A METHOD OF CLONING PROTEINS IN YEAST AND CELLULASE FROM HUMICOLA INSOLENS

Abstract: 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; b) transforming suitable yeast host cells with said vectors; 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). An enzyme which exhibits cellulase activity and has been isolated from DNA library of Humicola insolens. The enzyme has a cellulose binding domain and exhibits endocellulase activity in the presence of linear alkyl benzene sulfonate.

Description: A method of cloning proteins in yeast and cellulase from Humicola insolens . FIELD OF INVENTION 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 . BACKGROUND OF THE INVENTION The advent of recombinant DNA techniques has made it possible to select single protein components with interesting properties and produce them on a large scale. This represents an improvement over the previously employed production process using microorganisms isolated from nature and producing a mixture of proteins which would either be used as such or separated after the production step. However, the conventional cloning techniques have the drawback that each protein component has to be purified and characterized by its (partial) amino acid sequence before it is possible to prepare synthetic oligonucleotide probes for hybridization experiments. Since this is a rather time-consuming process, the cloning of novel proteins might be considerably expedited by using a screening method involving selecting clones expressing a desired protein activity. Such a screening method has previously been devised for the cloning of prokaryotic gene products in Bacillus, cf. US 4,469,791; P. Cornelis et al., Mol. Gen. Genet. 186, 1982, pp. 507-511; I. Palva, Gene 19, 1982, pp. 81-87; S.A. Ortlepp, Gene 23, 1983, pp. 267-276; H. Yamazaki et al., J. Bacteriol. 156, 1983, pp. 327-337; N. Tsukagoshi et al. , Mol. Gen. Genet. 193, 1984, pp. 58-63; M. Sibakov and I. Palva, Eur. J. Bioche . 145, 1984, pp. 567-572; and J.R. Mielenz, Proc. Natl. Acad. Sci. USA {?(), 1983, pp. 5975-5979. A screening method based on expression cloning of eukaryotic genes in mammalian cells has been described, e.g. in D.P. Gearing et al., The EMBO J. 8., 1989, pp. 3667-3676; N. Harada et al., Proc. Natl. Acad. Sci. USA 87, 1990, pp. 857-861; and R. Fukunaga et al., Cell 61, 1990, pp. 341-350. SUMMARY OF THE INVENTION It has now been found possible to screen for yeast clones expressing protein activities of interest with a view to isolating DNA coding for single protein components. Accordingly, 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 interes , (b) transforming suitable yeast host cells with said vectors, (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) . As indicated above, expression cloning of prokaryotic genes in Bacillus has previously been described. The prokaryotic systems devised for expression cloning, however, are not operable for the cloning of eukaryotic genes which are generally difficult to express in Bacillus. While 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. Furthermore, 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. Unlike the mammalian system, 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. According to the present invention, it has surprisingly been found that 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. Although 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), it has not been generally useful as it is based on complementation of essential genes and therefore is dependent on yeast host strains which have been mutated to lack these essential genes. In the present screening method, no such requirement is necessary for the yeast host strain to be used in the method. Besides, 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. In another aspect, 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. In a further aspect, 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. (b) said DNA sequence comprises at least one of the following' partial sequences (i) TGGCAGCAGT GTGGTGGCGT TGGCTTCTCG GGCTCTACGT CCTGTGTGTC CGGTTACACG TGCGTGTACT TGAACGACTG GTACAGCCAA TGC (SEQ ID#1) ATCGTGGCTT CA (SEQ ID#3) (iv) CTGACGTGAA CGTGACCAAC AACAACTTGG CCGTAGCGAC CGAGAACAAG CTGTGTACCA GATGCATCA (SEQ ID#4) (V) GGACGGTCCG GCACGAGCAC GGCCTGCGTC AGCACCCAGG TCGGCCTTCA GCGCGTCATT GGCGCGACCA ACTGGCTCAG GCAAAACGGC AAGGTTGGAC TGCTCGCGAC TTGCCGCGGC (SEQ ID#5) (Vi) GCCAAGTGGG TTTGCCAGCA GGCCATTGAG GGCATGCTGA ACCACCTCCA GGAGAATAGC GATGTCTGGA CAGGTGCGCT CTGGTGGGCG GGAGGCCCGT GGTGGGGTTG ACTATATCTA (SEQ ID#6) (c) the enzyme comprises a cellulose-binding domain, and (d) 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. In the present context, the term "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 (cf. the discussion