WO1998006858A1 - BETA-1,4-ENDOGLUCANASE ISSUE D'$i(ASPERGILLUS NIGER) - Google Patents

BETA-1,4-ENDOGLUCANASE ISSUE D'$i(ASPERGILLUS NIGER) Download PDF

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Publication number
WO1998006858A1
WO1998006858A1 PCT/EP1997/004415 EP9704415W WO9806858A1 WO 1998006858 A1 WO1998006858 A1 WO 1998006858A1 EP 9704415 W EP9704415 W EP 9704415W WO 9806858 A1 WO9806858 A1 WO 9806858A1
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WO
WIPO (PCT)
Prior art keywords
seq
enzyme
sequence
promoter
nucleotide sequence
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PCT/EP1997/004415
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English (en)
Inventor
Preben Rasmussen
Troels Nørgard GRAVESEN
Original Assignee
Danisco A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Danisco A/S filed Critical Danisco A/S
Priority to GB9901905A priority Critical patent/GB2332675A/en
Priority to AU41182/97A priority patent/AU4118297A/en
Publication of WO1998006858A1 publication Critical patent/WO1998006858A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • the present invention relates to an enzyme.
  • the present invention relates to a nucleotide sequence coding for the enzyme.
  • the present invention also relates to one or more uses of the enzyme.
  • the present invention also relates to a promoter suitable for expressing that nucleotide sequence, as well as other nucleotide sequences.
  • the enzyme of the present invention is a glucanase enzyme, more especially an enzyme that can degrade ]S-l ,4-glucosidic bonds.
  • Fruit and vegetable cell walls largely consist of polysaccharide, the major components being pectin, cellulose and xyloglucan (R.R. Selvendran and J.A. Robertson, IFR Report 1989). Numerous cell wall models have been proposed which attempt to incorporate the essential properties of strength and flexibility (P. Albersheim, Sci.
  • composition of the plant cell wall is complex and variable.
  • Polysaccharides are mainly found in the form of long chains of cellulose which is the main structural component of the plant cell wall; hemicellulose which comprises various ⁇ -xylan chains, such as xyloglucans; and pectic substances consisting of galacturonans, rhamnogalacturonans, arabinans, galactans and arabinogalactans.
  • glucans are polysaccharides made up exclusively of glucose subunits.
  • Typical examples of glucans are starch and cellulose.
  • glucanases The enzymes that degrade glucans are collectively referred to as glucanases.
  • a typical glucanase is /3-1,4-endoglucanase.
  • j3-l ,4-endoglucanases have uses in many industries.
  • ⁇ -1 ,4-endoglucanases are used in the pulp industry, the textile industry, and in the formulation of detergents, such as laundry detergents.
  • 0-1 ,4-endoglucanases are used in the food industry, such as the brewing industry.
  • barley is used for production of malt, and, in recent years, as adjunct in the brewing process.
  • problems with high viscosity in the wort can arise because of ⁇ -glucans from the barley .
  • barley contains large quantities of mixed 0-1,3/1 ,4- glucans of very high molecular weight. When dissolved, these glucans produce high viscosity solutions, which can cause troubles in some applications.
  • the high viscosity reduces the filterability of the wort and can lead to unacceptable long filtration times.
  • ⁇ - glucanase has been traditionally added to wort to avoid such problems - i.e. the problem with glucans can be avoided by addition of enzymes, in particular, glucanases, which degrade the polymers.
  • barley can be used for chicken feed because it is cheap, but again the 0-glucan can give problems for the digestion of the chicken.
  • the faeces of chickens feeding on feed containing barley can be very sticky making it difficult to remove and results in dirty eggs.
  • ⁇ -glucanase to the feed the digestibility of the feed can be increased. This, in turn, makes the faeces less sticky.
  • hydrolysis of primary cell wall xyloglucan has also been demonstrated in segments of dark grown squash hypocotyl, during IAA induced growth (K. Wakabayashi el al, Plant Physiol. , 95, 1070-1076, 1991). Furthermore, endohydrolysis of wall xyloglucan is thought to contribute to wall loosening which accompanies cell expansion (T. Hyashi. Ann. Rev. Plant Physiol. & Plant Mol. Biol., 40, 139-168, 1989). In addition, the average molecular weight of xyloglucan has also been shown to decrease during tomato fruit ripening and this may contribute to the tissue softening which accompanies the ripening process (D.J.
  • certain seeds - e.g. Nasturtium - contain up to 30% by weight of xyloglucan, stored in thickened cotyledonary cell walls, which serves as a reserve polysaccharide and is rapidly depolymerised during germination.
  • xyloglucan stored in thickened cotyledonary cell walls, which serves as a reserve polysaccharide and is rapidly depolymerised during germination.
  • it would be useful to increase glucanase activity for example to have a plant with high concentration of glucanase for use in feed.
  • WO 93/20193 discusses endo- ⁇ -l,4-glucanases (EC No. 3.2.1.4). According to WO 93/20193, these glucanases are a group of hydrolases which catalyse endo hydrolysis of 1 ,4- ⁇ -D-glycosidic linkages in cellulose, lichenin, cereal ⁇ -D-glucans and other plant material containing cellulosic parts. Endo-l,4- ⁇ -D-glucan 4-glucano hydrolase is sometimes called endo- ⁇ -l,4-glucanase.
  • the endo- ⁇ -l,4-glucanase of WO 93/20193 exhibits a pH-optimum of 2.0 to 4.0, an isoelectric point of 2.0 to 3.5, a molecular weight of between 30,000 and 50,000, and a temperature optimum between 30 and 70°C.
  • glucans may be found in WO 93/17101, in particular xyloglucans.
  • xyloglucans are 1 ,4- ⁇ -glucans that have been extensively substituted with ⁇ -l,6-xylosyl side chains, some of which are 1,2- ⁇ - galactosylated. They are found in large amounts in the primary cell walls of dicots but also in certain seeds, where they serve different roles.
  • xyloglucan Primary cell wall xyloglucan is fucosylated. Xyloglucan is tightly hydrogen bonded to cellulose microfibrils and requires concentrated alkali or strong swelling agents to release it. Xyloglucan is thought to form cross-bridges between cellulose microfibrils, the cellulose/xyloglucan network forming the major load-bearing/elastic network of the wall. DCB mutated suspension culture cells (cell walls lacking cellulose) release xyloglucan into their media, suggesting that xyloglucan is normally rightly bound to cellulose.
  • EP-A-0339550 reports on an alkaline cellulase produced by Bacillus sp. No sequence data for either the enzyme or the coding sequence are provided. The enzyme is said to be effective for laundry detergent compositions.
  • EP-A-0458162 reports on an proteinase-resistant cellulase produced by Aspergillus niger. No sequence data for either the enzyme or the coding sequence are provided. The enzyme is said to be effective for laundry detergent compositions.
