspect of the present invention there is provided an enzyme having α(l ,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof.
According to a second aspect of the present invention there is provided a recombinant enzyme having α(l ,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof.
According to a third aspect of the present invention there is provided a recombinant enzyme having α(l,4) glucan acetyl-transferase activity, wherein the enzyme has the amino acid sequence shown as SEQ ID No. 1.
According to a fourth aspect of the present invention there is provided a recombinant enzyme having α(l ,4) glucan acetyl-transferase activity, wherein the recombinant enzyme is immunologically reactive with an antibody raised against a purified recombinant enzyme according to the above-mentioned aspect of the present invention.
According to a fifth aspect of the present invention there is provided a nucleotide sequence coding for the enzyme of the present invention or a sequence that is complementary thereto.
According to a sixth aspect of the present invention there is provided a nucleotide sequence comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto. According to a seventh aspect of the present invention there is provided a nucleotide sequence having the sequence shown as SEQ ID No. 2.
According to an eighth aspect of the present invention there is provided a construct comprising or expressing the nucleotide sequence or the enzyme of the present invention.
According to a ninth aspect of the present invention there is provided a vector comprising or expressing the construct or the nucleotide sequence or the enzyme according to the present invention.
According to a tenth aspect of the present invention there is provided a plasmid comprising or expressing the vector, the construct or the nucleotide sequence or the enzyme according to the present invention.
According to an eleventh aspect of the present invention there is provided a transgenic organism comprising or expressing the plasmid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention. According to a twelfth aspect of the present invention there is provided a modified carbohydrate (preferably starch) prepared by a method comprising or expressing or using the present invention.
The enzyme of the present invention may be obtainable from any one of a bacterium, a fungus, an alga, a yeast, or a plant. Preferably, the enzyme is obtainable from E. coli.
The α(l,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac. The gene coding for the α(l,4) glucan acetyl-transferase of the present invention is also sometimes referred to as the mac gene. According to a seventh aspect of the present invention there is provided a nucleotide sequence having the sequence shown as SEQ ID No. 2.
According to an eighth aspect of the present invention there is provided a construct comprising or expressing the nucleotide sequence or the enzyme of the present invention.
According to a ninth aspect of the present invention there is provided a vector comprising or expressing the construct or the nucleotide sequence or the enzyme according to the present invention.
According to a tenth aspect of the present invention there is provided a plasmid comprising or expressing the vector, the construct or the nucleotide sequence or the enzyme according to the present invention.
According to an eleventh aspect of the present invention there is provided a transgenic organism comprising or expressing the plasmid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention. According to a twelfth aspect of the present invention there is provided a modified carbohydrate (preferably starch) prepared by a method comprising or expressing or using the present invention.
The enzyme of the present invention may be obtainable from any one of a bacterium, a fungus, an alga, a yeast, or a plant. Preferably, the enzyme is obtainable from E.coli.
The α(1 ,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac. The gene coding for the α(l,4) glucan acetyl-transferase of the present invention is also sometimes referred to as the mac gene. Preferably, the enzyme comprises the amino acid sequence shown as SEQ ID No 1 , or a variant, homologue or fragment thereof.
Preferably, the enzyme has the amino acid sequence shown as SEQ ID No 1.
Preferably, the enzyme is encoded by a nucleotide sequence comprising the nucleotide sequence shown as SEQ ID No 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto. Preferably, the enzyme is encoded by the nucleotide sequence shown as SEQ ID No 2.
Preferably, the organism is a plant. Preferably, the nucleotide sequence is a DNA sequence.
The enzyme or nucleotide sequence(s) coding for same may be used in vitro or in vivo in combination with one or more other enzymes or nucleotide sequence(s) coding for same, which enzymes or nucleotide sequence(s) coding for same are preferably prepared by use of recombinant DNA techniques.
Thus, according to one aspect of the present invention, an in vivo enzymatic modification process can be followed by an in vitro enzymatic modification process. In these modification steps, the enzymes used need not necessarily be the same enzymes.
The terms "variant", "homologue" or "fragment" in relation to the enzyme include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has α(l,4) glucan acetyl-transferase activity, preferably having at least the same activity of the enzyme shown as SEQ ID No. 1. In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant enzyme has α(l ,4) glucan acetyl-transferase activity. With respect to sequence homology, preferably there is at least 75 % . more preferably at least 85% , more preferably at least 90% homology to the sequence shown as SEQ ID No. 1. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 1.
