New Zealand No. 331426 International No PCT/EP97/01 117
TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION
Priority dates 13 03 1996,
Complete Specification Filed 07 03 1997
Classification (6) C12N9/10, C12N15/54.82, C12P19/04
Publication date 28 October 1999 Journal No 1445
NEW ZEALAND PATENTS ACT 1953
COMPLETE SPECIFICATION
Title of Invention An enzyme
Name, address and nationality of applicant(s) as in international application form
DANISCO A/S, Langebrogade 1, P O Box 17, DK-1001 Copenhegan K, Denmark
1
PCT/EP97/Q1117
AN ISOLATED ENZYME
The present invention relates to an isolated enzyme The present invention also relates to an isolated nucleotide sequence coding for the enzyme
Boos and coworkers in 1981 and 1982 (1, 2) presented evidence tor the existence of an enzvme capable of acetylating maltose via transfer of the acecyl group from Acetyl-coenzyme A to maltose in E coli In particular, Boos et al (1) observed the formation of acetyl-maltose and acetyl-oligomaltosides after accumulation of maltose 10 or maltoohgosides in E coli They also observed the formation of acetyl-maltose and acetvl-oligomaltosides in vitro when maltose or maltotriose, acetyl-coenzyme A and a cytosohc E coli extract were mixed together Boos et al (2)
Boos et al in 1981 stated that the activity responsible for maltose and maltodextnn 15 acetylation was unknown However, m their further studies of 1982 (2), Feundlieb and Boos named the unknown enzyme "maltose transacetylase" but then said that the function of maltose transacetylase in E coli was unclear
Later Brand and Boos (3) isolated an E coli mutant lacking the gene encoding 20 maltose transacetylase This mutant enabled them to map the gene at 10 4 mm on the E coli linkage map In addition, they cloned a 3 4 kb DNA fragment containing the gene in a high copy plasmid Over-expressed maltose transacetylase was then purified to homogeneity from cell free extracts of an E coli strain harbouring the above mentioned plasmid The enzyme was shown to be a homodimer with two 25 identical subunits of 20 kDa The km (mM) and Vmax (ptmol/mm x mg enzyme) values of this enzyme for the substrates glucose, maltose and acetyl-coenzyme A were t
62 and 200, 90 and 110, and 0 018 and 166 respectively Maltotriose and other oligosaccharides were found to be acetylated with a rate of 2% of the rate determined for glucose In addition. Brand and Boos presented the following relative 30 transacetylation rates glucose 1, maltose 0 55, mannose 0 2, fructose 0 07, galactose 0 04, maltotriose and other malto-oligosaccharides 0 02 Oligosaccharides are saccharides having less than ten sugar units
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Despite of these findings Brand and Boos did not sequence the enzyme or the nucleotide sequence coding for the maltose transacetylase enzyme
According to a first aspect of the present invention there is provided an isolated enzyme having g:(1,-t) glucan acetvl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No 1, or a variant, homologue or fragment thereof
According to a second aspect of the present invention there is provided a recombinant enzyme having a(l,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No 1, or a variant, homologue or fragment thereof
According to a third aspect of the present invention there is provided a recombinant enzyme having a(l,4) glucan acetyl-transferase activity, wherein the enzyme has the ammo acid sequence shown as SEQ ID No 1
According to a fourth aspect of the present invention there is provided a recombinant enzyme having a(l,4) glucan acetyl-transferase activity, wherein the recombinant enzyme is immunologically reactive with an antibody raised against a purified recombinant enzyme according to the above-mentioned aspect of the present invention
According to a fifth aspect of the present invention there is provided an isolated nucleotide sequence coding for the enzyme of the present invention or a sequence that is complementary thereto
According to a sixth aspect of the present invention there is provided an isolated 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
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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 enzvme 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 plasmid comprising or expressing the vector, the construct or the nucleotide sequence or the enzyme according to the present invention
According to an eleventh aspect of the present invention there is provided a transgenic organism comprising or expressing the plasrnid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention
According to a twelfth aspect of the present invention there is provided a modified carbohydrate (preferably starch) prepared by a method comprising or expressing or using the present invention
The enzyme of the present invention may be obtainable from any one of a bacterium, 25 a fungus an alga, a yeast, or a plant Preferably, the enzyme is obtainable from E coli
The a(l ,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac The gene coding for the c*(l,4) glucan acetyl-transferase of the present 30 invention is also sometimes referred to as the mac gene
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Preferably ihe 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
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 that is complementary thereto
Preferably the enzyme is encoded by the nucleotide sequence shown as SEQ ID No 2
Preferably the organism is a plant
Preferablv the nucleotide* sequence is a DNA sequence
The enzyme or nucleotide sequence(s) coding for same may be used in vitro or in vivo in combination with one or more other enzymes or nucleotide sequence(s) coding for same which enzymes or nucleotide sequence(s) coding for same are preferably 20 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 necessarily be the same 25 enzymes
The terms "variant", "homologue" or "fragment" in relation to the enzyme include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant 30 amino acid sequence has a(l,4) glucan acetyl-transferase activity, preferably having at least the same activity of the enzyme shown as SEQ ID No 1 In particular, the term "homologue' covers homology with respect to structure and/or function
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providing the resultant enzyme has a(l,4) glucan acetyl-transferase activity With respect to sequence homology, preferably there is at least 75% more preferably at least 85% more preferably at least 90% homology to tht sequence shown as SEQ ID No 1 More preferably there is at least 95%, more preferablv at least 98%, 5 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 ot 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 a(l,4) glucan acetyl-transferase activity, preferably having at least the same activity ot 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 codLs for an enzvme having a(l,4) 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% homology to the sequence shown as SEQ ID No 2 More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No 2
The above terms are svnonymous with allelic variations of the sequences
The term complementary" means that the present invention also covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention
The term "nucleotide" in relation to the present invention includes genomic DNA, lDNA, synthetic DNA and RNA Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention
Preferably the nucleotide sequence is not a native nucleotide sequence In this 30 regard, the term native nucleotide sequence' means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment
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Thus, the enzyme of the present invention can be expresc~d by a nucleotide sequence in its native organism but wherein the nucleotide sequence, is not under the control of the promoter with which it is naturally associated within that organism
The enzyme of the present invention may be used in conjunction with other enzymes
Preferably the enzyme is not a native enzyme In this regard the term "native enzyme" means an entire enzyme that is m its native environment and when it has been expressed by its native nucleotide sequence
The term "construct" - which is synonymous with terms such as 'conjugate", "cassette" and "hybrid" - includes the nucleotide sequence directly or indirectly attached or fused to a promoter An example of an indirect attachment is the provision ot a suitable spacer group such as an intron sequence such as the Shl-15 intron or the ADH intron, intermediate the promoter and the nucleotide sequence
In each case, it is highly preferred that the terms do not cover the natural combination of the t>ene 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 m 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 isomerasc (especially for plants) or those markers that provide for antibiotic resistance - e g resistance to G418 hygromycin, bleomycin, kanamycin and gentamycin
The term "vector" includes expression vectors and transformation vectors
The term "expression vcctor" means a construct capable of in vivo or in vitro
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FCT/EP97/01117
expression
The term " transformation vector" means a construct capable of being transferred from one species to another - such as from an E coli plasmid to an Agrobacterium to a 5 plant
The term "tissue" includes tissue and organ, which tissue and organ can be isolated tissue and isolated organ, as well as tissue and organ when within an organism
The term 'organism" in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be expressed when present in the organism
Preferably the organism is a plant
The term "transgenic organism" in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the enzyme according to 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
Preferably the transgenic organism is a plant
Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present 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