  • WO 90/09436 discloses a thermostable Bacillus (l,3-l,4)- ⁇ -glucanase.
  • WO 97/13862 discloses two cDNA clones coding for cellulase enzymes that are said to possess ⁇ -glucanase activity.
  • the nucleotide sequences of these clones are presented as SEQ ID NO: l: and as SEQ ID NO:3: in WO 97/13862.
  • the amino acid sequences of these cellulase enzymes are presented as SEQ ID NO:2: and as SEQ ID NO:4: in WO 97/13862.
  • a gene of interest (“GOI")
  • GOI gene of interest
  • tissue of an organism such as a filamentous fungus (e.g. Aspergillus niger) or even a plant crop.
  • the resultant protein or enzyme may then be used in industry.
  • the resultant protein or enzyme may be useful for the organism itself.
  • the crop may be made more useful as a feed.
  • the resultant protein or enzyme can be a component of the food composition or it can be used to prepare food compositions, including altering the characteristics or appearance of food compositions. It may even be desirable to use the organism, such as a filamentous fungus or a crop plant, to express non-plant genes, such as for the same purposes.
  • an organism such as a filamentous fungus or a crop plant, to express mammalian genes.
  • mammalian genes include interferons, insulin, blood factors and plasminogen activators.
  • micro-organisms such as filamentous fungi
  • filamentous fungi it is also desirable to use micro-organisms, such as filamentous fungi, to prepare products from GOIs by use of promoters that are active in the micro-organisms.
  • the present invention seeks to provide an enzyme that is useful for industry and also a nucleotide sequence coding for same.
  • the present invention also seeks to provide a promoter that is useful for expressing GOIs.
  • a promoter that is useful for expressing GOIs.
  • an enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 or SEQ ID No. 4.
  • an enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 and the sequence shown as SEQ ID No. 4.
  • an enzyme obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 and the sequence shown as SEQ ID No. 4, wherein SEQ ID No. 3 is nearer the N terminal end than SEQ ID No. 4.
  • the enzyme according to any one of the above-mentioned aspects is capable of exhibiting 3-1,4-endoglucanase activity.
  • an enzyme capable of exhibiting ⁇ -l,4-endoglucanase activity and being obtainable from Aspergillus, wherein the enzyme comprises at least the sequence shown as SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • the enzyme comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably all of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,
  • any two or more of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 may be separated by with another sequence or other sequences.
  • the location of SEQ ID No. 3 is nearer the N terminal end than SEQ ID No. 4 which is nearer the N terminal end than SEQ ID No. 5 which is nearer the N terminal end than SEQ ID No. 6 which is nearer the N terminal end than SEQ ID No. 7 which is nearer the N terminal end than SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics:
  • glucanase activity is endo 3-1,4-glucanase activity.
  • an enzyme having a sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof, but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • an enzyme capable of exhibiting j3-l,4-endoglucanase activity and being encoded by at least any one or more of the nucleotide sequences shown as: SEQ. I.D. No. 2, a variant, homologue or fragment thereof, SEQ. I.D. No. 9, SEQ. I.D. No. 10, SEQ. I.D. No. 11 , SEQ. I.D. No. 12, SEQ. I.D. No. 13, SEQ. I.D. No. 14, but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • nucleotide sequence coding for the enzyme according to the present invention there is provided a nucleotide sequence coding for the enzyme according to the present invention.
  • nucleotide sequence comprising at least the sequence shown as SEQ ID No. 9 or SEQ ID No. 10.
  • nucleotide sequence comprising at least the sequences shown as SEQ ID No. 9 and SEQ ID No. 10.
  • SEQ ID No. 9 is nearer the 5' end than SEQ ID No. 10.
  • nucleotide sequence codes for an enzyme capable of exhibiting 0-1 ,4- endoglucanase activity.
  • nucleotide sequence coding for an enzyme capable of exhibiting 0-1 ,4-endoglucanase activity wherein the nucleotide sequence comprises at least the sequence shown as SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • the nucleotide nucleotide sequence comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably all of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14 may be separated by another sequence or other sequences.
  • SEQ ID No. 12 SEQ ID No. 13, and SEQ ID No. 14 are present then the location of SEQ ID No. 9 is nearer the 5' end than SEQ ID No. 10 which is nearer the 5' end than SEQ ID No. 11 which is nearer the 5' end than SEQ ID No. 12 which is nearer the 5' end than SEQ ID No. 13 which is nearer the 5' end than SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • nucleotide sequence having the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • nucleotide sequence according to the present invention operatively linked to a promoter.
  • nucleotide(s) may be present to ensure expression of the nucleotide sequence - such as a start codon and/or a signal sequence.
  • a fourteenth aspect of the present invention there is provided a process of preparing an enzyme according to the present invention comprising expressing a nucleotide sequence according to the present invention.
  • an enzyme according to the present invention or an enzyme prepared by a process according to the present invention to degrade a glucan there is provided the use of an enzyme according to the present invention or an enzyme prepared by a process according to the present invention to degrade a glucan.
  • an enzyme according to the present invention or an enzyme prepared by a process according to the present invention to prepare a foodstuff (such as a feed).
  • a foodstuff comprising or prepared from the enzyme according to the present invention or an enzyme prepared by a process according to the present invention.
  • a promoter having the sequence shown as or contained within SEQ. I.D. No. 15 or a variant, homologue or fragment thereof.
  • a glucanase enzyme having the ability to degrade 0-1 ,4-glucosidic bonds, which is immunologically reactive with an antibody raised against a purified glucanase enzyme having the sequence shown as SEQ. I.D. No. 1.
  • nucleotide sequence comprising at least the sequence shown as SEQ ID No. 19, or the sequence shown as SEQ ID No. 20, or the sequence shown as SEQ ID No. 21 , or the sequence shown as SEQ ID No. 22.
  • aspects of the present invention include constructs, vectors, plasmids, cells, tissues, organs and transgenic organisms comprising or expressing the aforementioned aspects of the present invention.
  • aspects of the present invention include methods of expressing or allowing expression or transforming any one of the nucleotide sequence, the promoter, the construct, the plasmid, the vector, the cell, the tissue, the organ or the organism, as well as the products thereof.
  • Additional aspects of the present invention include uses of the promoter for expressing GOIs in culture media such as a broth or in a transgenic organism.
  • Further aspects of the present invention include uses of the enzyme for preparing or treating foodstuffs, including animal feed.
  • the enzyme can be prepared in certain or specific cells or tissues, such as in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant.
  • the present invention also provides a GOI coding for the enzyme that can be expressed preferably in specific cells or tissues, such as those of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant.