The terms "variant" , "homologue" or "fragment" in relation to the nucleotide sequence coding for the 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 codes for an enzyme having α(l ,4) glucan acetyl-transferase activity, preferably having at least the same activity of the enzyme shown as SEQ ID No. 1. In particular, the term "homologue" covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for an enzyme having α(l,4) glucan acetyl-transferase activity. With respect to sequence homology, preferably there is at least 75 % , more preferably at least 85% , more preferably at least 90% homology to the sequence shown as SEQ ID No. 2. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 2. The above terms are synonymous with allelic variations of the sequences.
The term "complementary" means that the present invention also covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention. The term "nucleotide" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention.
Preferably the nucleotide sequence is not a native nucleotide sequence. In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. Thus, the enzyme of the present invention can be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
The enzyme of the present invention may be used in conjunction with other enzymes.
Preferably the enzyme is not a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.
The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes the nucleotide sequence directly or indirectly attached or fused to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl- intron or the ADH intron, intermediate the promoter and the nucleotide sequence.
In each case, it is highly preferred that the terms do not cover the natural combination of the gene coding for the enzyme ordinarily associated with the wild type gene promoter and when they are both in their natural environment. One highly preferred embodiment of the present invention therefore relates to the nucleotide sequence of the present invention operatively linked to a heterologous promoter.
The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant, such as potato, into which it has been transferred. Various markers exist which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin. The term "vector" includes expression vectors and transformation vectors.
The term "expression vector" means a construct capable of in vivo or in vitro expression.
The term "transformation vector" means a construct capable of being transferred from one species to another - such as from an E.coli plasmid to an Agrobacterium to a plant.
The term "tissue" includes tissue and organ, which tissue and organ can be isolated tissue and isolated organ, as well as tissue and organ when within an organism. The term "organism" in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be e;κpressed when present in the organism.
Preferably the organism is a plant.
The term "transgenic organism" in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the enzyme according to the present invention and/or the products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism. Preferably the transgenic organism is a plant.
Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present invention, piasmids according to the present invention, cells according to the present invention, tissues according to the present invention, or the products thereof. For example the transgenic organism can also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a heterologous promoter. The transgenic organism does not comprise the combination of a promoter and the nucleotide sequence coding for the enzyme according to the present invention, wherein both the promoter and the nucleotide sequence are native to that organism and are in their natural environment.
The term "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. For example, 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. For example, suitable other sequences include the 5Λ7-intron or an ADH intron. Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' signal sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
Thus, in one aspect, the nucleotide sequence according to the present invention is under the control of a promoter that allows expression of the nucleotide sequence. In this aspect, 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, tuber, sprout, root and leaf tissues.
General teachings of recombinant DNA techniques may be found in Sambrook, J., Fritsch, E.F. , Maniatis T. (Editors) Molecular Cloning. A laboratory manual. Second edition. Cold Spring Harbour Laboratory Press. New York 1989. 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. Some of these background teachings are 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.
Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205- 225) and Christou (Agro-Food-Industry Hi-Tech March/ April 1994 17-27).
Thus, in one aspect, 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 rhizo genes 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. Several different Ti and Ri piasmids 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 appear to be essential for insertion of modified T-DNA into the plant genome. As will be understood from the above explanation, if the organism is a plant, then the vector system of the present invention is preferably one which contains the sequences necessary to infect me 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.
Furthermore, the vector system is preferably an Agrobacterium tumefaciens Ti- plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these piasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these piasmids or derivatives thereof.
In the construction of a transgenic plant 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. When 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. As reported in CA-A-2006454, a large amount of cloning vectors are available 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. M13 mp series, pACYC 184 etc. In this way, 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. As a method of 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.
After each introduction method of the construct or nucleotide sequence according to the present invention in the plants 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. The use of 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. Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds. : D.S. Ingrams and J.P. Helgeson, 203- 208. For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/ April 1994 17-27). With this technique, infection of a plant may be done on a certain pan or tissue of the plant, i.e. on a part of a leaf, a tuber, a root, a stem or another part of the plant. Typically, with direct infection of plant tissues by Agrobacterium carrying the nucleotide sequence of the present invention, 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.
