WO1993011249A1 - PROCEDE DE CLONAGE DE PROTEINES DANS LA LEVURE ET CELLULASE PROVENANT DE $i(HUMICOLA INSOLENS) - Google Patents

PROCEDE DE CLONAGE DE PROTEINES DANS LA LEVURE ET CELLULASE PROVENANT DE $i(HUMICOLA INSOLENS) Download PDF

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WO1993011249A1
WO1993011249A1 PCT/DK1992/000360 DK9200360W WO9311249A1 WO 1993011249 A1 WO1993011249 A1 WO 1993011249A1 DK 9200360 W DK9200360 W DK 9200360W WO 9311249 A1 WO9311249 A1 WO 9311249A1
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Prior art keywords
enzyme
protein
interest
seq
dna
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PCT/DK1992/000360
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English (en)
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Henrik DALBØGE
Hans Peter Heldt-Hansen
Grethe Rasmussen
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Novo Nordisk A/S
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Priority to JP5509731A priority Critical patent/JPH08504560A/ja
Priority to CA002124939A priority patent/CA2124939A1/fr
Priority to BR9206866A priority patent/BR9206866A/pt
Priority to EP93900092A priority patent/EP0618974A1/fr
Publication of WO1993011249A1 publication Critical patent/WO1993011249A1/fr
Priority to FI942644A priority patent/FI942644A0/fi

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—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)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38654—Preparations containing enzymes, e.g. protease or amylase containing oxidase or reductase
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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 a method of screening for DNA sequences coding for proteins of interest, as well as to a process for producing such proteins of interest .
  • the present invention relates to a method of screening for a DNA sequence coding for a protein of interest, the method comprising (a) cloning, in suitable vectors, a DNA library from an organism suspected of producing one or more proteins of interest,
  • step (c) culturing the host cells under suitable conditions to express any protein of interest encoded by a clone in the DNA library, and (d) screening for positive clones by determining any activity of a protein expressed in step (c).
  • yeast cloning of prokaryotic genes in Bacillus has previously been described.
  • the prokaryotic systems devised for expression cloning are not operable for the cloning of eukaryotic genes which are generally difficult to express in Bacillus.
  • expression cloning of eukaryotic genes in mammalian cells has been described, it is more advantageous to use yeast as a host organism as it is possible to obtain a much higher transformation frequency than with mammalian cells, and as yeast is far easier to cultivate.
  • the yeast clones are stable whereas the mammalian expression cloning system described in the references cited above is based on transient expression in COS cells.
  • the yeast system results in pure clones after the initial screening and, therefore, they need not be screened in pools and subpools as in the mammalian system. Apart from this conventional selection systems may be used to select yeast transformants.
  • yeast cells appear to be able to express heterologous genes extracellularly by means of heterologous secretion signals in amounts which are sufficient for screening purposes.
  • expression cloning of certain proteins in yeast has been described previously (G.L. McKnight and B.L. McConaughy, Proc. Nat. Acad. Sci. USA 80, 1983, pp. 4412-4416)
  • no such requirement is necessary for the yeast host strain to be used in the method.
  • the gene products of the previously described method are intracellular rather than extracellular as in the present method.
  • the advantage presented by the present screening method is primarily that it requires no prior knowledge of the structure of the protein of interest. This means that the rate at which novel genes may be isolated and, consequently, novel products be developed may be greatly increased. Furthermore, the method permits screening for multiple protein activities and may even result in the isolation of several different genes coding for the same type of proteins.
  • the present invention relates to a process for producing a protein of interest in a heterologous host cell, the process comprising transforming a suitable heterologous host cell with a DNA sequence coding for a protein of interest, which DNA sequence has been isolated by the screening method of the invention, culturing the transformed cells under suitable conditions to express the protein, and recovering the expressed protein from the culture.
  • the present invention relates to an enzyme which exhibits cellulase activity, and which has the following characteristics (a) the DNA sequence encoding the enzyme has been isolated from a DNA library of Humicola insolens.
  • said DNA sequence comprises at least one of the following partial sequences
  • the enzyme comprises a cellulose-binding domain
  • the enzyme exhibits endocellulase activity in the presence of linear alkyl benzene sulfonate.