  • the present invention provides a promoter that is capable of directing expression of a GOI, such as a nucleotide sequence coding for the enzyme according to the present invention, preferably in certain specific cells or tissues, such as in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant.
  • a promoter is used in Aspergillus wherein the product encoded by the GOI is excreted from the host organism into the surrounding medium.
  • the present invention provides constructs, vectors, plasmids, cells, tissues, organs and organisms comprising the GOI and/or the promoter and methods of expressing the same, preferably in specific cells or tissues, such as expression in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, or even a plant.
  • the enzyme of the present invention is sometimes referred to as the endo-0-l,4-glucanase II enzyme or the glucanase II enzyme or the egl-B enzyme or the egl-2 enzyme; whereas the coding sequence therefor is sometimes referred to as the eglB gene or the glucanase II gene or the gene encoding the egl-B enzyme or gene encoding the egl-2 enzyme.
  • the enzyme does not have a cellulose binding domain.
  • the transgenic organism is a fungus.
  • the transgenic organism is a filamentous fungus, preferably Aspergillus.
  • the transgenic organism is a plant.
  • the transgenic organism is a yeast.
  • the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof, and the nucleotide sequence has the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof.
  • the promoter of the present invention is operatively linked to a GOI.
  • the promoter is operatively linked to a GOI, wherein the GOI comprises a nucleotide sequence according to the present invention.
  • the enzyme of the present invention is used in the preparation of a foodstuff - particularly a foodstuff comprising a 0-glucan.
  • Typical foodstuffs include dairy products, meat products, poultry products, fish products and bakery products.
  • the foodstuff is a feed.
  • the foodstuff is a beverage.
  • variant in relation to the enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has 0-1,4-endoglucanase activity, preferably having at least the same activity as the enzyme shown as SEQ
  • 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. l in the attached sequence listings. More preferably there is at least 95 %, such as at least 98% , homology to the sequence shown as SEQ ID No. 1 in the attached sequence listings.
  • variant in relation to the nucleotide sequence coding for the enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for an enzyme having 0-1 ,4-endoglucanase activity, preferably having at least the same activity as the enzyme shown as SEQ I.D. No. 1 - but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • homologue covers homology with respect to sequence and/or structure and/or function providing the resultant nucleotide sequence codes for an enzyme having 0- 1,4-endoglucanase activity.
  • sequence homology i.e. similarity
  • variant in relation to the promoter include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence has the ability to act as a promoter in an expression system - such as the transformed cell or the transgenic organism according to the present invention, preferably having at least the same ability to act as a promoter as the promoter contained within or as shown as SEQ ID No. 15 in the attached sequence listings.
  • homologue covers homology with respect to sequence and/or structure and/or function providing the resultant nucleotide sequence has the ability to act as a promoter.
  • sequence homology preferably there is at least 75 % , more preferably at least 85 %, more preferably at least 90% homology to the promoter contained within or as shown as SEQ ID NO. 15 shown in the attached sequence listings. More preferably there is at least 95 %, such as at least 98% , homology to SEQ ID NO. 15 shown in the attached sequence listings.
  • the promoter comprises at least the nucleotides 567 to 1 136 as shown in Figure 1. More preferably, the promoter comprises at least the nucleotides 369 to 1136 as shown in Figure 1.
  • the present invention also covers sequences that are complementary to the above- mentioned sequences.
  • nucleotide sequences that can hybridise - preferably under stringent conditions - to the nucleotide sequences of the coding sequence or the promoter sequence, respectively.
  • nucleotide in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably it means cDNA.
  • construct which is synonymous with terms such as “conjugate” , “cassette” and “hybrid” - includes the nucleotide sequence according to the present invention directly or indirectly attached to a promoter. It also includes a GOI directly or indirectly attached to the promoter of the present invention.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the S ⁇ 7-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention or the GOI.
  • fused in relation to the present invention which includes direct or indirect attachment.
  • the terms do not cover the natural combination of the gene coding for the enzyme ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
  • a highly preferred embodiment is the or a GOI being operably linked to a or the promoter.
  • the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a filamentous fungus, preferably of the genus Aspergillus. such as Aspergillus niger, or plants, such as potatoes, sugar beet etc. , into which it has been transferred.
  • the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant seed, such as corn, wheat or barley, into which it has been transferred.
  • a marker which allows for the selection of the genetic construct in, for example, a plant seed, such as corn, wheat or barley, into which it has been transferred.
  • markers exist which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
  • vector includes expression vectors and transformation vectors.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • transformation vector means a construct capable of being transferred from one species to another - such as from an E.coli plasmid to a filamentous fungus, preferably of the genus Aspergillus. It may even be a construct capable of being transferred from an E.coli plasmid to an Agrobacterium to a plant.
  • tissue includes isolated tissue and tissue within an organ.
  • organism in relation to the present invention includes any organism that could comprise the promoter according to the present invention and/or the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, wherein the promoter can allow expression of a GOI and/or wherein the nucleotide sequence according to the present invention can be expressed when present in the organism.
  • the organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
  • Other preferred organisms include any one of Bacillus, Aspergillus oryzae, A. tubigensis, A. awamori, Trichoderma reesei, T. viride and T. longibrachiatum.
  • transgenic organism in relation to the present invention includes any organism that comprises the promoter according to the present invention and/or the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, wherein the promoter can allow expression of a GOI and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism.
  • the promoter and/or the nucleotide sequence is (are) incorporated in the genome of the organism.
  • the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
  • transgenic organisms include any one of Bacillus, Aspergillus oryzae,
  • A. tubigensis A. awamori, Trichoderma reesei, T. viride and T. longibrachiatum.
  • the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the promoter according to the present invention, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention or the products thereof.
  • the transgenic organism can comprise a GOI, preferably an exogenous nucleotide sequence, under the control of the promoter according to the present invention.
  • the transgenic organism can also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a promoter, which may be the promoter according to the present invention.
  • transgenic organism does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment.
  • present invention does not cover the native enzyme according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment.
  • the transformed cell or organism could prepare acceptable quantities of the desired compound which would be easily retrievable from, the cell or organism.
  • the construct of the present invention comprises the nucleotide sequence of the present invention and a promoter.
  • promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression.
  • the promoter of the present invention is capable of expressing a GOI, which can be the nucleotide sequence coding for the enzyme of the present invention.
  • the nucleotide sequence according to the present invention is under the control of a promoter that allows expression of the nucleotide sequence.
  • the promoter need not necessarily be the same promoter as that of the present invention.
  • the promoter may be a cell or tissue specific promoter. If, for example, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, stem, sprout, root and leaf tissues.
  • the promoter for the nucleotide sequence of the present invention can be the ⁇ -Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the ⁇ -Amy 637 promoter) as described in PCT patent application PCT/EP95/02195 (incorporated herein by reference).