When plant cells are constructed, these cells may be grown and maintained in accordance with well-known 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.
Even further useful teachings on the transformation of plants can be found in Danish patent application No. 940662 (filed 10 June 1994) and/or United Kingdom patent application No. 9702592.8 (filed 7 February 1997).
Reference may even be made to Spngstad et al (1995 Plant Cell Tissue Organ Culture 40 pp 1-15) as these authors present a general overview on transgenic plant construction.
In summation, the present invention relates to an enzyme having α(l,4) glucan acetyl- transferase activity and a nucleotide coding for same. The present invention also provides a modified carbohydrate (preferably starch) obtainable from use of the same.
The following sample was deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen. Scotland, United Kingdom. AB2 1RY on 7 March 1996:
DH5α-pM AC3 (which contains a 3.2 kb EcoPΛ-Pstl fragment from E. coli comprising the mac gene).
The deposit number is NCIMB 40789. This deposit concerns the plasmid pMAC3.
The following sample was deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1RY on 7 March 1996:
NF1830-pMAC5 (which contains the E.coli mac gene).
The deposit number is NCIMB 40790.
This deposit concerns the plasmid pMAC5.
A highly preferred aspect of the present invention therefore relates to an enzyme having α(l,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof; and wherein the enzyme is expressed by a nucleotide sequence obtainable from either deposit number NCIMB 40789 or deposit number NCIMB 40790.
Another highly preferred aspect of the present invention therefore relates to a nucleotide sequence comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto, and wherein the nucleotide sequence is obtainable from either deposit number NCIMB 40789 or deposit number NCIMB 40790.
The present invention also provides a modified carbohydrate (preferably starch) obtainable from use of this same plasmid.
The present invention will now be described only by way of example in which reference is made to the following Figures:
Figure 1 which shows the nucleotide sequence corresponding to SEQ ID No. 2:
Figure 2 which shows the amino acid sequence corresponding to SEQ ID No. 1 ;
Figure 3 which shows a nucleotide sequence comprising the sequence
corresponding to SEQ ID No. 2;
Figure 4 which is a plasmid map of pMAC1;
Figure 5 which is a plasmid map of pMAC2; Figure 6 which is a plasmid map of pMAC3;
Figure 7 which is a plasmid map of pMAC5 ;
Figure 8 which is a plasmid map of pMAC8;
Figure 9 which is a plasmid map of pMAC9; and
Figure 10 which is a plasmid map of pMAClO. Some details on the Figures are as follows: Figure 1
Nucleotide sequence corresponding to Seq ID No 2
Figure 2
Amino acid sequence corresponding to Seq ID No 1
183 amino acids
20073 MW
Figure 4
Plasmid name: pMAC1
Plasmid size: 7.26 kb
Comments: Insertion of a 4.3 kb EcoR1 fragment from lambda 151 into the EcoR1 site of pBluescript II SK +. Figure 5
Plasmid name: pMAC2
Plasmid size: 7.26 kb
Comments: Insertion of a 4.3 kb EcoR1 fragment from lambda 151 (Kohara collection) into the EcoR1 site of pBluescript II SK + .
Figure 6
Plasmid name: pMAC3
Plasmid size: 7.26 kb
Comments: Deletion of the 1.1 kb Pst1 fragment from pMAC2.
Figure 7
Plasmid name: pMAC5
Plasmid size: 4060 bp
Comments:
The E coli mac gene was amplified with primers:
#B411 (upper primer with EcoR1 site)
CGG AAT TCC GCC ATG AAG ACA TAC CC #B412 (lower primer with HindlII site)
CAC AAG CTT ATT TTG CAT AAC AGT TGC
using pMAC3 as template.
The 704 bp PCR product was digested with EcoRl and HindlU and inserted in pUHΕ21-2 digested with the same restriction enzymes.
Figure 8
Plasmid Name: pMAC8
Plasmid size: 4935 bp
Comments: The E coli mac gene was amplified with primers
# B 478 CGG GAT CCG AGC ACA GAA AAA GAA AAG ATG (upper primer with BamHI site) # B 479 AAC TGC AGA TTT TGC ATA ACA GTT GC (lower primer with
PstI site) and pMAC3 as template. The PCR product was digested with BamHI and PstI and inserted in pBETP5 digested with the same enzymes.