in PCT/DK91/00124) . The term "endocellulase activity" refers to the ability of the enzyme to degrade cellulose to glucose, cellobiose, triose and other cello- oligosaccharides, as determined by the formation of clearing zones in a carboxymethyl cellulose (CMC) gel under the conditions specified below. Unlike the endocellulase described in PCT/DK91/00123) , 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. DETAILED DISCLOSURE OF THE INVENTION According to the invention, the DNA library is preferably a cDNA library prepared from the mRNA of an organism suspected of producing one or more proteins of interest. Although it may also be possible to screen genomic libraries in this manner, at least some potential 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. To ensure a more accurate result, it may be an advantage to subject positive clones isolated in step (d) of the present method to rescreening, reisolation and recloning. 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. In this respect, screening of yeast colonies for different protein activities is far more e ficient 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. One type of industrially useful proteins currently obtained from fungi is enzymes. Thus, yeast clones may be screened by the method of the invention for expression of one or more enzyme activities by means of appropriate assays. Examples of enzymes which may be identified by this method are carbohydrases, e.g. cellulytic enzymes such as endocellulases, cellobiohydrolaseε, /.-glucanases or 3-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 (polygalacturonases and pectin lyases) 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, α-galactosidases may be detected by the release of p-nitrophenol (yellow) from p- nitrophenol- -galactopyranoside or by coupling released naphthole or naphthole derivatives from, e.g., 1-naphthole-α- galactopyranoside to azo dyes; similar methods may be used to detect 3-galactosidases, α- and 3-glycosidases, .-xylosidase and .-mannosidase. Numerous methods are available for the detection of proteases, e.g. clearing zones in casein after precipitation with trichloroacetic acid. 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) . 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. Thus, the yeast strain may suitably be selected from Saccharomyces sp., such as Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum or Schizosaccharomyces po be, Hansenula sp. Pichia sp. , Yarrowia sp. such as Yarrowia lipolvtica, 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. In each vector, 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 (Hitzemaή et al. , J. Biol. Chem. 255, 1980, pp. 12073-12080; Alber and Kawasaki, . Mol. Appl. Gen. 1, 1982, pp. 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering: of Microorganisms for Chemicals (Hollaender et al, eds.) , Plenum Press, New York, 1982) , or the TPI1 (US 4, 599, 311) or ADH2-4c (Russell et al. , Nature 304, 1983, pp. 652-654) promoters. 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 MFα terminators. The vector may further comprise a DNA sequence enabling the vector to replicate in yeast cell. An example of such a sequence is the yeast plasmid 2μ replication genes REP 1-3 and origin of replication. If the vector is a yeast/E. coli shuttle vector, it will also include an origin of replication region which is functional in ________ 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) . The procedures used to ligate the DNA library sequences, the promoter and the terminator, respectively, and to introduce them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf. , for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) . The transformation of yeast cells may for instance be effected by protoplast formation followed by transformation or by the LiAc method in a manner known per se. In the process of the invention of producing a protein of interest after the DNA coding for the protein has been isolated by the screening method described above, 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 _______ oryzae. The use of A^ . oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023. In particular when the host organism is A_;_ oryzae, 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 μl) 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 de¬ fined above) . The appropriate DNA sequence may then be isolated from the clone by standard procedures, e.g. as described in Example 1. In a further aspect, 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 granula- te, 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. It will be understood that the detergent additive may further include one or more other enzymes, such as a protease, lipase, peroxidase or amylase, conventionally included in detergent additives. In a still further aspect, the present invention relates to 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. It may also contain 0-40% non- ionic surfactant such as nonyl phenol ethoxylate or alcohol ethoxylate. Furthermore, it may contain a polyhydroxy fatty acid amide surfactant (e.g. as described in WO 92/06154) . 