  • the promoter for the nucleotide sequence of the present invention can be the ⁇ -Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the ⁇ -Amy 351 promoter) as described in PCT patent application PCT/EP95/02196 (incorporated herein by reference).
  • the promoter could be the glucanase promoter - sometimes referred to as the egla promoter - as described in PCT patent application PCT/EP96/01008 (incorporated herein by reference).
  • the promoter could be the arabinofuranosidase promoter as described in PCT patent application
  • the promoter is the promoter of the present invention.
  • the promoters could additionally include features to ensure or to increase expression in a suitable host.
  • the features can be conserved regions such as a Pribnow Box or a TATA box.
  • the promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the GOI.
  • suitable other sequences include the 5 ⁇ /-intron or an ADH intron.
  • Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
  • suitable elements to enhance transcription or translation may be present.
  • An example of the latter element is the TMV 5' signal sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
  • the present invention also encompasses combinations of promoters and/or nucleotide sequences coding for proteins or recombinant enzymes and/or elements.
  • the present invention also encompasses the use of promoters to express a nucleotide sequence coding for the enzyme according to the present invention or the GOI, wherein a part of the promoter is inactivated but wherein the promoter can still function as a promoter. Partial inactivation of a promoter in some instances is advantageous. In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a part of it so that the partially inactivated promoter expresses the nucleotide of the present invention or a GOI in a more specific manner such as in just one specific tissue type or organ.
  • partial inactivation means that the expression pattern of the promoter is modified but wherein the partially inactivated promoter still functions as a promoter.
  • the modified promoter is capable of expressing the nucleotide of the present invention or a GOI in at least one (but not all) specific tissue of the original promoter.
  • One such promoter is the Amy 351 promoter described above.
  • partial inactivation include altering the folding pattern of the promoter sequence, or binding species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognised by, for example, RNA polymerase.
  • Another, and preferable, way of partially inactivating the promoter is to truncate it to form fragments thereof. Another way would be to mutate at least a part of the sequence so that the RNA polymerase can not bind to that part or another part.
  • Another modification is to mutate the binding sites for regulatory proteins for example the CreA protein known from filamentous fungi to exert carbon catabolite repression, and thus abolish the catabolite repression of the native promoter.
  • GOI with reference to the combination of constructs according to the present invention means any gene of interest.
  • a GOI can be any nucleotide that is either foreign or natural to the organism (e.g. filamentous fungus, preferably of the genus Aspergillus, or a plant) in question.
  • Typical examples of a GOI include genes encoding for proteins and enzymes that modify metabolic and catabolic processes.
  • the GOI may code for an agent for introducing or increasing pathogen resistance.
  • the GOI may even be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues.
  • the GOI may even code for a non-native protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans.
  • Examples of GOIs include pectinases, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, rhamno- galacturonases, hemicellulases, endo-0-glucanases.
  • the GOI may be a protein giving nutritional value to a food or crop.
  • Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non-transgenic plant).
  • the GOI may even code for an enzyme that can be used in food processing such as chymosin, thaumatin and ⁇ -galactosidase.
  • the GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for patatin or ⁇ -amylase, ADP-glucose pyrophosphorylase (e.g. see EP-A-0455316), a protease antisense, a glucanase or genomic 0-1,4-endoglucanase.
  • the GOI may even code for an intron of a particular enzyme but wherein the intron can be in sense or antisense orientation.
  • the particular enzyme could be genomic 0-1 ,4-endoglucanase.
  • Antisense expression of genomic exon or intron sequences as the GOI would mean that the natural 0-1 ,4-endoglucanase expression would be reduced or eliminated but wherein the expression of the 0-1,4- endoglucanase gene according to the present invention would not be affected.
  • the GOI can be the nucleotide sequence coding for the arabinofuranosidase enzyme which is the subject of PCT patent application PCT/EP96/01009 (incorporated herein by reference).
  • the GOI can be any of the nucleotide sequences coding for the ADP- glucose pyrophosphorylase enzymes which are the subject of PCT patent application PCT/EP94/01082 (incorporated herein by reference).
  • the GOI can be any of the nucleotide sequences coding for the ⁇ -glucan lyase enzyme which are described in PCT patent application PCT/EP94/03397 (incorporated herein by reference).
  • the GOI can be any of the nucleotide sequences coding for the glucanse enzyme which are described in PCT patent application PCT/EP96/01008 (incorporated herein by reference) .
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see
  • the vector such as an expression vector or a transformation vector
  • the cell, the tissue, the organ, the organism or the transgenic organism is present in combination with at least one GOI.
  • the promoter and the GOI are stably maintained within the transgenic organism.
  • the promoter and/or the GOI (such as the nucleotide sequence according to the present invention) may be maintained within the transgenic organism in a stable extrachromosomal construct. This is preferred for transgenic bacteria and yeast, or even some filamentous fungi.
  • the promoter and/or the GOI (such as the nucleotide sequence according to the present invention) may be stably incorporated within the transgenic organism's genome. This is preferred for some transgenic bacteria and yeast, and most filamentous fungi.
  • the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
  • the transgenic organism can be a yeast.
  • the transgenic organism can even be a plant, such as a monocot or dicot plant.
  • a preferred embodiment according to the present invention is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ⁇ 1000 D (as determined by using an SDS gel); and glucanase activity; wherein the glucanase activity is endo 0-1,4-glucanase activity; and wherein the enzyme comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably all of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 - but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • Another preferred embodiment according to the present invention is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ⁇ 1000 D (as determined by using an SDS gel); and glucanase activity; wherein the glucanase activity is endo 0-1 ,4-glucanase activity; and wherein the enzyme is encoded by a nucleotide nucleotide sequence which comprises at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably all of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14 - but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • a highly preferred embodiment according to the present invention is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ⁇ 1000 D (as determined by using an SDS gel); and glucanase activity; wherein the glucanase activity is endo 0-1 ,4-glucanase activity; and wherein the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof - but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • Another highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ⁇ 1000 D
  • glucanase activity is endo 0-1 ,4-glucanase activity; wherein the enzyme is coded by the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof - but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • Another highly preferred embodiment is an enzyme obtainable from Aspergillus, wherein the enzyme has the following characteristics: a MW of 37,000 D ⁇ 1000 D (as determined by using an SDS gel); and glucanase activity; and wherein the glucanase activity is endo 0-1 ,4-glucanase activity; wherein the enzyme has the sequence shown as SEQ. I.D. No. 1 or a variant, homologue or fragment thereof; and wherein the enzyme is coded by the nucleotide sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof - but wherein the enzyme is not the cellulase sequence of WO 97/13862 and wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • the enzyme sequence is that shown as SEQ. I.D. No. 1.