  • the enzyme of the invention may be isolated by the method of the invention.
  • cellulose-binding domain is intended to indicate an amino acid sequence capable of effecting binding of the enzyme to a cellulosic substrate.
  • Cellulose-binding domains have been found to be important for the endoglucanase activity of cellulytic enzymes on substrates
  • endocellulase activity refers to the ability of the enzyme to degrade cellulose to glucose, cellobiose, triose and other cellooligosaccharides, as determined by the formation of clearing zones in a carboxymethyl cellulose (CMC) gel under the conditions specified below.
  • CMC carboxymethyl cellulose
  • the enzyme of the present invention shows substantially unchanged stability in the presence of linear alkyl benzene sulfonates. This is an important advantage as linear alkyl benzene sulfonates are commonly used in detergent compositions.
  • the DNA library is preferably a cDNA library prepared from the mRNA of an organism suspected of producing one or more proteins of interest.
  • a cDNA library prepared from the mRNA of an organism suspected of producing one or more proteins of interest.
  • yeast hosts may not be able to splice eukaryotic genomic DNA correctly, and therefore a positive result of the screening may more often be obtained by using cDNA instead.
  • the organism suspected of producing one or more proteins of interest is typically a eukaryotic organism, in particular a fungus since fungi are known to produce a large number of different proteins which makes the traditional process of isolating a gene coding for a particular protein product by initially purifying each protein separately particularly cumbersome.
  • This makes it particularly advantageous to screen fungal DNA libraries by the method of the invention because a large number of different protein activities (and DNAs coding for them) may be identified within a relatively short time-span using the same library.
  • screening of yeast colonies for different protein activities is far more efficient than screening of filamentous fungi as a large number (i.e. about 500-1000) of yeast colonies may be grown on each plate, compared to 10-50 filamentous fungi/plate.
  • yeast clones may be screened by the method of the invention for expression of one or more enzyme activities by means of appropriate assays.
  • enzymes which may be identified by this method are carbohydrases, e.g.
  • cellulytic enzymes such as endocellulases, cellobiohydrolases , ⁇ -glucanases or ⁇ -glucosidases, hemicellulases or pectinolytic enzymes such as galactanases, galactosidases, mannanases, xylanases, pectinases, xylosidases, arabanases, rhamnogalacturonases or amylases; esterases, e.g. lipolytic enzymes such as lipases; proteases; oxidoreductases, e.g. peroxidases, oxidases or laccases; or isomerases, e.g.
  • glucose isomerase A wide range of indicator systems for the different types of enzymes may be used for the screening of yeast colonies on agar plates. For instance, endocellulases may be identified by clearing zones in carboxymethyl cellulose after staining with Congo Red; similar methods may be used to detect glucanases, xylanases and galactanases. Endoarabanases may be identified by blue zones obtained after dissolution of azurine-crosslinked araban. This principle is general and may be used to detect, e.g., mannanases, xylanases and cellulases.
  • Pectinases may be identified by clearing zones in pectin after precipitation with quaternary ammonium ions.
  • Amylases may be identified by clearing zones in starch after visualisation with iodine, ⁇ -galactosidases may be detected by the release of p-nitrophenol (yellow) from pnitrophenol- ⁇ -galactopyranoside or by coupling released naphthole or naphthole derivatives from, e.g., 1-naphthole- ⁇ - galactopyranoside to azo dyes; similar methods may be used to detect ⁇ -galactosidases, ⁇ - and ⁇ -glycosidases, ⁇ -xylosidase and ⁇ -mannosidase.
  • Peroxidases and oxidases may be detected by the reaction of 4-aminoantipyrine with ESBT (N-ethyl-N-sulfobutyl-m-toluidine) in the presence of hydrogen peroxide (generating a purple colour).
  • ESBT N-ethyl-N-sulfobutyl-m-toluidine
  • Lipases may be detected by the formation of clearing zones in tributyrine emulsions.
  • the yeast strain selected to be the host cell for the DNA library may be any yeast strain conventionally used for the cloning of heterologous DNA sequences.
  • the yeast strain may suitably be selected from Saccharomyces sp., such as Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum or Schizosaccharomyces pombe, Hansenula sp. Pichia sp., Yarrowia sp. such as Yarrowia lipolytica, or Kluyveromvces sp. such as Kluweromyces lactis.
