CA2248540A1 - Dna encoding alpha-1(1,4)-glucan acetyl-transferase - Google Patents
Dna encoding alpha-1(1,4)-glucan acetyl-transferase Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- 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
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Abstract
An enzyme is described. The enzyme has .alpha.(1,4) glucan acetyl-transferase activity.
Description
CA 02248~40 1998-09-10 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 (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 (1) observed the formation of acetyl-m~ltose and acetyl-oligomaltosides after accum~ tion of maltose 10 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), Fel-n-ilieb and Boos named the umcnown 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 enro~inv 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 fragmem cont~ining the gene in a high copy plasmid. Over-expressed maltose transacetylase was then purified to homogeneity from cell free extracts of an ~. coli strain harbouring the above mentioned plasmid. The enzyme was shown to be a homodimer with two i-lentic~l 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 62 and 200, 90 and 110, and 0.018 and 166 respectively. Maltotriose and other oligos~cch~rides were found to be acetylated with a rate of 2% of the rate detel.llil~ed for glucose. In addition, Brand and Boos presented the following relative transacetylation rates: glucose 1, maltose 0.55, m,.nnose 0.2, fructose 0.07, galactose 0.04, maltotriose and other malto-oligosaccharides 0.02. Oligosaccharides are saccharides having less than ten sugar units.
CA 02248~40 l998-09-lO
W O 97~3974 PCTAEP97/01117 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 enzyme5 having ~(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.
According to a second aspect of the present invention there is provided a recombinant 10 enzyme having ~x(1,4) glucan acetyl-transferase activity, wherein the enzymecomprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologueor fragment thereof.
According to a third aspect of the present invention there is provided a recombinant 15 enzyme having CY(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 enzyme having ~(1,4) glucan acetyl-transferase activity, wherein the recombinant20 enzyme is imml-nologically reactive with an antibody raised against a purified recombinant enzyme according ~o the above-mentioned aspect of the present invention.
According to a fifth aspect of the present invention there is provided a nucleotide 25 sequence coding for the enzyme of the present invention or a sequence that is complementary there~o.
According to a sixth aspect of the present invention there is provided a nucleotide sequence Cc).~ illg the sequence shown as SEQ ID No. 2, or a variant, homologue 30 or fragment thereof or a sequence that is complem~nr~ry thereto.
CA 02248~40 1998-09-10 Wo 97/33974 PCT/EP97/01117 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 S 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 e~r~s~ing the construct or the nucleotide sequence or the enzyme 10 according to the present invention.
According to a tenth aspect of the present invention there is provided a plasmidcomprising or e~pressillg 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 e~ essilg the plasmid, the vector, the construct or the nucleotide seque~re or enzyme according to the present invention.
20 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, 25 a fungus, an alga, a yeast, or a plant. Preferably, the enzyme is obtainable from E. coli.
~r The cY(1,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac. The gene coding for the ~(1,4) glucan acetyl-lldhsreldse of the present 30 invention is also somptimps referred to as the mac gene.
CA 02248~40 1998-09-10 WO 97/33974 PCT/EPg7/01117 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 5 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 10 according to the present invention.
According to a tenth aspect of the present invention there is provided a plasmidcomprising or e~pressing 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 transgerlic organism comprising or e~lcssillg the plasmid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention.
20 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, 25 a fungus, an alga, a yeast, or a plant. Preferably, the enzyme is obtainable from E. coli.
The c~(1,4) glucan acetyl-transferase of the present invention is som~tim~s referred to as Mac. The gene coding for the o~(1,4) glucan acetyl-transferase of the present 30 invention is also sometimes referred to as the mac gene.
CA 02248~40 1998-09-10 WO 97/33974 PCT/EPg7/01117 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.
S
Preferablv. 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 ~hat is complementary thereto.
10 Preferably. the enzyme is encoded by the nucleotide sequence shown as SEQ ID No 2.
Preferably, the organism is a plant.
15 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 preferably20 prepared bv 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 n~cess~rily be the same 25 enzymes.
The terms "variant", "homologue" or "fragment" in relation to the enzyme includeany substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the seq~le~re providing the res~lt~nr 30 amino acid sequence has cY(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 CA 02248=740 l998-09-lO
S
providing the resultant enzyme has ~(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 IDNo. 1. More preferably there is at least 95%, more preferably at least 98%, S 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 10 the sequence providing the resultant nucleotide sequence codes for an enzyme having ol(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 c~ ) glucan acetyl-transferase activity. With 15 respect to sequence homology, preferably there is at least 75%, more preferably at least 85%. more preferably at least 90% ~omology to the sequence shown as SEQ IDNo. 2. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 2.
20 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.
25 The term "nucleotide" in relation to the present invention includes genomic DNA, cDNA, synthe~ic 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 30 regard, the term "native nucleotide se4uence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire l.,ullloter with which it is narurally associated, which promoter is also in its native environrnent.
CA 02248~40 1998-09-10 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 comrol of the promoter wilh which it is naturally associated within that organism.
~ S The enzyme of the present invention may be used in conjunction with other enzymes.
Preferably the enzyme is not a native enzyme. In this re~ard. the term "native enzyme" means an entire enzyme that is in its native environment and when it hasbeen expressed by its native nucleotide sequence.
The term "construct" - which is synonymous with terrns such as "conjuga~e", "cassette" and "hybrid" - includes the nucleotide sec~uence 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-15 intron or the ADH intron, interrnecii~te the promoter and the nucleotide sequence.
In each case, it is highly preferred that the terms do not cover the natural combinationof 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 20 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 25 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 resict~nre - e.g. resistance to G418. hygromycin, bleomycin, kanamycin and gentamycin.
30 The term "vector" includes expression vectors and transformation vectors.
The term "expression vector" means a construct capable of ~n vivo or ~n vZtro W O 97/33974 PCTAEr97/01117 expression.
The term "transforrnalion vector" means a construct capable of being transferred from one species to another - such as from an E.coli plasmid to an Agrobacterium to a5 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.
10 The lerrn "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 theorganism.
Preferably the organism is a plant.
The term "transgenic organism" in relation to the present invention includes anyorganism that comprises the nucleotide sequence coding for the enzyme according to 20 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.
25 Preferably the transgenic organism is a plant.
Therefore, the tran~genir organism of the present invention includes an organismcomprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present 30 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 , . ,, ,, ~
CA 02248~40 l998-09-lO
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 promoler and the nucleotide sequence coding for the enzyme according to the present invention, wherein both the promoter and the S nucleotide sequence are native to that organism and are in their natural environment.
The term "promoter" is used in the norrnal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Mond theory of gene expression.
10 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 m~inr~in, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention. For example, suitable other15 sequences include the Shl-intron or an ADH intron. Other sequences include inducible elements - such as temperature, ch~rnic~l, light or stress inducible elements.
Also, suitable elements to enhance transcription or translation may be present. An example of the latter elemem is the TMV S' signal sequence (see Sleat Gene 217 20 [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 25 example, the or~anism is a plant then the promoter can be one that affec~s expression of the nucleotide sequence in any one or more of seed, stem, tuber, sprout, root and leaf tissues.
General te~chingc of recombinant DNA techniques may be found in Sambrook,J., 30 Fritsch, E.F., Maniatis T. (Editors) Molecular Cloning. A laboratory manual. Second edition. Cold Spring Harbour Laboratory Press. New York 1989.
CA 02248~40 1998-09-10 W O 97133974 PCT~EP97/01117 Even thou~h 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 cornrnentary on the types of techniques thal 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 modi~1ed 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 inforrnation and introduction of the genetic information by use of a vector system. A review of the general techniques may befound in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech MarchlApril 1994 17-Z7).
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 iscapable 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 reterred to as a binary vector system. 13inary 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 Agrobaaerium rhizogenes An et al.(1986), PlantPhysiol. 81, 301-305andButcherD.N. etal. (1980), TissueCulture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
.. .. ..
CA 02248~40 1998-09-10 wo 97/33974 PCT/EPg7/01117 Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell construc~s described above.
The nucleotide sequence or construct of the present invention should preferably be 5 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.
10 As will be understood from the above explanation, if the organism is a plant, then the vector svstem of the present invention is preferably one which contains the sequences n~ceSsary to infect tne 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 rhi~ogenes 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 20 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 2S of a useful microorganism is E. coli, but other microorganicm~ 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 n~cessa1y, into a suitable Agrobaclerium strain, e.g. Agrobacterium tumefaciens. The Ti-plasmid harbouring the nucleotidesequence or construct of the invention is thus preferably transferred into a suitable 30 Agrobacterium strain. e.g. A. tumefaciens, so as to obtain an Agrobaclerium cell harbouring the nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
. ~ . . ~ . .
CA 02248~40 1998-09-lO
As reported in CA-A-~006454, 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 transforrned cells. The vectors contain for example pBR 3". 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 arecultivated in a suitable nutrient medium and then harvested and Iysed. The plasmid is then recovered. As a method of analysis there is generally used sequence analysis, restriction analvsis, electrophoresis and further biochemical-molecular biological 10 methods. After each manipulation, the used DNA sequence can be restricted andconnected with the ne~ct DNA sequence. Each sequence can be cloned in the same or different plasmid.