The SBE TV-mac fusion was control sequenced with primer # C028
The 35S terminator-mαc fusion was sequenced with primer # B456 og # C027.
Figure 9
Plasmid name: pMAC9
Plasmid size: 9.37 kb
Comments: Insertion of the 2294 bp EcoRI fragment (Patatin promoter-SBΕ TP-mac - 35S terminator) from pMAC8 in the ΕcoRI site of pVictor IV Man. Figure 10
Plasmid name: pMAC10
Plasmid size: 9.37 kb
Comments: Insertion of a 2294 bp EcoR1 fragment (Patatin promoter-SBΕ TP-mac - 35S terminator) from pMAC8 in the EcoR1 site of pVictor IV Man.
Cloning and sequencing of the mac gene from E. coli.
Following, initially the teachings of Boos and Brand (3), the mac gene was isolated from the 4.3 kb EcoRI fragment from λ phage 8C4 (151) from the Kohara collection (4). The fragment was inserted into the EcoRI site of plasmid pBluescript II SK (+) in both orientations yielding piasmids pMAC1 and pMAC2 (Figures 4 and 5). When harboured in E. coli these piasmids gave rise to highly elevated maltose acetyltransferase levels indicating that the 4.3 kb EcoRI fragment contains the mac gene.
In order to localise the mac gene on the 4.3 kb EcoRI fragment, the 1.1 kb PstI fragment was deleted from plasmid pMAC2. This plasmid construction pMAC3 (Figure 6) also gave rise to increased maltose acetyltransferase levels in strains containing this plasmid, thus demonstrating that the mac gene is present on the 3.2 kb EcoRl-Pstl fragment.
The nucleotide sequence of the 3.2 kb EcoRI-PstI insert in pMAC3 was then determined by automated sequencing on an A.L.F. sequencer. The 3137 bp DNA sequence revealed a 372 bp region of the 3' end of the E. coli acrB gene and three open reading frames potentially encoding proteins of 124, 126, and 183 amino acids (Figure 3).
In accordance, 35S-methionine labelling experiments with E. coli minicells containing pMAC3 showed the synthesis of proteins having molecular weights corresponding to these sizes. The 183 codon orf which encodes a protein of a predicted molecular weight of 20073 (Figure 2) is the mac gene, since the E. coli maltose acetyl-transferase has an estimated subunit molecular weight of 20.000 (3). Over-expression of the Mac enzyme in E. coli.
In order to purify the Mac enzyme, the mac gene was inserted after an isopropylthiogalactosidase (IPTG) inducible phage T7-promoter Al in pUHE21-2 to give pMAC5 (Figure 7). Cultures of E coli strain NF1830 (MC1000. recAl . F' lacIqlZ: :tm5, a gift from Niels Fiil, University of Copenhagen) harbouring pMAC5 was found to have highly elevated levels of maltose acetyltransferase, when expression of the mac gene is induced by addition of IPTG to the growth medium.
Growth Conditions
A 1 L LB culture of NF1830-pMAC5 supplemented with ampicillin (100 μg/ml) and kanamycin (25 μg/ml) was grown at 37°C with vigorous shaking until the A600 reached 0.7. IPTG was added to a final concentration of 2mM and growth was continued for four hours. The cells were harvested by centrifugation (10 min. at 4 000 x g) and washed by resuspension in 200 ml 0.9% NaCl. The cell pellet was then resuspended in 250 ml 20 mM potassium phosphate pH 7.5 containing 0.4 mM PMSF, 0.4 mg/ml pepstatin and 1.6 mM EDTA. The suspension was sonicated 5 x 1 min. using a Vibra Cell VC 600 with a 19 mm High Gain Horn and extender (all from Sonics and Materials Inc. , USA). The homogenate was clarified by centrifugation for 60 min. at 90 000 x g at 4°C and subsequent filtration through a 0.22 μm filter. Purification of Recombinant Mac