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also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a heterologous promoter The transgenic organism does not comprise the combination of a promoter and the nucleotide sequence coding for the enzyme according to the present invention, wherein both the promoter and the 5 nucleotide sequence are native to that organism and are in their natural environment
The term promoter' is used in the normal sense of the an, e g an RNA polymerase binding sue in the Jacob-Mond theorv of gene expression
The promoter could additionally include one or more features to ensure or to increase expression in a suitable host For example, the features can be conserved regions such as a Pribnow Box or a TATA box The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention For example suitable other 15 sequences include the STiy-intron or an ADH intron Other sequences include inducible elements - such as temperature, chemical light or stress inducible elements
Also, suitable elements to enhance transcription or translation mav be present An example of the latter element is the TMV 5' 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 organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, stem, tuber, sprout root and leaf tissues
General teachings of recombinant DNA techniques may be found in Sambrook.J , 30 Fntsch E F , Maniatis T (Editors) Molecular Cloning A laboratory manual Second edition Cold Spring Harbour Laboratory Press New York 1989
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Even though the enzyme arid the nucleotide sequence of the present invention are not disclosed in EP-B-0470143 and CA-A-2006454, those two documents do provide some useful background commentary on the types of techniques that may be employed to prepare transgenic plants according to the present invention Some ot 5 these background teachings are now included in the following commentary
The basic principle in Uie construction of genetically modified plants is to insert genetic information ir tlic plant genome so as to obtain a stable maintenance of the inserted genetic material
Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use ot a vector system A review of the general techniques may be tound in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42 205-1:> 225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27)
Thus, in one aspect, the present invention relates to a vector system which carries the nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an 20 organism, such as a plant
The vector system may comprise one vector, but it can comprise two vectors In the case of two vectors, the vector system is normally referred to as a binary vector system Binary vector systems are described in turther detail in Gynheung An et al 25 (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19
One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium rumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al 30 (1986), Plant Physiol 81, 301-305 and Butcher D N et al (1980), Tissue Culture Methods for Plant Pathologists eds D S Ingrams and J P Helgeson. 203-208
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Several different Ti and Ri plasmids have been constructed which are suitable for the construction ot the plant or plant cell constructs described above
The nucleotide sequence or construct of the present invention should preferably be 5 inserted into the Ti-plasmid between the terminal sequences ot 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 ot these regions appear to be essential for insertion of modified T-DNA into the plant genome
As will be understood from the above explanation if the organism is a plant, then the vector system of the present invention is preferably one which contains the sequences necessary to infect tne plant (e g the vir region) and at least one border part ol 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 rhtzogenes Ri-plasmid or a derivative thereof, as these plasmids are well-known and widely employed in the construction ot transgenic pla.its, tnanv vector svstems 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 easv to manipulate before insertion into the plant An example 25 of a useful microorganism is E coli, but other microorganisms having the above properties may be used When a vector of a vector system as defined above has been constructed in E colt, it is transferred, if necessary, into a suitable Agrobacterium strain, e g Agrobacterium tumefaciens The Tt-plasmid harbouring the nucleotide sequence or construct of the invention is thus preferably transferred into a suitable 30 Agrobacterium strain e g A tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or construct of the invention which DNA is subsequently transferred into the plant cell to be modified
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As reported in CA-A-2006454, a large amount of cloning vcctors are available which contain a replication svstem in E colt and a marker which allows a selection of the transformed cells The vcctors contain for example pBR 322 plJC series M13 mp series, pACYC 184 etc In this way, the nucleotide or construct ot the present 5 invention can be introduced into a suitable restriction position in the vrctor The contained plasmid is used for the transformation in E coli The E coli cells are cultivated in a suitable nutrient medium and then harvested and Ivsed The plasmid is then recovered As a method of analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological 10 methods ^tter each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence Each sequence can be cloned in the same or different plasmid
After each introduction method of the construct or nucleotide sequence according to 15 the present invention in the plants the presence and/or insertion of further DNA sequences may be necessary IF, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected The use of T-DNA for the transformation of 20 plant cells has been intensively studied and is described in EP-V120516, Hoekema, in The Binary Plant Vector System Offset-drukkenj Kanters B B , Alblasserdam, 1985, Chapter V, Fraley et al, Cnt Rev Plant Sci ,4 1-46 and An et al , EMBO J (1985) 4 277-284
Direct infection of plant tissues by Agrobactenum 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 Pathologistv, 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-TechMarchyApril 30 1994 17-27) With this technique, infection of a plant may be done on a certain pan or tissue of the plant i e on a pan of a leaf, a tuber a root, a stem or another part of the plant
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Typically with direct infection of plant tissues by Agrobacteruun carrying the nucleotide sequence of the present invention, a plant to be intected is wounded, e g b\ cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive The wound is then inoculated with the -Iqrobactemwi The 5 inoculated plant or plant pan is then grown on a suitable culture medium and allowed to develop into mature plants
When plant cells are constructed, these cells nny be grown and maintained in accordance with well-known tissut. cultunng methods such as by cultunng the cells 10 in a suitable culture medium supplied with the necessary growth factors such as amino acids plant hormones, vitamins etc
Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue 15 cultures for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones etc
Even tunher useful teachings on the transformation of plants can be found in Danish 20 patent application No 940662 (filed 10 June 1994) and/or United Kingdom patent application No 9702592 8 (filed 7 February 1997)
Reference may even be made to Spngstad et al (1995 Plant Cell Tissue Organ Culture 40 pp 1-15) as these authors present a general overview on transgenic plant 25 construction
In summation the present invention relates to an enzyme having a(l ,4) glucan acetyl-transferase activitv and a nucleotide coding for same The present invention also provides a modified carbohydrate (preferably starch) obtainable from use of the same
The following sample was deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria
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Limited (NCIMB) at 23 St Machar Drive, Aberdeen Scotland, United Kingdom, AB2 IRY on 7 March 1996
DH5a-pMAC3 (which contains a 3 2 kb EcaRl-Pstl fragment from E coli comprising the mac gene)
The deposit number is NCIMB 40789
This deposit concerns the plasmid pMAC3
The following sample was deposited in accordance with the Budapest Treatv at the recognised depositary The National Collections of Industrial and Marine Bactcna Limited (NCIMB) at 23 St Machar Drive, Aberdeen. Scotland, United Kingdom, 15 AB2 1RY on 7 March 1996
NF1830-pMAC5 (which contains the E coli mac gene)
The deposit number is NCIMB 40790
This deposit concerns the plasmid pMAC5
A highly preferred aspect of the present invention therefore relates to an enzyme having a(l,4) glucan acetyl-transferase 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 enzvme is expressed by a nucleotide sequence obtainable from either deposit number NCIMB 40789 or deposit number NCIMB 4075K)
Another highly preferred aspect of the present invention therefore relates to a 30 nucleotide sequence comprising the sequence shown as SEQ ID No 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto, and wherein the nucleotide sequence is obtainable from either deposit number NCIMB
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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 ot example in which reference is made to the following Figure";
Figure 1 which shows the nucleotide sequence corresponding to SEQ ID No 2,
Figure 2 which shows the ammo acid sequt-nce corresponding to SEQ ID No 1,
Figure 3 which shows a nucleotide sequence comprising the sequence corresponding to SEQ ID No 2,
Figure 4 which is a plasmid map of pMACl Figure 5 which is a plasmid map of pMAC2.