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 1-40% of 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. perborate, percarbonate, tetraacetyl ethylene diamine (TAED) , or nonanoyloxybenzene sulfonate (NOBS) , anti-corrosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, stabilizing agents for the enzyme(s) , foam depressors, dyes, bactericides, optical brighteners or perfumes. Particular forms of detergent composition within the scope of the invention include: a) A detergent composition formulated as a detergent powder containing phosphate builder, anionic surfactant, nonionic surfactant, silicate, alkali to adjust to desired pH in use, and neutral inorganic salt. b) A detergent composition formulated as a detergent powder containing zeolite builder, anionic surfactant, nonionic surfactant, acrylic or equivalent polymer, silicate, alkali to adjust to desired pH in use, and neutral inorganic salt. c) 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. d) A detergent composition formulated as a nonaqueous deter- gent 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. e) A detergent composition formulated as a detergent powder in the form of a granulate having a bulk density of at least 600 g/1, containing anionic surfactant and nonionic surfactant, low or substantially zero neutral inorganic salt, phosphate builder, and sodium silicate. f) A detergent composition formulated as a detergent powder in the form of a granulate having a bulk density of at least 600 g/1, containing anionic surfactant and nonionic surfactant, low or substantially zero neutral inorganic salt, zeolite builder, and sodium silicate. g) 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. h) A liquid compact detergent comprising 5-65% by weight of surfactant, 0-50% by weight of builder and 0-30% by weight of electrolyte. Apart from these ingredients, 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 g of enzyme protein per liter. The detergent composition of the invention is typically employed in concentrations of 0.5 - 20 g/1 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. BRIEF DESCRIPTION OF THE DRAWINGS 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 a picillin resistance, "2μ ori" indicates the yeast plasmid 2μ origin of replication, and "URA3 11 indicates a gene 5 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 0 Promoter" indicates the A. oryzae TAKA amylase promoter; The present invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed. EXAMPLES Materials and Methods Donor organism: mRNA was isolated from the following organisms: H. insolens, DSM 1800, grown in a cellulose-rich fermentation medium with agitation to ensure sufficient aeration. 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. Kawasaki, J. Mol. Appl. Genet. 1, 1982, pp. 419-434) was slightly modified: an internal SphI site was removed by deleting the four bp constituting the core of this site. ■ Furthermore, redundant sequences upstream of the promoter were removed by Ball exonuclease treatment followed by addition of a SphI linker. Finally, an EcoRI linker was added at position - 10. After these modifications, the promoter is included in a Sphl-EcoRI fragment. Its effeciency compared to the original promoter appears to be unaffected by the modifications. The resulting plasmid pYHD17 is shown in Fig. 1. Isolation of mRNA: Total 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 guanidiniu 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: 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 -80"C 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 μl -80°C bacterial stock propagated over night. Construction of 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"C overnight in 10 ml YPD (this culture can be stored for several days at 5°C) . 10, 30, and 60 μl of this culture were added to 3 shaker flasks containing 100 ml YPD, and incubated with shaking overnight at 30°C. The culture with an OD 600 closest to 0.3-0.4 was selected. The cells were harvested in 50 ml tubes in a Beckman centrifuge (speed 6, 10 minutes) , the cells were resuspended in 2 x 5 ml H 2 0, centrifuged as described above, resuspended in 5 ml buffer containing 0.1 M LiAc, 10 mM Tris-Cl, 1 mM EDTA, pH 7.5, and centrifuged again. The cells were resuspended in 500 μl of the above buffer and incubated for 60 minutes at 30°C. 250 μg carrier DNA (sterile salmon-sperm DNA 10 mg/ml) was added and aliquots of 100 μl were prepared. The DNA to be transformed (approx. 