The resulting crude extract was applied to a Q-Sepharose 26/10 column (Pharmacia Biotech) equilibrated with 20 mM potassium phosphate pH 7.5 (hereinafter called "buffer A") at a flow rate of 2 ml/min. The column was washed with 300 ml of buffer A and the bound protein was eluted by applying a 0 to 0.3 M NaCl linear gradient in buffer A (300 ml). The fractions containing enzyme activity were pooled and applied to a 8 ml Affi-Gel Blue (Biorad) column (16 mm x 26 cm) equilibrated with buffer A at a flow rate of 1 ml/min. The column was washed with 50 ml of the same buffer containing 0.4 M NaCl. The enzyme was then eluted with the same buffer containing 2 M NaCl. The active pool was dialysed overnight against buffer A and subsequently concentrated to approximately 3 ml in a Centriprep-30 (Amicon). This fraction was applied to a 6 ml Acetyl-coA-Minileak column equilibrated with buffer A at a flow rate of 0.3 ml/min. This affinity resin was made by coupling 200 mg of Acetyl-coA to 5 g (dry weight) of Minileak High (Kem-En-Tek, Denmark) in 10 ml of 1 M NaCO3 pH 11 for 20h at room temperature. The column was washed with 20 ml of buffer A. It was then turned upside down and the pure enzyme was eluted in less than 20 ml with buffer A containing 0.5 M NaCl. The purification of the maltose acetyltransferase to homogeneity was achieved after three chromatographic steps. From 11 culture we were able to get 5.8 mg pure Mac. The yield was 29% and the enzyme was purified 80-fold. The purity of the enzyme was assessed both by SDS-PAGE and mass spectrometry. The latter revealed a molecular mass of 19,982 Da.
Determination of enzyme concentration and activity
The concentration of pure Mac solutions was estimated spectrophotometrically at 280 nm using an extinction coefficient of 0.66 as determined from the amino acid composition of Mac according to (5). The acetyl-transferase activity of Mac was assayed spectrophotometrically according to a modified Alpers' assay (6). A Perkin Elmer Lambda 18 spectrophotometer was used. The assay mixture of a total volume of 1 ml contained a 50 mM potassium phosphate, 2 mM EDTA buffer at pH 7.5, 100 μl of maltose 1M, 100 μl of Acetyl-coA 0.4 mM, 10 μl 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 40 mM dissolved in methanol and 10 μl enzyme. The reaction was started by the addition of enzyme or maltose and was monitored at 412 nm at 25°C. One activity unit was defined as the amount of enzyme that produced an increase in absorbance of 1 per minute at 25°C. An extinction coefficient of 13 600 M-1 x cm-1 was used for DTNB in order to calculate the consumption of acetyl coenzyme A.
N-terminal sequencing of Recombinant Mac
N-terminal sequencing of pure Mac was performed using an Applied Biosystems 476A protein sequencer. One nanomole of protein was desalted by RP-HPLC on a C2 column (4.6/30) prior to loading onto the sequencer. The N-terminal sequence of Mac was determined up to residue was determined up to residue 48 and was in complete concordance with the nucleotide sequence of the mac gene (Figure 1). Furthermore, the N-terminal methionine residue was not present on the mature protein (Figure 2).
Production of polyclonal antibodies against Recombinant Mac
Rabbits were immunised subcutaneously at 2-week intervals during 6 weeks and at 4- week intervals thereafter with 90 μg of pure protein emulsified (1 : 1 , vol/vol) with Freund's adjuvant. Antisera were tested against Mac in immunoblots and were found highly specific. Characterisation and activity profile of recombinant Mac
Mass spectrometry studies indicated that Mac may be a trimer. The isoelectric point of Mac was determined by isoelectric focusing on a PhastGel IEF 4-6.5 (Pharmacia) and was found to be 5.7.
The pH profile of Mac was investigated between pH 5 and 8.5 at a 100 mM maltose concentration in 50 mM buffers containing 100 mM NaCl. Under these conditions, the pH optimum was 7.7.
The pH stability of Mac was examined at 25 °C between pH 3.0 and 10.0. Mac was instantaneously inactivated at pH 3.0 but was stable between pH 4.0 and 10.0 for at least six hours.
The thermostability of Mac was investigated at pH 7.5 between 40 and 70°C. After incubation for four hours at 40°C and 50°C, the remaining activity of Mac was 100% and 75%, respectively. Its half-life was 70 min, and 22 min at 60°C and 70°C, respectively.
