FI120097B - Novel enzyme preparations and processes for their preparation - Google Patents

Description

Novel enzyme preparations and processes for their preparation

The present invention relates to isolated nucleic acid molecules, recombinant vectors, recombinant hosts, and to a process for producing enzyme-enriched enzyme activity, culture medium and product. The invention further relates to the use of products in the pulp, paper and feed industries. The invention also discloses the production of such enzyme preparations by members of Trichoderma species that have been subjected to genetic engineering. These preparations are rich in xylanase enzymes.

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Cellulose is a linear polysaccharide formed by glucose units linked by β-1,4 bonds. In nature, cellulose is usually combined with lignin together with hemicelluloses such as xylans and glucomannans. In the pulp and paper industry, most of the lignin is extracted in the manufacture of chemical pulp 15 (cooked) to obtain an acceptable pulp product. However, the resulting pulp is predominantly brown because a small amount of lignin remains in the pulp after cooking. This residual lignin is traditionally removed in a multi-step bleaching process, typically using a combination of chlorine chemicals and extraction steps. When it comes to reducing or avoiding the use of chlorine chemicals, 20 peroxides, oxygen and ozone are also used.

The hemicellulases can also be used in enzyme-assisted bleaching of pulps to reduce the chemical dose in subsequent bleaching or to increase pulp. blue reflection index (Kantelinen et al., International Pulp Bleaching Conference, Tapp 25 Proceedings, 1-5 (1988); Viikari et al., Paper and Timber 7: 384-389 (1991)), and • · ·

Kantelinen et al., "Enzymes in Bleaching of Kraft Pulp," Dissertation for the Degree of. :. Doctor of Technology, Technical Research Center of Finland, VTT Publications 114, • · ·. ···. Espoo, 1992). Kim hemicellulase is used in this way, of course, it should not contain • · • ··. *. cellulases that would damage the pulp fibers.

• · · • ·· 3o • ·

The use of hydrolyzing enzymes in hemicellulose in various bleaching formulations is known from WO 89/08738, EP 383 999, WO 91/02791, EP 395 792, EP 386 888 and WO '· 91 91 055 by.

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Other industrial applications of hemicellulose-degrading enzymes include the production of thermo-pulp pulps, where the use of hemicellulose-degrading enzymes is to reduce energy consumption. The hemicellulose-degrading enzymes can be used to improve the drying of the recycled pulp or to make soluble pulps (Viikari et al. 5 "Hemicellulases for Industrial Applications," in Bioconversion of Forest and

Agricultural Wastes, Saddler, J., Publisher, CAB International, USA (1993)).

The use of hemicellulolytic enzymes for improved dewatering of mechanical pulp is known from EP 262 040, EP 334 739 and EP 351 655 and DE 4 000 558.

When considering the hydrolysis of biomass to liquid fuels or chemicals, conversion of both cellulose and hemicellulose is essential to obtain high yields (Viikari et al., "Hemicellulases for Industrial Applications," 15 Bioconversion of Forest and Agricultural Wastes, Saddler, J.). CAB International, USA (1993)).

There is also a need in the feed industry to use an appropriate combination of enzyme activities to cleave a substrate containing β-glucan and hemicellulose.

In order to make the use of hemicellulolytic enzymes economically viable, the production costs of enzymes must be reduced. This means that the production volumes must be high and the manufacturing process cannot involve any expensive purification steps. The most economical way would be to select production strains and conditions in such a way that the cultivated 25 has a lot of required activities and only secondary activities. minor components.

Thus, there is a clear need for enzyme preparations that contain unique 'enzyme profiles and are tailor-made, i.e. specifically designed for the industry in which they are to be used, and which can be obtained at low cost, such as directly from a culture medium of a microorganism modified to produce the desired enzyme but which does not produce appreciable amounts of the undesired enzymes. .

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Xylans are complex heteropolymers consisting mainly of xylose and arabinose. Xylans are composed of B1, 4-linked xylopyranose units which may be substituted by acetyl as well as arabinose and methyl glucuronic acid residues (Timell, TE, et al., Wood Sci. Technol. 1: 45-70 (1967). Xylans are 5 cellulose The complete hydrolysis of xylan requires a number of enzymes, the most important of which belongs to the group of endo-B-glucanases (Biely, P., Trends Biotechnol 3: 286-260 (1985); Dekker, RFH, in Hignehi, T). (Publisher), Biosynthesis and Biodegradation of Wood Components (Academic Press Inc., Orlando), pp. 505-533 (1985); Woodward, J., Top Enzyme Fer-10 Ment., Biotechnol 8: 9-30 (1984). )).