Figure 6 which is a plasmid map of pMAC3,
Figure 7 which is a plasmid map of pMAC5,
Figure 8 which is a plasmid map of pMAC8,
Figure 9 which is a plasmid map of pMAC9, and Figure 10 which is a plasmid map of pMACIO 30 Some details on the Figures are as follows
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Figure 1
Nucleotide sequence corresponding to Seq ID No 2 Figure 2
Amino acid sequence corresponding to Seq ID No 1 183 ammo acids 20073 MW
Figure 4 10 Plasmid name pMACl Plasmid size 7 26 kb
Comments Insertion of a 4 3 kb EcoRl fragment from lambda 151 into the EcoR 1 site of pBluescript II SK -f-
Figure 5
Plasmid name pMAC2 Plasmid size 7 26 kb
Comments Insertion of a 4 3 kb EcoRl fragment from lambda 151 (Kohara collection) into the EcoRl site of pBlutscnpt II SK +
Figure 6
Plasmid name pMAC3 Plasmid size 7 26 kb
Comments Deletion of the 1 1 kb Rrrl fragment from pMAC2
Figure 7
Plasmid name pMAC5 Plasmid size 4060 bp Comments
The E coh mac gene was amplified with primers 0B411 (upper primer with EcoR 1 site)
CGG AAT TCC GCC ATG AAG ACA TAC CC
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PCT/EP97/01H7
0B412 (lower primer with HindlU site)
CAC AAG CTT ATT TTG CAT AAC AGT TGC
using pMAC3 as template
The 704 bp PCR product was digested with EcoR 1 and Hindlll and inserted in 5 pUHE21-2 digested with the same restriction enzymes
Figure 8
Plasmid Name pMAC8 Plasmid size 4935 bp 10 Comments The £ coli mac eene was amplified with primers
# B 478 CGG GAT CCG AGC ACA GAA AAA GAA AAG ATG (upper primer with BamHl site)
tf B 479 AAC TGC AGA TTT TGC ATA ACA GTT GC (lower primer with Pstl site)
and pMAC3 as template The PCR product was digested with BamHl and Pstl and inserted in pBETP5 digested with the same enzymes
The SBE TP-mac fusion was control sequenced with primer # C028
The 35S terminator-mac fusion was sequenced with primer tt B456 og # C027
Figure 9
Plasmid name pMAC9 Plasmid size 9 37 kb
Comments Insertion of the 2294 bp EcoRl fragment (Patatin promoter-SBE TP-mac -35S terminator) from pMAC8 in the EcoRl site of pVictor IV Man
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Figure 10
Plasmid name pMACIO Plasmid size 9 37 kb
Comments Insertion of a 2294 bp EcoRl fragment (Patatin promoter-SBE TP-mac-5 35S terminator) from pMAC8 in the EcoRl site of pVictor IV Man
Cloning and sequencing of the mac Rene from E coli
Following. Initially the teachings of Boos and Brand (3), the mac gene was isolated 10 trom the 4 3 kb EcoRl fragment from X phage 8C4 (151) from the Kohara collection (4) The fragment was inserted into the EcoRl sue of plasmid pBluescripi II SK (+) in both orientations yielding plasmids pMACl and pMAC2 (Figures 4 and 5) When harboured in E coli these plasmids gave rise to highly elevated maltose acetyitransferase levels indicating that the 4 3 kb EcoRl fragment contains the mac 15 gene
In order to localise the mac gene on the 4 3 kb EcoRl fragment, the 1 1 kb Pj/I fragment was deleted from plasmid pMAC2 This plasmid construction pMAC3 (Figure 6) also gave rise to increased maltose acetyitransferase levels in strains 20 containing this plasmid, thus demonstrating that the mac gene is present on the 3 2 kb EcoRl-Pstl fragment
The nucleotide sequence of the 3 2 kb Ecofll-Pjrl insert in pMAC3 was then determined 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 proteins of 124, 126, and 183 amino acids *
(Figure 3)
In accordance, 35S-methionine labelling experiments with E coli minicells containing 30 pMAC3 showed the synthesis of proteins having molecular weights corresponding to these sizes
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The 183 codon ort which encodes a protein of a predicted molecular weight of 20073 (Figure 2) is the mac gene, since the E coli maltose acetyl-transterase has an estimated subunit molecular weight of 20 000 (3)
Over-expression of the Mac enzvme in E coli
In order to purify the Mac enzyme, the mac gene was inserted after an isopropylthiogalactosidase (IPTG) inducible phage T7-promoter Al in pUHE21-2 to give pMAC5 (Figure 7) Cultures of E coli strain NF1830 (MC1000 recAl F' 10 lacIqlZ tm5, a gift from Niels Fill, University of Copenhagen) harbouring pMAC5 was found to have highly elevated levels ot maltose acetyitransferase, when expression ot the mac gene is induced by addition of IPTG to the growth medium
Growth Conditions
A 1 LLB culture of NF1830-pM4C5 supplemented withampicillin (100 /*g/ml) and kanamyctn (25 /jg/ml) was grown at 37 °C with vigorous shaking until the A600 reached 0 7 IPTG was added to a final concentration of 2ntM and growth was continued for four hours The cells were harvested by centrifugation (10 mm at 4 20 000 x g) and washed by resuspension in 200 ml 0 9% NaCl The cell pellet was then resuspended in 250 ml 20 mM potassium phosphate pH 7 5 containing 0 4 mM PMSF, 0 4 mg/ml pepstatm and 1 6 mM EDTA The suspension was sonicated 5 x 1 mm using a Vibra Cell VC 600 with a 19 mm High Gain Horn and extender (all from Sorucs and Materials Inc , USA) The homogenate was clarified by 25 centrifugation for 60 min at 90 000 x g at 4°C and subsequent filtration through a 0 22 nm filter
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Purification of Recombinant Mac
The resulting crude extract was applied to a Q-Sepharose 26/10 column (Pharmacia Biotech) equilibrated with 20 mM potassium phosphate pH 7 5 (hereinafter called "buffer A") at a flow rate of 2 ml/min The column was washed with 300 ml of buffer A and the bound protein was eluted by applying a 0 to 0 3 M NaCl linear gradient in buffer A (300 ml) The fractions containing enzyme activity were pooled and applied to a 8 ml Affi-Gel Blue (Biorad) column (16 mm x 26 cm) equilibrated with buffer A at a flow rate of 1 ml/nun The column was washed with 50 ml of the same buffer containing 04 M NaCl The enzyme was then eluted with the same buffer containing 2 M NaCl The active pool was dialysed overnight against buffer A and subsequently concentrated to approximately 3 ml in a Centnprep-30 (Amicon) This fraction was applied to a 6 ml Acetyl-coA-Minileak column equilibrated with buffer A at a flow rate of 0 3 ml/min This affinity resin was made by coupling 200 mg of Acetvl-coA to 5 g (dry weight) of Minileak High (Kem-En-Tek, Denmark) in 10 ml of 1 M NaCOj pH 11 for 20h at room temperature The column was washed with 20 ml of buffer A It was then turned upside down and the pure enryme was eluted in less than 20 ml with buffer A containing 0 5 M NaCl
