AU720991B2 - An enzyme - Google Patents

Description

WO 97/33974 PCT/EP97/01117 1 AN ENZYME The present invention relates to an enzyme. The present invention also relates to a nucleotide sequence coding for the enzyme.

Boos and coworkers in 1981 and 1982 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 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 Boos et al in 1981 stated that the activity responsible for maltose and maltodextrin acetylation was unknown. However, in their further studies of 1982 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 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 (A/mol/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 transacetylation 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 isolated or purified enzyme having a(1,4) 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 said variant, homologue or fragment has at least 75% sequence homology to the amino acid sequence shown as SEQ ID No. 1.

According to a second aspect of the present invention there is provided a recombinant enzyme having ca(1,4) 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 said variant, homologue or fragment has at least 75% sequence homology to the amino acid sequence shown in SEQ ID No. 1.

According to a third aspect of the present invention there is provided a recombinant enzyme having a(1,4) 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 ;i enzyme having a(1,4) glucan acetyl-transferase activity, wherein the recombinant I 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 :o 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.

WO 97/33974 PCT/EP97/01117 3 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 a(1,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac. The gene coding for the a(1,4) glucan acetyl-transferase of the present invention is also sometimes referred to as the mac gene.

WO 97/33974 PCT/EP97/01117 4 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 ct(1,4) 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 PCT/lEP97/0117 WO 97/33974 providing the resultant enzyme has 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 a(1,4) 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 a(1,4) 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.

WO 97/33974 PCT/EP97/017 6 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 Shlintron 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 PCT/EP97/01117 WO 97/33974 7 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 expressed 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, plasmids 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 WO 97/33974 PCT/EP97/01117 8 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 Shl-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, Maniatis T. (Editors) Molecular Cloning. A laboratory manual. Second edition. Cold Spring Harbour Laboratory Press. New York 1989.

WO 97/33974 PCT/EP97/01117 9 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 rumefaciens 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 Methodsfor Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.

WO 97/33974 PCT/EP97/01117 Several different 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 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 tne plant 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 Tiplasmid 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.

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.

WO 97/33974 PCT/EP97/01117 11 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 Riplasmid 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 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 part 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.

WO 97/33974 PCT/EP97/01117 12 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 pp 1-15) as these authors present a general overview on transgenic plant construction.

In summation. the present invention relates to an enzyme having a(1,4) glucan acetyltransferase 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 WO 97/33974 PCT/EP97/01117 13 Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom.

AB2 1RY on 7 March 1996: DH5ca-pMAC3 (which contains a 3.2 kb EcoRI-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 A highly preferred aspect of the present invention therefore relates to an enzyme having a(1,4) 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 WO 97/33974 PCT/lEP97/0117 14 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 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 Some details on the Figures are as follows: WO 97/33974 PCT/EP97/01117 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 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 Pstl fragment from pMAC2.

Figure 7 Plasmid name: 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 WO 97/33974 PCT/EP97/01117 16 #B412 (lower primer with HindIII site) CAC AAG CTT ATT TTG CAT AAC AGT TGC using pMAC3 as template.

The 704 bp PCR product was digested with EcoR1 and HindIII and inserted in pUHE21-2 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 TP-mac fusion was control sequenced with primer C028 The 35S terminator-mac 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-SBE TP-mac terminator) from pMAC8 in the EcoRI site of pVictor IV Man.

WO 97/33974 PCT/EP97/01117 17 Figure Plasmid name: Plasmid size: 9.37 kb Comments: Insertion of a 2294 bp EcoR1 fragment (Patatin promoter-SBE TP-mac- 35S terminator) from pMAC8 in the EcoRl site of pVictor IV Man.

Cloning and sequencing of the mac gene from E. coli.

Following, initially the teachings of Boos and Brand the mac gene was isolated from the 4.3 kb EcoRI fragment from X phage 8C4 (151) from the Kohara collection The fragment was inserted into the EcoRI site of plasmid pBluescript II SK in both orientations yielding plasmids pMAC1 and pMAC2 (Figures 4 and When harboured in E. coli these plasmids 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 EcoRI-PstI 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, "S-methionine labelling experiments with E. coli minicells containing pMAC3 showed the synthesis of proteins having molecular weights corresponding to these sizes.