  • nucleotide sequence is that shown as SEQ. I.D. No. 2.
  • a preferred host organism for the expression of the nucleotide sequence of the present invention and/or for the preparation of the enzyme according to the present invention is an organism of the genus Aspergillus, such as Aspergillus niger.
  • a transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In:
  • filamentous fungi have been widely used in many types of industry for the production of organic compounds and enzymes. For example, traditional Japanese koji and soy fermentations have used Aspergillus sp. Also, in this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry. There are two major reasons why filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracelluar products, for example enzymes and organic compounds such as antibiotics or organic acids. Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous expression according to the present invention.
  • expression constructs are prepared by inserting a GOI (such as a nucleotide sequence coding for an amylase enzyme or even SEQ. I.D. No. 2) into a construct designed for expression in filamentous fungi.
  • a GOI such as a nucleotide sequence coding for an amylase enzyme or even SEQ. I.D. No. 2
  • the constructs contain the promoter according to the present invention (or if desired another promoter if the GOI codes for the enzyme according to the present invention) which is active in fungi.
  • promoters other than that of the present invention include a fungal promoter for a highly expressed extracellulary enzyme, such as the glucoamylase promoter or the ⁇ -amylase promoter.
  • the GOI can be fused to a signal sequence which directs the protein encoded by the GOI to be secreted. Usually a signal sequence of fungal origin is used, such as that of the present invention.
  • a terminator active in fungi ends the expression system, such as that of the present invention.
  • nucleotide sequence according to the present invention or even the GOI
  • This aspect can stabilize the protein encoded by the nucleotide sequence according to the present invention (or even another GOI which encodes a protein of interest (POI)).
  • POI protein of interest
  • a cleavage site recognized by a specific protease, can be introduced between the fungal protein and the protein encoded by the nucleotide sequence according to the present invention (or even the GOI), so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein encoded by the nucleotide sequence according to the present invention (or even another GOI).
  • a site which is recognized by a KEX-2 like peptidase found in at least some Aspergilli (Broekhuijsen et al 1993 J Biotechnol 31 135-145).
  • a fusion leads to cleavage in vivo resulting in protection of the expressed product and not a larger fusion protein.
  • proteins can be deposited intracellularly if the nucleotide sequence according to the present invention (or another GOI) is not fused to a signal sequence. Such proteins will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins. If the nucleotide sequence according to the present invention (or another GOI) is equipped with a signal sequence the protein will accumulate extracelluarly.
  • heterologous proteins are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracelluar proteases which degrade heterologous proteins. To avoid this problem special fungal strains with reduced protease production have been used as host for heterologous production.
  • filamentous fungi For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi (Ballance 1991 , ibid). Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca 2+ ions. The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A commonly used transformation marker is the amdS gene of A.
  • auxotrophic markers such as argB, trpC, niaD and pyrG
  • antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance.
  • a commonly used transformation marker is the amdS gene of A.
  • the transgenic organism can be a yeast.
  • yeast have also been widely used as a vehicle for heterologous gene expression.
  • the species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast
  • Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
  • yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
  • expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast.
  • the constructs contain a promoter active in yeast fused to the nucleotide sequence of the present invention, usually a promoter of yeast origin, such as the GAL1 promoter, is used.
  • a promoter of yeast origin such as the GAL1 promoter
  • a signal sequence of yeast origin such as the sequence encoding the SUC2 signal peptide, is used.
  • a terminator active in yeast ends the expression system.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
  • the transformed yeast cells are selected using various selective markers.
  • markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1 , and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.
  • Another host organism is a plant.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • the present invention relates to a vector system which carries a nucleotide sequence or promoter or construct according to the present invention and which is capable of introducing the nucleotide sequence or promoter or construct into the genome of an organism, such as a plant.
  • the vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system.
  • Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3 , 1-19.
  • One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
  • Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above.
  • a non-limiting example of such a Ti plasmid is pGV3850.
  • the nucleotide sequence or promoter or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T- DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.
  • the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T- DNA sequence, the border part being located on the same vector as the genetic construct.
  • the vector system is an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
  • the promoter or nucleotide sequence or construct of the. present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant.
  • a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant.
  • An example of a useful microorganism is E. coli. , but other microorganisms having the above properties may be used.
  • a vector of a vector system as defined above has been constructed in E. coli. it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.
  • Ti-plasmid harbouring the promoter or nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the promoter or nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
  • a suitable Agrobacterium strain e.g. A. tumefaciens
  • cloning vectors which contain a replication system in E. coli and a marker which allows a selection of the transformed cells.
  • the vectors contain for example pBR 322, the pUC series, the M13 mp series, pACYC 184 etc.
  • the promoter or nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector.
  • the contained plasmid is used for the transformation in E. coli.
  • the E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed.
  • the plasmid is then recovered and then analysed - such as by any one or more of the following techniques: sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
  • the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected.
  • T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A- 120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B. , Alblasserdam, 1985, Chapter V; Fraley, et al , Crit. Rev. Plant Sci. , 4: 1-46; and An et al. , EMBO J. (1985) 4:277-284.
  • infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
  • a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive.
  • the wound is then inoculated with the Agrobacterium.
  • the inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
  • tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
  • Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
  • the present invention provides a glucanase enzyme and a nucleotide sequence coding for the same. In addition, it provides a promoter that can control the expression of that, or another, nucleotide sequence.
  • E.coli containing plasmid pEglB-1 The deposit number is NCIMB 40825.
  • E.coli containing plasmid pEglB-2 The deposit number is NCIMB 40826.
  • nucleotide coding sequences and promoter sequences obtainable from those deposits, including expression vectors, constructs, organisms and transgenic organisms comprising those same sequences or plasmids.
  • Figure 1 presents nucleotide and enzyme sequences
  • Figure 2 presents nucleotide and enzyme sequences
  • Figure 3 presents nucleotide and enzyme sequences
  • Figure 4 presents a plasmid map of pEgl-Bl
  • Figure 5 presents a plasmid map of pEgl-B2
  • Figure 6 presents a genomic map of Egl-B
  • Figure 7 presents a plasmid map of pPR59
  • Figure 8 presents a graph
  • Figure 9 presents a graph.
  • the enzyme was purified as described as follows.
  • the egl-B enzyme was prurified from the crude fermentation broth by using successively Sephadex G25 M gel filtration media for desalting, Q-Sepharose BB,
  • the N-terminal sequence was obtained by sequencing the purified enzyme which gave following sequence:
  • the enzyme was digested using endopeptidase lys-C and three peptides were purified and sequenced. The following sequences were found:
  • the localisation of the primers can be described schematically as follows:
  • the PCR amplification was done using the same program as in first round.
  • the complete sequence of the PCR fragment is shown in Figure 3.