The purification of the maltose acetvltransferase to homogeneity was achieved after three chromatographic steps From 11 culture we were able to get 5 8 mg pure Mac The yield was 29% and the enzyme was purified 80-fold The purity of the enzyme was assessed both by SDS-PAGE and mass spectrometry The latter revealed a molecular mass of 19,982 Da
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Determination of enzyme concentration and activity
The concentration of pure Mac solutions was estimated spectrophotometrically at 280 am using an extinction coefficient ol 0 66 as determined from the amino acid 5 composition of Mac according to (5) The acetyl-transferase activity of Mac was assayed spectrophotometrically according to a modified Alpers' assay (6) A Perkin Elmer Lambda 18 spectrophotometer was used The assay mixture of a total volume of 1 ml contained a 50 mM potassium phosphate, 2 mM EDTA buffer at pH 7 5, 100 n\ of maltose 1M 100 /*1 of Acetyl-coA 0 4 mM, 10 /il 5,5'-dithiobis(2-nurobenzoic 10 acid) (DTNB) 40 mM dissolved in methanol and 10 /il enzyme The reaction was started bv the addition of enzyme or maltose and was monitored at 412 nm at 25°C One activity unit was defined as the amount of enzyme that produced an increase in absorbance of 1 per minute at 25"C An extinction coefficient of 13 600 M 1 x cm 1 was used for DTN'B in order to calculate the consumption of acetyl coenzyme A
iVterminal sequencing of Recombinant Mac
N-terminal sequencing of pure Mac was performed using an Applied Biosystems 476A protein sequencer One nanornole of protein was desalted by RP-HPLC on a 20 C2 column (4 6/30) prior to loading onto the sequencer The N-termina! sequence of Mac was determined up to residue was determined up to residue 48 and was in complete concordance with the nucleotide sequence of the mac gene (Figure 1) Furthermore, the N-terminal methionine residue was not present on the mature protein (Figure 2)
Production of polyclonal antibodies against Recombinant Mac
Rabbits were immunised subcutaneously at 2-week intervals during 6 weeks and at 4-week intervals thereafter with 90 fig of pure protein emulsified (1 1, vol/vol) with 30 Freund s adjuvant Antisera were tested against Mac m immunoblots and were found highly specific
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Characterisation and activity profile of recombinant Mac
Mass spectrometry studies indicated that Mac may he a trimer
The isoelectric point of Mac was determined by isoelectric focusing on a PhastGel IEF 4-6 5 (Pharmacia) and was found to be 5 7
The pH profile of Mac was investigated between pH 5 and 8 5 at a 100 mM maltose concentration in 50 mM buffers containing 100 mM NaCl Under these conditions, 10 the pH optimum was 7 7
The pH stability of Mac was examined at 25 °C between pH 3 0 and 10 0 Mac was instantaneously inactivated at pH 3 0 but was stable between pH 4 0 and 10 0 for at least six hours
The thermostability of Mac was investigated at pH 7 5 between 40 and 70°C After incubation for four hours at 40°C and 50°C, the remaining activity of Mac was 100% and 75%, respectively Its half-life was 70 min, and 22 mm 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 disacchandes tested maltose and isomaltose were the best substrates
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Table 1 Comparison of the relative activuv of Mac towards various monosaccharides as acetvl-acceptors
— - — 1 - ■ 1 ■ -
SJVaWdl
Relative Activity
(j5?0
(% of activity on glucose)
Glucose
100
Mannose
38
Fructose
17
Galactose
0 9
Table 2 Comparison of the relacive activity ot Mac towards various disacchandes as acetyl-acceptors
Substrate
Relative Activity
(100 mM)
(% of activity on maltose)
Maltose (a-elucose(l,4) a-glucose)
100
Isomaltose (a-glucose(l,4) a-glucose)
110
Lactose (^-galactose 0-(l,6) a-glucose)
04
Sucrose (a-glucose a-(l,4) 0-fructose)
04
Cellobiose (/3-glucose /?-(!,4) /3-glucose)
0
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Table 3 Comparison of the relative activity of Mac towards various maltoohgosaccharides as acetyl-acceptors
Substrate
—
Relative Activity
(50 mM)
(% of activity on maltose)
Maltose
100
Maltotriose
7 5
Maltotetraose
0 5
Maltopentaose
0 9
Maltohexaose
1 2
Maltoheptaose
1 1
Kinetic studies
Kinetic studies of Mac catalysed acetylation reactions revealed that the Km for the acceptor substrate is in the mM range whereas it ts in the fiM range for acetyl-coenzyme A Thus, Mac has about a 1000 fold more affinity for acetyl-coenzyme A than for the acceptor
NMR studies
'H-NMR structure determination of the products of acetylation of glucose and maltose by Mac was investigated
In order to investigate the substrate regio-specificity of Mac regarding the acetylation site of the acceptor substrate, we prepared milligram amounts of acetylated glucose and maltose by incubating 10 mg of glucose or maltose with E coli Mac and 1 mg acetyl-coenzyme A in phosphate buffer at pH / 5 for 48 hours Additional ahquots
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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 us non-reducing glucose moiety These results reveal that Mac acetylates hexoses at their C6 position
Activity of the SBE-Mac fusion in E. coli
Because the 27 amino acid SBE portion of the SBE-Mac fusion in pMAC9 and pMACIO described below mav interfere with the acetyitransferase activity, the SBE-Mac fusion was insened in the E coli expression vector pAL7Sl (Invitrogene San Diego USA) in order to over-express the fusion enzyme in E coli and analyse the ictivitv 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
IN VIVO MODIFICATION OF STARCH IN POTATO
General teachings on potato transformation may be found in our copending patent applications PCT/EP96/03053, PCT/EP96/03052 and PCT/EP94/01082 (the contents 25 of each of which are incorporated herein bv reference)
For the present studies, the following protocol was adopted
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Construction or nlasmids for the expression of the E coh mac eene in potato
The E coli mac gene was amplified -with primers
5'-CGG GAT CCG AGC ACA GAA AAA GAA AAG ATG-3' (upper primer with BamHl site)