After each introduction method of the ccnstruct or nucleotide sequence according to 15 the present invention in the plants the presence and/or insertion of further DNA
sequences may be n~cess~ry. 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 conn~cted. The use of T-DNA for the transformation of 20 plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binarv Plant Vector System Offset-drukXerij Kamers B.B., Alblasserdam, 1985, Chapter ~/; Fraley. et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO
J. (1985) 4:277-284.
25 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/April30 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.
.
CA 02248~40 1998-09-10 Wo 9~1~3974 PCT/EPg7/01117 Typically, with direct infection of plant tissues by Agrobacten~cm carr,ving thenucleotide sequence of the present invention, a plant to be infected is wounded, e.g.
by cuttina the plant with a razor or puncturing the plant with a needle or mbbing the plant with an abrasive. The wound is then inoculated with the Agrobaclenum. The S inoc~ tecl 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 m:linr~in~ in accordance with well-known tissue culturing methods such as by culturing the cells 10 in a suitable culture medium supplied with the nPcess~ry 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 15 cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium cont~ining the approp-iate nutrients, plant hormones, etc.
Even further useful teachings on the transforrnation of plants can be found in Danish 20 patent application No. 940662 (filed 10 June 1994) and/or United Kinodom 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 25 construction.
In sl~rnm~tion. the present invention relates to an enzyme having ~(1,4) 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 therecognised depositary The National Collections of Industrial and Marine Bacteria .
CA 02248~40 1998-09-lO
~.imitec1 (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom.
AB2 lRY on 7 March 1996:
5 DH5~-pMAC3 (which contains a 3.2 kb ~coRI-Pstl fragment from E. coli comprising the mac gene).
The deposit number is NCIMB 40789.
10 This deposit concerns the plasmid pMAC3.
The following sample was deposi~ed in accordance with the Budapest Treaty at therecognised depositary The National Collections of Industrial and Marine Bacteriar imit~ (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 lRY on 7 March 1996:
NF1830-pMAC5 (which contains the E.coli mac gene).
The deposil number is NCIMB 40790.
This deposit concerns the plasmid pMAC5.
A highly preferred aspect of the present invention therefore relates to an enzyme having ~(1,4) glucan acetyl-~lan~r.lase activity, wherein the enzyme comprises the 25 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 obtainablefrom either deposit number NCIMB 40789 or deposit number NCIMB 40790.
Another highly pleft~ d aspec~ of the present invention therefore relates to a 30 nucleotide seq~en~e comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complement~t-v thereto, and wherein the nucleotide sequence is obtainable from either deposit number NCIMB
CA 02248~40 1998-09-lO
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 followin~ 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 Ihe sequence corresponding ~o SEQ ID No. 2;
Figure 4 which is a plasmid map of pMAC1;
Figure 5 which is a plasmid map of pMAC2;
20 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 pMAC10.
30 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 Figure 4 Plasmid name: pMACl Plasmid size: 7.26 kb Commems: Insertion of a 4.3 kb EcoRl fragment from lambda 151 into the EcoR1 site of pBluescript II SK +.
15 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: pMAC5 Plasmid size: 4060 bp Comments:
30 The E coli mac gene was amplified with primers:
#B411 (upperprimerwithEcoR1 site) CGG AAT TCC GCC ATG AAG ACA TAC CC
... .
#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 HindlII and inserted in 5 pUHE21-2 digested with the same restriction enzymes.
Figure 8 Plasmid Name: pMAC8 Plasmid size: 4935 bp 10 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) 15 # 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 prirner # B456 og # C027.
Figure 9 25 Plasmid name: pMAC9 Plasmid size: 9.37 kb Co~ llen~: Insertion of the 2294 bp EcoRI fragment (Patatin promoter-SBE TP-mac -35S terminator) from pMAC8 in the EcoRI site of pVictor IV Man.
CA 02248~40 1998-09-10 W O 97/33974 PCT~EP97/01117 Figure 10 Plasmid name: pMAC10 Plasmid size: 9.37 kb Comments: Insertion of a 2294 bp EcoR1 fragment (Patatin promoter-SBE TP-mac-5 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 ~ 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 plasmids pMAC1 and pMAC2 (Figures 4 and 5). When harboured in E. co~i these plasmids gave rise to highly elevated maltose acetyltransferase levels indicating that the 4.3 kb EcoRI fragment contains the mac 15 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 20 cont~ining this plasmid, thus demonstrating that the mac gene is present on the 3.2 kb EcoRI-PstI fr~gm~slt The nucleotide sequence of the 3.2 kb EcoRI-PstI insert in pMAC3 was then deterrnined by automated sequencing on an A.L.F. sequencer. The 3137 bp DNA
25 sequence revealed a 372 bp region of the 3' end of the E. coli acrB gene and three open reading frames potentially encoding ~Ol~iJlS of 124, 126, and 183 amino acids (Figure 3).
In accordance, 35S-methionine labelling experiments with ~. coli minicells Cont~ining 30 pMAC3 showed the synthesis of proteins having molecular weights corresponding to these sizes.
CA 02248540 1998-09-lO
W O 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 (3).
Over-expression of the ~Iac ~"~ e in E. coli.
In order to purify the Mac enzyme, the mac gene was inserted after an isopropylthiogalactosidase (IPTG) inducible phage T7-promoter A1 in pUHE21-2 to give pMACS (Figure 7). Cultures of E coli strain NF1830 (MC1000. recA1. F' lacIqlZ::tm5, a gift from Niels Fiil, University of Copenhagen) harbouring pMAC5was found to have highly elevated levels of maltose acetyltransferase, when expression of the mac gene is in~ ced by addition of IPTG to the growth medium.
Growth Conditions 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 sh~kinP 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 p~I 7.5 cont~ininP 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.
CA 02248~40 1998-09-10 W O 97/33974 PCT~EP97/01117 Purification of Recombinant Mac The resulting crude extract was applied to a Q-Sepharose 26/10 column (pharmaciaBiotech) equilibrated with 20 mM potassium phosphate pH 7.5 (hereinafter called 5 "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 applyin~ a 0 to 0.3 M NaCl linear gradient in buffer A (300 ml). The fractions cont~ininP enzyme activity were pooled and applied to a 8 ml Affi-Gel Blue (Biorad) column (16 mm x ~6 cm) e~uilibratedwith buffer A at a flow rate of 1 ml/min. The column was washed with 50 ml of the 10 same buffer cont~ining 0.4 M NaCl. The enzyme was then eluted with the same buffer conr~ining 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 withbuffer A at a flow rate of 0.3 ml/min. This affinity resin was made by coupling 200 15 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 te~ ldture. 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 cont~ining 0.5 M NaCI.
20 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 a~sessed both by SDS-PAGE and mass spectrometry. The latter revealed a molecular mass of 19,982 Da.
CA 02248~40 l998-09-lO
WO 97/339?4 PCT~EP97tO1117 Determination of enzyme conce"t.~Lion and activity The concentration of pure Mac solutions was estimated spectrophotometrically at 280 nm using an extinction coefficient of 0.66 as deterrnined 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 rnl contained a 50 mM potassium phosphate, 2 mM EDTA buffer at pH 7.5, 100 ~1 of maltose lM, 100 ~1 of Acetyl-coA 0.4 mM, 10 ,ul 5,5'-dithiobis(2-nitrobenzoic 10 acid) (DTNB) 40 mM dissolved in methanol and 10 ,ul enzyme. The reaction was started b,v the addition of enzyme or maltose and was monitored at 412 nrn 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 ' x cm-' was used for DTNB in order to calculate the consumption of acetyl coenzyme A.
lS
~-terminal seqll~ncin~ of Recombinant Mac N-terminal se~uencing of pure Mac was performed using an Applied Biosystems 476A protein sequencer. One nanomole of protein was desalted by RP-HPLC on a 20 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 r,ucleotide 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 imn~--ni.~e~ subcutaneously at 2-week intervals during 6 weeks and at 4-week intervals thereafter with 90 ~g of pure protein emulsified (1:1, vol/vol) with 30 Freund s adjuvant. Antisera were tested against Mac in immunoblots and were found highly specific.
., . , .. ... ~ ... .. ... ...
CA 02248~40 1998-09-10 W O 97/33974 PCT~EP97/01117 Characterisation and activity profile of recombinant Mac Mass speclrometry studies indicated that Mac may be a trimer.
5 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 cont~ining 100 mM NaCl. Under these conditions, 10 the pH optimum was 7.7.
The pH stability of Mac was e~rninP(i at 25~C between pH 3.0 and 10Ø 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 investi~te~l at pH 7.5 between 40 and 70~C. Afterincubation for four hours at 40~C and 50~C, the r~m~ining 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 25 presented in Tables 1, 2 and 3. Among the monosaccharides tested, glucose was the best substrate and among the ~ic~ch~rides tested, maltose and isomaltose were the best substrates.
W O 97/33974 PCT~EP97/01117 Table 1. Comparison of the relative activity of Mac towards various monosaccharides as acetyl-acceptors.
\~ Relative Activity C ~ ( % 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 (c~-glucose(1,4) ~-glucose) 100 Isomaltose (~x-glucose(1,4) a!-glucose) 110 Lactose (~-galactose ~-(1,6) cY-glucose) 0.4 Sucrose (cx-glucose c~-(1,4) ,B-fructose) 0.4 Cellobiose (~-glucose ,B-(1,4) ~-glucose) 0 CA 02248~40 l998-09-lO
W O 97~3974 PCT~EP97/01117 Table 3. Comparison of the relative activity of Mac lowards various maltooligosaccharides as acetyl-acceptors.