  • the vector used for cloning the DNA library may be any vector which may conveniently be subjected to recombinant DNA procedures.
  • the DNA sequence derived from the library should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the yeast cell. Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255, 1980, pp. 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp.
  • Each DNA library sequence may also be operably connected to a suitable terminator, such as the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) or yeast MF ⁇ terminators.
  • a suitable terminator such as the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) or yeast MF ⁇ terminators.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in yeast cell.
  • a DNA sequence enabling the vector to replicate in yeast cell.
  • An example of such a sequence is the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
  • the vector is a yeast/E. coli shuttle vector, it will also include an origin of replication region which is functional in E. coli.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell such as URA3 , or one which confers resistance to a drug, e.g. ampicillin, kanamycin, chloramphenicol, tetracyclin, etc., or the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40, 1985, pp. 125-130).
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell such as URA3 , or
  • yeast cells may for instance be effected by protoplast formation followed by transformation or by the LiAc method in a manner known per se.
  • the heterologous host cell transformed with the isolated DNA sequence may be a strain of a filamentous fungus, e.g. fungi belonging to the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes or Fungi Imperfecti, including Hyphomycetes such as the genera Aspergillus, Trichoderma, Penicillium, Fusarium or Humicola.
  • the filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recombinant proteins, e.g. a strain of Aspergillus sp., such as A. niger, A. nidulans or A. oryzae.
  • a strain of Aspergillus sp. such as A. niger, A. nidulans or A. oryzae.
  • A. oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023.
  • a preferred promoter for use in the process of the present invention is the A. oryzae TAKA amylase promoter as it exhibits a strong transcriptional activity in A. oryzae.
  • the sequence of the TAKA amylase promoter appears from EP 238 023.
  • Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the techniques used to transform a fungal host cell may suitably be as described in EP 238 023.
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing Aspergillus cells.
  • the mature protein secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • a preferred endocellulase enzyme according to the invention is an enzyme, a crude extract (15 ⁇ l) of which diluted with one volume of 0.15% linear alkyl benzene sulfonate and added to a 2% agarose gel containing 1% carboxymethyl cellulose in 50 mM sodium phosphate buffer, pH 7, mixed with one volume of 0.15% linear alkyl sulfonate forms a clearing zone in said agarose gel after 18 hours of incubation, which clearing zone is equal to (less 3 mm) the clearing zone formed in a similar carboxymethyl cellulose gel not containing any linear alkyl benzene sulfonate, provided that the concentration of enzyme in the extract is such that a clearing zone of at least 10 mm is formed in a carboxymethyl cellulose gel (with no linear alkyl benzene sulfonate) under the conditions specified above.
  • the DNA sequence coding for the enzyme may for instance be isolated by screening a cDNA library of Humicola insolens, e.g strain DSM 1800, deposited on 1 October 1981 at the Deutsche Sammlung von Mikroorganismen in accordance with the provisions of the Budapest Treaty and selecting for clones expressing the appropriate enzyme activity (i.e. endocellulase activity as defined above).
  • the appropriate DNA sequence may then be isolated from the clone by standard procedures, e.g. as described in Example 1.
  • the invention relates to a detergent additive comprising the enzyme of the invention.
  • the detergent additive may suitably be in the form of a non-dusting granulate, stabilized liquid or protected enzyme.
  • Non-dusting granulates may be produced e.g. according to US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art.
  • Protected enzymes may be prepared according to the method disclosed in EP 238 216.
  • detergent additive may further include one or more other enzymes, such as a protease, lipase, peroxidase or amylase, conventionally included in detergent additives.
  • enzymes such as a protease, lipase, peroxidase or amylase, conventionally included in detergent additives.
  • the present invention relates to a detergent composition
  • a detergent composition comprising the enzyme of the invention.
  • the detergent composition of the invention may be in any convenient form, e.g. as powder, granules or liquid.
  • a liquid detergent may be aqueous, typically containing up to 90% water and 0-20% organic solvent.
  • the detergent composition comprises a surfactant which may be anionic, non-ionic, cationic, amphoteric or a mixture of these types.