The substrate preference of Mac towards the carbohydrate acetyl-acceptor substrate was investigated by measuring the initial rate of the acetylation of various carbohydrates (used at 50 and 100 mM concentrations) following the procedure described in "Determination of enzyme concentration and activity". The results are presented in Tables 1 , 2 and 3. Among the monosaccharides tested, glucose was the best substrate and among the disaccharides tested, maltose and isomaltose were the best substrates.
Figure imgf000025_0001
Table 3. Comparison of the relative activity of Mac towards various maltooligosaccharides as acetyl-acceptors.
Figure imgf000026_0001
Kinetic studies
Kinetic studies of Mac catalysed acetylation reactions revealed that the Km for the acceptor substrate is in the mM range whereas it is in the μM range for acetyl-coenzyme A. Thus, Mac has about a 1000 fold more affinity for acetyl-coenzyme A than for the acceptor.
NMR studies
'H-NMR structure determination of the products of acetylation of glucose and maltose by Mac was investigated.
In order to investigate the substrate regio-specificity of Mac regarding the acetylation site of the acceptor substrate, we prepared milligram amounts of acetylated glucose and maltose by incubating 10 mg of glucose or maltose with E. coli Mac and 1 mg acetyl-coenzyme A in phosphate buffer at pH 7.5 for 48 hours. Additional aliquots of 1 mg acetyl-coenzyme A were added during the incubation. The reaction products were separated by thin layer chromatography and the acetylated glucose and maltose were isolated from the chromatogram and freeze dried. The structures of these acetylated sugars were determined by 'H-NMR. Glucose was only acetylated at the C6 position, and maltose was acetylated at the C6 position of its non-reducing glucose moiety. These results reveal that Mac acetylates hexoses at their C6 position.
Activity of the SBE-Mac fusion in E. coli. Because the 27 amino acid SBE portion of the SBE-Mac fusion in pMAC9 and pMACIO described below may interfere with the acetyltransferase activity, the SBE-Mac fusion was inserted in the E. coli expression vector pAL781 (Invitrogene, San Diego. USA) in order to over-express the fusion enzyme in E. coli and analyse the activity. A comparison of the highly over-expressed SBE-Mac fusion and the purified wild type Mac enzyme on SDS gels showed that the fusion migrated slightly slower due to the 27 amino acid extension. Moreover, the fusion retained the ability to use maltose as a substrate for acetylation. Thus, the fusion enzyme appears to be intact and is fully active in E. coli. Therefore, it may be assumed, that the SBE-Mac fusion enzyme will be active in potatoes.
IN VIVO MODIFICATION OF STARCH IN POTATO
General teachings on potato transformation may be found in our copending patent applications PCT/EP96/03053, PCT/EP96/03052 and PCT/EP94/01082 (the contents of each of which are incorporated herein by reference).
For the present studies, the following protocol was adopted. Construction of piasmids for the expression of the E coli mac gene in potato.
The E coli mac gene was amplified with primers: 5'-CGG GAT CCG AGC ACA GAA AAA GAA AAG ATG-3' (upper primer with BamHI site) and 5' -AAC TGC AGA TTT TGC ATA ACA GTT GC-3' (lower primer with PstI site) and pMAC3 as template.
The PCR product was digested with BamHI and PstI and inserted in pBETP5 (see PCT patent application No. WO 94/24292, the contents of which are incorporated herein by reference) digested with the same enzymes yielding pMAC8. Thereby, the mac gene is inserted in an expression cassette that provides tuber specific expression from a patatin promoter and transcription termination at a CaMV 35S terminator. Moreover, the Mac enzyme is fused to 102 amino acids of the N-terminus of the potato starch branching enzyme including a 75 amino acid transit peptide that directs the mac gene product to the potato tuber amyloplasts. Upon import to the amyloplast the 75 amino acid transit peptide is cleaved off, to give a Mac fusionprotein that has the 27 amino acids from the mature starch branching enzyme N-terrninus. The 2294 bp EcoRI expression cassette was isolated from pMAC8 and inserted in the EcoRI site of the plant transformation vector pVictor IV Man (see PCT patent application No. WO 94/24292 and British patent application No. 951443.8, the contents of each of which are incorporated herein by reference) giving piasmids pMAC9 and pMAC10 (Figures 9 and 10, respectively). Preparation of potato minitubers
A segment containing the nodium - i.e. a segment taken from 2 mm above and 5 mm below the nodium - was cut from in vitro grown potato plants or mannose selected shoots (for mannose selection see our earlier patent applications WO 93/05163 and/or WO 94/20627). The leaf was removed from the nodium segment, and the segment was placed vertically on agar plates with MS medium (Sigma) supplemented with 60 g sucrose/1 and 2 mg 6-benzyl-aminopurine/l. The nodium segments were grown for 7 days at 20°C with a 16 hour light period and an 8 hour dark period. Subsequently, the plates were wrapped in alu-foil and placed in the dark at 20°C. The minitubers were harvested after about 28 days and applied for western analysis in order to detect Mac expression.