Substrate Relative Activily (50 mM) (% of activity on maltose) Maltose 100 Maltotriose 7 . 5 Maltotetraose 0.5 Maltopentaose 0. 9 Maltohexaose i.2 Maltohepeaose 1. 1 Kinetic studies Kinetic studies of Mac catalysed acetylation reactions revealed that the Km for the acceptor substrate is in Ihe mM range whereas it is in the ~M range for acetyl-coenzyme A. Thus, Mac has about a 1000 fold more affiniey for acetyl-coenzyme A than for the acceptor.
NMR s~eudies IH-NMR struceure determination of the products of acetylation of glucose and maltose by Mac was in-~esrig~tt~.
In order to investigate the substrate regio-specificity of Mac regarding the acetylation site of the accepeor substrate, we prepared milligram amounts of aceeylated glucose and maltose by in~lbating 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 CA 02248~40 1998-09-lO
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 5 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.
10 Because the 2/ amino acid SBE portion of the SBE-Mac fusion in pMAC9 and pMAC10 described below may inte,relc with the acetyltransferase activity, the SBE-Mac f~sion was inserted in the ~. coli exp~ession vector pAL781 (Invitrogene, San Die_o. USA) in order to over-express the fusion enzyme in E. coli and analyse the activit~. A comparison of the highly over-expressed SBE-Mac fusion and the 15 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 enzvme will be active in potatoes.
lN 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 25 of each of which are incorporated herein bv reference).
For the present studies, the following protocol was adopted.
CA 02248~40 1998-09-10 W O 97133974 PCT~EP97/01117 Construction of Dlasmids for the expression of the E coli mac ~ene in potato.
The E coli mac gene was amplified with primers:
S S'-CGG GAT CCG AGC ACA GAA AAA GAA AAG ATG-3' (upper primer with BamHI site) and 10 S'-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 BamH~ and PstI and inserted in pBETPS (see 15PCT patent application No. WO 94/24292, the contents of which are incorporatedherein by reference) digested with the same enzymes yielding pMAC8. Thereby, themac gene is inserted in an expression cassette that provides tuber specific expression from a patatin promoter and transcription terrnination at a CaMV 35S termin~tor.Moreover, the Mac enzyme is fused to 102 amino acids of the N-terminus of the 20 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 ~:coRI site 25 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 ofwhich are incorporated herein by reference) giving plasmids pMAC9 and pMAC10 (Figures 9 and 10, respectively).
CA 02248540 1998-09-lO
Preparation of potato minitllhers 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 selectedshoots (for mannose selection see our earlier patent applications WO 93/05163 and/or WO 94/20627). The leaf was removed from the nodium se~ment~ and the se_ment was placed vertically on agar plates with MS medium (Sigma) supplemented with 60g sucrosell and 2 mg 6-benzyl-aminopurine/1. 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.
E~pression of the SBE-Mac fusion in potato minitllhers Potato minit~ ers transformed with the pMAC9 or pMAC10 constructs were ex~min,~ by Western analysis for expression of the E. coli mac gene with antibodies raised towards the ~. coli maltose acetyltransferase. The analysis clearly demonstrated that ~ out of 5 MAC9 minitubers and 5 out of 7 MACI0 minitubers gave a distinct expression of the E. coli maltose acetyltransferase. The positive mininlkers expressed a 209 amino acid SBE-Mac fusion that co-migrates with a similar construction expressed in E. coli. These results in~ic~te that the 75 amino acid SBE transit peptide, that was originally fused to the 209 amino acid SBE-Mac fusion, has been removed from ~he SBE-fusion. ~urthermore. this implies that thetransit peptide was conectly processed by the signal peptidase in the amyloplastmembrane, and that the SBE-Mac fusion has been directed to the amyloplast.
. . ..
CA 02248~40 1998-09-10 W 0 97~3974 PCT~EP97/01117 T~ -.loblots on potato tuber e~tracts 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 ~l of SDS-PAGE sample buffer. 25 ,~1 were subsequently loaded onto 15 %
polyacrylamide gels. After electrophoresis proteins were transferred onto Problot PVDF membranes by semi-dry blotting. For irnmunodetection ~lac antiserum was diluted 1:' 000 and secondary antibody was coupled to ~ Iin~o phosphatase.
10 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 20 for 30 min and the insoluble material was removed by centrifugalion. Protein conceMration was measured using the BCA reagent (Pierce).
Mac activity was measured in duplicates or triplicates as follows: 0, 50, 100 or 200 ~I potato extract, 10 ,ul of 1 mM acetyl-coenzyme A, 25 ~l of 1 M glucose and assay 25 buffer (50 mM potassium phosphate, 2 mM EDTA, pH 7 . 5) were mixed per microtiter plate well to give a total volume of 250 ,ul. The reaction was started by the addition of acetyl-coenzyme A. After 10 min. reaction at room temperature, 25 ~L~l of freshly made 4 mM DTNB was added and A405 was measured irnmediately.
Two wells were prepared for each single assay, one with glucose and one without.30 Activity was calc~ t~cl by subtracting the absorbance of the well without glucose (background absorbance) from that of the well with glucose.
CA 02248~40 1998-09-10 Relatively high levels of Mac activity could be measured in eight out of nine transgenic tubers. Some of ~he tubers had a Mac activity that was 15 to 20 fold above the almost negligible activity found in non-transformed tubers.
5 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 ~apid Visco 10 Analyser 3C. The results showed that the starch from the transformed potatoes had a differen~ viscometric profile to the starch from the non-transformed potato.
DSC studies lS Samples of starch obtained from tubers of non-transformed potatoes and from transforrned potatoes according to the present invention were analysed bv dirrerellLial sc~nning colometry (using a 10% w/w aqueous starch suspension). The samples wereheated from 20 to 100~C at a velocity of 10~C per minute. The results showed that the starch from the transforrned potatoes had a different enthalpy to the starch from 20 the non-transforrned potato. We additionally found a difference in gel~tini.~rion temperarure 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 25 art.
CA 02248540 lsss-os-lo W O 97/33974 PCT~EP97101117 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.
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 (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 (1) observed the formation of acetyl-m~ltose and acetyl-oligomaltosides after accum~ tion of maltose 10 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), Fel-n-ilieb and Boos named the umcnown 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 enro~inv 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 fragmem cont~ining the gene in a high copy plasmid. Over-expressed maltose transacetylase was then purified to homogeneity from cell free extracts of an ~. coli strain harbouring the above mentioned plasmid. The enzyme was shown to be a homodimer with two i-lentic~l 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 62 and 200, 90 and 110, and 0.018 and 166 respectively. Maltotriose and other oligos~cch~rides were found to be acetylated with a rate of 2% of the rate detel.llil~ed for glucose. In addition, Brand and Boos presented the following relative transacetylation rates: glucose 1, maltose 0.55, m,.nnose 0.2, fructose 0.07, galactose 0.04, maltotriose and other malto-oligosaccharides 0.02. Oligosaccharides are saccharides having less than ten sugar units.
CA 02248~40 l998-09-lO
W O 97~3974 PCTAEP97/01117 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 enzyme5 having ~(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.
According to a second aspect of the present invention there is provided a recombinant 10 enzyme having ~x(1,4) glucan acetyl-transferase activity, wherein the enzymecomprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologueor fragment thereof.
According to a third aspect of the present invention there is provided a recombinant 15 enzyme having CY(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 enzyme having ~(1,4) glucan acetyl-transferase activity, wherein the recombinant20 enzyme is imml-nologically reactive with an antibody raised against a purified recombinant enzyme according ~o the above-mentioned aspect of the present invention.
According to a fifth aspect of the present invention there is provided a nucleotide 25 sequence coding for the enzyme of the present invention or a sequence that is complementary there~o.
According to a sixth aspect of the present invention there is provided a nucleotide sequence Cc).~ illg the sequence shown as SEQ ID No. 2, or a variant, homologue 30 or fragment thereof or a sequence that is complem~nr~ry thereto.
CA 02248~40 1998-09-10 Wo 97/33974 PCT/EP97/01117 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 S 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 e~r~s~ing the construct or the nucleotide sequence or the enzyme 10 according to the present invention.
According to a tenth aspect of the present invention there is provided a plasmidcomprising or e~pressillg 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 e~ essilg the plasmid, the vector, the construct or the nucleotide seque~re or enzyme according to the present invention.
20 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, 25 a fungus, an alga, a yeast, or a plant. Preferably, the enzyme is obtainable from E. coli.
~r The cY(1,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac. The gene coding for the ~(1,4) glucan acetyl-lldhsreldse of the present 30 invention is also somptimps referred to as the mac gene.
CA 02248~40 1998-09-10 WO 97/33974 PCT/EPg7/01117 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 5 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 10 according to the present invention.
According to a tenth aspect of the present invention there is provided a plasmidcomprising or e~pressing 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 transgerlic organism comprising or e~lcssillg the plasmid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention.
20 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, 25 a fungus, an alga, a yeast, or a plant. Preferably, the enzyme is obtainable from E. coli.
The c~(1,4) glucan acetyl-transferase of the present invention is som~tim~s referred to as Mac. The gene coding for the o~(1,4) glucan acetyl-transferase of the present 30 invention is also sometimes referred to as the mac gene.
CA 02248~40 1998-09-10 WO 97/33974 PCT/EPg7/01117 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.
S
Preferablv. 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 ~hat is complementary thereto.