5 μg) was added to the 100 μl aliquot, mixed gently, and incubated for 30 minutes at 30°C 700 μl 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 μl H 2 0, and plated on SC plates without uracil, followed by incubation for three days at 30 β C. Preparation of carrier DNA: 100 mg salmon-sperm DNA was weighed out and dissolved overnight in 10 ml 10 mM Tris-Cl, 1 mM EDTA, pH 7,5 (TE) . The solution was then sonicated in a plastic container in ice water until it was no longer viscous. The solution was then phenole extracted and EtOH precipitated, and the pellet was washed and resuspended in 5 ml TE. The suspension was EtOH precipitated, and the pellet was washed and resuspend in 5 ml TE. The OD 260 was measured, and the suspension was diluted with TE to 10 mg/ml. Media: YPD: 10 g yeast extract, 20 g peptone, H 2 0 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 0 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 0 ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml 20% glucose added. SC-H agar: 7.5 g/1 yeast nitrogen base without amino acids, 11.3 g/1 succinic acid, 6.8 g/1 NaOH, 5.6 g/1 casamino acids without vitamins, 0.1 g/1 tryptophan and 20 g/1 agar (Bacto) . Autoclaved for 20 min. at 121°C. After autoclaving, 55 ml of a 22% galactose solution and 1.8 ml of a 5% threonine solution were added per 450 ml agar. SC-H broth: 7.5 g/1 yeast nitrogen base without amino acids, 11.3 g/1 succinic acid, 6.8 g/1 NaOH, 5.6 g/1 casamino acids without vitamins, 0.1 g/1 tryptophan. Autoclaved for 20 min. at 121°C. After autoclaving, 10 ml of a 30% galactose solution, 5 ml of a 30% glucose solution and 0.4 ml of a 5% threonine solution were added per 100 ml medium. YNB-1 agar: 3.3 g/1 KH 2 P0 4 , 16.7 g/1 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-l 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. Construction of an Aspergillus expression vector: 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 poly erase +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. Example l A library from H. insolens consisting of approx. 300,000 individual clones in 50 pools was constructed. 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. At higher stringency (0.1 x SSC, 75°C ) one band corresponding to the expected size for 43kD cellulase was detected in 5 out of 10 pools. Similar results were obtained with the CBH 2 probe. Here 10 out of 10 pools were found to have a band corresponding to the expected size for CBH 2. In addition, 4 pools contained bands with a higher molecular weight. These bands were seen even under stringent conditions, demonstrating that the library is of an appropriately high quality. DNA from the Humicola library, pools 1-10, was transformed into yeast, and plates containing 20-25,000 colonies were obtained from each pool. The colonies were scraped off and stored in glycerol at -80"C. 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 plates were then washed once or twice by pouring 10-15 ml of 2M NaCl onto the plates. The NaCl solution was removed after 15-25 min. Cellulase-positive colonies were identified on the plates with the CMC overlayer as colonies with colourless or pale red clearing zones on a red background. Xylanase-positive colonies identified on the plates with oat spelt xylan overlayers as colourless or pale red clearing zones on a red background. Cellulase-positive colonies were also identified on plates with oat spelt xylan overlayers as pale red or blue clearing zones on a red background. Cells from enzyme-positive colonies were spread for single colony isolation on agar, and an enzyme-producing single colony was selected for each of the cellulase- or xylanase-producing colonies identified. Each of the 133 cellulase-producing colonies and 147 of the xylanase-producing colonies were isolated. Some of these colonies were inoculated into 20 ml YNB-1 broth in a 50 ml glass test tube. The tube was shaken for 2 days at 30°C. The cells were harvested by centrifugation for 10 min. at 3000 rpm. 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 /3-mercaptoethanol. 100 μl 2 mg/ml Zy olase was added, and the suspension was incubated at 37"C for 30 minutes and spun for 30 seconds. The pellet (spheroplasts) was resuspended in 0.4 ml TE. 90 μl of (1.5 ml 0.5 M EDTA pH 8.0, 0.6 ml 2 M Tris-Cl pH 8.0, 0.6 ml 10% SDS) was added, and the suspension was incubated at 65"C for 30 minutes. 