  • the detergent will usually contain 0-50% anionic surfactant such as linear alkyl benzene sulphonate (LAS), alpha-olefin sulphonate (AOS), alkyl sulphate (AS), alcohol ethoxy sulphate (AES) or soap.
  • LAS linear alkyl benzene sulphonate
  • AOS alpha-olefin sulphonate
  • AS alkyl sulphate
  • AES alcohol ethoxy sulphate
  • the detergent composition may additionally comprise one or more other enzymes, such as an amylase, lipase, peroxidase, oxidase or protease.
  • the pH (measured in aqueous detergent solution) will usually be neutral or alkaline, e.g. 7-11.
  • the detergent may contain
  • a detergent builder such as zeolite, phosphate, phosphonate, citrate, NTA, EDTA or DTPA, alkenyl succinic anhydride, or silicate, or it may be unbuilt (i.e. essentially free from a detergent builder). It may also contain other conventional detergent ingredients, e.g. fabric conditioners, foam boosters, bleaching agents, e.g.
  • TAED tetraacetyl ethylene diamine
  • NOBS nonanoyloxybenzene sulfonate
  • anti-corrosion agents soil-suspending agents
  • sequestering agents anti-soil redeposition agents
  • foam depressors dyes, bactericides, optical brighteners or perfumes.
  • a detergent composition formulated as an aqueous detergent liquid comprising anionic surfactant, nonionic surfactant, humectant, organic acid, caustic alkali, with a pH in use adjusted to a value between 7 and 10.5.
  • a detergent composition formulated as a nonaqueous detergent liquid comprising a liquid nonionic surfactant consisting essentially of linear alkoxylated primary alcohol, phosphate builder, caustic alkali, with a pH in use adjusted to a value between about 7 and 10.5.
  • a detergent composition formulated as a detergent powder containing anionic surfactant, nonionic surfactant, acrylic polymer, fatty acid soap, sodium carbonate, sodium sulphate, clay particles, and sodium silicate.
  • a liquid compact detergent comprising 5-65% by weight of surfactant, 0-50% by weight of builder and 0-30% by weight of electrolyte.
  • the detergent compositions a)-h) include the cellulase of the invention and optionally one or more other enzymes, as indicated above.
  • the softening, soil removal and colour clarification effects obtainable by means of the enzyme of the invention generally require a concentration of the enzyme in the washing solution of 0.0001 - 100, preferably 0.0005 - 60, and most preferably 0.01 - 20 mg of enzyme protein per liter.
  • the detergent composition of the invention is typically employed in concentrations of 0.5 - 20 g/l in the washing solution. In general, it is most convenient to add the detergent additive in amounts of 0.1 - 5% w/w or, preferably, in amounts of 0.2 - 2% of the detergent composition.
  • Fig. 1 is a map of plasmid pYHD17, wherein "TPI promoter” indicates the S. cerevisiae triose phosphate isomerase promoter, “Terminator” indicates the S . cerevisiae triose phosphate isomerase terminator, “Amp” indicates the gene mediating ampicillin resistance, “2 ⁇ ori” indicates the yeast plasmid 2 ⁇ origin of replication, and “URA3'' indicates a gene encoding a selection marker complementing a uracil deficiency in the host strain; and
  • Fig. 2 is a map of plasmid pHD414, wherein "AMG Terminator” indicates the A. niger glucoamylase terminator, and "TAKA Promoter” indicates the A. oryzae TAKA amylase promoter;
  • mRNA was isolated from the following organisms: H. insolens, DSM 1800, grown in a cellulose-rich fermentation medium with agitation to ensure sufficient aeration.
  • Plasmid Construction of an expression plasmid The commercially available plasmid pYES II (Invitrogen) was cut with Spel, filled in with Klenow DNA polymerase + dNTP and cut with Clal. The DNA was size fractionated on an agarose gel, and a fragment of about 2000 bp was purified by electroelution. The same plasmid was cut with Clal/PvuII, and a fragment of about 3400 bp was purified by electroelution. The two fragments were ligated to a blunt-ended Sphl/EcoRI fragment containing the yeast TPI promoter. This fragment was isolated from a plasmid in which the TPI promoter from S. cerevisiae (cf. T. Albers and G.