  • the sequence shown in lower case letters is the pT7blue sequence.
  • the underlined nucleic acid sequences are the primer sequences.
  • the double underlined amino acid sequences are peptides 1 and 2/3.
  • the sequence is a combination of two independent pT7-blue clones each containing the PCR fragment in opposite direction.
  • the cloned PCR fragment was radiolabelled and used as a probe to screen an A. niger 4M147 library (see below), and two independent cloned were found. Both clones contained the entire coding sequence.
  • SEQ ID No. 15 comprises the glucanase II promoter.
  • the inoculated NZY plates were incubated over night at 37°C and plaque lifts of the plates were made. Two lifts were made on each plate on Hybond N (Amersham) sheets.
  • the DNA was fixed using UV radiation for 3 min and the sheets were hybridised, using radiolabelled PCR clone as probe.
  • the filters were prehybridised in 25 ml prehybridisation buffer (6.25 ml 20XSSC, 1.25ml 100X Denhard (2% Bovine serum albumin, 2% FicolTM, 2% polyvinylpyrrolidone), 1.25 ml 10 % SDS and 16.25 ml water. 150 ⁇ l 10 mg/ml denatured Salmon sperm DNA was added immediately before use for one hour at 65°C.
  • the prehybridisation buffer was discarded and the filters hybridised over night at 65°C in 25 ml prehybridisation buffed with the PCR fragment labelled with 32 P-dCTP using Ready-to-Go labelling kit (Pharmacia).
  • SDS sodium dodecyl sulphate
  • SSC saline sodium citrate made from a stock solution of 20 x SSC (3M NaCl, 0.3M sodium citrate))
  • the positive clones were isolated using a Pasteur pipette and the phages were then eluted.
  • the clones were purified by plating them on to small petri dishes with NZY and hybridising plaquelifts by essentially the same procedure.
  • genomic DNA sequence for the enzyme of the present invention is shown in FIG. 1
  • Figure 1 and Figure 2 also show the coding sequence and the enzyme sequence.
  • the full enzyme sequence according to the present invention is also shown as SEQ ID No. 1 and the nucleotide coding sequence according to the present invention is shown as SEQ ID No. 2.
  • SEQ ID No. 16 presents the full genomic sequence.
  • SEQ ID No. 17 presents the N-terminal sequence.
  • the endo-0-1 ,4-glucanase II precursor polypeptide presents the endo-0-1 ,4-glucanase II precursor polypeptide.
  • the first 28 amino acids are believed to act as a signal and/or secretory sequence.
  • the mature enzyme comprises 304 amino acids.
  • the ATG start codon is underlined.
  • the N-terminal of the mature enzyme is double underlined.
  • the sequences of the peptides are written in bold.
  • a possible signal sequence cleavage site is marked by an arrow. Further details on the nucleotide sequence are as follows:
  • the molecular weight of the glucanase II enzyme was determined to be 37 kDa using SDS-PAGE. This measured molecular weight is higher than the calculated molecular weight of 33.8 kDa. We believe that the difference is due glycosylation (possibly to up to 10% glycosylation) of the glucanase as evidenced by rapid periodic acid-Schiff staining - i.e. PAS reaction (Van Seuningen and DVeronica 1992 Electrophoresis L3 97- 99) - of the purified enzyme.
  • the purified protein was assayed for endo 0-1,4 glucanase activity using AZO-CM- Cellulose (supplied by Megazyme, Australia) by the instructions given by the manufacturer.
  • the purified enzyme gave a high activity on this substrate.
  • the egl-B (or egl-2) enzyme is that of the present invention.
  • the egl-A (or egl-1) enzyme is that of PCT/EP96/01008.
  • the method comprised measuring the number of reducing end-groups produced by the action of 0-glucanase enzymes on barley-0-glucan. Potassium ferricyanide was used as a colouring agent.
  • Egl-1 and egl-2 were diluted in acetate buffer, pH 5.5, and pre-treated at 50, 60, 70 and 80°C for 30 minutes. After cooling to ambient temperature the residual activity is determined. 3. Results
  • the enzyme of the present invention is heat stable, even though it is produced by a mesophile organism.
  • the trpC terminator was purified from the pBARGTEl (Pall, M.L and Brunelli, J.P. (1993) A series of six fungal transformtion vectors containing polylinkers with multiple unique restriction sites. Fungal genetic Newsletter 40, 59-63).
  • pBARGPEl was digested with Notl and Kpnl and a fragment on ca. 700 bp was purified. The fragment was bluntended using Mung Bean nuclease.
  • pBluescriptIISK+ was digested with Notl and bluntended using Klenow Polymerase and the TrpC terminator fragment was inserted into this vector.
  • the plasmid pEglA-2 was digested with Bglil, and the ends completely filled in using Klenow polymerase. Next the plasmid was digested with Pstl and a fragment on 583 nucleotides was purified. The eglB gene was isolated by amplification of the coding sequence from pEglB-1. A PCR reaction was performed using primer:
  • the PCR amplification was done using the Pfu polymeraseXNew England) according to the instructions of the manufacturer.
  • this reaction amplified a fragment on 1279 nucleotides containing the entire eglB gene (nucleotides 1124-2395 in Figure 1).
  • the PCR fragment was inserted into pBluescript-TrpC construct digested with Smal and dephosphorylates with alkaline phosphatase.
  • the resultant construct was digested with EcoRl and the ends completely filled in using Klenow polymerase, and subsequently digested with Pstl, and the vector purified.
  • the fragment containing the egl-A promoter was inserted into this vector which gave the expression plasmid pPR59.
  • expression plasmid pPR59 - as shown in Figure 7 - was constructed where egl- B was expressed under control of the egl-a promoter. Transformation of Aspergillus Niger
  • Gerlinger, C Blaiseau, P.L. , Cassan, M. , Lebrun, M.H., Parisot, D.. Brygoo, Y. 1989 (Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans. Curr. Genet. 15:453-456) and Punt, P.J., van den Hondel, C. A.M. J.J. 1992 (Transformation of filamentous fungi based on hygromycin B and Phleomycin resistance markers. Meth. Enzym. 216:447-457).
  • spores from one PDA (Potato Dextrose Agar - from Difco Lab. Detroit) plate of fresh sporulated N400 (CBS 120.49, Centraalbureau voor Schimmelcultures, Baarn) (7 days old) were washed off in 5-10 ml water.
  • the mycelium was harvested using Miracloth paper and 3-4 g wet mycelium were transferred to a sterile petri dish with 10 ml STC (1.2 M sorbitol, 10 mM Tris HC1 pH 7,5, 50 mM CaCL) with 75 mg lysing enzymes (Sigma L-2265) and 4500 units lyticase (Sigma L-8012).