80 μl 5 M KOAc was added, and the suspension was incubated on ice for at least 60 minutes and spun for 15 minutes at full speed. The supernatant was transferred to a fresh tube which was filled with EtOH (room temp.) followed by thorough but gentle mixing and spinning for 30 seconds. The pellet was washed with cold 70% ETOH, spun for 30 seconds and dried at room temperature. The pellet was resuspended in 50 μl TE and spun for 15 minutes. The supernatant was transferred to a fresh tube. 2.5 μl 10 mg/ml RNase was added, followed by incubation at 37°C for 30 minutes and addition of 500 μl isopropanol with gentle mixing. The mixture was spun for 30 seconds, and the supernatant was removed. The pellet was rinsed with cold 96% EtOH and dried at room temperature. The DNA was dissolved in 50 μl water to final concentration of approximately 100 μl/ml. The DNA was transformed into E.coli. by standard procedures. Two EL. 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 (MATα; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-113; prcl::HIS3; prbl:: LEU2) and rescreened for enzyme activity. The DNA sequences of several of the positive clones were partially determined. The partial DNA sequences are shown in Sequence Listings SEQ ID#7-15. Based on the DNA sequence, the clones were classified as follows: Endocellulases: CMC 1: C3, 26, 27, XY33, XY46 250 amino acids (SEQ ID#7) CMC 4: C46, 47, 50, 51, 54, 101, 102, 103, 104 " 1400 bp (the enzyme of the invention) (SEQ ID#8) CMC 5: XY49 " 1050 bp (SEQ ID#9) CMC 6: C49 " 1000 bp (SEQ ID#10) CMC 38K: C13 (SEQ ID#11) CMC EG1: C6, 11, 15, 16, 17, 21, 22, 23, 25, XY34, 41, 145 (SEQ ID#12) Xylanases: XYL 1: XY30, 31, 40, 42, 101, 102, 110, 117, 119, 123, 125, 136, XY56, 60, 137 22 kD (SEQ ID#13) XYL 2: XY103, 104, 107, 108, 109, 113, 114, 118, 120, 121, 124, 126, 128, 130, 134, 142, 143 (SEQ ID#14) XYL 3: XY115, 116, 132, 146 (SEQ ID#15) In order to express the genes in Aspergillus 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 Aj_ niger according to the general procedure described below. Transformation of Aspergillus oryzae or Aspergillus niger (general procedure) 100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) is inoculated with spores of A. oryzae or A. niger and incubated with shaking at 37°C for about 2 days. The mycelium is harvested by filtration through miracloth and washed with 200 ml of 0.6 M MgS0 4 . The mycelium is suspended in 15 ml of 1.2 M MgS0 4 . 10 mM NaH 2 P0 4 , pH = 5.8. The suspension is cooled on ice and 1 ml of buffer containing 120 mg of Novozym 234, batch 1687 is added. After 5 minutes 1 ml of 12 mg/ml BSA (Sigma type H25) is added and incubation with gentle agitation continued for 1.5-2.5 hours at 37°C until a large number of protoplasts is visible in a sample inspected under the microscope. The suspension is filtered through miracloth, the filtrate transferred to a sterile tube and overlayered with 5 ml of 0.6 M sorbitol, 100 mM Tris-HCl, pH = 7.0. Centrifugation is performed for 15 minutes at 100 g and the protoplasts are collected from the top of the MgS0 4 cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH = 7.5. 10 mM CaCl 2 ) are added to the protoplast suspension and the mixture is centrifugated for 5 minutes at 1000 g. The protoplast pellet is resuspended in 3 ml of STC and repelleted. This is repeated. Finally the protoplasts are resuspended in 0.2-1 ml of STC. 100 μl of protoplast suspension is mixed with 5-25 μg of the appropriate DNA in 10 μ.1 of STC. Protoplasts are mixed with p3SR2 (an A. nidulans amdS gene carrying plasmid) . The mixture is left at room temperature for 25 minutes. 0.2 ml of 60% PEG 4000 (BDH 29576). 10 mM CaCl 2 and 10 mM Tris-HCl, pH = 7.5 is added and carefully mixed (twice) and finally 0.85 ml of the same solution is added and carefully mixed. The mixture is left at room temperature for 25 minutes, spun at 2500 g for 15 minutes and the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one more sedimentation the protoplasts are spread on the appropriate plates. Protoplasts are spread on minimal plates (Cove Biochem.Biophys.Acta 113 (1966) 51-56) containing 1.0 M sucrose, pH = 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit background growth. After incubation for 4-7 days at 37"C spores are picked and spread for single colonies. This procedure is repeated and spores of a single colony after the second reisolation is stored as a defined transformant. Example 2 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 [isolated as described in PCT/DK91/000123]) were inoculated in 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 SC-H broth. The shake flasks were agitated for 4 days at 30°C. 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 3.3 g of glass beads (420-500 μm in diameter) in 10 ml glass test tubes. The crude cell extracts were collected after about 8 minutes of agitation by means of a IKA vibrax VXR (available from IKA Labortechnik) . The cellulase activity of the crude cell extracts from the yeast clones C46, C51 and 43 kD were measured under different conditions by the size of the clearing zones formed in CMC containing gels. 