Various microorganisms are capable of cleaving xylans, and xylanases have been found in both prokaryotes and native (Dekker, R.F.H., Richards, G.N., Adv.

Carbohydrate Chem. Biochem. 32: 277-352 (1976)). Xylan-cleaving microorganisms often produce many xylanases so that they can cleave different bonds on the xylan molecules. In a recent report, Bastawde (Bastawde, K.B., World J.

Microbiol. Biotechnol. 8: 353-368 (1992)) presented a compilation of current knowledge on the properties and modes of action of microbial endoxylases. There are several reports concerning the molecular cloning of these enzymes from bacteria (e.g., Ghangas, GS et al., J. Bacteriol. 171: 2963-2969 (1989); Lin, L.-L., Thomson, ···• J. Mol, Gen. Gen. Genet 228: 55-61 (1991); Shareck, F. et al., Gene 107: 75-82 (1991), Scheirlinck T. et al. , Appl. Microbiol Biotechnol. 33: 534-541 (1990); Whitehead, · · ·: * V TR, Lee, DA, Curr. Microbiol. 23: 15-19 (1991)) but rare in fungi (Boucher, Nucleic Acids Res. 16: 9874 (1988); Ito, K. et al., Editors, Xylans and Xylanases (Elsevier Science, Amsterdam), p. 349-360 (1992), van den Broeck, H. et al

.:. aJL "Cloning and expression of xylanase genes from fungal origin," EP 463,706 AI

(1992)).

. Trichoderma reesei is a potent producer of cellulolytic and xylanolytic enzymes (Suominen, P., et al., Kuwahara, M., Shimada, M. (eds.), Biotech. Pulp and Paper). Industry (Uni Publishers Co., Ltd., Tokyo), pp. 439-445 (1992); Tenkanen, M. et al. (Enzyme Microb. Technol. 14: 566-574 (1992)).

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Trichoderma reesei also produces all the enzymes required for the complete hydrolysis of native substituted xylans (Poutanen, K., et al., J. Biotechnol. 6: 49-60 (1987)). Multiple endo-β-1,4-xylanases have been purified from filtrates of Trichoderma cultures (Baker, C.J., et al. Phytopathology 67: 1250-1258 (1977)); Chromo-5 va, M., et al., Arch. Microbiol. 144: 307-311 (1986); John and Schmidt, Methods Enzymol. 160A: 662-671 (1988); Lappalainen, A., Biotechnol. Appl. Biochem. 8: 437-448 (1986); Sinner and Dietrichs, Holzforschung 29: 207-214 (1975); Tan, L.U.L. et al. Enzyme Microb. Technol. 7: 425-430 (1985); Wood and McCrae, Carbohydr. Res. 148: 321-330 (1986)). Two specific endoxylanases of T. reesei have been characterized with isoelectric points at pH 5.5 (endoxylanase I) and pH 9.0 (endoxylanase II), respectively (Tenkanen, M., et al.) Enzyme Microb. Technol. 14: 566-574 (1992)).

There is a need for efficient producers of myrobic and especially fungal xylanase, for example producers of enzyme mixtures for enzyme-assisted bleaching of pulp (Kantelinen, A., et al.) "Hemicellulases and Their Potential Role in Bleaching" Int. Pulp

Bleaching Confrence, Tappi Proceedings 1-9 (1988); Lahtinen, T., et al .. "Using

Selective Trichoderma Enzyme Preparations in Kraft Pulp Bleaching, "

Biotechnology in the Pulp and Paper Industry, Kuwahara and Shimada, editors, Uni 20 1 · Publishers Co., Ltd., Tokyo, Japan, p. 129-137 (1992); Viikari, L., et al .. "Bleaching ·· · • · ·: · 1 with Enzymes," Biotechnology in the Pulp and Paper Industry, Proc. 3rd Int. Conf., • · · / ·· 1 Stockholm, ss. 67-69 (1986); Viikari, L., et al., Proc. 4th Int. Symp. Wood and Pulp- · · ·: 1V ing Chemistry, Paris 1: 151-154 (1987).

The inventors have realized the importance of producing large amounts of hemicellulases economically and ideally in combination with other enzymes. decompose wood or plant material and have therefore studied recombinant DNA. the use of technology to construct hosts that would be useful in combining • ·. as a source of enzymes produced on a large scale by the use of DNA technology, which are advantageous for the use of hemicellulolytic enzymes.