and
5'-AAC TGC AGA TTT TGC ATA ACA GTT GC-3' (lower primer with PstI site) and pMAC3 as template
The PCR product was digested with BamHl and Pstl and inserted in pBETPS (see 15 PCT patent application No WO 94/24292, the contents of which are incorporated herein by reference) digested with the same enzymes yielding pMAC8 Thereby, the mac gene is insened in an expression cassette that provides tuber specific expression from a patatin promoter and transcription termination at a CaMV 35S terminator Moreover the Mac enzyme is fused to 102 amino acids of the N-termmus 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 EcoRl expression cassette was isolated from pMAC8 and insened in the EcoRl sue 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 of which are incorporated herein by reference) giving plasmids pMAC9 and pMACIO (Figures 9 and 10, respectively)
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Preparation of potato mmitubers
A segment containing the nodium - i e a segment taken from 2 mm above and 5 mm belovv the nodium - was cut from in vitro grown potato plants or mannose selected 5 shoots (for mannose selection see our earlier patent applications WO 93/05163 and/or WO 94/20627) The leat was removed from the nodium segment and the segment was placed vertically on agar plates with MS medium (Sigma) supplemented with 60 g sucrose'l and 2 mg 6-beazvl-aminopurine/l The nodium segments were grown for 7 days at 20°C wich a 16 hour light period and an 8 hour dark period Subsequently, 10 the plates were wrapped in alu-foil and placed in the dark at 20°C The minirubers were harvested after about 28 days and applied for western analvsis in order to detect Mac expression
Expression of the SBE-Mac fusion in potato mmitubers
Potato mmitubers transformed with the pMAC9 or pMACIO constructs were examined by Western analysis for expression of the E coli mac gene with antibodies raised towards the E colt maltose acetyitransferase The analvsis clearly demonstrated that 3 out of 5 MA.C9 mmitubers and 5 out of 7 MAC10 mmitubers 20 gave a distinct expression of the E coli maltose acetyitransferase The positive mmitubers expressed a 209 ammo acid SBE-Mac fusion that co-migrates with a similar construction expressed in E coli These results indicate that the 75 ammo acid SBE transit peptide, that was originally fused to the 209 amino acid SBE-Mac fusion, has been removed from the SBE-fusion Furthermore, this implies that the 25 transit peptide was correctly processed by the signal peptidase in the amyloplast membrane, and that the SBE-Mac fusion has been directed to the amyloplast
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Immunoblots on potato tuber extracts
0 5 ml potato protein extract was precipitated with 20% TCA tor 30 min on ice Protein precipitates were recovered after centrifugation and resuspended in 50 of 5 SDS-PAGE sample buffer 25 jtl were subsequently loaded onto 15 % polyacrylamide gels After electrophoresis proteins were transferred onto Problot PVDF membranes by semi-dry blotting For immunodetection Mac antiserum was diluted 1 2 000 and secondary antibody was coupled to alkaline phosphatase
In accordance with the Western Blot analysis of the mmitubers described above, the western analysis ot 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 centrifugation Protein concentration was measured using the BCA reagent (Pierce)
Mac activity was measured in duplicates or triplicates as follows 0, 50, 100 or 200 potato extract, 10 /xl of 1 mM acetyl-coenzyme A, 25 jtl of 1 M glucose and assay 25 buffer (50 mM potassium phosphate, 2 mM EDTA, pH 7 5) were mixed per microliter plate well to give a total volume of 250 jil The reaction was started by the addition of acetyl-coenzyme A After 10 mm reaction at room temperature, 25 lil of freshly made 4 mM DTNB was added and A^j was measured immediately Two wells were prepared for each single assay, one with glucose and one without 30 Activity was calculated by subtracting the absorbance of the well without glucose (background absorbance) from that of the well with glucose
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Relatively high levels of Mac activity could be measured in eight out of nine transgenic tubers Some of the tubers had a Mac activity that was 15 to 20 fold above the almost negligible activity found in non-transformed tubers
Viscometric studies
Samples ot starch obtained trom tubers of non-transformed potatoes and from tranr,rormed potatoes according to the present invention were analysed by viscoamvlograph of an aqueous suspension using a Newport Scientific Rapid Visco 10 Analyser 3C The results showed that the starch from the transformed potatoes had a different viscometric profile to the starch from the non-transtormed potato
DSC studies
Samples of starch obtained from tubers of non-transformed potatoes and from transformed potatoes according to the present invention were analysed b\ differential scanning colometry (using a 10% w/w aqueous starch suspension) The samples were heated from 20 to 100°C at a velocity of 10°C per minute The results showed that the starch from the transformed potatoes had a different enthalpy to the starch from 20 the non-transformed potato We additionally found a difference in gelatinisatton temperature for The transformed potatoes compared to the starch from the non-transformed potatoes
Other modifications of the present invention will be apparent to those skilled in the 25 art
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REFERENCES
1 Boos W , Ferenci T & Shuman H A 1981 / Bacteriol 146. 725-732
2 Freundlieb S & Boos \V 1982 Ann Microbiol (Inst Pasteur) 133 A, 181-189
3 Brand B & Boos W 1991 J Biol Chem 266, 14113-14118 10 4 Kohara et al 1987 Cell 50 July 31 issue
Gill S C & von Hippel P H 1989 Anal Biochem 182, 319-326
6 Alpers D H , Appel S H &. Tomkrins G M 1965 J Biol Chem 240, 10-15 13
7 Ogasawara N , Nakai S & Yoshikawa H 1994, DNA Research I, 1-14
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FCT/EP97/01117
SEQUENCER
srouFNrr 'o no \
Amino acid sequence
MSTEKEKHIAGELYRSADETLSRDRLRARQLIHRYNHSLAEEHTLRQQIL 5(1
ADLFGQVTEAY1EPTRCDYGYNIFLGNNFFANFOCVMLDVCPIR1GDNI 100
MLAPGVH 1 YTATIIP I DP VARNSGAELGKPVTI GNNVW I GGRAV INPGVTI 150
GDNVVVASGAVVTKDVPDNVVVGGNP4RI IkKL 193
SEQUENCE ' D NO ?