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 μl 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 40"C by staining with Congo Red as described above. The results are shown in the following table. Clone C46 C51 43 kD CMC 14 14 17 CMC LAS 15 13 0 Activities are shown as mm clearing zones. It appears from the table that the enzyme produced by C46/C51 is LAS resistant. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Novo Nordisk A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: Denmark (F) POSTAL CODE (ZIP) : DK-2880 (G) TELEPHONE: +45 4444 8888 (H) TELEFAX: +45 4449 3256 (I) TELEX: 37304 (ii) TITLE OF INVENTION: A Method of Cloning Proteins in Yeast (iii) NUMBER OF SEQUENCES: 15 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (Vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TCGCAGCAGT G__C___GGOGT TGGCITCTOG GXSCTCIACGT CCTGIGΣGIC OGGTTACACG 60 TCOSI -TIACΓ TGAACGACTG GΠΆCAGCCAA TGC 93 (i) SEQUENCE CHARACTERISTICS: (A) lENCΞflH: 92 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIOGY: linear (ϋ) MO_-__ LE TYPE: CDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (xi) SEQUENCE DES-__π__TON: SEQ ID NO: 2: (__J3-3GCAGCC GAOGAOG__TA OGGACAACAC AACAACGACC AGGGCAACAT OGACAACAAG 60 GK_AGCCCOG G_3GC__A.C_T CAACCACDCC GG 92 (2) INFORMATiα. FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTTCS: (A) IENSIH: 132 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIOGY: linear (ii) MOIECDIE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (xi) SEQUENCE DESCRIPπON: SEQ ID NO: 3: CCAAGGOGAA G__TCAACTGG TIGGCATCAA CO-CJTCCTGC GCTGACTTOG GCAAGGAGAG 60 TATCOGGCIA _X_ _X_ -__AGCA _3__ACTTCCT TOGCGAOGIC GTOGATTCAA GOGC_A -__TCA 120 A_E_3_TGGCΓΓ CA 132 (2) INFOEMATTCN FOR SEQ ID NO: 4 : (i) SEQUENCE CHARACDΕE-ESTICS: (A) IENGTH: 69 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ϋ) MOIECUIE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (Xi) SEQUENCE DESCRIPπON: SEQ ID NO: 4: CTGACX-IGAA CX IGAO-AAC AAC-AACΠGG COSTAGOGAC OGAGAACAAG CI__I___ACeA 60 GATGCBTCA 69 (2) INPORMATI-S- FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) IENGIH: 120 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MOIECUIE TYPE: cDNA (Vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: G__AC_ _TCOG GCACGAGCAC GGCCTGCGTC ΑGCACCCM-G TCGG_X_TTCA _-___C_?I_-ATT 60 GGCGCGACCA ACTCGCTCAG GCAAAACGGC AAGGTTGGAC TGCIOGOGAC TTCCOGOGGC 120 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCCAAGTGGG T_?TGCCAGCA GGCCAITGAG G__C__IGCI___ ACCACCTCCA GGAGAATAGC 60 G__IGIC__GGA ChGσ∑GCGCT CTGGTGGGOG GGAGGCCOGT GX3IGGGGTTG ACTATATCTA 120 (2) INPOI-MATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1027 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIOGY: linear (ii) MOIECUIE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (xi) SEQUENCE DESCKEPITON: SEQ ID NO: 7: C3_rα_CC_-___. TACCACCAGC TCmCIGCAG ACCETGTCCA ATTTCΓCGGA TCACCGCCAT 6 GC__CAAGΓCT GX^ -XCΓCC TOGGGCCCGC GG OJJTICG GΓICAGTOGG C ΓOGATOCC 12 GACXATCCCG ____ _____TG AGCCTCGCCA GLA-!?__ _--_C__Α-. C_K3_?I_-AGC G CGGCT 18 O-GGTCOGGC AATGGCIATG AGCTCTIGAA CAACCICTGG GGCAAGGACA CCGCCACATC 24 OGGCTGGCAA TGCACCIACC TOGACGGCAC CAACAAOGGT GGC-ATTCACT 03AGCACOGC 30 GTGGGASDGG C&SGGCGCTC CGGACAAOGT (_AAGAGCI_AC -__CIATGTrG GCAAGCAGAT 36 (3CAGOGCGGC OC_CAAGATCA GCϊGACATCAA CAGCATGOGC ACCTOGGTGT CSSIGGAOGTA 420 OGATOGGACC GkCKICCGTG CCAATCICGC T__ATGATGTC TTCAOGGCTC G G__TOCGGA 480 C__ATCCCAAC TGGGGCGGCG ACTACGAGCT C__I_ ___-TGG CTrøXOGCT ATCGCGGCAT 540 cEACcccATc GX-CAOGTΓCC ACΆGCCAGCT CAACCΓΓGCΓ GGECGTACCT G__GATCTCTG βoo GACTGGCTAC AACGGCAACA TGCJ3IGTCIA CAGC__TCC__C (__XX_OGTCCG α_X_ΑC__r__CG 660 TGACITCAGC TGOGACATCA AGGLACTTCTT CAACTACCIT GAGCGCAACC AIGGCTACCC 720 GGCACGGGAG CAGAATCIGA T∞TCTACCA AGITGGAACC __ASTGCTTCA CEGGCGGTCC 780 GGCCAGGTTC ACGIGCAGGG ACTICAGGGC TGACODCTGG TAAGAGGGGT CATIGGAGTA 840 GGGIGΣk π ~~ GCCAGGAAGC GGAGAGGAAC AAGGTAGATA TAATGACAGA C___AGa__A_7I_. 900 G__3__CAG___A ACAC-_CIT0G TCCTTCA-DGG GCAAGACTIG CAAGTGCCTA GACAACAGIG 960 CTX-AGGGGAC ACAGGGACGA AG__TGCATCC TCCAAAACTC AGaAATCGGT AGCAATCGCC 1020 TITAGAG 1027 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) IENGIH: 872 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIOGY: linear (ii) MOIECUIE TYPE: cDNA (Vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens ( i) SEQUENCE DESCRIFTICN: SEQ ID NO: 8: AAAGCCTGAA CACTAITACC ATGTTGCACA G__GTCC_7IGC _X3G_X_-CTrC GCGAC3GGAG 60 αSCTCGCCCA OSGCXΞ__GCAT GGCAGCASIG TGGTGGCSTT GGCTTCEOGG GCTCTACSTC 120 C__GIG_GTCC ___I__AC__OGT GCGTGTACIT GAAOGACTGG TACΑGCCAAT GCCAGCGCAG 180 CCGACGACGT TACGGACAAC ACAACAACGC CAGGGCAACA TCGACAACAA G__ CAGCCCC 240 GGCΓGCCACΓ TCAACCACΓC CGGCCAAGGC GAAG ΓCAAG TGGTIGGCAT