These studies have led to the development work that has resulted in fungal hosts that express large amounts of the desired enzymes and preferably large amounts of hemicellulolytic enzymes.

These studies have also led to the development of fungal hosts which not only express large amounts of the desired enzymes, preferably hemicellulolytic enzymes, but lack at least one component of the cellulase lysing system.

It is an object of the invention to provide fungal hosts belonging to the genus Trichoderma 10 which are capable of expressing large amounts of xylanase activity, or fungal hosts which additionally partially or completely lack cellulase activity. It is a further object of the invention to provide fungal hosts which produce large amounts of xylanolytic and certain cellulolytic, preferably endoglucanases, but which also partially or completely lack any other cellulase activity and preferably cellobiohydrolase. It is a further object of the present invention to provide novel hosts (plant, yeast, fungal and bacterial hosts) containing and expressing such xylanases. More particularly, the subject matter of the invention is characterized by what is stated in the characterizing part of claims 1,5,6, 9, 11,14 and 15.

. . Furthermore, the process for producing the enzyme preparation according to the invention is characterized by what is stated in the characterizing part of claim 18. The culture medium or product of the invention is characterized by what is said in the characterizing part of the corresponding claims 24 and 25. Further, the use of a culture medium or product according to the invention is characterized by what is stated in the claims. ·: ·. 25 26 and 27 in the character section.

• · ·; * j *. It is a further object of this invention to isolate and clone xylanase genes from Trichoderma.

• · · • • • • • a. ·! ; Figure 1 shows the general principle of gene deletion.

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Figure 2 shows a restriction map prepared at 5.7 kb (kilobase) • · · · l ”of pALK475. size insert (Kpnl). The location of the xln2 gene is indicated by an arrow. Separate lines t t · represent inserts in the subclones pALK573, pALK574, pALK570 and pALK476. The SmaI site at the 5 'end of the fragment is derived from the polylinker pUC19.

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Figure 3 shows the nucleotide sequence of the T. reesei xln2 gene [SEQ ID NO: 1]. The coding regions are indicated by the upper large kernels [SEQ ID NO: 2]. The peptide sequences obtained from the purified protein are indicated by underlining; the double underlined sequence was used to prepare the PCR-5 primer. The TATA box is indicated by dots. The putative 58 cleavage sites are indicated by the arrow (s) and the putative N-glycosylation sites by the triangle (▼). The N-terminus of the mature protein is represented by the arrow (). The two glutamic acids which have been proposed to belong to the active site have been framed.

Figure 4 shows plasmid pALK476 used to target the xln2 gene to the cbh1 locus. The xln2 gene is under the control of its own promoter.

Figure 5 shows the pALK476 fragment used for transformations. The 1.9 kb cbhl 5 'flanking region (Scal-EcoRI) is above the 2.2 kb cbhl coding region, the 1.8 kb 3' region (BamHI- Eco RI) is 1.4 kb cbh1 below the end of the coding region. The amdS gene was from p3SR2 (3.1 kb SpeI-XbaI fragment), and the xln2 gene was from pALK475 (5.0 kb SmaI fragment, see Figure 2). The promoter region of the xln2 gene is 2.3 kb in the fragment.

Figure 6 shows the relative levels of xylanase production of the transformants of Figure 5 which are · · · · · *; calculated for T. reesei yield levels of VTT-D-79125. The bars indicate the average • · · • ·. **! values and range of yield levels among the transformants of each host strain.

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“V One bottle of each transformant was grown. The numbers of transformants examined were as follows (CBHI + /): T. reesei VTT-D-79125 19/38, ALK02221 16/26 and • · · 25 ALK02721 22/31.

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Figure 7 shows the construction of pALK174 (xln2 gene is fused to the cbh1 promoter).

'. Only relevant restriction sites are shown.

Figure 8 shows a relative increase in the production of xylanase activity by pALK174- · · · f 'transformants.

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Figure 9 shows a restriction map of a 2.3 kb EcoRI insert of pALK572. The location of the xlnl gene is indicated by an arrow.

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Figure 10 shows the nucleotide sequence of the T.reesei xIII gene [SEQ ID NO: 5]. The coding regions are indicated by upper case letters [SEQ ID NO: 4]. The peptide sequences obtained from the purified protein are underlined .; the double underlined sequence was used to prepare the PCR primer. The TATA box is indicated by dots. The expected signal breakpoint is indicated by the arrow (s). The N-terminus of the mature protein is represented by the arrow (). The two glutamic acids are framed-10, which are expected to be present at the active site.