10 Preferably. the enzyme is encoded by the nucleotide sequence shown as SEQ ID No 2.
Preferably, the organism is a plant.
15 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 preferably20 prepared bv 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 n~cess~rily be the same 25 enzymes.
The terms "variant", "homologue" or "fragment" in relation to the enzyme includeany substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the seq~le~re providing the res~lt~nr 30 amino acid sequence has cY(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 CA 02248=740 l998-09-lO
S
providing the resultant enzyme has ~(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 IDNo. 1. More preferably there is at least 95%, more preferably at least 98%, S 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 10 the sequence providing the resultant nucleotide sequence codes for an enzyme having ol(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 c~ ) glucan acetyl-transferase activity. With 15 respect to sequence homology, preferably there is at least 75%, more preferably at least 85%. more preferably at least 90% ~omology to the sequence shown as SEQ IDNo. 2. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 2.
20 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.
25 The term "nucleotide" in relation to the present invention includes genomic DNA, cDNA, synthe~ic 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 30 regard, the term "native nucleotide se4uence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire l.,ullloter with which it is narurally associated, which promoter is also in its native environrnent.
CA 02248~40 1998-09-10 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 comrol of the promoter wilh which it is naturally associated within that organism.
~ S The enzyme of the present invention may be used in conjunction with other enzymes.
Preferably the enzyme is not a native enzyme. In this re~ard. the term "native enzyme" means an entire enzyme that is in its native environment and when it hasbeen expressed by its native nucleotide sequence.
The term "construct" - which is synonymous with terrns such as "conjuga~e", "cassette" and "hybrid" - includes the nucleotide sec~uence 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-15 intron or the ADH intron, interrnecii~te the promoter and the nucleotide sequence.
In each case, it is highly preferred that the terms do not cover the natural combinationof 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 20 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 25 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 resict~nre - e.g. resistance to G418. hygromycin, bleomycin, kanamycin and gentamycin.
30 The term "vector" includes expression vectors and transformation vectors.
The term "expression vector" means a construct capable of ~n vivo or ~n vZtro W O 97/33974 PCTAEr97/01117 expression.
The term "transforrnalion vector" means a construct capable of being transferred from one species to another - such as from an E.coli plasmid to an Agrobacterium to a5 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.
10 The lerrn "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 theorganism.
Preferably the organism is a plant.
The term "transgenic organism" in relation to the present invention includes anyorganism that comprises the nucleotide sequence coding for the enzyme according to 20 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.
25 Preferably the transgenic organism is a plant.
Therefore, the tran~genir organism of the present invention includes an organismcomprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present 30 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 , . ,, ,, ~
CA 02248~40 l998-09-lO
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 promoler and the nucleotide sequence coding for the enzyme according to the present invention, wherein both the promoter and the S nucleotide sequence are native to that organism and are in their natural environment.
The term "promoter" is used in the norrnal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Mond theory of gene expression.
10 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 m~inr~in, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention. For example, suitable other15 sequences include the Shl-intron or an ADH intron. Other sequences include inducible elements - such as temperature, ch~rnic~l, light or stress inducible elements.
Also, suitable elements to enhance transcription or translation may be present. An example of the latter elemem is the TMV S' signal sequence (see Sleat Gene 217 20 [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 25 example, the or~anism is a plant then the promoter can be one that affec~s expression of the nucleotide sequence in any one or more of seed, stem, tuber, sprout, root and leaf tissues.
General te~chingc of recombinant DNA techniques may be found in Sambrook,J., 30 Fritsch, E.F., Maniatis T. (Editors) Molecular Cloning. A laboratory manual. Second edition. Cold Spring Harbour Laboratory Press. New York 1989.
CA 02248~40 1998-09-10 W O 97133974 PCT~EP97/01117 Even thou~h 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 cornrnentary on the types of techniques thal 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 modi~1ed 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 inforrnation and introduction of the genetic information by use of a vector system. A review of the general techniques may befound in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech MarchlApril 1994 17-Z7).
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 iscapable 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 reterred to as a binary vector system. 13inary 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 Agrobaaerium rhizogenes An et al.(1986), PlantPhysiol. 81, 301-305andButcherD.N. etal. (1980), TissueCulture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
.. .. ..
CA 02248~40 1998-09-10 wo 97/33974 PCT/EPg7/01117 Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell construc~s described above.
The nucleotide sequence or construct of the present invention should preferably be 5 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.
10 As will be understood from the above explanation, if the organism is a plant, then the vector svstem of the present invention is preferably one which contains the sequences n~ceSsary to infect tne 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 rhi~ogenes 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 20 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 2S of a useful microorganism is E. coli, but other microorganicm~ 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 n~cessa1y, into a suitable Agrobaclerium strain, e.g. Agrobacterium tumefaciens. The Ti-plasmid harbouring the nucleotidesequence or construct of the invention is thus preferably transferred into a suitable 30 Agrobacterium strain. e.g. A. tumefaciens, so as to obtain an Agrobaclerium cell harbouring the nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
. ~ . . ~ . .
CA 02248~40 1998-09-lO
As reported in CA-A-~006454, 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 transforrned cells. The vectors contain for example pBR 3". 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 arecultivated in a suitable nutrient medium and then harvested and Iysed. The plasmid is then recovered. As a method of analysis there is generally used sequence analysis, restriction analvsis, electrophoresis and further biochemical-molecular biological 10 methods. After each manipulation, the used DNA sequence can be restricted andconnected with the ne~ct DNA sequence. Each sequence can be cloned in the same or different plasmid.
After each introduction method of the ccnstruct or nucleotide sequence according to 15 the present invention in the plants the presence and/or insertion of further DNA
sequences may be n~cess~ry. 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 conn~cted. The use of T-DNA for the transformation of 20 plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binarv Plant Vector System Offset-drukXerij Kamers B.B., Alblasserdam, 1985, Chapter ~/; Fraley. et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO
J. (1985) 4:277-284.
25 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/April30 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.
.
CA 02248~40 1998-09-10 Wo 9~1~3974 PCT/EPg7/01117 Typically, with direct infection of plant tissues by Agrobacten~cm carr,ving thenucleotide sequence of the present invention, a plant to be infected is wounded, e.g.
by cuttina the plant with a razor or puncturing the plant with a needle or mbbing the plant with an abrasive. The wound is then inoculated with the Agrobaclenum. The S inoc~ tecl 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 m:linr~in~ in accordance with well-known tissue culturing methods such as by culturing the cells 10 in a suitable culture medium supplied with the nPcess~ry 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 15 cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium cont~ining the approp-iate nutrients, plant hormones, etc.
Even further useful teachings on the transforrnation of plants can be found in Danish 20 patent application No. 940662 (filed 10 June 1994) and/or United Kinodom 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 25 construction.
In sl~rnm~tion. the present invention relates to an enzyme having ~(1,4) 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 therecognised depositary The National Collections of Industrial and Marine Bacteria .
CA 02248~40 1998-09-lO
~.imitec1 (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom.
AB2 lRY on 7 March 1996:
5 DH5~-pMAC3 (which contains a 3.2 kb ~coRI-Pstl fragment from E. coli comprising the mac gene).
The deposit number is NCIMB 40789.
10 This deposit concerns the plasmid pMAC3.
The following sample was deposi~ed in accordance with the Budapest Treaty at therecognised depositary The National Collections of Industrial and Marine Bacteriar imit~ (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 lRY on 7 March 1996:
NF1830-pMAC5 (which contains the E.coli mac gene).
The deposil number is NCIMB 40790.
This deposit concerns the plasmid pMAC5.
A highly preferred aspect of the present invention therefore relates to an enzyme having ~(1,4) glucan acetyl-~lan~r.lase activity, wherein the enzyme comprises the 25 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 obtainablefrom either deposit number NCIMB 40789 or deposit number NCIMB 40790.
Another highly pleft~ d aspec~ of the present invention therefore relates to a 30 nucleotide seq~en~e comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complement~t-v thereto, and wherein the nucleotide sequence is obtainable from either deposit number NCIMB
CA 02248~40 1998-09-lO
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 followin~ 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 Ihe sequence corresponding ~o SEQ ID No. 2;
Figure 4 which is a plasmid map of pMAC1;
Figure 5 which is a plasmid map of pMAC2;
20 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 pMAC10.
30 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 Figure 4 Plasmid name: pMACl Plasmid size: 7.26 kb Commems: Insertion of a 4.3 kb EcoRl fragment from lambda 151 into the EcoR1 site of pBluescript II SK +.
15 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: pMAC5 Plasmid size: 4060 bp Comments:
30 The E coli mac gene was amplified with primers:
#B411 (upperprimerwithEcoR1 site) CGG AAT TCC GCC ATG AAG ACA TAC CC
... .
#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 HindlII and inserted in 5 pUHE21-2 digested with the same restriction enzymes.
Figure 8 Plasmid Name: pMAC8 Plasmid size: 4935 bp 10 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) 15 # 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 prirner # B456 og # C027.
Figure 9 25 Plasmid name: pMAC9 Plasmid size: 9.37 kb Co~ llen~: Insertion of the 2294 bp EcoRI fragment (Patatin promoter-SBE TP-mac -35S terminator) from pMAC8 in the EcoRI site of pVictor IV Man.