WO 97/33974 PCT/EP97/01117 18 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 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 Cultures of E coli strain NF1830 (MC1000, recAl, F' laclqlZ::tm5, a gift from Niels Fiil, University of Copenhagen) harbouring 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 Ag/ml) and kanamycin (25 g/ml) was grown at 37 0 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% NaCI. 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 pzm filter.

WO 97/33974 PCT/EP97/01117 19 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 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 NaCI 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 NaCI. The enzyme was then eluted with the same buffer containing 2 M NaCI. 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 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 NaCI.

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.

WO 97/33974 PCT/EP97/01117 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 The acetyl-transferase activity of Mac was assayed spectrophotometrically according to a modified Alpers' assay 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 pl of maltose 1M, 100 tl of Acetyl-coA 0.4 mM, 10 /l 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 40 mM dissolved in methanol and 10 1l enzyme. The reaction was started by the addition of enzyme or maltose and was monitored at 412 nm at 25 0

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 x cm' 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 gg of pure protein emulsified vol/vol) with Freund's adjuvant. Antisera were tested against Mac in immunoblots and were found highly specific.

WO 97/33974 PCT/EP97/0117 21 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 70C. 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 0

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.

WO 97/33974 PCT/EP97/01117 22 Table 1. Comparison of the relative activity of Mac towards various monosaccharides as acetyl-acceptors.

Z o ?A 1%) Relative Activity of activity on glucose) Glucose 100 Mannose 38 Fructose 17 Galactose 0.9 Table 2. Comparison of the relative activity of Mac towards various disaccharides as acetyl-acceptors.

Substrate Relative Activity (100 mM) of activity on maltose) Maltose (a-glucose(1,4) a-glucose) 100 Isomaltose (a-glucose(1, 4 a-glucose) 110 Lactose (P-galactose a-glucose) 0.4 Sucrose (a-glucose P-fructose) 0.4 Cellobiose (0-glucose -glucose) 0 WO 97/33974 PCT/EP97/01117 23 Table 3. Comparison of the relative activity of Mac towards various maltooligosaccharides as acetyl-acceptors.

Substrate Relative Activity mM) of activity on maltose) Maltose 100 Maltotriose Maltotetraose Maltopentaose 0.9 Maltohexaose 1.2 Maltoheptaose 1.1 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 iM 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 WO 97/33974 PCT/EP97/01117 24 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.

WO 97/33974 PCT/EP97/01117 Construction of plasmids 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-terminus. 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 plasmids pMAC9 and (Figures 9 and 10, respectively).

WO 97/33974 PCT/EP97/01117 26 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 g sucrose!l and 2 mg 6-benzyl-aminopurine/l. The nodium segments were grown for 7 days at 20 0 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 0 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 MAC10 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.

WO 97/33974 PCT/EP97/01117 27 Immunoblots on potato tuber extracts ml potato protein extract was precipitated with 20% TCA for 30 min on ice.

Protein precipitates were recovered after centrifugation and resuspended in 50 /l of SDS-PAGE sample buffer. 25 pl 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 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, 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 jl potato extract, 10 1 l of 1 mM acetyl-coenzyme A, 25 1l 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 Il. The reaction was started by the addition of acetyl-coenzyme A. After 10 min. reaction at room temperature, /l of freshly made 4 mM DTNB was added and A 4 0 s 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 *i heated from 20 to 100°C at a velocity of 10 0 C per minute. The results showed that the starch from the transformed potatoes had a different enthalpy to the starch from 20 the non-transformed potato. We additionally found a difference in gelatinisation temperature for the transformed potatoes compared to the starch from the nontransformed potatoes.

Other modifications of the present invention will be apparent to those skilled in the art.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

WO 97/33974 PCT/EP97/01117 29

REFERENCES

1. Boos 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.

Gill S. C. von Hippel P. H. 1989. Anal. Biochem. 182, 3 19-326.