Figure 11 shows the construction of plasmid pALK807 (the xln1 gene is fused to the cbh1 promoter). Only the relevant restriction sites are shown.

Figure 12 shows FPLC analysis of the CBHI negative transformant VTT-D-87312.

Figure 12A shows its comparison with an untransformed host.

Figure 13 shows a diagram of plasmid pALK99.

Figure 2 shows the replacement of the chromosomal cbh2 gene with the aergB gene.

Figure 15 shows the structure of plasmid pALK412.

Figure 16 shows a diagram of plasmid pPLE3.

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Figure 17 shows the construction of the Acbh1-Acbh2 strain; (UV) trpC mutant of VTT-D-79125.

Fig. 18 shows a (re )i hypertellulolytic mutant strain of T. reesei VTT-D-79125 (UV); . enzyme profile and a genetic modification of this strain lacking the cellobiohydrolase • · · • · · ''.

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Figure 19 shows the modified enzyme profiles of T. reesei strains # 1, # 2 and # 3 engineered.

The following description uses a number of terms that are widely known in recombinant DNA (rDNA) technology. In order that the specification, the claims, and the scope of the invention as set forth in such terms will be understood, the following definitions are provided.

Cellulase. Cellulase is a collective name for enzymes that catalyze reactions that are involved in the degradation of insoluble cellulose to soluble carbohydrate. The term "cellulase" is known in the art, which refers to said enzymes. For efficient digestion of cellulose to glucose, at least three cellulases (three types of cellulase enzyme activity) are required: randomly cleaving endoglucanases (1,4-BD-glucan glucanohydrolase, EC 3.2.1.4), which are usually active on substituted, soluble substrates and cellulose, cellobiohydrolase (1,4-BD-glucan cellobiohydrolase, EC 3.2.1.91), which is able to degrade crystalline cellulose but has no activity against cellulose derivatives, and B-glucosidase (β-D-glucosidiglyohydrolase), 3.2 degrades cellobiose and cello-oligosaccharides to produce glucose. Each of the three major types of enzymes occurs in multiple forms. For example, two immunologically distinct cellobiohydrolases, CBHI and CBHII, are known.

• * · • · '! In addition, five to eight distinct endoglucanases are known which differ electrophoretically. Synergistic action between some of these enthusiasts has been illustrated. Cellulase activity is synonymous with cellulolytic activity.

Cellulose, cellobiose, soforose, and lactose stimulate or induce cellulase biosynthesis and are inhibited by glucose or other readily used carbon sources.

«· ·« • · · · · · · ·

Trichoderma host. which is "substantially incapable" of synthesizing one or * · more cellulase enzymes means a Trichoderma host. wherein one or more of the 3Q cellulase enzymes are depressed, deficient, or absent compared to the organic (transformed) Trichoderma type.

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Hemi-cellulolytic enzymes Chemi-cellulases A variety of enzymes, each termed "hemicellulase", are required for enzymatic cleavage and conversion of hemicelluloses. The two main glycanases that depolymerize the hemicellulose backbone are endo-1,4-B-xylanase and endo-1,4-B-D-mannanase. Small 5 oligosaccharides are further hydrolyzed by 1,4-β-D-xylosidase, 1,4-β-D-mannosidase and 1,4-β-D-glucosidase. The side groups are eliminated by α-L-arabinosidase, α-D-glucuronidase and α-D-galactosidase. The esterified side groups are produced by various esterases.

The definition of hemicellulolytic enzymes is taken from Viikari et al., He-10 Micellulases for Industrial Applications, published in Bioconversion of

Forest and Agricultural Wastes (1992).

Entsvvmivalmiste. "Enzyme preparation" means a composition containing enzymes extracted (either partially or completely purified) from fungi or from culture media used to grow such fungi. Therefore, the term "enzyme preparation" refers to a composition comprising a medium previously used for the cultivation of said fungi and any enzymes secreted by the fungi during culture.

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1201 Grain casting. By culture medium is meant a composition previously used for cultivation of fungi ("used" culture medium), a culture medium containing enzymes secreted by the fungus during culture. The cereal medium can be used as such or partially, completely, cleaned, concentrated, dried or immobilized, ie immobilized. If desired, the expressed endoglucanase protein can be further purified under conventional conditions such as extraction, ··· precipitation, chromatography, affinity chromatography, electrophoresis, etc.