CA 02248~40 1998-09-10 W O 97/33974 PCT~EP97/01117 Figure 10 Plasmid name: pMAC10 Plasmid size: 9.37 kb Comments: Insertion of a 2294 bp EcoR1 fragment (Patatin promoter-SBE TP-mac-5 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 ~ 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 plasmids pMAC1 and pMAC2 (Figures 4 and 5). When harboured in E. co~i these plasmids gave rise to highly elevated maltose acetyltransferase levels indicating that the 4.3 kb EcoRI fragment contains the mac 15 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 20 cont~ining this plasmid, thus demonstrating that the mac gene is present on the 3.2 kb EcoRI-PstI fr~gm~slt The nucleotide sequence of the 3.2 kb EcoRI-PstI insert in pMAC3 was then deterrnined by automated sequencing on an A.L.F. sequencer. The 3137 bp DNA
25 sequence revealed a 372 bp region of the 3' end of the E. coli acrB gene and three open reading frames potentially encoding ~Ol~iJlS of 124, 126, and 183 amino acids (Figure 3).
In accordance, 35S-methionine labelling experiments with ~. coli minicells Cont~ining 30 pMAC3 showed the synthesis of proteins having molecular weights corresponding to these sizes.
CA 02248540 1998-09-lO
W O 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 (3).
Over-expression of the ~Iac ~"~ e in E. coli.
In order to purify the Mac enzyme, the mac gene was inserted after an isopropylthiogalactosidase (IPTG) inducible phage T7-promoter A1 in pUHE21-2 to give pMACS (Figure 7). Cultures of E coli strain NF1830 (MC1000. recA1. F' lacIqlZ::tm5, a gift from Niels Fiil, University of Copenhagen) harbouring pMAC5was found to have highly elevated levels of maltose acetyltransferase, when expression of the mac gene is in~ ced by addition of IPTG to the growth medium.
Growth Conditions 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 sh~kinP 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 p~I 7.5 cont~ininP 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.
CA 02248~40 1998-09-10 W O 97/33974 PCT~EP97/01117 Purification of Recombinant Mac The resulting crude extract was applied to a Q-Sepharose 26/10 column (pharmaciaBiotech) equilibrated with 20 mM potassium phosphate pH 7.5 (hereinafter called 5 "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 applyin~ a 0 to 0.3 M NaCl linear gradient in buffer A (300 ml). The fractions cont~ininP enzyme activity were pooled and applied to a 8 ml Affi-Gel Blue (Biorad) column (16 mm x ~6 cm) e~uilibratedwith buffer A at a flow rate of 1 ml/min. The column was washed with 50 ml of the 10 same buffer cont~ining 0.4 M NaCl. The enzyme was then eluted with the same buffer conr~ining 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 withbuffer A at a flow rate of 0.3 ml/min. This affinity resin was made by coupling 200 15 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 te~ ldture. 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 cont~ining 0.5 M NaCI.
20 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 a~sessed both by SDS-PAGE and mass spectrometry. The latter revealed a molecular mass of 19,982 Da.
CA 02248~40 l998-09-lO
WO 97/339?4 PCT~EP97tO1117 Determination of enzyme conce"t.~Lion and activity The concentration of pure Mac solutions was estimated spectrophotometrically at 280 nm using an extinction coefficient of 0.66 as deterrnined 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 rnl contained a 50 mM potassium phosphate, 2 mM EDTA buffer at pH 7.5, 100 ~1 of maltose lM, 100 ~1 of Acetyl-coA 0.4 mM, 10 ,ul 5,5'-dithiobis(2-nitrobenzoic 10 acid) (DTNB) 40 mM dissolved in methanol and 10 ,ul enzyme. The reaction was started b,v the addition of enzyme or maltose and was monitored at 412 nrn 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 ' x cm-' was used for DTNB in order to calculate the consumption of acetyl coenzyme A.
lS
~-terminal seqll~ncin~ of Recombinant Mac N-terminal se~uencing of pure Mac was performed using an Applied Biosystems 476A protein sequencer. One nanomole of protein was desalted by RP-HPLC on a 20 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 r,ucleotide 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 imn~--ni.~e~ subcutaneously at 2-week intervals during 6 weeks and at 4-week intervals thereafter with 90 ~g of pure protein emulsified (1:1, vol/vol) with 30 Freund s adjuvant. Antisera were tested against Mac in immunoblots and were found highly specific.
., . , .. ... ~ ... .. ... ...
CA 02248~40 1998-09-10 W O 97/33974 PCT~EP97/01117 Characterisation and activity profile of recombinant Mac Mass speclrometry studies indicated that Mac may be a trimer.
5 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 cont~ining 100 mM NaCl. Under these conditions, 10 the pH optimum was 7.7.
The pH stability of Mac was e~rninP(i at 25~C between pH 3.0 and 10Ø 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 investi~te~l at pH 7.5 between 40 and 70~C. Afterincubation for four hours at 40~C and 50~C, the r~m~ining 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 25 presented in Tables 1, 2 and 3. Among the monosaccharides tested, glucose was the best substrate and among the ~ic~ch~rides tested, maltose and isomaltose were the best substrates.
W O 97/33974 PCT~EP97/01117 Table 1. Comparison of the relative activity of Mac towards various monosaccharides as acetyl-acceptors.
\~ Relative Activity C ~ ( % 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 (c~-glucose(1,4) ~-glucose) 100 Isomaltose (~x-glucose(1,4) a!-glucose) 110 Lactose (~-galactose ~-(1,6) cY-glucose) 0.4 Sucrose (cx-glucose c~-(1,4) ,B-fructose) 0.4 Cellobiose (~-glucose ,B-(1,4) ~-glucose) 0 CA 02248~40 l998-09-lO
W O 97~3974 PCT~EP97/01117 Table 3. Comparison of the relative activity of Mac lowards various maltooligosaccharides as acetyl-acceptors.
Substrate Relative Activily (50 mM) (% of activity on maltose) Maltose 100 Maltotriose 7 . 5 Maltotetraose 0.5 Maltopentaose 0. 9 Maltohexaose i.2 Maltohepeaose 1. 1 Kinetic studies Kinetic studies of Mac catalysed acetylation reactions revealed that the Km for the acceptor substrate is in Ihe mM range whereas it is in the ~M range for acetyl-coenzyme A. Thus, Mac has about a 1000 fold more affiniey for acetyl-coenzyme A than for the acceptor.
NMR s~eudies IH-NMR struceure determination of the products of acetylation of glucose and maltose by Mac was in-~esrig~tt~.
In order to investigate the substrate regio-specificity of Mac regarding the acetylation site of the accepeor substrate, we prepared milligram amounts of aceeylated glucose and maltose by in~lbating 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 CA 02248~40 1998-09-lO
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 5 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.
10 Because the 2/ amino acid SBE portion of the SBE-Mac fusion in pMAC9 and pMAC10 described below may inte,relc with the acetyltransferase activity, the SBE-Mac f~sion was inserted in the ~. coli exp~ession vector pAL781 (Invitrogene, San Die_o. USA) in order to over-express the fusion enzyme in E. coli and analyse the activit~. A comparison of the highly over-expressed SBE-Mac fusion and the 15 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 enzvme will be active in potatoes.
lN 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 25 of each of which are incorporated herein bv reference).
For the present studies, the following protocol was adopted.
CA 02248~40 1998-09-10 W O 97133974 PCT~EP97/01117 Construction of Dlasmids for the expression of the E coli mac ~ene in potato.
The E coli mac gene was amplified with primers:
S S'-CGG GAT CCG AGC ACA GAA AAA GAA AAG ATG-3' (upper primer with BamHI site) and 10 S'-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 BamH~ and PstI and inserted in pBETPS (see 15PCT patent application No. WO 94/24292, the contents of which are incorporatedherein by reference) digested with the same enzymes yielding pMAC8. Thereby, themac gene is inserted in an expression cassette that provides tuber specific expression from a patatin promoter and transcription terrnination at a CaMV 35S termin~tor.Moreover, the Mac enzyme is fused to 102 amino acids of the N-terminus of the 20 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 ~:coRI site 25 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 ofwhich are incorporated herein by reference) giving plasmids pMAC9 and pMAC10 (Figures 9 and 10, respectively).
CA 02248540 1998-09-lO
Preparation of potato minitllhers 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 selectedshoots (for mannose selection see our earlier patent applications WO 93/05163 and/or WO 94/20627). The leaf was removed from the nodium se~ment~ and the se_ment was placed vertically on agar plates with MS medium (Sigma) supplemented with 60g sucrosell and 2 mg 6-benzyl-aminopurine/1. 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.
E~pression of the SBE-Mac fusion in potato minitllhers Potato minit~ ers transformed with the pMAC9 or pMAC10 constructs were ex~min,~ by Western analysis for expression of the E. coli mac gene with antibodies raised towards the ~. coli maltose acetyltransferase. The analysis clearly demonstrated that ~ out of 5 MAC9 minitubers and 5 out of 7 MACI0 minitubers gave a distinct expression of the E. coli maltose acetyltransferase. The positive mininlkers expressed a 209 amino acid SBE-Mac fusion that co-migrates with a similar construction expressed in E. coli. These results in~ic~te that the 75 amino acid SBE transit peptide, that was originally fused to the 209 amino acid SBE-Mac fusion, has been removed from ~he SBE-fusion. ~urthermore. this implies that thetransit peptide was conectly processed by the signal peptidase in the amyloplastmembrane, and that the SBE-Mac fusion has been directed to the amyloplast.
. . ..