  • the mycelium was incubated with the enzyme until the mycelium was degraded and the protoplasts were released.
  • the degraded mycelium was then filtered through a sterile 60 ⁇ m mesh filter.
  • the protoplasts were harvested by centrifugation 10 min at 2000 ⁇ m in a swing out rotor. The supernatant was discarded and the pellet was dissolved in 8 ml 1.5 M MgSO 4 and then centrifuged at 3000 m for 10 min.
  • the upper band, containing the protoplasts was transferred to another tube, using a transfer pipette and 2 ml 0.6 M KC1 was added. Carefully 5 ml 30% sucrose was added on the top and the tube was centrifuged 15 min at 3000 ⁇ m. The protoplasts, lying in the interface band, were transferred to a new tube and diluted with 1 vol. STC. The solution was centrifuged 10 min at 3000 ⁇ m. The pellet was washed twice with STC, and finally solubilised in 1 ml STC. The protoplasts were counted and eventually concentrated before transformation.
  • the supernatant was removed and the protoplasts were solubilized in the remaining of the supernatant. 3-5 ml topagarose was added and the protoplasts were quickly spread on selective plates.
  • Transformants of A. niger 3M43 were prepared (as described above) and analysed for glucanase II expression. Those transformants expressed the heat stable glucanase enzyme of the present invention.
  • An expression vector was made by fusing fragments of the glucanase promoter to the gene encoding 0-glucuronidase from E. Coli. These constructs were used in a co- transformation experiment with the hygromycin resistance gene as the selectable marker. 0-glucuronidase activity was detected when the promoter fragments comprised at least the nucleotides 567 to 1136, such as fragments comprising at least the nucleotides 369 to 1136 as shown in Figure 1.
  • the enzyme was produced efficiently in solid state fermentation.
  • a transformed organism such as transformed A. niger (e.g. transformed A. niger as mentioned above)
  • the enzyme can be produced in a more convenient way - such as by use of submerge fermentation techniques.
  • Antibodies were raised against the enzyme of the present invention by injecting rabbits with the purified enzyme and isolating the immunoglobulins from antiserum according to procedures described according to N Harboe and A Ingild ("Immunization, Isolation of Immunoglobulins, Estimation of Antibody Titre" In A Manual of Quantitative Immunoelectrophoresis, Methods and Applications, N H Axelsen, et al (eds.), Universitetsforlaget, Oslo, 1973) and by T G Cooper
  • a foodstuff (such as a feed) was prepared and tested as described in the following text.
  • the egl-B (or egl-2) enzyme is that of the present invention.
  • the egl-A (or egl-1) enzyme is that of PCT/EP96/01008.
  • the heat stability of the enzyme of the present invention makes it attractive for feed use since feeds often have to be heat treated and pelleted before use to avoid
  • the present invention provides a novel and inventive 0-1,4- endoglucanase, as well as the coding sequence therefor.
  • An important advantage of the present invention is that the enzyme can be produced in high amounts.
  • the present invention therefore provides an enzyme having glucanase activity wherein the enzyme can be prepared in certain or specific cells or tissues, such as in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger.
  • the enzyme may even be prepared in a plant.
  • the present invention provides a promoter that is capable of directing expression of a GOI, such as a nucleotide sequence coding for the enzyme according to the present invention, preferably in certain specific cells or tissues, such as in just a specific cell or tissue, of an organism, typically a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, or even a plant.
  • a GOI such as a nucleotide sequence coding for the enzyme according to the present invention
  • the promoter is used in Aspergillus wherein the product encoded by the GOI is excreted from the host organism into the surrounding medium.
  • the promoter may even be tailored (if necessary) to express a GOI in a plant.
  • Some of the advantages of the present invention are that it provides a means for preparing a glucanase enzyme and the nucleotide sequence coding for the same. In addition, it provides a promoter that can control the expression of that, or another, nucleotide sequence.
  • the enzyme of the present invention is advantageous as it has a high fibre-conversion potential. In addition, there are fewer processing problems when the enzyme is used, particularly with non-starchy polysaccharides. In addition, the enzyme efficiently degrades 0-glucans. Thus, the enzyme of the present invention is advantageous for preparing or for use as feed supplements.
  • the enzyme of the present invention is advantageous for use in the brewing industry as it can be used to lower the viscosity of beer, and also to improve the filterability of beer. This is important as large molecular weight glucans in beer and the like can cause filtration difficulties and give rise to sediments, gels and hazes.
  • the enzyme can be used to prepare useful feeds containing cereals, such as barley, maize, rice etc.
  • useful feeds containing cereals such as barley, maize, rice etc.
  • the present invention will be described by way of numbered paragraphs.
  • An enzyme obtainable from Aspergillus wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 and the sequence shown as SEQ ID No. 4.
  • An enzyme obtainable from Aspergillus wherein the enzyme comprises at least the sequence shown as SEQ ID No. 3 and the sequence shown as SEQ ID No. 4, wherein SEQ ID No. 3 is nearer the N terminal end than SEQ ID No. 4.
  • An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus wherein the enzyme comprises at least the sequence shown as SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus wherein the enzyme comprises at least two of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus wherein the enzyme comprises at least three of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.
  • An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus wherein the enzyme comprises at least four of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.
  • An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being obtainable from Aspergillus wherein the enzyme comprises at least five of the sequences shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.
  • glucanase activity is endo 0-1,4-glucanase activity.
  • An enzyme capable of exhibiting 0-1,4-endoglucanase activity and being encoded by at least any one or more of the nucleotide sequences shown as: SEQ. I.D. No. 2, a variant, homologue or fragment thereof, SEQ. I.D. No. 9, SEQ. I.D. No. 10, SEQ. I.D. No. 11, SEQ. I.D. No. 12, SEQ. I.D. No. 13, SEQ. I.D. No. 14, but wherein the enzyme is not the cellulase sequence of WO 97/13862.
  • a nucleotide sequence comprising at least the sequence shown as SEQ ID No. 10.
  • a nucleotide sequence comprising at least the sequences shown as SEQ ID No. 9 and SEQ ID No. 10.
  • a nucleotide sequence comprising at least the sequences shown as SEQ ID No.
  • SEQ ID No. 9 and SEQ ID No. 10, wherein SEQ ID No. 9 is nearer the 5' end than SEQ ID No. 10.
  • nucleotide sequence according to any one of paragraphs 21 to 24 wherein the nucleotide sequence codes for an enzyme capable of exhibiting 0-1,4-endoglucanase activity.
  • nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity comprises at least the sequence shown as SEQ ID No. 11.
  • a nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity wherein the nucleotide sequence comprises at least the sequence shown as SEQ ID No. 12, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity comprises at least the sequence shown as SEQ ID No. 13.