6. Alpers D. Appel S. H. Tomkrins G. M. 1965. J1 Bio. Chem. 240, 13.

7. Ogasawara Nakai S. Yoshikawa H. 1994, DNA Research 1, 1-14.

WO 97/33974 WO 9733974PCT/EP97O1117

SEQUENCES

SEQUENCE 10 NO 1 Amino acid sequence MSTEKEKMIAGELYRSADETLSRDRLRARQLIHRYNHSLAEEHTLRQQI

L

ADLFGQVTEAY I EPTFRCDYGYN I FLGNNFFANFDC VMLDVCP I RIGDNC MLAPGVHIYTATHPIDPVARNSGAELGKPVTIGNNVWIGGRAV

INPGVTI

GDNVVVASGAVVTKDVPDNVVVGGNPAR II KKL SEQUENCE PIO. 2 NUCl1ecti de sequence

ATGAGCACAG

ATCGCTCGGC

CGCTCGTCAG

GAAGAGCACA

TCGGTCAGGT

CTGTGACTAT

TTCGCCMACT

TTCGCATCGG

TATCTACACG

AATAGCGGTG

ATAACGTCTG

TGTGACCATT

GTTGTCACkA

GTAATCCAGC

AAAAAGAAAA

AGATGAGACG

CTTATTCACC

CATTACGCCA

GACAGAGGCT

GGCTATAACA

TCGATTGCGT

TGATMACTGT

GCMACATC

CTGMCTGGG

GATTGGCGGA

GGTGATMACG

MSrATGTCCC

CAGAATAATT

GATGATTGCT

TTATCTCGCG

GATACAATCA

GCAAATTCTC

TATATTGAGC

TTTTTCTCGG

GATGCTTGAT

ATGTTGGCAC

CCATCGACCC

GAAACCCGTC

CGCGCGGTCA

TC-GTGGTAGCU

GGACAACGTT

AAAAAATTGT

GGTGAGTTGT

ATCGCCTGCG

TTCCCTGGCG

GCTGATCTAT

CMCGTTTCG

TAATAATTTr

GTCTGCCCTA

CAGGCGTTCA

TGTAGCACGT

ACCATCGGTA

TTAACCCTGG

CTCAGGTGCA

GTCGTGGGCG

AA

WO 97/33974 WO 9733974PCT/EP97O1117 SEQUENCE 1.0. NO. 3 Nucleotide sequence Complete nucleotide sequence of the 3.2 kb ECORI-Pstl fragment in pMAC3.

GMATTCGCCA AAGACTTGAT GCGGTGCGGA TGCGTMTACG ATGCCGCTGG TTATCAGTAC GTAATGGGCG

GGATGGTGAC

GTGGTGGTTC GCCGCCGCTT GATCATCATT GATACAACGT ACCTACGAAC ATTMAGGAGTj TTAACCACGG ATAAMATTCA CTAATAGATT

TAATMATCCA

TTTATTCATT CC.TCGGTTAT

GGATAMAGM

TCCGATCCTG

TGGTGCTGGT

CGC.AACGGTA

TAGCCGCAAG

GTAATCACTA

MTTGAACCA

TAAAAAATAC

TAATCATTA

TACGTCATAT

SGTAAAGGTC

4TGACCTCGC

TCCGGCGCGC

CTGGCAATCT

AATGAAGATA

AGGCCGCGTA

CCAACTCAGG

TC-ATTGTTAG

GAGGCTATTC

TCAGAGCAAT

GAATAGTTCG

TCCTGTCCCG

GAGTGGCTTA

ATCCCGAAAG

CACCCAAAAG

GCCTTGCAAA

ACCTCCAGTT

AGTATAATGA

TGATTGMGC

TGGCGTTTAT

AGAACGCAGT

TCTTCGTTCC

TCGAGCACAG

AGCGGCCTT

ATCTCATACG

TTMTTTATA

TTAATTATT

CCTGGTATTA

AGCAAA-T GC

CTTCCTTOCT

TAGTTMAGCT

GTGTCCGT TA

ACATGATATC

CCTTGMAGAA

GAATGMACTG

AGACAATMAG

TAGTTATGGT

CCGTTGTTTTr TTTCATCTCG CU^ATAATCCT GAAATGTTAT TGCGGGTTAG TGCTAGTATG TTTCGTAATA ATGCGATGAA AGGGCAAGTC CAGGTCAGTA TAAGAAGGGG ACGCGTTATG AGTTCTCTG TGAAACCCTG GCTGGGTAAA. CGACCCAACC TTGCGACCTT CGCACTTMAT