Expression of the SBE-Mac fusion in potato minitubers
Potato minitubers transformed with the pMAC9 or pMAC10 constructs were examined by Western analysis for expression of the E. coli mac gene with antibodies raised towards the E. coli maltose acetyltransferase. The analysis clearly demonstrated that 3 out of 5 MAC9 minitubers and 5 out of 7 MAC 10 minitubers gave a distinct expression of the E. coli maltose acetyltransferase. The positive minitubers expressed a 209 amino acid SBE-Mac fusion that co-migrates with a similar construction expressed in E. coli. These results indicate that the 75 amino acid SBE transit peptide, that was originally fused to the 209 amino acid SBE-Mac fusion, has been removed from the SBE-fusion. Furthermore, this implies that the transit peptide was correctly processed by the signal peptidase in the amyloplast membrane, and that the SBE-Mac fusion has been directed to the amyloplast. Immunoblots on potato tuber extracts
0.5 ml potato protein extract was precipitated with 20% TCA for 30 min on ice. Protein precipitates were recovered after centrifugation and resuspended in 50 μl of SDS-PAGE sample buffer. 25 μl were subsequently loaded onto 15 % polyacrylamide gels. After electrophoresis proteins were transferred onto Problot PVDF membranes by semi-dry blotting. For immunodetection Mac antiserum was diluted 1 :2 000 and secondary antibody was coupled to alkaline phosphatase. In accordance with the Western Blot analysis of the minitubers described above, the western analysis of the transgenic tubers clearly demonstrated that the 209 a SBE-Mac fusion is expressed in the tubers.
Analysis of potato tubers for Mac activity
Potato tubers of comparable sizes were chosen and cut into pieces and homogenised in extraction buffer and Dowex (1 % , w/vol) using a mortar and pestle or an electric blender. 5 ml extraction buffer (50 mM potassium phosphate pH 7.5, 2 mM EDTA, 0.5 mM PMSF) was used per gram potato. The mixture was allowed to stand on ice for 30 min and the insoluble material was removed by centrifugation. Protein concentration was measured using the BCA reagent (Pierce).
Mac activity was measured in duplicates or triplicates as follows: 0, 50, 100 or 200 μl potato extract, 10 μl of 1 mM acetyl-coenzyme A, 25 μl of 1 M glucose and assay buffer (50 mM potassium phosphate, 2 mM EDTA, pH 7.5) were mixed per microtiter plate well to give a total volume of 250 μl. The reaction was started by the addition of acetyl-coenzyme A. After 10 min. reaction at room temperature, 25 μl of freshly made 4 mM DTNB was added and A405 was measured immediately. Two wells were prepared for each single assay, one with glucose and one without. Activity was calculated by subtracting the absorbance of the well without glucose (background absorbance) from that of the well with glucose. Relatively high levels of Mac activity could be measured in eight out of nine transgenic tubers. Some of the tubers had a Mac activity that was 15 to 20 fold above the almost negligible activity found in non-transformed tubers. Viscometric studies
Samples of starch obtained from tubers of non-transformed potatoes and from transformed potatoes according to the present invention were analysed by viscoamylograph of an aqueous suspension using a Newport Scientific Rapid Visco Analyser 3C. The results showed that the starch from the transformed potatoes had a different viscometric profile to the starch from the non-transformed potato.
DSC studies Samples of starch obtained from tubers of non-transformed potatoes and from transformed potatoes according to the present invention were analysed by differential scanning colometry (using a 10% w/w aqueous starch suspension). The samples were heated from 20 to 100°C at a velocity of 10°C per minute. The results showed that the starch from the transformed potatoes had a different enthalpy to the starch from the non- transformed potato. We additionally found a difference in gelatinisation temperature for the transformed potatoes compared to the starch from the non- transformed potatoes.