CA 02248~40 1998-09-10 W 0 97~3974 PCT~EP97/01117 T~ -.loblots on potato tuber e~tracts 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 ~l of SDS-PAGE sample buffer. 25 ,~1 were subsequently loaded onto 15 %
polyacrylamide gels. After electrophoresis proteins were transferred onto Problot PVDF membranes by semi-dry blotting. For irnmunodetection ~lac antiserum was diluted 1:' 000 and secondary antibody was coupled to ~ Iin~o phosphatase.
10 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 20 for 30 min and the insoluble material was removed by centrifugalion. Protein conceMration was measured using the BCA reagent (Pierce).
Mac activity was measured in duplicates or triplicates as follows: 0, 50, 100 or 200 ~I potato extract, 10 ,ul of 1 mM acetyl-coenzyme A, 25 ~l of 1 M glucose and assay 25 buffer (50 mM potassium phosphate, 2 mM EDTA, pH 7 . 5) were mixed per microtiter plate well to give a total volume of 250 ,ul. The reaction was started by the addition of acetyl-coenzyme A. After 10 min. reaction at room temperature, 25 ~L~l of freshly made 4 mM DTNB was added and A405 was measured irnmediately.
Two wells were prepared for each single assay, one with glucose and one without.30 Activity was calc~ t~cl by subtracting the absorbance of the well without glucose (background absorbance) from that of the well with glucose.
CA 02248~40 1998-09-10 Relatively high levels of Mac activity could be measured in eight out of nine transgenic tubers. Some of ~he tubers had a Mac activity that was 15 to 20 fold above the almost negligible activity found in non-transformed tubers.
5 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 ~apid Visco 10 Analyser 3C. The results showed that the starch from the transformed potatoes had a differen~ viscometric profile to the starch from the non-transformed potato.
DSC studies lS Samples of starch obtained from tubers of non-transformed potatoes and from transforrned potatoes according to the present invention were analysed bv dirrerellLial sc~nning colometry (using a 10% w/w aqueous starch suspension). The samples wereheated from 20 to 100~C at a velocity of 10~C per minute. The results showed that the starch from the transforrned potatoes had a different enthalpy to the starch from 20 the non-transforrned potato. We additionally found a difference in gel~tini.~rion temperarure 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 25 art.
CA 02248540 lsss-os-lo W O 97/33974 PCT~EP97101117 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 SO: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. Bio~. Chem. 240, 10-13.
7. Ogasawara N., Nakai S. & Yoshikawa H. 1994, DNA Research 1, 1-14.
_ .
CA 02248~40 1998-09-lO
W O 97/33974 PCT~EP97/01117 SEQUENCES
SEQUENCE ID N0 l Amino acid sequence MSTEKEKMIAGELYRSADETLSRDRLRARQLIHRYNHSLAEEHTLRaQIL 50 ADLFGQVTEAYIEPTFRCDYGYNIFLGNNFFANFDCVMLaVCP~RlGDNC 100 GDNVVVASGhVVTKDVPDNVVVGGNPARIIKKL lS3 SEQUENCE ! D N0. ~
Nuc1e~tide sequence ATGAGCACAG M AM G M AA GATGATTGCT GGTGAGTTGT
ATCGCTCGGC AGATGAGACG TTATCTCGCG ATCGCCTGCG
CGCTCGTCAG CTTATTCACC GATAC M TCA TTCCCTGGCG
GAAGAGCACA CATTACGCCA GCAM TTCTC GCTGATCTAT
TCGGTCAGGT GACAGAGGCT TATATTGAGC C M CGTTTCG
CTGTGACTAT GGCTATAACA I~ GG TAAT M TTTT
TTCGCC M CT TCGATTGCGT GATGCTTGAT GTCTGCCCTA
TTCGCATCGG TGAT M CTGT ATGTTGGCAC CAGGCGTTCA
TATCTACACG GC M CACATC CCATCGACCC TGTAGCACGT
M TAGCGGTG CTG M CTGGG G MM CCCGTC ACCATCGGTA
TGTGACCATT GGTGATAACG TCGTGGTAGC CTCAGGTGCA
GTTGTCAC~A AA ATGTCCC GGAC M CGTT GTCGTGGGCG
GTAATCCAGC CAG M T M TT AA M M TTGT AA
....
CA 02248540 l998-09-lO
SECUENCE 1 2 N0. 3 Nucleotide sequence Complete nucleotide sequence of the 3.2 kb EcoRI-Pstl fragmen~ in pMAC3.
GCGGTGCGGA TGCG m ACG TCCGATCCTG ATGACCTCGC TGGCGTTTAT CCTCGGCGTT 120 GT M TGGGCG GGATGGTGAC CGC M CGGTA CTGGC M TCT TCTTCGTTCC GGTATTC m 240 GATCATCATT GATACM CGT GT M TCACTA AGGCCGCGTA AGCGGCC m m ATGCATA 360 CTAATAGATT TAAT M TCCA TAATCA m A GAGGCTATTC TT M TTA m GCGGT M TTC 540 lS TTTATTCATT CCTCGGTTAT TACGTCATAT TCAGAGC M T CCTGGTATTA GTGTCACC M 600 TTTCATCTGG ~ATM TCCT G M ATGTTAT GAATAGTTCG AGC MM CTGC TTTTACCTGC 660 TGCGGGTTAG TGCTAGTATG AA MM GTG~G TCCTGTCCCG CTTCCTTCCT M TTGTA~TT ,20 AGGGC M GTC CAGGTCAGTh AGTTT m CC ATCCCG MM G GTGTCCGTTA GTTC M CCGC 840 AG m CTCTG TG MACCCTG TATCATGACT GCCTTGCA M CCTTG M G M AGC M TCATG 960 AGATCGACGA ATATCTGGAT GACACCTTTA TGTTGTTCAG TAGTThTGGT ATT M TATGC 1140 AGGATCTTCA G MM TGGCGG M GTCAGGTA AHCGACTATH CCG~ ! GTC M TGCGA 1200 TCCGAAAAAC CTTT MC& M M CCGATTAT TT M TGCGTT TACGTCGTTG CCAGACAATT 1320 GACACGCTGG AGCGGTTTAW TCGAG M AAA TA M TACG M TTATCAGATA ATG M CrGGC 1380 CTTTATCACA CAGATGTAAT GGG M CGTTC TCTTCACTGA ~ lcG~cT TA~ IIG 1620 CCGCATTTTC A&C M CCGGA GTCAGT M TG AGChCAGA M M G M M GAT GATTGCTGGT 1680 GAGTTGTATC GCTCGGCAGA rGAGACGTTA TCTCGCGATC GCCTGCGCGC TCGTCAGCTT 1740 TATAACA m TTCTCGGT M T M m TTTC GCC M CTTCG ATTGCGTGAT GCTTGATGTC 1920 GTGGGCGGTA ATCCAGCCAG M T M TTA M M ATTGT M T CGGTT m CG C M CTGTTAT 2220 CGA M CGATT GAGTCTCTGA ATACCCGCGA AA M CGCGAC M C MACCCC GC m AGTAT 2400 . , ., .. ... . . _ . ...
CA 02248~40 1998-09-10 CAGTTTTATC CGTAMCATC CGGGGCTGTT TATCGGTATG TACGTTGCTT TTTTTGCCAC 2~60 TATCGACGTG CTGGATTTCC GCGTTTGCTA TAACGGCGM TGGTACMCA CGCGCTTTGT .640 ACCTGCCGCG CTGGTTGMG CCATCTTGM CTCTCCGTGT CGCGGATGTT CATMGGMC 2,00 TCGCGCCGAA TCAACATCTT MGTTA&GGT TACATACCAG GCGTMA&CT CTGCGCCTGG 2320 GGMMGAT~,C GCTGCAG 3137 ~EOUENCE ! D. NO. 4 Complete nllcleotlde sequence of the 3.2 kb ~coRI-Fstl fragment in pMAC3. The Mac enzyme 2mino acid sequence is also shown below t~e mdc gene coding sequence.
ATGCCGCTGGTTATCAGTACTGGTGCTGGTTCCr~rlCGCGCAGMCGCAGTAGGTACCGGT 180 GATCATCATTGATACMCGTGTMTCACTMGGCCGCGTMGCGGC(;I1111IATGCATA 360 TTTCATCTGGCGATMTCCTGMMTGTTATGMTAGTTCGAGCAAACTGCTmACCTGC660 AGGGCMGTCCAGGTCAGTM~illllllCCATCCCGAMGGTGTCCGTTAGTTCMCCGC 840 AGTTTcTcTGTGMMcccTGTATcATGAcTGccTTGcMMccTTGMGMMGcMTcATG960 TTG~ CCTTCGCACTTMTTACMAATTMGTATMTGMGACMTMGCTCATTGAGC 1080 AGATCGACGMTATCTGGATGACACCmAl(illGIIt;AGTAGTTATGGTATTMTATGC 1140 AGGATCTTCAGMMTGGCGGMGTCAGGTMHCGACTATHCC[il I ~i I III(ilCMTGCGA 1200 GACACGCTGGAGCGGmAWTCGAGMMMTMMTACGMTTATCAGATMTGMCTGGC 1380 TMCCGîaCTGTTTATTMGMTmATACTTmCGCCATGMGACATACCCTATGTGAT 1560 ~ . .. . ... . .......