AAAAAGTGAG

PACCTGCA

AGTTTTTTCC

GATGMATACT

TATCATGACT

TrCGGCGATCA

TACMMATTA

AGATCGACGA

AGGATCTTCA

CGMAGAGAA

TCCGAAAAAC

GACACGCTG'G

GGTATTTTAC

CAAGATCCCT

TAACCGGC'IG

CTTTATCACA

CCGCATTTTC

GAGTTGTATC

ATTCACCGAT

GATCTATTCG

TATAACATTT

TGCCCTATTC

ACACATCCCA

ATCGGTAATA

GATAACGTCG

GTGGGCGGTA

GCAAAATTGT

CCCTGGAAAG

CGAAACGATT

TATCTGGAT GACACCTTTA AAATGGCGG MAGTCAGGTA FCCTGCGAGT TTATCTTGTT -TTTAACGAA AACCGATTAT AGCGGTTTAW TCGAGAAAAA VCAGCCGCAG ATCACCGCCOT TCCTCAGTAT1

GGAAATTTAT

TTTATTAAGA ATTTTATACT CAGATGTAAT

GGGAACGTTC

AGCMACCGGA GTCAGTMATG GCTCGGCAGA TGAGACGTTA ACAATCATTC CCTGGCGGAA GTCAGGTGAC AGAGGCTTAT TTCTCGGTMA

TAATTTTTTC

GCATCGGTGA TMACTGTATG TCGACCCTGT AGCACGTMAT ACGTCTGGAT TGGCGGACGC TGGTAGCCTC AGGTGCAGTT ATCCAGCCAG AATMATTAAA GGTAGATCTG TTACTTCCCC TTGCAGATAC

CACGAAGGCA

GAGTCTCTGA ATACCCGCGA

FGTTGTTCAG

kHCGACTATH GACGCTT GAT

CCTCGGCGTT

AGGTACCGGT

GGTATTCTT

CCATACTGTC

TTATGCATA

AAAACCAGTA

TTAAGTAGCG

GCGGTMATTC

GTGTCACCAA

TTTTACCTGC

AATTGTMATT

AACAAACGAG

GTTCAACCGC

GCACAGCTTA

AGCAATCATG

ATTGAGCATA

CTCATTGAGC

ATTAATATGC

GTCAATGCGA

TAGMAGTATG

CCAGACAATT

ATGMACTGGC

AACTGTACGA

TTCGGAGCTA

CCTATGTGAT

TACTGTGTTG

GAY VGCTGGT

TCGTCAGCTT

AATTCTCGCT

TGACTATGGC

*GCTTGATGTC

-CTACACGGCA

ACCCGTCACC

FGACCATTGGT

k CAACGTTGTC 3 CMACTGTTAT

SATAGGGTGTT

C GCCTGCTGAC

CGCTTTAGTAT

120 180 240 300 360 420 480 540 600 660 7 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 %GAATTATTA CMACCATAGG TMATGCGTT TACGTCGTTG TAMATACGAA TTATCAGATA CGCCGAATTG ACCATGMATA TCGCTAATMA ATAATTCGCT TTTTCGCCAT