  • RNA was isolated from approximately 7 g of mycelium. The mycelium was frozen in liquid nitrogen and ground in a mortar with 1 g of quartz sand to a consistency of flour. The RNA was extracted with guanidinium thiocyanate and centrifuged through CsCl essentially as described in Sambrook et al., 1989, op. cit.. Poly A RNA was isolated from total RNA by chromatrography on oligo dT cellulose.
  • cDNA synthesis was carried out by means of a cDNA synthesis kit from Invitrogen according to the manufacturer's specifications.
  • the DNA was adapted to the expression vectors by addition of a Bstxl linker (Invitrogen) and size fractionated on an agarose gel. Only DNA larger than 5-600 bp was used in the library construction. The adapted cDNA was ligated into an appropriate vector cut with Bstxl. Following test ligations (in order to determine the size of the library) the library was plated onto 50 agar plates. To each plate containing from approximately 500 to 5000 individual clones (dependent on the library size) was added 3 ml medium. The bacteria were scraped off, 1 ml glycerol was added, and stored at -80oC as 50 pools. The remaining 2 ml were used for DNA isolation. If the amount of DNA was insufficient to give the required number of yeast transformants (see below), large scale DNA was prepared from 500ml medium (TB) inoculated with 50 ⁇ l -80°C bacterial stock propagated over night.
  • Bstxl linker Invitrogen
  • Yeast Libraries DNA from one or more pools was transformed into yeast as described below. To ensure that all the bacterial clones were tested in yeast a number of yeast transformants 5 x larger than the number of bacteria clones in the original pools was set as a limit.
  • Transformation of yeast The yeast strain used was yNG231. (MAT alpha, leu2, ura3-52, his4-539, pep4-delta 1, cir+). One colony was grown at 30 oC overnight in 10 ml YPD (this culture can be stored for several days at 5°C).
  • carrier DNA sterile salmon-sperm DNA 10 mg/ml
  • carrier DNA 250 ⁇ g carrier DNA (sterile salmon-sperm DNA 10 mg/ml) was added and aliquots of 100 ⁇ l were prepared.
  • the DNA to be transformed (approx. 5 ⁇ g) was added to the 100 ⁇ l aliquot, mixed gently, and incubated for 30 minutes at 30°C 700 ⁇ l 40% PEG 4000, 0.1 M LiAc, 10 mM Tris-Cl, 1 mM EDTA, pH 7.5 was added, and incubation was continued for 60 minutes at 30°C.
  • the transformation mixture was subjected to heat shock for 5 minutes at 42°C, spun briefly in a micro centrifuge, resuspended in 100-200 ⁇ l H 2 O, and plated on SC plates without uracil, followed by incubation for three days at 30oC.
  • YPD 10 g yeast extract, 20 g peptone, H 2 O to 810 ml. Autoclaved, 90 ml 20% glucose (sterile filtered) added. 10 x Basal salt: 66.8 g yeast nitrogen base, 100 g succinic acid, 60 g NaOH, H 2 O ad 1000 ml, sterile filtered.
  • SC-URA 90 ml 10 x Basal salt, 22.5 ml 20 % casamino acids, 9 ml 1% tryptophane, H 2 O ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml 20% glucose added.
  • SC-H agar 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/l succinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins, 0.1 g/l tryptophan and 20 g/l agar (Bacto).
  • SC-H broth 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/l succinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins, 0.1 g/l tryptophan.
  • YNB-1 agar 3.3 g/l KH 2 PO 4 , 16.7 g/l agar, pH adjusted to 7. Autoclaved for 20 min. at 121°C. After autoclaving, 25 ml of a 13.6% yeast nitrogen base without amino acids, 25 ml of a 40% glucose solution, 1.5 ml of a 1% L-leucine solution and 1.5 ml of a 1% histidine solution were added per 450 ml agar.
  • YNB-1 broth Composition as YNB-1 agar, but without the agar.
  • CMC overlayer gel 1% agarose, 1% carboxymethyl cellulose in Tris-malate buffer, pH 7. The gel was boiled and then cooled to 55°C before the overlayer was poured onto agar plates.