Nuclectice sequence
ATGAGCACAG
AAAAAGAAAA
GATGATTGCT
GGTGAGTTGT
ATCGCTCGGC
AGATGAGACG
TTATCTCGCG
ATCGCCTGCG
CGCTGTCAG
CTTATTCACC
GATACAATCA
TTCCCTGGCG
GAAGAGCACA
CATTACGCCA
GCAAATTCTC
GCTGATCTAT
TCGGTCAG5T
GACAGAGGCT
TATATTGAGC
CAACGTTTCG
CTGTGACTAT
GGCTATAACA
TTTTTCTCGG
TAATAATTTT
TTCGCCAACT
TCGATTGCuT
GATGCTTGAT
GTCTGCCCTA
TTCGCATCGG
T5ATAACTGT
ATGTTGGCAC
CAGGCGTTCA
TATCTACACG
GCAACACATC
CCATCGACCC
TGTAGCACGT
AATAGCGGTG
CTGAACTGGG
GAAACCCGTC
ACCATCGGTA
ATAACGTCTG
G/>TTGGCGGA
CGCGCGGTCA
TTAACCCTGG
TGTGACCA7T
GGTGATAACG
TCGTGGTAGC
CTCAGGTGCA
GTTGTCACAA
AAGATGTCCC
GGACAACGTT
GTCGTGGGCG
GTAATCCAGC
CAGAATAATT
AAAAAATTGT
AA
Printed from Mimosa 19 00 07
31
rCT/Er97/0U17
SEQUENCE ■ ^ MO 1 Nur I entitle sequence
Complete ruclpotide sequenue of the 3 2 kb EcoRl f'atl fraqmer* in pHAC3
CaAATTCGCCA AAbACTTGAT GGATAAAGAA GCilAAAGGTC 7GATTGAAGC GACGCTGAT 60
GCGGTGCGGA 7GCGTTTACG TCCGATCCTG ATGACCTCGC TGGCGTTTAT CCTCGGCGTT 120
ATGCCGCTGG TTATCAGTAC TGGTGCTGGT TCCGGCGCGC AGAACGCAGT AGGTACCGGT 130
GTAATGGGCG GGATGGTGAC CGCAAfGGTA CTGGCAATCT TCTTCGTTCC GGTArTCTTT 240
GTGGTGGTTC GCCGCCGCT7 TAGCCGCAAG A.ATGAAGATA TCGAGCACAG CCATACT3rC 300
GATCATCATT GATAfAACGT GTAATCACTA AG5CCGCGTA AGCGGCCTTT TTTATGCATA 360
ACCTACGAAC A'TAAGU.T AAT7GAACCA CCAACTCAGG ATCTCATACG AAMCCAGTA 420
TTAACCACGG ATAAAATTCA TAAAAAATAC TCATTGTTAG TTAATTTATA TTAAGTAGCG 480
CTAATAGATT TAATAATCCA TMTCATTTA GAGGCTATTC TTAATTATTT GCGGTAATTC 540
TTTATTCATT CCTCGGTTAT TACGTCATAT TCAGAGCAAT CCTCGTATTA GTGTCACCAA 600
TTTCATCTCC CGATAATCCT GMAIGTTAT GAATAGTTCG AGLAAACTGC 'TTTArCTf.r b60
TGCGGGTTAG TGCTAGTATG AAAAAGTGAG T(.(JGTCCCG CPTCTT'CT AATTG~AATT 70
TTTCGTAATA ATGCGATGAA *ACCTGCAAA GAGrGGCTTA TAGTTAAGCT AACAAACGAG 780
AGGGCAAGTC CAGGTCAGT« AGTTTTTTCC ATCCCGAAAG GTGTCCG^A GTTCAACCGC 340
TAAGAAGGGG ACGCGTTATG GATGAATACT CACCCAAAAG ACATGATATC GCACAGCTTA 900
AGTTTCTCTG TGAAACCCTG TATCATGACT GCCTTGCAAA CCTTGAAGAA AGCAATCATG 960
GCTGGGTAAA CGACCCAACC TCGGCGATCA ACCTCCAGTT GAATGAACTG ATTGAGCATA 1020
TTGCGACCTT CGCACTTAAT TACAAAATTA AGTATAATGA AGACAATAAG CTCATTGAGC 1080
AGATCGACGA ATATCTGGAT GACACCTTTA TGTTGTTCAG TAGTTATGGT ATTAATATGC 1140
AGGATCTTCA GAAATGGCGG AAGTCAGGTA AHCGACTATU CCGTTGTTTT GTCAATGCGA 1200
CGAAAGAGAA TCCTGCGAGT TTATCTTGTT AGAATTATTA CAACCATAGG TAGAAGTATG 1260
TCCGAAAAAC C'TTAACGAA AACCGATTAT TTAATGCGTT TACGTCGTTG CCAGACAATT 1320
GACACGCTGG AGCGGTTTAW TCGAGAAAAA TAAATACGAA TTATCAGATA ATGAACTGGC 1380
GGTATTTTAC TCAGCCGCAl. A'CACCGCCT CGCCGAATT", ACCATGAATA AACTGTACGA 1440
CAAGATCCCT TCCTCAGTA7 GGAAATTTAT TCGCTAATAA ATAATTCGCT TTCGGAGCTA 1SOO
TAACCGGCTG TTTATTAAGA ATTTTATACT TTTTCGCCAT GAAGACATAC CCTATGTGAT 1560
CTTTATCACA CAGATGTAAT GGGAACGTTC TCTTCACTGA CTTTTCGTCT TACTGTGTTG 1620
CCGCATTTTC 4GCAACCGGA GTCAGTAATG AGCACAGAAA AAGAAAAGAT GATTGCTGGT 1630
uAGTTGTATC CCTCGGCAGA TGAGACGTTA TCTCGCGATf GCCTGCGCGC TCGTCAGCTT 1740
ATTCACCGAT ACAATCATTC CCTGGCGGAA GAGCACACAT TACGCCAGCA AATTCTCGCT 18C0
GATCTATTCG GTAGGTGAC AGAGGCTTAT ArTGAGCCAA CGTTTCGCTG TGACTATGGC 1860
TATAACATTT TTCTCGGTAA TAATTTTTTC GfCAACTTCG ATTGCGTGAT GCTTGATGTC 1920
TGCCCTATTC GCATCGGTGA TAACTGTATG TTGGCACCAG GCGTTCATAT CTACACGGCA 1980
ACACATCCCA TCGACCCTGI AbLACGTAAT AGCGGTGCTG AACTGGGGAA ACCCGTCACC 2Q40
40 ATCGGTAATA ACGTCTGGAT TGGCGGACGC GCGGTCATTA ACCCTGGTGT GACCATTGGT 2100
GATAACGTCG 7GGTAGCCTC AGGTGCAGTT GTCACAAMG ATGTCCCGGA CAACGTTGTC 2160
G'GGGCGGTA ATCCAGCCAG AAtAATTAAA AAATTGIAAT CGGTTTTTCG CAACTGTTAT 2220
GCAAAATTGT G5TAGATCTG TTACTTCCCC TCTACTATTC CCACGTTAAA ATAGGGTGTT 2280
CCCTGGAAAG TTGCAGATAC CACGAAGGCA AACCATGACC GAAATACAAC GCCTGCTGAC 2340
45 CGAAACGA1T GAGTCTCTGA ATACCCGCGA AAAACGCGAC AAfAAACCCC GCTTTAGTAT 2400
Printed from Mimosa 19 00 07
32
CAGTTTTATC CGTAAACATC CGGGGCTGTT TATCGGTATG TACGTTGCTT TTTTTGCCAC 2-460
CCTGGCGGTG ATGTTGCAGT CCGAAACGCT GTCAGGCT2T GTCTGGCTAC TGGTTGTATT 2520
ATTTATCCT3 fTTAATGGTT TCTTCTTUT CGATGTCTAC CCACGCTACr GCTATGAAGA 2580
TATCG4CGTC CTGGATTTCC GCGTTTGCTA TAACGGCGAA TGGTACAACA CGCGCTTTGT 2640
ACCTGCCGC3 CTGGTTGAALi LCATCTTGAA CTCTCCGTGT CGCGGATGIT CATAAGGAAC 2700