GAAGACATAC

TCTTCACTGA CTTTTCGTCT AGCACAGPAA AAGAAAAGAT TCTCGCGATC

GCCTGCGCCC

GAGGACACAT TACGCCAGCA ATTGAGCCAA

CGTTCGCTG

GCCAACTTCG ATTGCGTGNI TTGGCACCAG GCGTTCATA1 AGCGGTGCTG AACTGGGGAI GCGGTCATTA ACC'CTGGTG1 GTCACAAAAG ATGTCCCGG~ AAATTGTAAT CGGTTTTC( TCTACTATTC CCACGTTAA' MACGATGACC GAMATACAA( AAAACGCGAC AACAAACCCI WO 97/33974 PCT/EP97/01117 32 CAGTTTTATC CGTAMACATC CGGGGCTGTT TATCGGTATG TACGTTGCTT TTTTTGCCAC 2460 CCTGGCGGTG ATGTTGCAGT CCGAAACGCT GTCAGGCTCT GTCTGGCTAC TGGTTGTATT 2520 ATTTATCCTG CTTAATGGTT TCTTCTTTTT CGATGTCTAC CCACGCTACC GCTATGAAGA 2580 TATCGACGTG CTGGATTTCC GCGTTTGCTA TAACGGCGAA TGGTACAACA CGCGCTTTGT 2640 ACCTGCCGCG CTGGTTGMAG CCATCTTGAA CTCTCCGTGT CGCGGATGTT CATAAGGAAC 2700 AACTGCAAAA AATGATCGTC CGTAAAGGTG AACTGTCWT TTACGATATT TTTACCCTCS 2760 TCGCGCCGAA TCAACATCTT AAGTTAGGGT TACATACCAG GCGTAAAGCT CTGCGCCTGG 2-820 TCAMATGACA ATGATCGTTT CCACCCATCA CTTCATGAAA TACCAGCTCT ACCTCCTTAT 2880 CTCCAGCCAG CCTTTTTCCA CAATCAGATA TACTTTCCCT ACACTGTGTT MTAAGGATA 2940 TGCTGGTGAG AACACGACAT CTGGTCGGCC TTATTCGGG AGTACTGATT CTTTCAGTAT 3000 TGCTGCCTGT CGGCTTAAGC ATCTGGCTGG CCCATCAGCA GGTAGMAACA TCGTTTATTG 3060 AAGAGCTGGA TACCTATTCC TCCCGCGTCG CTATTCGAGC CAATAAGGTG GCGACACMAG 3120 GGAAAGATGC GCTGCAG 3137 SEQUENCE 1.D. NO. 4 Complete nucleotide sequence of the 3.2 kb EcoRI-PstI fragment in pMAC3. The Mac enzyme amino acid sequence is also shown below the mac gene coding sequence.