CA 02248~40 1998-09-10 M S T E K E K M I A G
GAGTTGTATCGCTCGGCAGATGAGACGTTATCTCGCGATCGCCTGCGC&CTCGTCAGCTT 1740 E L Y R S A D E T L S R D R L R A R Q L
I H R Y N H S L A E E H T L R Q Q I L A
D L F G Q V T E A Y I E P T F R C D Y G
TATMCATTTTTCTCGGTMTAAT~ lrGCCMCTTCGATTGCGTGATGCTTGATGTC 1920 Y N I F L G N N F F A N F D C V M L D V
C P I R I G D N C M L A P G V H I Y T A
ACACATCCCATCGACCCTGTAGCACGTMTAGCGGTGCTGMCTG&GGAMCCCGTCACC 2040 lS T H P I D P V A R N S G A E L G K P V T
ATCGGTMTMCC~TCTGGATTGGCGGACGCGCGGTCATTMCCCTGîJ~GTGACCATTGG' 2100 I G N N V W I G G R A V I N P G V T I G
D N V V V A S G A V V T K D V P D N V V
V G G N P A R I I K K L
GCMMTTGTGGTAGATCTGTTACTTCCCCTCTACTATTCCCACGTTA,4MTAGGGTGTT 2280 CAGTmATCCGTAMCATCCGGGGCTGTTTATCGGTATGTACGTTG(;I11111IGCCAC 2460 ATTTATCCTGCTTAATGGIll~ lcGATGTCTACCCAcGcTACcGCTATGMGA 2580 TATCGACGTGCTGGATTTCCGCGTTTGCTATMCGGCGMTGGTACMCACGCGCmGT 2640 ACCTGCCGCGCTGGTTGMGCCATCTTGMCTCTCCGTGTCGCGGATGTTCArMGGMC 2700 MCTGCMAMATGATCGTCCGTMMGGTGM(,T(il(;lllllACGATATTTTTACCCTCS 2760 TCMMTGACMTGATCGmCCACCCATCACTTCATGAMTACCaGCTCTACCTCCTTAT 2880 CTCCAGCCAGCCIllllccAcMTcAGATATACmCcCTACAcTGTGTTAATMGGATA 2940 TGCTGGTGAGMCACGACATCTGGTCGGCCTTAmCGGGAGTACTGATTCmCAGTAT 3000 , CA 02248~40 l998-09-l0 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: DANISCO A/S
(B) STREET: LANGEBROGADE 1 (C) CITY: COPENHAGEN
(D) STATE: COPENHAGEN
(E) COUNTRY: DENMARK
(F) POSTAL CODE (ZIP): DK-1001 (ii) TITLE OF INVENTION: AN ENZYME
(iii) NUMBER OF SEQUENCES: 5 (iv) CORRESPONDENCE ADDRESS
BENNETT JONES VERCHERE
855 - 2ND STREET S.W.
CALGARY, ALBERTA
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) CURRENT APPLICATION DATA:
APPLICATION NUMBER:
FILING DATE: 7 MARCH 1997 CLASSIFICATION: C12N
(vii) PRIOR APPLICATION DATA:
APPLICATION NUMBER: PCT/EP97/01117 FILING DATE: 7 MARCH 1997 CLASSIFICATION: C12N
(viii) PATENT AGENT INFORMATION:
BENNETT JONES VERCHERE (ROSEANN CALDWELL) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 183 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide CA 02248~40 l998-09-l0 .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Glu Leu Gly Lys Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro Ala Arg Ile Ile Lys Lys Leu (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 552 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CA 02248~40 l998-09-l0 (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CA 02248~40 l998-09-lO
AGGGCAAGTC CAGGTCAGTA A~ll"llll'CC ATCCCGAAAG GTGTCCGTTA GTTCAACCGC 840 CA 02248~40 1998-09-10 (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CA 02248~40 l998-09-lO
AGGGCAAGTC CAGGTCAGTA A~llllllCC ATCCCGA~AG GTGTCCGTTA GTTCAACCGC 840 AGGATCTTCA GAAATGGCGG AAGTCAGGTA AHCGACTATH CC~l"l'~l"l"l'l' GTCAATGCGA 1200 CA 02248~40 l998-09-l0 GCA~AATTGT GGTAGATCTG TTACTTCCCC TCTACTATTC CCACGTTAAA ATAGGGTGTT 2280 GGA~AGATGC GCTGCAG 3137 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 183 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide CA 02248~40 l998-09-l0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser ~la Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe ~he Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile ~ly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Glu Leu Gly Lys Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala ~al Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro Ala Arg Ile Ile Lys Lys Leu
_ .
CA 02248~40 1998-09-lO
W O 97/33974 PCT~EP97/01117 SEQUENCES
SEQUENCE ID N0 l Amino acid sequence MSTEKEKMIAGELYRSADETLSRDRLRARQLIHRYNHSLAEEHTLRaQIL 50 ADLFGQVTEAYIEPTFRCDYGYNIFLGNNFFANFDCVMLaVCP~RlGDNC 100 GDNVVVASGhVVTKDVPDNVVVGGNPARIIKKL lS3 SEQUENCE ! D N0. ~
Nuc1e~tide sequence ATGAGCACAG M AM G M AA GATGATTGCT GGTGAGTTGT
ATCGCTCGGC AGATGAGACG TTATCTCGCG ATCGCCTGCG
CGCTCGTCAG CTTATTCACC GATAC M TCA TTCCCTGGCG
GAAGAGCACA CATTACGCCA GCAM TTCTC GCTGATCTAT
TCGGTCAGGT GACAGAGGCT TATATTGAGC C M CGTTTCG
CTGTGACTAT GGCTATAACA I~ GG TAAT M TTTT
TTCGCC M CT TCGATTGCGT GATGCTTGAT GTCTGCCCTA
TTCGCATCGG TGAT M CTGT ATGTTGGCAC CAGGCGTTCA
TATCTACACG GC M CACATC CCATCGACCC TGTAGCACGT
M TAGCGGTG CTG M CTGGG G MM CCCGTC ACCATCGGTA
TGTGACCATT GGTGATAACG TCGTGGTAGC CTCAGGTGCA
GTTGTCAC~A AA ATGTCCC GGAC M CGTT GTCGTGGGCG
GTAATCCAGC CAG M T M TT AA M M TTGT AA
....
CA 02248540 l998-09-lO
SECUENCE 1 2 N0. 3 Nucleotide sequence Complete nucleotide sequence of the 3.2 kb EcoRI-Pstl fragmen~ in pMAC3.
GCGGTGCGGA TGCG m ACG TCCGATCCTG ATGACCTCGC TGGCGTTTAT CCTCGGCGTT 120 GT M TGGGCG GGATGGTGAC CGC M CGGTA CTGGC M TCT TCTTCGTTCC GGTATTC m 240 GATCATCATT GATACM CGT GT M TCACTA AGGCCGCGTA AGCGGCC m m ATGCATA 360 CTAATAGATT TAAT M TCCA TAATCA m A GAGGCTATTC TT M TTA m GCGGT M TTC 540 lS TTTATTCATT CCTCGGTTAT TACGTCATAT TCAGAGC M T CCTGGTATTA GTGTCACC M 600 TTTCATCTGG ~ATM TCCT G M ATGTTAT GAATAGTTCG AGC MM CTGC TTTTACCTGC 660 TGCGGGTTAG TGCTAGTATG AA MM GTG~G TCCTGTCCCG CTTCCTTCCT M TTGTA~TT ,20 AGGGC M GTC CAGGTCAGTh AGTTT m CC ATCCCG MM G GTGTCCGTTA GTTC M CCGC 840 AG m CTCTG TG MACCCTG TATCATGACT GCCTTGCA M CCTTG M G M AGC M TCATG 960 AGATCGACGA ATATCTGGAT GACACCTTTA TGTTGTTCAG TAGTThTGGT ATT M TATGC 1140 AGGATCTTCA G MM TGGCGG M GTCAGGTA AHCGACTATH CCG~ ! GTC M TGCGA 1200 TCCGAAAAAC CTTT MC& M M CCGATTAT TT M TGCGTT TACGTCGTTG CCAGACAATT 1320 GACACGCTGG AGCGGTTTAW TCGAG M AAA TA M TACG M TTATCAGATA ATG M CrGGC 1380 CTTTATCACA CAGATGTAAT GGG M CGTTC TCTTCACTGA ~ lcG~cT TA~ IIG 1620 CCGCATTTTC A&C M CCGGA GTCAGT M TG AGChCAGA M M G M M GAT GATTGCTGGT 1680 GAGTTGTATC GCTCGGCAGA rGAGACGTTA TCTCGCGATC GCCTGCGCGC TCGTCAGCTT 1740 TATAACA m TTCTCGGT M T M m TTTC GCC M CTTCG ATTGCGTGAT GCTTGATGTC 1920 GTGGGCGGTA ATCCAGCCAG M T M TTA M M ATTGT M T CGGTT m CG C M CTGTTAT 2220 CGA M CGATT GAGTCTCTGA ATACCCGCGA AA M CGCGAC M C MACCCC GC m AGTAT 2400 . , ., .. ... . . _ . ...