AACTGCAAAA AATGATCGTC CGTAAAGGTG AACTGTCTTT TTACGATATT TTTACCCTCS 27bO
TCGCGCCGAA TCAACATCTT AAGTTAGGGT TACATACCAu GCGTAAAGCT CTGCGCCTGG 2920
'CAAATGACA ATGATCGTTT CCACCCATCA CTTCATGAAA TACCAGCTCT ACCTCCTTAT 2380
CTCCAGCCAG CCTTTTTCCA CAATCAGATA TACTTTCCCT ACACTGTGTT AATAAGGATA 2940
TGCTGGTGAG AACACGACAT CTGGTCGGCC TTATTTCGGG AGTACTGATT CTTICAGTAT 3000
TGCTGCCTGT CGGCTTAAGC ATCTGGCTGG CCCATCAGCA GGTAGAAACA TCGTTTATTG 3UoO
4AGAGCTGGA TACCTATTCC TCCCGCGTCG CTATTCGAGC CAATAAGGTG GCGACACAAG 3120
GGAAAGATGC GCTGCAG 3137
SEQUENC! ! D NO 4
Complete nucleot'de sequence of tne 3 2 kb EcoR I Fst I fragment m DhUC3 The Ma-enzyme amino acid sequence is also shown below the mac gene coding sequence
(jAATTCGCCAAAGACTTGATGGATAAAGAAGGTAAAGGTCTGAPGAAGCGACGCTTGAT 60
GCGGTGCGGATGCGTTTACGTCCGATCCTGATGACCTCGCTGGCGTTTATCCTCGGCGTT 120
MGCCGC7GGTTAiCAGTACTGGTGCTGGTTCCGGCGCGCAGAACGCAGTAGGTACCGGT 1B 0
GTAATGGGCGGGATGGTGACCGCAACGGTACTGGCAATCTTCTTCGTTCCGGTATTCTTT 24 0
GTGGTGGTTCGCCGCCGCTTTAGCCGCAAGAATGAAGATATCGAGCACAGCCATACTGTC 30 0
GATCATCATTGATACAACGTGTAATCACTAAGGCCGCGTAAGCGGCCTTTTTTATGCATA 360
ACCT ACGAACATT AAGGAGT AATT GAACCACC AACT CAGGAT CT CAT Af GAAAACCAGT A 420
TTAACCAC3GATAAAATTfATAAAAAATACTGATTGTTAGTTAATTTATATTAAGTAGCG 480
CTAATAGATTTAATAATCCATAATCATTTAGAGGCTATTCTTAATTATTTGCGGTAATTC 540
TTTATTCATTCCTCGGTTATTACGTCATATTCAGAGCAATCCTGGTATTAGTGTCACCAA 600
TTT CAT CT GGCGAT AAT CCT GAAATGTT ATGAAT AGTT CGAGCAAACT GC TTTT ACCTGC 660
TGCGGGTT AGT GCT AGT AT GAAAAAGTGAGT CCTGT CCCGCTT CCTTCCT AATT GT AATT 720
TTTCGT AAT AATGCGAT GAAAACCT GCAAAGAGT GGCTT AT AGTT AAGCT AAC AAACGAG 780
AGGGCAAGT CCAGGTCAGT AAGTTTTTTCCATCCCGAAAGGT GT CCGTTAGTT C AACCGC 840
TAAGAAGGGGACGCGTTATGGATGAATACTCACCCAAAAGACATGATATCGCACAGCFA 900
AGTTTCTCTGTGAAACCCTGTATCATGACTGCCTTGCAAACCTTGAAGAAAGCAATCATG 960
GCTGGGT AAACGACCCAACCT CGGCGAT CAACCT CC AGTTGAATGAACTG ATT GAGC AT A 1020
TTGCGACCTTCGCACTTAATTACAAAATTAAGTATAATGAAGACAATAAGCTCATTGAGC 1080
AGATCGACGAATATCTGGATGACACCTTTATGTTGTTCAGTAGTTATGGTATTAATATGC 1140
AGGATCTTCAGAAATGGCGGAAGTCAGGTAAHCGACTATHCCGTTGTTTTGTCAATGCGA 1200
40 CGAAAGAGAAT CCTGCGAGTTT ATCTT GTT AGAATT ATT ACAACCAT ACGT AGAAGTATG 1260
TCCGAAAAACCTTTAACGAAAACCGATTATTTAATGCGTTTACGTCGTTGCCAGACAATT 1320
G ACACGCTGGAGCGGTTTAWTC GAGAAAAAT AAAT ACG AATT AT CAGAT AAT GAACT GGC 1380
GGTATTTTACTCAGCCGCAGATCACCGCCTCGCCGAATTGACCATGAATAAACTGTACGA 1440
CAAGATCCCTTCCTCAGTATGGAAATTTATTCGCTAATAAATAATTCGCTTTCGGAGCTA 1500
45 TAACCGG:TGTTTATTAAGAATTTTATACTTTTTCGCCATGAAGACATACCCTATGTGAT 1560
Printed from Mimosa 19 OO 07
33
CnTATCACArAGATGTAATGGGAACGTTCTCTTCACTGACTTTTCGTCTTACTGTGTTC 1620
CCGCATTTTCAGCAACCGGAGTCAGTAAiGAGCACAGAAAAAGAAAAGATGATTGCTGGT 168C
M S T E < E K M I A G
GAGTTGTATCGCTCGGCAaMGAGACGTrATCTCGCGATCGCCrGCGCGCTCGTCAGCTT 740 *> E l > RSADETLbRDRLRARQL
ATTCACCGATACAATCATTfCCTGGCGGAAGAGCACACATTACGCCAGCAAATTCTCGCT 1800 i HRYNHSLAE THTLROQ I LA
GATCTATTCGGTCAGGTGACAGAGGCTTATATTGAGCCAArGTTTCGCTGTGACTATGGC 1360 OlFGQVTEAY ie^tfrcdyg
"ATAACATTTTTCTCGGTAATAATTTTTTCGCCAACTTCGATTGCGTGATGCTTGATGTC 1920 YNlFlGNNFFANFDCVMLOV
TGCCCTATTCGCATCGGTGATAACTGTATGTTGGCACCAGGCGTTCATATCTACACGGCA 1380 cpirigdncmlapgvhiyta
ACACATCCCATCCACCCTGTAGCACGTAATAGCGGTGCTGAACTGGGGAAACCCGTCACC 2040 15 r H P I DPVARNSGAELGKPVT
ATLGGTAATAACGTCTGGATTGGCGGACGCGCGGTCATTAACCCTGG7GTGACCAtTGG" 2100 I GN NVW IGGRAV INPCj VT IG
GAT AACGTCGTGGTAGCCTCAGGTGCAGTTGTCACAAAAGATGTCCCGGACAACCjTTGTC 2160 D V V VVASGAVVTkOVPDNVV
GTGGGCGGTAATCCAGCCAGAATAATTAAAAMTTGTAATCGGTTTT"tCGCAACTGTTAI 2220 VGGNPARIIKKL
GCAAAATTGTGGTAGATCTGTTACTTCCCCTCTACTATTCCCACGTTAAAATAGGGTGTT 2280
CCCTGGAAAGTTGCAGATACCACGAAGGCAAACGATGACCGAAATACAACGCCTGCTGAC 2340
CGAAACGATTGAGTCTCTGAATACCCGCGAAAAACGCGACAACAAACCCCGCTTTAGTAT 2400
CAGTTTTATCCGTAAACATCCGGGGCTGTTTATCGGTATGTACGTTGCrTTrmGCCAC 2460
CCTGCCGGTGATGTTGCAGTCCGAAACGCTGTCAGGCTCTGTCTGGCTACTGGTTGTATT 2520
ATTTATCCTGCTTAATGGTTTCTTCTTTTTCGATGTCTACCCACGCTACCGCTATGAAGA 2530
TATCGACGTGCTGGATTTCCGCGTTTGCTATAACGGCGAArGGTACAACACGCGCTTTGT 2640
ACCTGCCGCGCTGGTTGAAGCCATCTTGAACTCTCCGTGTCGCGGATGTTCATAAGGAAC 2700
AACTGCAAAAAATGATCGTCCGTAAAGGTGAALTGTCTTTTTACGATATTTTTACCCTCS 2760