• · · · • i · »· · i ·

Biological bleaching. By "biological bleaching" is meant the extraction of lignin from the pulp after having been treated with hemicellulose-degrading enzymes with or without .30 lignin-degrading enzymes. • · · may be limited by hemicelluloses. removal of lignin, which is performed either physically (by re-precipitation on the fiber * ** surface during cooking) or chemically (via lignin-carbohydrate complexes). The hemicellulase activity partially degrades the hemicellulose, which increases the extractability of lignins with conventional bleaching chemicals (such as chlorine, chlorine dioxide, peroxide, etc.) (Viikari et al., "Bleaching with Enzymes" in Biotechnology in the Pulp and Paper Industry, Proc. Conf., Stockholm, pp. 67-69 (1986); Viikari et al., "Applications of Enzymes in Bleaching" in Proc. 4th Int. Symp. Wood 5 and Pulping Chemistry, Paris, Volume 1, pp. 151-154 (1986). (1987); Kantelinen et al., "Hemi-cellulases and their Potential Role in Bleaching", International Pulp Bleaching Conference, Tappi Proceedings, pp. 1-9 (1988). The advantages of this improved bleaching are lower consumption of bleaching chemicals and lower environmental loads or higher final diffuse reflectance values. The hemicellulolytic enzymes improve unbleaching by facilitating the removal of lignin by conventional bleaching chemicals.

Gene. The gene is a DNA sequence containing a template for RNA polymerase. The RNA encoding the protein is called messenger RNA (mRNA) and is transcribed by RNA polymerase II in eukaryotes. However, it is also known to construct a gene containing a template of RNA polymerase II which transcribes an RNA sequence having a complementary sequence to a specific mRNA, but which does not normally translate. Such a gene construct is combined here. is referred to as an "antisense RNA gene" and such an RNA transcript is referred to as "i. ant." V.2iJ "antisense RNA". Translation of antisense RNAs is normally not possible due to the presence of translation stop codons in the antisense RNA sequence.

The "complementary DNA" or "cDNA" gene contains recombinant genes synthesized by reverse transcription of mRNA, with intermediate sequences (introns) » 25 have been deleted.

• «· • · ·«

By an enzyme homologous to the Trichoderma host of the invention, it is meant that the untransformed Trichoderma of the host species produces naturally the same amount of organic protein; a 3D gene homologous to the Trichoderma host of the invention refers to a gene present in such an untransformed Trichoderma I t f ^ "" "" · · ^. genome of the same species as the host species.

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By a heterologous enzyme to the Trichoderma host of the invention is meant that such is an untransformed Trichoderma. the same species as the host species does not produce the same amount of organic enzyme; by a Trichoderma host heterologous gene of the invention is meant a gene which is not present in the genome of an untransformed Trichoderma man of the same species as the host species.

Hybridization. By hybridization is meant the conditions under which all Trichoderma xylanase genes hybridize to the nucleic acid sequence encoding the T. reesei xylanase p1.5 amino acid sequence or the nucleic acid sequence encoding the 10 T. reesei xylanase p1 9 amino acid sequence. These conditions are characterized by preferably hybridizing at 60-68 ° C with 6x SSC + 0.1% SDS and washing at 68 ° C with 6x SSC + 0.1 percent SDS.

The cloning. By cloning vehicle is meant a plasmid or phage DNA or other DNA sequence (such as linear DNA) which provides a suitable nucleic acid environment for the transfer of the gene of interest into the host cell. The cloning vehicles of the invention can be engineered to replicate autonomously in prokaryotic or eukaryotic hosts. Cloning vehicles generally do not replicate. autonomously in Trichoderma. and, instead, exclusively provide a transporter for transferring the gene of interest to the Trichoderma host for subsequent insertion into the Trichoderma genome. In addition, the cloning vehicle may be characterized by a single endonuclease recognition site or a small number of such recognition sites, 4 * · «· ·, i, in which these DNA sequences can be cleaved in a specific manner without loss of the biological function of the vehicle. to provide replication and cloning of such DNA. The cloning vehicle may additionally contain a ·· «marker suitable for identifying cells transformed with the cloning vehicle 4 · · · s': *. Markers include, for example, tetracycline resistance or ampicillin resistance. The word "vector" is sometimes used for "cloning vehicle". Alternatively, such markers may be provided by a separate cloning vehicle from the vehicle in which the gene of interest is obtained.