CA 02248~40 1998-09-10 CAGTTTTATC CGTAMCATC CGGGGCTGTT TATCGGTATG TACGTTGCTT TTTTTGCCAC 2~60 TATCGACGTG CTGGATTTCC GCGTTTGCTA TAACGGCGM TGGTACMCA CGCGCTTTGT .640 ACCTGCCGCG CTGGTTGMG CCATCTTGM CTCTCCGTGT CGCGGATGTT CATMGGMC 2,00 TCGCGCCGAA TCAACATCTT MGTTA&GGT TACATACCAG GCGTMA&CT CTGCGCCTGG 2320 GGMMGAT~,C GCTGCAG 3137 ~EOUENCE ! D. NO. 4 Complete nllcleotlde sequence of the 3.2 kb ~coRI-Fstl fragment in pMAC3. The Mac enzyme 2mino acid sequence is also shown below t~e mdc gene coding sequence.
ATGCCGCTGGTTATCAGTACTGGTGCTGGTTCCr~rlCGCGCAGMCGCAGTAGGTACCGGT 180 GATCATCATTGATACMCGTGTMTCACTMGGCCGCGTMGCGGC(;I1111IATGCATA 360 TTTCATCTGGCGATMTCCTGMMTGTTATGMTAGTTCGAGCAAACTGCTmACCTGC660 AGGGCMGTCCAGGTCAGTM~illllllCCATCCCGAMGGTGTCCGTTAGTTCMCCGC 840 AGTTTcTcTGTGMMcccTGTATcATGAcTGccTTGcMMccTTGMGMMGcMTcATG960 TTG~ CCTTCGCACTTMTTACMAATTMGTATMTGMGACMTMGCTCATTGAGC 1080 AGATCGACGMTATCTGGATGACACCmAl(illGIIt;AGTAGTTATGGTATTMTATGC 1140 AGGATCTTCAGMMTGGCGGMGTCAGGTMHCGACTATHCC[il I ~i I III(ilCMTGCGA 1200 GACACGCTGGAGCGGmAWTCGAGMMMTMMTACGMTTATCAGATMTGMCTGGC 1380 TMCCGîaCTGTTTATTMGMTmATACTTmCGCCATGMGACATACCCTATGTGAT 1560 ~ . .. . ... . .......
CA 02248~40 1998-09-10 M S T E K E K M I A G
GAGTTGTATCGCTCGGCAGATGAGACGTTATCTCGCGATCGCCTGCGC&CTCGTCAGCTT 1740 E L Y R S A D E T L S R D R L R A R Q L
I H R Y N H S L A E E H T L R Q Q I L A
D L F G Q V T E A Y I E P T F R C D Y G
TATMCATTTTTCTCGGTMTAAT~ lrGCCMCTTCGATTGCGTGATGCTTGATGTC 1920 Y N I F L G N N F F A N F D C V M L D V
C P I R I G D N C M L A P G V H I Y T A
ACACATCCCATCGACCCTGTAGCACGTMTAGCGGTGCTGMCTG&GGAMCCCGTCACC 2040 lS T H P I D P V A R N S G A E L G K P V T
ATCGGTMTMCC~TCTGGATTGGCGGACGCGCGGTCATTMCCCTGîJ~GTGACCATTGG' 2100 I G N N V W I G G R A V I N P G V T I G
D N V V V A S G A V V T K D V P D N V V
V G G N P A R I I K K L
GCMMTTGTGGTAGATCTGTTACTTCCCCTCTACTATTCCCACGTTA,4MTAGGGTGTT 2280 CAGTmATCCGTAMCATCCGGGGCTGTTTATCGGTATGTACGTTG(;I11111IGCCAC 2460 ATTTATCCTGCTTAATGGIll~ lcGATGTCTACCCAcGcTACcGCTATGMGA 2580 TATCGACGTGCTGGATTTCCGCGTTTGCTATMCGGCGMTGGTACMCACGCGCmGT 2640 ACCTGCCGCGCTGGTTGMGCCATCTTGMCTCTCCGTGTCGCGGATGTTCArMGGMC 2700 MCTGCMAMATGATCGTCCGTMMGGTGM(,T(il(;lllllACGATATTTTTACCCTCS 2760 TCMMTGACMTGATCGmCCACCCATCACTTCATGAMTACCaGCTCTACCTCCTTAT 2880 CTCCAGCCAGCCIllllccAcMTcAGATATACmCcCTACAcTGTGTTAATMGGATA 2940 TGCTGGTGAGMCACGACATCTGGTCGGCCTTAmCGGGAGTACTGATTCmCAGTAT 3000 , CA 02248~40 l998-09-l0 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: DANISCO A/S
(B) STREET: LANGEBROGADE 1 (C) CITY: COPENHAGEN
(D) STATE: COPENHAGEN
(E) COUNTRY: DENMARK
(F) POSTAL CODE (ZIP): DK-1001 (ii) TITLE OF INVENTION: AN ENZYME
(iii) NUMBER OF SEQUENCES: 5 (iv) CORRESPONDENCE ADDRESS
BENNETT JONES VERCHERE
855 - 2ND STREET S.W.
CALGARY, ALBERTA
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) CURRENT APPLICATION DATA:
APPLICATION NUMBER:
FILING DATE: 7 MARCH 1997 CLASSIFICATION: C12N
(vii) PRIOR APPLICATION DATA:
APPLICATION NUMBER: PCT/EP97/01117 FILING DATE: 7 MARCH 1997 CLASSIFICATION: C12N
(viii) PATENT AGENT INFORMATION:
BENNETT JONES VERCHERE (ROSEANN CALDWELL) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 183 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide CA 02248~40 l998-09-l0 .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Glu Leu Gly Lys Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro Ala Arg Ile Ile Lys Lys Leu (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 552 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CA 02248~40 l998-09-l0 (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CA 02248~40 l998-09-lO
AGGGCAAGTC CAGGTCAGTA A~ll"llll'CC ATCCCGAAAG GTGTCCGTTA GTTCAACCGC 840 CA 02248~40 1998-09-10 (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CA 02248~40 l998-09-lO
AGGGCAAGTC CAGGTCAGTA A~llllllCC ATCCCGA~AG GTGTCCGTTA GTTCAACCGC 840 AGGATCTTCA GAAATGGCGG AAGTCAGGTA AHCGACTATH CC~l"l'~l"l"l'l' GTCAATGCGA 1200 CA 02248~40 l998-09-l0 GCA~AATTGT GGTAGATCTG TTACTTCCCC TCTACTATTC CCACGTTAAA ATAGGGTGTT 2280 GGA~AGATGC GCTGCAG 3137 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 183 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide CA 02248~40 l998-09-l0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser ~la Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe ~he Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile ~ly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Glu Leu Gly Lys Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala ~al Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro Ala Arg Ile Ile Lys Lys Leu
Claims (15)
1. An enzyme having .alpha.(1,4) glucan acetyl-transferase activity, wherein theenzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof.
2. A recombinant enzyme having .alpha.(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.
3. A recombinant enzyme having .alpha.(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 .alpha.(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.
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 organismis 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9605274.1 | 1996-03-13 | ||
| GBGB9605274.1A GB9605274D0 (en) | 1996-03-13 | 1996-03-13 | An enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2248540A1 true CA2248540A1 (en) | 1997-09-18 |
Family
ID=10790311
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002248540A Abandoned CA2248540A1 (en) | 1996-03-13 | 1997-03-07 | Dna encoding alpha-1(1,4)-glucan acetyl-transferase |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0906413A2 (en) |
| JP (1) | JP2000506023A (en) |
| CN (1) | CN1259997A (en) |
| AU (1) | AU720991B2 (en) |
| BR (1) | BR9708029A (en) |
| CA (1) | CA2248540A1 (en) |
| GB (1) | GB9605274D0 (en) |
| NZ (1) | NZ331426A (en) |
| PL (1) | PL328829A1 (en) |
| WO (1) | WO1997033974A2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AUPO069996A0 (en) | 1996-06-27 | 1996-07-18 | Australian National University, The | Manipulation of plant cellulose |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4425688A1 (en) * | 1994-07-14 | 1996-01-18 | A U F Analytik Umwelttechnik F | High acetylated starch prodn. for biodegradable film etc. mfr. |
-
1996
- 1996-03-13 GB GBGB9605274.1A patent/GB9605274D0/en active Pending
-
1997
- 1997-03-07 CA CA002248540A patent/CA2248540A1/en not_active Abandoned
- 1997-03-07 AU AU20243/97A patent/AU720991B2/en not_active Ceased
- 1997-03-07 WO PCT/EP1997/001117 patent/WO1997033974A2/en not_active Application Discontinuation
- 1997-03-07 EP EP97908181A patent/EP0906413A2/en not_active Withdrawn
- 1997-03-07 NZ NZ331426A patent/NZ331426A/en unknown
- 1997-03-07 PL PL97328829A patent/PL328829A1/en unknown
- 1997-03-07 CN CN97194352A patent/CN1259997A/en active Pending
- 1997-03-07 JP JP9532251A patent/JP2000506023A/en active Pending
- 1997-03-07 BR BR9708029-2A patent/BR9708029A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| GB9605274D0 (en) | 1996-05-15 |
| AU720991B2 (en) | 2000-06-22 |
| WO1997033974A3 (en) | 1997-10-30 |
| AU2024397A (en) | 1997-10-01 |
| CN1259997A (en) | 2000-07-12 |
| NZ331426A (en) | 1999-10-28 |
| WO1997033974A2 (en) | 1997-09-18 |
| EP0906413A2 (en) | 1999-04-07 |
| BR9708029A (en) | 2000-02-01 |
| PL328829A1 (en) | 1999-02-15 |
| JP2000506023A (en) | 2000-05-23 |
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