TCGCGCCGAATCAACATCTTAAGTTAGGGTTACATACCAGGCGTAAAGCTCTGCGCCTGG 2820
TCAAATGACAATGATCGTTTCCACCCATCACTTCATGAAATACCAGCTCTACCTCCTrAT 2880
CTCCAGCCAGCCTTTTTCCACAATCAGATATACTTTCCCTACACTGTCTTAATAAGGATA 294 0
TGCTGGTGAGAACACGACATCTGGTCGGCCTTATTTCGGGAGTACTGATTfTTTCAGTAT 3000
TGCTGCCrGTCGGCTTAAGCATCTGGCTGGCCCATCAGCAGGTAGAAACATCGTTTATTG 3060
AAGAGCTGGATACCTATTCCTCCCGCGTCGCTATTCGAGCCAATAAGGTGGCGACACAAG 3120
GGAAAGATGCGCTGCAG 3137
40
Printed from Mimosa 19 00 07
WO 9703974 PCT/EP97/01U7
34
- | " iniemiiurui U9H<1'">" s
INDICATIONS RELATING TO \ DEPOSITED MtCKOORCxMSM (PCT Rule Ijitf)
\ nwitaciani -ulc 2e o • o pc Tuc-ours-ium rt s-.a to m ns acscnptiun on-jas 12 13 tne
~ i - ■ . . '■ ' . wi'—
— - 1 1 . ■=—BWCK=3
8 IDC^TlFtCATtO.N OF DEPOSIT r-»ru< acsorni _
- gct i -a on — ciiiomi toes* Qj
41 -"3uj ^rt inj tuuson
r~e Sa^iorai Collest.ons cf Incus -.al ana Harms Sacte-
a L-Tiued * NCIMB)
a,, rtj 3 «esusu±"' nniiuooft 'it Mm nf sostat com nacowmrsi
^3 51, ^iac-t- Drive
^Derccen
AB2 I'V
oni t»; Mmjeon
C« j j <*«.2uiu
7 Harci l99o
Awu>iion Numoc
•NCIM8 40709
C AOOfTlONAL INDICATIONS tUa*t oian*. tft oopttcooiti
This inrorrrution u continued on an Additional met j" ]
in rs3Dtc. zr those cessations in which a Curooean oatsnt Is soutpi and any ofie" Sesi^natea state navma equivalent legislation, a samole of tie oeoosited m croo-gansn mil onlv Cj mace available either until tie oi.olica ion of tne wincun of tne g-ant of tne patent o- after twenty years frc* the cate of filmo if ti; lool.ca'ion nas o«sn refused or withdrawn c" is deeme: to Oe «itndrawn cnlv r/ c-e issue of si.cn a sande to an exaert nominatec Dy tne oe-son rsauestmc tne sa'ncle Hule 23T — ] JPC)
0 DESICNaTED states FOR which INDICATIONS are MaOE f'fmt tnaicsrtcns an nor 'or ail 3,j,jnci,aSiarui e. separate Ft. rn1shi.ng of indications /lien wcnk tfxo, aophcabit)
io«inc^jijontla *3 9e»ow will m tuormnea to toe IfttcnauoruJ Bureau liter of jvtw-zsonxt.1 "hccowt lumnt*
-or r*c*i»fn| Office um only
[XI rnutne*twis rrcnT«] wrthincmccnozMiui appUciiion
Auutoruad oifxtr
Wr3 T BrdcAer Tazeltar for (mcr-iiuonaJ Bureau iac onlv
□ This sheet v»u rectivca bv the InimiuonaJ 9urou on
Autnonxed officcr
Form KT/W>ij4<juiy i*H)
Printed from Mimosa 19 00 07
WO 97/33974 PCT/EP97/01117
ADOiiuiiDr j,nii nlc ^ llnicmanonai acoiiciiiyr s w -w iun?c L 3*6 1 C7H |
INOICaTIONS RELaTINC TO a DEPOSITED \HCftOORC-vMSM (PCT Rule 1 jbts)
A -tg inuc«*,om m-Ct oe o** rsiatr to tne inc.our$anism *c nnoaci line 1?
e-ed to in the ueicnptiun - 71
9 IDENTIFICATION OF DEP05IT
Funhc 300$IU vt ide-
rt on i- cdtiiorui th«
□
NaJ-« 31 —•"/ IfUUtUllOfi
Th- Vauc-al Collections of Incostrial and Marine Bacteria L
."uted {NCTHET
•> .crm oi aeroi irv institution (mc udi/ij pox -i ~oat ana country)
23 St Haca" D-iv-Aaeraee*
AS 2 1 =*v
Uni fcC2 K.iCOCi*
c*t. ul cejuv |7 Marci 1996
Awucuion Sunoer
NCIMB 40790
C ADDITIONAL INDICATIONS fitavt otaut ifntn avohcabttj Thil miomuttan ts co-ni rued on in adUiituiuJ Jhect □
In resDe: :f those desianaticns in wnicn a turoDean patent s souam, ana any o:n-r designates state havna equivalen- legislation, a samols cf tie deoosited ncroorqan-sm uiH onlv be made available either urtil the o-oiication or the Tertian cr vie grant of tie oatent or after twenty years frcn tie cate of filino .f the aca-.ca.ion has &sen refused o- withdrawn o- is deemec -o Oe withdrawn only av ie issue cf sucn a samcle to an exoer, naminateC tv ie De'S^n reauestinq tie s*no'e 3ule 23C> ZPZ )
ft DESIGNATED 5TaTE5 FOR WHICH INDICATIONS are MaDE f\f bt metcaiiom art at for a ts\fnctti Sta it)
E. SEPARATE rt;R.SISHINC or 1!>DICaT10H5 rttuv* ilwrk i/rw atpUiuh)
Tic inciuiioni I it aciox »nl se Jusmmcs u -it (nunatioul Bureau iiicrr.iptryvi/vfTnrrnner.iTii/iMr Mcamii; Accmon V ynbtr u DtjanO
"or rtceivmj Office ux onlv
(S Thu snc* wu r*ccmo withtfve micmwionai ippltoxion
Aumonied ofTtct-
Mrs T Brftckor TazelMr
For IntCTOUonai Bureau tmoniv
] Thu jneei rrcetvea b" ihc ImnuaonaJ Bureau on
AuUionzcd officcr
Form PCT/ftO/lH (July 1991)
Printed from Mimosa 19.00 07
36