CAACCAGTCC - 300 TGCGCIGAGT TCEGCAAGGA GAGEFLCOGG CTATGGGCAA GCA< ~~ ~~ T CXTTTCGOGAC 360 G___X3TO_AIT CAAGOGCACA TCAATCGTGG CITCACATGT CCNNNNNGGC AACTCTGGAC 420 G__ IGAOGTG CX-TGACCA ACAACAACTT GGCOGTAGOG ACCGAGAACA AGCTSIGTAC 480 C-____TGCATC AG__AC_-TOGA <_ _XX_ __AOGGT C__3G2A(_ __AG CΑα_X_CCTGC GTCAGCACCC 540 AGGΓCGGCCΓ TCAGCGCGTC ATTGGOGOGA 0_AACTGGCT CAGGGAAAAC GG ΑPiGGYIG 600 GAOPGCTCGC GACETGCCGC GGCGCCAAGT GX___I__IGCCA GCAGGC NTΣ CΑGGGCATGC 660 TGAACCACCΓ CCAGGAGAAT AGCGATSΓCT GGACAGCΞΠGC GC_TCTGGTGG GOGGGAGGCC 720 CCTGGTGGGG TGACTATATC TAGTIKCTITG AACCTCTTOG G^TATTGGCT ACACCTACTA 780 C_ A__[Ό_CTT CΓCAAGAAAT ACGTGCCATA GGTGCTATAA GAC__G_ -__TC CΓGACΓCAAG 840 AOΞGTΓIGAC AGGAACGCAG CXXΠGAGGCΓ TΓ 872 (2) INFORMATION FOR SEQ ID NO: 9 : (I) SEQUENCE CHARACT__ -ISTICS : (A) LENGTH: 368 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIOGY: linear (ii) MDLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : GTIGAAGGCC CTCAACAGAG GACCTCACGT CCCGAAAATG TCCAGGGCTA CXΞGAGTATGT 60 ACAGAAIATC CACAACCAAA CAAAAGTCAA TTCACCTCOG ATCCCAACAT CACAACCCCT 120 TCAGGCTGGG TCGGAACCAA C-TACCTAGCC AGCC ~ ~ ~ CIC CATTI CCAT CTCCTAACAC 180 CAACCCCCC C___A_X_TC__G C_-CC_TAAAT CGGCTTGACC C_ _aATC__ATC (______ X__CA 240 AACACCACCT GOCAACCATC CXSCTGCGCCC GCCCIGGGTG AAGAACGACT TGGCCGGTCT 300 ∞ΠGCCGGA GCAGCCACTC GACCAGTAGC TGGCCGCOGT GCGAACTIGC CGTTGTAGTA 360 CCAIAGGT 368 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARA CTΞRISTICS : (A) IENGIH: 720 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sil gle (D) TOPOIOGY: linear (ii) MOIECUIE TYPE: cENA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: NAAAGGCACC AAGGTGACGG CGTCACCTOG GGOGAGTGGG AGACXSATCOG CATCACOGAG 6 AACCACIGGT CGCACGGCCC CGIGACGGAC ___-___-_T0GC CGGCCATGAC GTGCIACGAG 12 AAGACGCCCG GCCAGOGGGC CCNAAGAOGG TCAAOSTGCG GGCCGGCGCG ACCGTCACCT 180 TCACCG-TCTA CACGGAOGIG GGCCACCCGG GCGCTGCACT TCTACCTGGC C^AGGIGOOG 240 CGGCAAGACG GCCGCGACGT TTGACGGCAA OSGCGCCGTG TGGTTCAAGA TTTACCAGGA 300 GS-OCIGGIG GHTCGGACAG C 3GITGACC TGGCX-ATGCT TGGCAAGAAG AGKSPOGTCA 360 A-ENNNNNNAC GTGGCCEACG TIGGCAAGAA GAAGTCTCGT <_3^__TCCCC0G TG_X3TCCAGG 420 AOGGOGASTA CXXTOCOGOG TOGAGCACAT TGXXTGCACA GGOGOGAGCG TCGGCGGTGC 480 _K_AGCTC_2AC AITr-X-IGOG CECAAATCAA CGTCACGGCG GCACCGGCAC GCTCAACCOG 540 GG___AGC__CG ___IC_?I__CCC GGGCGCCIAC AAGCCCACOG ACCOGGGCAT CCTGTTCCAG 600 CTCTACTGGC CXJCOGCOGAC _X2\___ACATC A CCCCGGIC OGGOGCCGGT GAAGTGCTGA 660 _a__EGAσπx_: ATGASΓACΓC CAATGAAGSΓ TGCGCGGCGG CGAGGGTAGG TOGAIAGTIT 720 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARAC_3ERISTICS: (A) IENGIH: 724 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIDGY: linear (ii) _-D__-C0I__ TYPE: cENA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CCTAGCTCGC C ~ A~~7 ~~ ~CG _X______ TC__T GCT _ __ -X- _OG CCTACCTGCT (-ΑGCGCOGCC 60 C__X_-_CCTOGC CX_G _XXGGA GCIOGAGCCC OGGCAGTCOG GCAACTCCIT CTCOGGO-GC 120 ACOCTCCTGG TCAACTCGGA CTATAGCAGC AAGCICGACC ACΑCX-OGCCA GX_ _X_Η7rCCT 180 GICCOGCGGC GACCAGACCA ACGCTGCCAA GGTCAACTAC GTCCAGGAGA AGGTTCGCAC 240 C____I. _ _-A!__TG GACTTCCAAC ATCTTCCTCC TGOGCAGCAC TGA ~ ~TTGCC ATCCAGAATG 300 CXMGCCGCCA AGGCCGCGCG AGAACCCCAT CGTCGGTCTC _π _CTG__ACA ACX-TCCCCGA 360 CCGCGACTGC AGCGACGCGG CAGTACCTCT GGC-_ CX___TA AGCICTCCCA GAA(-__GCCTG 420 AAO-GGTACA AGAAOGAGTA CXJICAACCCG ITC-SCCCAGA AGCTCAAGGC 0-_- _TCC__AC 480 GIGCAGITOG CCGTCATCCT OGAGCCCGAT GCCATOGGCA ACAΓGGTCAC GGGCACCΆGC 540 GCCTICTGCC -X_AAC__CCOG CEGCCCTCAG AGGAGGCCAT CGGCTATGCT AT(_ __CTCCTC 600 GGCTGGGCCG ATAAGCTOGA CXX_AACTGCC CAGGAGGIGC C_A___ATCCTC CAAAAGGCCG 660 GIAACAACGC AAGATOGCGG CTTCTCAGCA AC__ _TCCAAC TACAACCTAT TCACGACAAC 720 OGCG 724 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE 0___RACTERISTICS: (A) IENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 12 : CCTCCTICCA GΠXΠTTGACT TCCTTOGGAC CTGCAGCCTC CTGAACAACT OGCTCTAGCT 60 CAACAACCAT G 71 (2) INFORMATION FOR SEQ ID NO : 13 : (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 572 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MOLECULE TYPE: cENA (Vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (xi) SEQUENCE DESOHPTION: SEQ ID NO: 13: GGCOΞO GCC GCT_X___CAG TTSTG-EAOGA TCATCCAGCA A<___ _X-X_A___T _ACC_ TGGTC 60 TCCKTCAAGT C__SrCCTCGC GGCCGCCACG GCTGIGAGCT CTGC_______- _- TGCCCCTTTT 120 CΑCTTCGITC C__CGGGACAA CTCGAOGGCC C__TCAGGCTC GCCAGGTCAC CCCCAACGCC 180 GAGGGCTGGC ACAAOGGCTA C__TCTACTOG TGG__O_TC0G ACGGCGGAGG CC G TCAG 240 TACACCAACC TOGAGGGCAG COGCIACCAG GTCAGATGGC GTAACACCGG CAACTTCCTC • 300 GGTGGIAAGG GTTGGAACCC GGGAACCGGC CGCACGATCA ACTACGGCGG CTACTTCAAC 360 CXrCAGGGCA A<__-GCTACCT GGCOGTCTAC GGCTGGACCC GCAACCCGCT OGTCGAGTAC 420 TA3X_IX_?