  • Oat spelt xylan overlayer gel 1% agarose, 1% oat spelt xylan (Sigma Chemical Company) in Tris-malate buffer, pH 7. The gel was boiled and then cooled to 55°C before the overlayer is poured onto agar plates.
  • the vector pHD414 is a derivative of the plasmid p775 (described in EP 238 023). In contrast to this plasmid, pHD 414 has a string of unique restriction sites between the promoter and the terminator. The plasmid was constructed by removal of an approximately 200 bp long fragment (containing undesirable RE sites) at the 3'end of the terminator, and subsequent removal of an approximately 250 bp long fragment at the 5'end of the promoter, also containing undesirable sites.
  • the 200 bp region was removed by cleavage with Narl (positioned in the pUC vector) and Xbal (just 3' to the terminator), subsequent filling in the generated ends with Klenow DNA polymerase +dNTP, purification of the vector fragment on gel and religation of the vector fragment.
  • This plasmid was called pHD413.
  • pHD413 was cut with StuI (positioned in the 5'end of the promoter) and PvuII (in the pUC vector), fractionated on gel and religated.
  • the plasmid pHD 414 is shown in Fig. 2.
  • DNA was isolated from 20 individual clones from the library and subjected to analysis for cDNA insertion.
  • the insertion frequency was found to be >90 % and the average insert size was approximately 1400bp.
  • DNA was isolated from 10 pools from the Humicola library (2ml from the original plate). An aliquot was digested with restriction enzymes in order to excise the cDNA insert and analyzed by Southern blot using a 43kD cellulase cDNA probe (the 43 kD enzyme is disclosed in PCT/DK91/00123) and a CBH 2 cDNA probe (the enzyme is disclosed in PCT/DK91/00124). Several bands were found to hybridize with the 43kD cellulase probe after a low stringency wash ( 2x SSC 65°C) in the 10 pools from the Humicola library.
  • Yeast cells from the library were spread onto YNB agar to a total of about 400,000 colonies. The number of colonies per plate varied from 50 to 500. After 4 or 5 days of growth, the agar plates were replica plated onto two sets of SC-H agar plates. These plates were then incubated for 2-4 days at 30°C before the two sets of agar plates were overlayered with a CMC indicator gel for detection of cellulase activity and oat spelt xylan indicator gel for the detection of xylanase and cellulase. After incubation overnight at 40°C, enzyme reactions were visualised with Congo Red. 10-15 ml of a 0.1% solution of Congo Red was poured onto the overlayer and removed after 10-20 min.
  • the cells were resuspended in 1 ml 0.9 M sorbitol, 0.1 M EDTA, pH 7.5. The pellet was transferred to an Eppendorf tube, and spun for 30 seconds at full speed. The cells were resuspended in 0.4 ml 0.9 M sorbitol, 0.1 M EDTA, 14 mM ⁇ -mercaptoethanol. 100 ⁇ l 2 mg/ml Zymolase was added, and the suspension was incubated at 37oC for 30 minutes and spun for 30 seconds. The pellet (spheroplasts) was resuspended in 0.4 ml TE.
  • the DNA was transformed into E.coli. by standard procedures. Two E. coli colonies were isolated from each of the transformations and analysed with the restriction enzymes Hindlll and Xbal which excised the DNA insert. DNA from one of these clones was retransformed into S. cerevisiae strain JG169 (MAT ⁇ ; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-113; prcl::HIS3; prb1:: LEU2) and rescreened for enzyme activity.
  • S. cerevisiae strain JG169 MAT ⁇ ; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-113; prcl::HIS3; prb1:: LEU2
  • CMC 1 C3, 26, 27, XY33, XY46 250 amino acids (SEQ ID#7)
  • CMC 4 C46, 47, 50, 51, 54,
  • XYL 1 XY30, 31, 40, 42, 101, 102,
  • XYL 2 XY103, 104, 107, 108, 109,
  • XYL 3 XY115, 116, 132, 146 (SEQ ID#15)
  • the cDNA insert is isolated from one or more representatives of each family and cloned into the vector pHD414 which is transformed into A. oryzae or A. niger according to the general procedure described below.
  • YPD Yeast et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981
  • the mycelium is harvested by filtration through miracloth and washed with 200 ml of 0.6 M MgSO 4 .