GMATTCGCCAAAGACTTGATGGATMAGAAGGTAAAGGTCTGATTGMGCGACGCTTGAT GCGGTGCGGATGCGTTTACGTCCGATCCTGATGACCTCGCTGGCGTMATCCTCGGCGTT 120 ATGCCGCTGGTTATCAGTACTGGTGCTGGTTCCGGCGCGCAGAACGCAGTAGGTACCGGT 180 GTAATGGGCGGGATGGGACCGCACGGTACTGGCTCTTCTCGTTCCGGTATCMf 240 GTGGTGGTTCGCCGCCGCTTTAGCCGCMAGAATGAAGATATCGAGCACAGCCATACTGTC 300 GATCATCATTGATACACGTGTATCACTMAGGCCGCGTAGCGGCCTTflATGCATA 360 ACCTACGAACATTMAGGAGTAATTGAACCACCMACTCAGGATCTCATACGMMACCAGTA 420 TTAACCACGGAIMMATTCATAAAAAATACTGATTGTTAGTTAATTTATATTMGTAGCG 480 CTAATAGATTTAATAMTCCATMATCATTTAGAGGCTATTCTTMATTATTTGCGGTMATTC 540 TTTATTCATTCC%'TCGGTTATTACGTCATATTCAGAGCMATCCTGGTATTAGTGTCACCMA 600 TTTCATCTGGCGATAATCTGAAATGTTATGAATAGTTCGAGCAACTGCTTACCTGC 660 TGCGGGTTAGTGCTAGTATGAAAAAGTGAGTCCTGTCCCGCTTCCTTCCTMUTGTATT 720 TTTCGTAATAATGCGATGAACCTGCAAAGAGTGGCTTATAGTTAGTCACG.G 780 AGGGCAAGTCCAGGTCAGTAAGTTTTTTCCATCCCGAAAGGTGTCCGTAGTCACCGC 840 TAAGAAGGuGGACGCGTTATGGATGAATACTCACCCAAAAGACATGATATCGCACAGCTU A 900 AGTTTCTC TGTGAAACCCTGTATCATGACTGCCTTGCAAACCTTGAGAGCAATCATG 960 GCTGGGTAAACGACCCAACCTCGGCGATCAACCTCCAGTTGAATGMACTGATTGAGCATA 1020 TTGCGAC'CTTCGCACTTAATTACAAAATTAAGTATAATGAAGACMATMGCTCATTGAGC 1080 AGATCGACGAATATCTGGATGACACCTTTATGTTGTTCAGTAGTTATGGTATATATGC 1140 AGGATCTrTCAGAMTGGCGGAGTCAGGTAAHCGACTATHCCGTGfGTCATGCGA 1200 CGMAAGAGM.L1TCCTGCGAGTTTATCTTGTTAGMTTATTACAACCATAGGTAGMAGTATG 1260 TCCGAAAAACCTTTMACGAAAACCGATTATTTAATGCGTTTACGTCGTTGCCAGACMUT 1320 GACACGCT'GAGCGGTTTAWTCGAGAAMAATAAATACGMATTATCAGATMATGMACTGGC 1380 GGTATTTTACTCAGCCG'CAGATCACCGCCTCGCCGAATTGACCATGATACTGTACGA 1440 CAAGATCCC" TTCCTCAGTATGGMAATTTATTCGCTATAATATTCGCTTTCGGAGCTA 1500 TAACCGGCTiGTTTATTMAGAATTTTATACTTTTCGCCATGAAGACATACCCTATGTGAT 1560 WO 97/33974 PCTIEP97/O1 117 33 CTTTATCACACAGATGTAATGGGACGTTCTCTTCACTGACTTTCGTC-TTACTGTGTTG 1620 CCGCATTTTCAGCAACCGGAGTCAGTAATGAGCACAGAAAAAGAAGATGATTGCTGGT 1680 M S TEK E K MI A G GAGTTGTATCGCTCGGCAGATGAGACGTTATCTCGCGATCGCCTGCGCGCTCGTCAGCTT 1740 E L YR SAD ET LS RD R LR A ROL ATTCACCGATACAATCATTCCCTGGCGGAAGAGCACACATTACGCCAGCATTCTCGCT 1800 I H RY N HS L AE E HTL R Q Q I L A GATCTATTCGGTCAGGTGACAGAGGCTTATATTGAGCCMACGTTTCGCTIGTGACTATGGC 1860 D L FG QV TEA Y I E P T FR CD Y G TATMACATTTTTCTCGGTAATAATTTTTTCGCCAACTTCGATTGCGTGUATGCTTGATGTC 1920 Y N I F L GN N FF AN F DC V M L DV TGCCCTATTCGCATCGGTGATMACTGTATGTTGGCACCAGGCGTTCATATCTACACGGCA 1980 C P IR 1GDN C ML AP G VH I YT A ACACATCCCATCGACCCTGTAGCACGTAATAGCGGGCTGACTGGGGAACCCGTCACC 2040 T H P I D P V A R N S G A E L G K P V T ATCGG'TAATMCG"TCTGGA TTGGCGGACGGGCGGTCATTMACCCTGGTGTGACCATTGGT 2100 I G N N V W I G G R A V I N P G V T I G GATMCGTCGTGGTAGCCTCAGGTGCAGTTGTCACAAAAGATGTCCCGGACACGTTGTC 2160 D N V V V A S G A V V T K D V P 0 N V V GTGGGCGGTMATCCAGCCAGMATMTTAAAMATTGTMTCGGTTTTTICGCMCTGTTAT 2220 V GG N P AR I I KK L GCAAAATTGTGGTAGATCTGTTACTTCCCCTCTACTATTCCACGTTAwATAGGGTGTT 2280 CCCTGGAAAGTTGCAGATACCACGAAGGCAAACGATGACCGATACACGCCTGCTGAC 2340 CGAAACGATTGAGTCTCTGAATACCCGCGAAAAACGCGACACACCLCCGCTTTAGTAT 2400 CAGTTTTATCCGTAAACATCCGGGGCTGTMATCGGTATGTACGTGCTTfTMGCCAC 2460 CCTGGCGGTGATGTTGCAGTCCGAAACGCTGTCAGGCTCTGTCTGGCTACTGGTTGTATT 2520 ATTTATCCTGCTTAATGGTTTCTTCTTTTTCGATGTCTACCCACGCTACCGCTATGMAGA 2580 TATCGACGTGCTGGATTTCCGCGTTTGCTATACGGCGATGGTACAACACGCGCTTTGT 2640 ACCTGCCGCGCTGGTTGMAGCCATCTTGAACTCTCCGTGTCGCGGATGTTCATMAGGMC 2700 AACTGCAAAAAATGATCGTCCGTAAAGGTGCTGTCTTTnArGATAnTTTACCCTCS 2760 TCGCGCCGMATCMCATCTTMGTTAGGGTTACATACCAGGCGIAAMGCTCTGCGCCTGG 2820 TCAAATGACAATGATCGTTTCCACCCATCACTTCATGAAATACCAGCTCTACCTCCUTAT 2880 CTCCAGCCAGCCiTTTCCACAATCAGATATACTTCCCTACACTGTGTTAATMGGATA 2940 TGCTGGTGAGAACACGACATCTGGTCGGCCTTATTTCGGAGTACTGA-CTMCAGTAT 3000 TGCTGCCTGTCGGCTTAAGCATCTGGCTGGCCCATCAGCAGGTAGAMACATCGTUTATTG 3060 MAGAGCTGGATACCTATTCCTCCCGCGTCGCTATrTCGAGCCMATMGGTGGCGACACMAG 3120 GGAAAGATGCGCTGCAG 33 WO 97/33974 PTE9/11 PCT/EP97/01117 -k or :iin, IcP 576 1CTH I l::c ufflcc INDICATIONS RELATINGC TO A DEPOSITED.N1ICROORGANISNI (PCT Rule l~bis) A. T'%c flu"ii ons ma;:c ccio'v 0rc mic~ourZZAIStn t o inl t1W deci1tiurt B. IDENTIFICATION OF DEPOSIT Further ac.osiLs or.