__ -__ AGTOGTAOGG CACGTACAAT CXXXΞGCAGCC AGGCTCACTA CAAGGGCACA 480 TTCTA-EACOG AO3G0GATCA G___1_ ACATC TTTGTGAGCA CCCGTCACAA CCAGCCCAGC 540 ATCAOGGCAC COGGACGTCC AGCTAGTACT GG 572 (2) INFORMA3_LON FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 173 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens ( i) SEQUENCE DESCRIPIION: SEQ ID NO: 14: AAAGEAGATC GTCXJDGCTCC CTAGAAACCA GTCACTCATT CACAATGOGT TC___TCX_CTC 60 TOSCTCEEGC TGCEGCTCOG G__CCTCCTOG CCCAGTCCCA GCTCTGGGCC AG_DGOGGTGG 120 CATOGGCTGG AAOGGCCTAC GACT_DG __-TC TOGGGOGCTA (XTGCACCAA GAT 173 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) IENGIH: 214 base pairs (B) TYPE: nucleic acid (C) STRANDEENESS: single (D) TOPOIOGY: linear (ii) MOLECULE TYPE: cENA (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: AAAGCCGCAG C_____CAAGAA CX_X»AGC__Gr CTGTCAAAAT GGTCGCCITC TC__ICCCTCT 60 _X-CTCGGTGC TTICCATOGCC GCCAOGCGTC G__OOGGCCCC G_π_?___-GCTG (XO_GCA__-_C 120 ACCTGAACAA Gα_TCAGACC TACACCCCAG AGOGCTACOG GCACTCACAA O-CCTACATC 180 T TCCΠCT GGACTGAOGG TCTGGCCGAA CGTC 214

Claims: CLAIMS 1. 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, (b) transforming suitable yeast host cells with said vectors, (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) . 2. A method according to claim 1, wherein the DNA library is a cDNA library prepared from the mRNA of an organism suspected of producing one or more proteins of interest. 3. A method according to claim 1 or 2, wherein positive clones isolated in step (d) are subjected to rescreening, reisolation and recloning. 4. A method according to claim 1 or 2, wherein the organism suspected of producing one or more proteins of interest is a eukaryotic organism. 5. A method according to claim 4, wherein the eukaryotic organism is a fungus. 6. A method according to claim 4, wherein the eukaryotic organism is a plant. 7. A method according to any of claims 1-6, wherein the protein of interest is an enzyme. 8. A method according to claim 1, wherein the yeast host cell is a strain of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula, Pichia, Yarrowia lipolytica or Kluyveromvces lactis. 9. 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 method of claim 1, culturing the transformed cells under suitable conditions to express the protein, and recovering the expressed protein from the culture. 10. A process according to claim 9, wherein the DNA sequence coding for the protein of interest has been isolated by the method of claim 3. 11. A process according to claim 9 or 10, wherein the protein of interest is an enzyme. 12. A process according to any of claims 9-11, wherein the host cell is a strain of Aspergillus, for instance a strain of Aspergillus oryzae or Aspergillus niger. 13. 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, (b) said DNA sequence comprises at least one of the following partial sequences (i) TGGCAGCAGT GTGGTGGCGT TGGCTTCTCG GGCTCTACGT CCTGTGTGTC CGGTTACACG TGCGTGTACT GAACGACTG GTACAGCCAA TGC (SEQ ID#1) (ii) CAGCGCAGCC GACGACGTTA CGGACAACAC AACAACGACC AGGGCAACAT CGACAACAAG GTCAGCCCCG GCTGCCACTT CAACCACTCCG G (SEQ ID#2) (iii) CCAAGGCGAA GTTCAAGTGG TTGGCATCAA CCAGTCCTGC GCTGAGTTCG GCAAGGAGAG TATCCGGCTA TGGGCAAGCA CTTACTTCCT TCGCGACGTC GTCGATTCAA GCGCACATCA ATCGTGGCTT CA (SEQ ID#3) (iv) CTGACGTGAA CGTGACCAAC AACAACTTGG CCGTAGCGAC CGAGAACAAG CTGTGTACCA GATGCATCA (SEQ ID#4) (V) GGACGGTCCG GCACGAGCAC GGCCTGCGTC AGCACCCAGG TCGGCCTTCA GCGCGTCATT GGCGCGACCA ACTGGCTCAG GCAAAACGGC AAGGTTGGAC TGCTCGCGAC TTGCCGCGGC (SEQ ID#5) (Vi) GCCAAGTGGG TTTGCCAGCA GGCCATTGAG GGCATGCTGA ACCACCTCCA GGAGAATAGC GATGTCTGGA CAGGTGCGCT CTGGTGGGCG GGAGGCCCGT GGTGGGGTTG ACTATATCTA (SEQ ID#6) (c) the enzyme comprises a cellulose-binding domain, and (d) the enzyme exhibits endocellulase activity in the presence of linear alkyl benzene sulfonate. 14. An enzyme according to claim 13, a crude extract (15 μl) 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. 15. A detergent additive comprising an enzyme exhibiting cellulase activity according to any of claims 13-14, preferably in the form of a non-dusting granulate, stabilised liquid or protected enzyme. 16. A detergent additive according to claim 15, which further comprises one or more other enzymes such as a protease, amylase, lipase or peroxidase. 17. A detergent composition comprising an enzyme exhibiting cellulase activity according to any of claims 13-14. 18. A detergent composition according to claim 17, which further comprises a linear alkyl benzene sulfonate surfactant. 19. A detergent composition according to claim 17 or 18, which further comprises one or more other enzymes such as a protease, amylase, lipase or peroxidase.

Inventors: DALBOEGE HENRIK, HELDT-HANSEN HANS PETER, RASMUSSEN GRETHE, DALBOEGE, HENRIK, HELDT-HANSEN, HANS, PETER, RASMUSSEN, GRETHE
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1992
Main classification: C12N1
IPC codes: C11D3, C12N1, C12N9, C12N15, C12R1