  • the suspension is cooled on ice and 1 ml of buffer containing 120 mg of Novozym ® 234, batch 1687 is added.
  • the suspension is filtered through miracloth, the filtrate transferred to a sterile tube and overlayered with 5 ml of 0.6
  • protoplasts are resuspended in 0.2-1 ml of STC.
  • 100 ⁇ l of protoplast suspension is mixed with 5-25 ⁇ g of the appropriate DNA in 10 ⁇ l of STC.
  • Protoplasts are mixed with p3SR2 (an A. nidulans amdS gene carrying plasmid). The mixture is left at room temperature for 25 minutes.
  • Cellulase type 4 clones C46 and C51 and a 43 kD cellulase control clone (obtained by transforming yeast strain JG169 with pYHD17 carrying a DNA sequence coding for the 43 kD cellulase
  • test tubes 100 ml test tubes with 15 ml YNB-1 broth. The tubes were agitated at 30°C for 2 days. 5 ml of broth from each tube were then used as seed material for shake flasks containing 100 ml
  • the cells from 20 ml of broth were collected by centrifugation and mixed with 1-2 ml 0.1 M sodium phosphate buffer, pH 7, and
  • CMC gel CMC overlayer gel as described above.
  • CMC LAS gel 2% agarose, 1% CMC in 50 mM sodium phosphate buffer, pH 7, boiled and mixed with one volume of 0.12% LAS.
  • the cellulase activity was measured by adding 15 ⁇ l crude cell extract to 4 mm (diameter) holes in the gel.
  • the crude cell extracts were diluted with one volume of 0.12% LAS before addition to the CMC LAS gel and with one volume of water before addition to the CMC gel.
  • the clearing zones were then visualised after 18 hours of incubation at 40oC by staining with Congo Red as described above.
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens
  • SEQUENCE DESCRIPTION SEQ ID NO: 7: CATCGCCTTA TACCACCAGC TCTACTGCA G ACCETGTCCA ATTTCTCGGA TCACCGCCAT 60
  • GACTGGCTAC AACGGCAACA TGCGTGTCTA CAGCTTCCTC CCCCCGTCCG GCGACATTCH 660
  • ORGANISM Humicola insolens
  • AAAGCCTGAA CACTATTACC ATGTTGCACA GTGTCCTTGC CGGTCTCTTC GCGACTGGAG 60 CGCTCGCCCA GGGCGTGCAT GGCAGCGTG TGGTGGCGTT GGCTTCTCGG GCTCTACGTC 120 CTGTGTCC GGTTACACGT GCGTGTACTT GAACGACTGG TAC ⁇ GCCAAT GCCAGCGCAG 180
  • ORGANISM Humicola insolens
  • NAAAGGCACC AAGGTGACGG CGTCACCTcG GGcGAGTGGG AGACGATCCG CATCACCGAG 60
  • TCACCGTCTA CACGGACGTG GGCCACCCGG GCGCTGCACT TCTACCTGGC CAAGGTGCCG 240 CGGCAAGACG GCCGCGACGT TTGACGGCAA CGGCGCCGTG TGGTTCAAGA TTTACCAGGA 300
  • GTTTGAGTTC ATGAGTACTC CAATGAAGG ⁇ TGCGCGGCGG CGAGGGTAGG TCGATAGTTT 720
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens (xi) SEQUENCE DESCRIPTION SEQ ID NO: 13:
  • ORGANISM Humicola insolens
  • ORGANISM Humicola insolens

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Abstract

Procédé de détection d'une séquence d'ADN codant pour une protéine à examiner. Il consiste (a) à cloner, dans des vecteurs appropriés, une banque d'ADN provenant d'un organisme dont on pense qu'il produit une ou plusieurs protéines à examiner; (b) à transformer, à l'aide desdits vecteurs, des cellules hôtes de levure appropriées; (c) à cultiver les cellules hôtes dans des conditions appropriées pour exprimer une éventuelle protéine à examiner codée par un clone dans la banque d'ADN; et (d) à détecter les clones positifs par la détermination de toute activité d'une protéine exprimée dans l'étape (c). On décrit également une enzyme dotée d'une activité cellulasique et isolée à partir de la banque d'ADN de Humicola insolens. Elle a un domaine de liaison de cellulose et présente une activité endocellulasique en présence de sulfonate de d'alkylbenzène linéaire.