~c -'acitiofi snc= C: NZro Ics~~nstiution T!Ie National Collections of Incustrial and Marine Bacteri-a Limited (,NCtMB) A,±C.rcSs :oinstitution lucc sdngpostai cod a nd counr-v) 7-3 St Machat Drive Abe rceen A82 1RY Unice.- Kingom Caw irs Access Numncc 7 Marcn 1996 NCIM8 48789 C. ADDITIONALE ID ICATI1ONS (leave blank if/mat aaohicabic, This intforrazon is coninued on an additional slieoc in respect of those designations in which a European patent is sought, and any other designated st~ate havino equivalent legislation, a samole of the deposited microorganism will only be made available either until the puolica~ion of the menc--on of ',he grant of the patent or after twenty years from the date or filino if the aoolication has oeen refused or withdrawn or is deemetd to be withdrawn, only ty tne issue of sucr a samole to an expert nominated by tne person reauestinc the, 3aMole. Rule 28(-1) EPC) D. DES(CNATED STATES FOR WHICH INDICATIONS ARE NIADE (if the indicaitom are nor fice ail deziynated Swici, SEPARATE FURNISHINC OF INDICATIONS Ileirv blank if not aplieblael The inicat~ofs Iis~ el ow wtit be Sumte tome intcCfonal Bur=u tatr (wpecq'tegen nrat~ oeofnazzaoan e.g. 'dccWzfl ;Vgjinor O(DepasiC,) :For receiving Office use only 71is Sheet was received with the inemtoflsl ;Miiccion Authont:4 officer Mrs- T. 1r6caer-Tazelaar Form PCT/RO/t34 (July 19M) For (ncetnianal Sum=~ use only 7 This sheet was received by the Irnemnaonal Sureu on: AuW10fizeU officeir WO 97/33974 PCTIEP97/01117 numcer Pt-, 376 1 CTH INDICATIONS RELATING TO A DEPOSITED ICROORGANISMI (PCT Rule I3bts) .1~c ineica::ons rn-at ocvo- r-tto ic.c nicroorzznismtc."erre ta in the dcscripciun onor v~c line 12 '?1 I IDE.N-TFICA.TIONJ OF DEPOSIT Furthc- acoosics am~::c on :Q1iditional sncc: The Naticna1 Collections of Industrial and Marine Bacteria Limited (NCIMBI .A~rsS oi accus;:ary instiution (inciuding por:_i code and~ country) 23 St Macnar Drive Aberdeen A82 IPY United Kincoom UtCCPUSI: Acvmsion Numuer 17 March 1996 NCIMB 40790 C. ADDITIONAL INDICATIONS (leave blank if nat applicable) This intormflucin is continued on an additiunal sheetD In resoec: of those desionations in whiCh a European patent is sougnt, and any othier oesicnated State having equivalent legislation, a sample of the deposited microorganism will only be made available either until the ouolication of the mrertion of the crant of the catent or after twenty years from the (late of filing if the aroi-ication has been refused or withdrawn or is deemec to0 be withdrawn, only ty th e issue of sucn a sample to an expert nominated by the person reouestinq the sanole. 10ule 2B(40) VC) Dl. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE lf:ht indicationj art :ut foraoizt:signctedstoaesj F. S EPA RAT E FUR.NIS HING OF IND ICATIO0NS fleave blank if not applicabl1e) The ini~iet~os -cio- -itl be suotmittedc to uic Lnentational Bureau later tecy'vLhw grmci=U atite inataloene.g. "Aewwn N'umber o eptorwi ror receivig Ofice use only For internauonai Bureu use oniv CKThis she::. was rec.-svea with thle interTiatiofai appliationt This sheet was received by the lotemnational Bureu on: Auttioneed officer Authonzed aficer Mrs. T. Brtcker-TaZeiaar Formt Prt/Ro (July 1992)