  • a nucleotide sequence coding for an enzyme capable of exhibiting 0-1 ,4- endoglucanase activity wherein the nucleotide sequence comprises at least the sequence shown as SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • a nucleotide sequence coding for an enzyme capable of exhibiting 0-1 ,4- endoglucanase activity wherein the nucleotide sequence comprises at least two of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • a nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity wherein the nucleotide sequence comprises at least three of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.
  • nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity comprises at least four of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.
  • nucleotide sequence coding for an enzyme capable of exhibiting 0-1,4- endoglucanase activity wherein the nucleotide sequence comprises at least five of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.
  • a nucleotide sequence coding for an enzyme capable of exhibiting 0-1 ,4- endoglucanase activity wherein the nucleotide sequence comprises at least all of the sequences shown as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 , SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.
  • nucleotide sequence having the sequence shown as SEQ. I.D. No. 2 or a variant, homologue or fragment thereof or a sequence complementary thereto, but wherein the nucleotide sequence is not the cellulase sequence of WO 97/13862.
  • a nucleotide sequence comprising at least the sequence shown as SEQ ID No. 20.
  • a nucleotide sequence comprising at least the sequence shown as SEQ ID No. 21.
  • a nucleotide sequence comprising at least the sequence shown as SEQ ID No. 22.
  • a vector comprising or capable of expressing the invention of any one of paragraphs 1 to 42.
  • a plasmid comprising or capable of expressing the invention of any one of paragraphs 1 to 43.
  • a transgenic organism comprising or capable of expressing the invention according to any one of paragraphs 1 to 44.
  • a process of preparing an enzyme according to any one of paragraphs 1 to 19 comprising expressing a nucleotide sequence according to any one of paragraphs 20 to 41.
  • a foodstuff comprising or prepared from the enzyme according to any one of paragraphs 1 to 19 or an enzyme prepared by a process according to paragraph 50 or 51.
  • a promoter according to paragraph 56 operatively linked to a GOI.
  • ATCCTCTCAG ATGTTGATCC TTCTTCTCCC GTCACGGACC TCATTTCCGT ATCAGTTTCA 840
  • SEQUENCE ID NO. 18 (i) SEQUENCE CHARACTERISTICS:
  • Nacre Signature (s) of person. s) havlr.5 the powe: to represent -the International Depositary Authority or of authorised official(s):

Abstract

Cette invention concerne une enzyme de type glucanase, ainsi qu'une séquence de nucléotide codant cette enzyme, et un promoteur.
PCT/EP1997/004415 1996-08-15 1997-08-11 BETA-1,4-ENDOGLUCANASE ISSUE D'$i(ASPERGILLUS NIGER) WO1998006858A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
GB9901905A GB2332675A (en) 1996-08-15 1997-08-11 Beta-1,4-endoglucanase from aspergillus niger
AU41182/97A AU4118297A (en) 1996-08-15 1997-08-11 Beta-1,4-endoglucanase from (aspergillus niger)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9617184.8A GB9617184D0 (en) 1996-08-15 1996-08-15 Enzyme
GB9617184.8 1996-08-15

Publications (1)

Publication Number Publication Date
WO1998006858A1 true WO1998006858A1 (fr) 1998-02-19

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PCT/EP1997/004415 WO1998006858A1 (fr) 1996-08-15 1997-08-11 BETA-1,4-ENDOGLUCANASE ISSUE D'$i(ASPERGILLUS NIGER)

Country Status (3)

Country Link
AU (1) AU4118297A (fr)
GB (2) GB9617184D0 (fr)
WO (1) WO1998006858A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070998A1 (fr) * 2000-03-20 2001-09-27 Dsm N.V. Bêta-glucanases issues de talaromyces emersonii
WO2009111692A2 (fr) * 2008-03-06 2009-09-11 Novozymes A/S Polypeptides possédant une activité d'endoglucanase et polynucleotides codant pour ceux-ci
EP2319920A1 (fr) 2005-12-22 2011-05-11 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
CN110564625A (zh) * 2019-08-13 2019-12-13 内蒙古世洪农业科技有限公司 一种抗盐碱黄柄曲霉菌及其分离方法和应用

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WO1993020193A1 (fr) * 1992-03-27 1993-10-14 Novo Nordisk A/S ENDO-β-1,4-GLUCANASE ET SEQUENCE D'ADN
WO1997013862A1 (fr) * 1995-10-13 1997-04-17 Gist-Brocades B.V. Cellulases fongiques

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AKIBA S. ET AL.: "Purification and characterization of a protease-resistant cellulase from Aspergillus niger", JOURNAL OF FERMENTATION AND BIOENGINEERING, vol. 79, no. 2, 1995, pages 125 - 130, XP002051165 *
SINGH A. ET AL.: "GENERAL AND KINETIC PROPERTIES OF ENDOGLUCANASE FROM ASPERGILLUS NIGER", FEMS MICROBIOLOGY LETTERS, vol. 71, no. 1/02, 1 September 1990 (1990-09-01), pages 221 - 224, XP000611394 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070998A1 (fr) * 2000-03-20 2001-09-27 Dsm N.V. Bêta-glucanases issues de talaromyces emersonii
US9593324B2 (en) 2005-12-22 2017-03-14 Roal Oy Treatment of cellulosic material and enzymes useful therein
EP2319920A1 (fr) 2005-12-22 2011-05-11 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
EP2453014A1 (fr) 2005-12-22 2012-05-16 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
EP2453013A1 (fr) 2005-12-22 2012-05-16 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
US8409836B2 (en) 2005-12-22 2013-04-02 Roal Oy Treatment of cellulosic material and enzymes useful therein
US9758777B2 (en) 2005-12-22 2017-09-12 Roal Oy Treatment of cellulosic material and enzymes useful therein
WO2009111692A3 (fr) * 2008-03-06 2009-11-26 Novozymes A/S Polypeptides possédant une activité d'endoglucanase et polynucleotides codant pour ceux-ci
US20110061135A1 (en) * 2008-03-06 2011-03-10 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
US8609932B2 (en) 2008-03-06 2013-12-17 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2009111692A2 (fr) * 2008-03-06 2009-09-11 Novozymes A/S Polypeptides possédant une activité d'endoglucanase et polynucleotides codant pour ceux-ci
CN110564625A (zh) * 2019-08-13 2019-12-13 内蒙古世洪农业科技有限公司 一种抗盐碱黄柄曲霉菌及其分离方法和应用
CN110564625B (zh) * 2019-08-13 2023-01-24 内蒙古世洪农业科技有限公司 一种抗盐碱黄柄曲霉菌及其分离方法和应用

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GB2332675A (en) 1999-06-30
GB9617184D0 (en) 1996-09-25
AU4118297A (en) 1998-03-06

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