PCT/DK1992/000360 1991-12-04 1992-12-02 PROCEDE DE CLONAGE DE PROTEINES DANS LA LEVURE ET CELLULASE PROVENANT DE $i(HUMICOLA INSOLENS) WO1993011249A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP5509731A JPH08504560A (ja) 1991-12-04 1992-12-02 フミコラ・インソレンスからのセルラーゼ及び酵母におけるタンパク質のクローニング方法
CA002124939A CA2124939A1 (fr) 1991-12-04 1992-12-02 Methode pour le clonage de proteines dans une levure, et cellulase d'humicola insolens
BR9206866A BR9206866A (pt) 1991-12-04 1992-12-02 Processo para a triagem de uma sequência de DNA que codifica uma proteína de interesse processo para produzir uma proteína de interesse em uma célula hospedeira heteróloga enzima aditivo detergente e composição detergente
EP93900092A EP0618974A1 (fr) 1991-12-04 1992-12-02 PROCEDE DE CLONAGE DE PROTEINES DANS LA LEVURE ET CELLULASE PROVENANT DE $i(HUMICOLA INSOLENS)
FI942644A FI942644A0 (fi) 1991-12-04 1994-06-03 Menetelmä proteiinien kloonaamiseksi hiivassa ja sellulaasi, joka on Humicola insolensista peräisin

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DK9100379 1991-12-04
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WO1996013580A1 (fr) * 1994-10-26 1996-05-09 Novo Nordisk A/S Enzyme a activite lipolytique
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WO1997028256A1 (fr) * 1996-01-29 1997-08-07 Novo Nordisk A/S Procede de desencollage d'un tissu contenant de la cellulose
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EP0869167A2 (fr) 1996-12-09 1998-10-07 Novo Nordisk A/S Réduction de substances contenant du phosphore dans les huiles comestibles à haute teneur en phosphore non-hydratable avec une phospholipase , une phospholipase issue d'un champignon filamenteux et présentant une activité de phospholipase A et/ou B
WO1999032617A2 (fr) * 1997-12-22 1999-07-01 Dsm N.V. Clonage d'expression dans les champignons filamenteux
WO1999045143A2 (fr) * 1998-03-06 1999-09-10 Novo Nordisk A/S Procede d'incineration de dechets et incinerateur pour la mise en oeuvre dudit procede
US6015783A (en) * 1996-01-29 2000-01-18 Novo Nordisk A/S Process for removal or bleaching of soiling or stains from cellulosic fabric
WO2000024882A1 (fr) * 1998-10-28 2000-05-04 Novozymes A/S Procede de creation d'une banque de genes
US6060274A (en) * 1996-10-28 2000-05-09 Novo Nordisk A/S Extracellular expression of cellulose binding domains (CBD) using Bacillus
EP1059351A1 (fr) * 1999-06-11 2000-12-13 The Procter & Gamble Company Compositions détergentes liquides non-aqueuses contenant un composé libérant du borate et mannanase
US6184019B1 (en) 1995-10-17 2001-02-06 Röhm Enzyme Finland OY Cellulases, the genes encoding them and uses thereof
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EP1272669A1 (fr) * 2000-04-13 2003-01-08 Emalfarb, Mark Aaron Criblage a debit eleve de bibliotheques d'adn exprimees dans des champignons filamenteux
WO2003070957A2 (fr) * 2002-02-20 2003-08-28 Novozymes A/S Production de polypeptides de plantes
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EP2261359A1 (fr) 1998-06-10 2010-12-15 Novozymes A/S Mannanases
USRE43135E1 (en) 2001-05-18 2012-01-24 Danisco A/S Method of improving dough and bread quality
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CA2124939A1 (fr) 1993-06-10
JPH08504560A (ja) 1996-05-21
MX9206979A (es) 1993-07-01
EP0618974A1 (fr) 1994-10-12
FI942644A (fi) 1994-06-03
BR9206866A (pt) 1995-11-21
FI942644A0 (fi) 1994-06-03

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