Claims (14)

1. An isolated or purified enzyme having a(1,4) 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 said variant, homologue or fragment has at least 75% sequence homology to the amino acid sequence shown as SEQ ID No. 1.

2. A recombinant enzyme having a(1,4) 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 said variant, homologue or fragment has at least 75% sequence homology to the amino acid sequence shown in SEQ ID No. 1.

3. A recombinant enzyme having a(1,4) glucan acetyl-transferase activity, wherein the enzyme has the amino acid sequence shown as SEQ ID No. 1.

4. A recombinant enzyme having a(1,4) glucan acetyl-transferase activity, wherein the recombinant enzyme is immunologically reactive with an antibody raised against a purified recombinant enzyme according to claim 3. *0 S* 5. A recombinant nucleotide sequence coding for the enzyme of any one of claims 1 to 4 or a sequence that is complementary thereto.

6. A recombinant nucleotide sequence according to claim 5, wherein the nucleotide sequence is a DNA sequence. S7. A recombinant 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 recombinant nucleotide sequence having the sequence shown as SEQ ID No. 2. 37

9. A construct comprising or expressing the invention according to any one of claims 1 to 8. A vector comprising, or capable of directing the expression of, the invention of any one of claims 1 to 9.

11. A plasmid comprising, or capable of directing the expression of, the invention of any one of claims 1 to

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 isolated or purified enzyme according to claim 1 substantially as hereinbefore described.

16. A recombinant enzyme according to claim 2 substantially as hereinbefore described. S.17. A transgenic organism according to claim 13 substantially as hereinbefore described.

18. A modified carbohydrate according to claim 14 substantially as hereinbefore described. Xk9AL