Other modifications of the present invention will be apparent to those skilled in the art. REFERENCES
1. Boos W. , Ferenci T. & Shuman H. A. 1981. J. Bacteriol. 146, 725-732. 2. Freundlieb S. & Boos W. 1982. Ann. Microbiol. (Inst. Pasteur) 133 A, 181- 189.
3. Brand B. & Boos W. 1991. J. Biol. Chem. 266, 14113-14118. 4. Kohara et al. 1987. Cell 50: July 31 issue.
5. Gill S. C. & von Hippel P. H. 1989. Anal. Biochem. 182, 319-326.
6. Alpers D. H. , Appel S. H. & Tomkrins G. M. 1965. J. Biol. Chem. 240, 10- 13.
7. Ogasawara N. , Nakai S. & Yoshikawa H. 1994, DNA Research 1 , 1-14.
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001

Claims

1. An enzyme having α(l ,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1 , or a variant, homologue or fragment thereof.
2. A recombinant enzyme having α(l ,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1 , or a variant, homologue or fragment thereof.
3. A recombinant enzyme having α(l ,4) glucan acetyl-transferase activity, wherein the enzyme has the amino acid sequence shown as SEQ ID No. 1.
4. A recombinant enzyme having α.(l ,4) glucan acetyl-transferase activity, wherein the recombinant enzyme is immunologically reactive with an antibody raised against a purified recombinant enzyme according to claim 3.
5. A nucleotide sequence coding for the enzyme of any one of claims 1 to 4 or a sequence that is complementary thereto.
6. A nucleotide sequence according to claim 5. wherein the nucleotide sequence is a DNA sequence.
7. A nucleotide sequence comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto.
8. A nucleotide sequence having the sequence shown as SEQ ID No. 2.
9. A construct comprising or expressing the invention according to any one of claims 1 to 8.
10. A vector comprising or expressing the invention of any one of claims 1 to 9.
11. A plasmid comprising or expressing the invention of any one of claims 1 to 10.
12. A transgenic organism comprising or expressing the invention according to any one of claims 1 to 11.
13. A transgenic organism according to claim 12, wherein the transgenic organism is a plant.
14. A modified carbohydrate (preferably starch) prepared by a method comprising or expressing or using the invention according to any one of claims 1 to 13.
15. An enzyme substantially as described herein.
PCT/EP1997/001117 1996-03-13 1997-03-07 Dna encoding alpha-1(1,4)-glucan acetyl-transferase WO1997033974A2 (en)

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PL97328829A PL328829A1 (en) 1996-03-13 1997-03-07 Enzyme
EP97908181A EP0906413A2 (en) 1996-03-13 1997-03-07 Dna encoding alpha-(1,4)-glucan acetyl-transferase
JP9532251A JP2000506023A (en) 1996-03-13 1997-03-07 DNA encoding α (1,4) glucan acetyl-transferase
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US6495740B1 (en) 1996-06-27 2002-12-17 The Austrailian National University Manipulation of cellulose and/or β-1,4-Glucan

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CN114686547B (en) * 2020-12-30 2024-05-14 中国医学科学院药物研究所 Method for enzymatic synthesis of acetyl-CoA by diacerein donor

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DE4425688A1 (en) * 1994-07-14 1996-01-18 A U F Analytik Umwelttechnik F High acetylated starch prodn. for biodegradable film etc. mfr.

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DE4425688A1 (en) * 1994-07-14 1996-01-18 A U F Analytik Umwelttechnik F High acetylated starch prodn. for biodegradable film etc. mfr.

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Title
BRAND B. AND BOOS W.: "Maltose transacetylase of Escherichia coli" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 21, 25 July 1991, pages 14113-14118, XP000676505 cited in the application *
ROBERTS D. ET AL.: "Sequence of minutes 4-25 of E. coli, AC U82664" EMBL DATABASE, 19 January 1997, HEIDELBERG, XP002036098 *

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US6495740B1 (en) 1996-06-27 2002-12-17 The Austrailian National University Manipulation of cellulose and/or β-1,4-Glucan
US7154026B2 (en) 1996-06-27 2006-12-26 The Australian National University Manipulation of cellulose and/or β-1,4,-glucan

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