• »i * · •« · • ··

Ilmentvmisvehikkeli. The expression vehicle is a vehicle or vector similar to the cloning vehicle but capable of expressing the gene of interest which has been cloned into it after being transformed into the desired host. In a preferred embodiment, such an expression vehicle, after being transformed by the desired host, provides for increased expression of the gene of interest cloned therein.

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In a preferred embodiment, the gene of interest provided to the fungal host as part of the cloning or expression vehicle is integrated into the fungal chromosome. Sequences derived from a cloning vehicle or expression vehicle may also be integrated with a gene of interest during an integration event.

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Preferably, the gene of interest may be placed under the control of certain control sequences (i.e., operably linked) such as promoter sequences provided by the vector (which integrates with the gene of interest). If desired, such a control sequence may be provided by the fungal host chromosome, which is the result of the insertion site.

The expression control sequences on the expression vector will vary depending on whether the vector is constructed to express a particular gene in a prokaryotic or eukaryotic host (for example, a shuttle vector may provide a gene for bacterial host selection), and the vector may further include transcription elements such as: end sequences and / or translation start and end points.

Genetic Construction of Trichoderma Hosts The genetic construction method of hosts of the invention has been made easy by: • · «cloned gene sequences capable of encoding desired enzyme activity,. as well as expressing such gene sequences. As used herein, the term "gene sequences" is intended to refer to a nucleic acid molecule (preferably DNA).

• · · • # Gene sequences capable of encoding the desired enzyme are derived from $ 0. from different sources. These sources include genomic DNA, cDNA, synthetic. DNA and combinations thereof.

• · · • · · · · · * 13

Mesophilic Imperfect fungus Trichoderma reesei (before T. viridae) is classified as a member of Fungi imperfect. Fungi imperfecti is a category of all kinds of fungi that do not reproduce sexually or have no obvious affinity with sexually reproductive genera such as the highly characteristic Aspergillus.

5 Although a poorly defined sexual grade, which is the Hypocrean Imperfect of the per fect ascomycete species, has been reported with Trichoderma, the genera Aspergillus and Trichoderma have been clearly taxonomically distinct.

Enhanced enzyme preparations of the present invention are produced by Trichoderma-10 sponge modified by recombinant DNA techniques. The Trichoderma hosts of the invention have been engineered to be capable of producing large amounts of enzymes, preferably hemicellulases. In addition, these hosts have been engineered to completely lack at least one cellulase enzyme (which is undesirable in pulp and paper production). While the remaining cellulase activities may thus be intact, the Trichoderma hosts of the invention lack, partially or completely, the necessary full amount of enzymes that completely degrade cellulose to glucose, and therefore such cellulose degradation is greatly reduced or completely inhibited.

Transformation of Trichoderma by a Novel Gene Construct In one embodiment, the Trichoderma host of the present invention is partially or completely lacking in at least one cellulase activity, with a gene construct capable of expressing at least one desired pulp and paper production enzyme homologous to Trichoderma to provide this · · · · · 125 increased levels of the enzyme in the Trichoderma host. Examples of homologous enzymes that are desired for pulp and paper production include, for example, hemicellulases and pectin-degrading enzymes. Trichoderma can • · · *. naturally producing a variety of hemicellulases including endoxylanases, • · 30 mannanases, β-xylosidases, α-arabinosidase, α-glucuronidase, and acetyl esters. each of which may be the enzyme desired in the formulations of the invention.

Organic Trichoderma also produces small amounts of pectin-degrading enzymes, such as polygalacturonidase, which can be classified as the enzyme 14 desired in the enzyme preparations of the invention. In addition, any other Trichoderma enzyme. which oxidizes cellulose may be utilized in the enzyme preparations of the invention and may be the desired enzyme.

5 xylanolytic production of Trichoderma reesei QM 9414, Aspergillus awamori VTT-D-75028, Fusarium oxvsporum VTT-D-80134, Bacillus subtilis ATCC 1271 l and Streptomyces olivochromomogenes ATCC 21713 has been demonstrated that T. reesei produced the highest xylanase activities. Therefore, under conditions where it is desired to maintain xylanolytic activity, T. reesei is the preferred host. (Poutanen, K., "Characterization of Xylanolytic Enzymes for Potential

Applications "in Dissertation for Doctor of Technology, Technical Research Center of Finland, Publications 47).

Although the above preparations from various microbial sources differed in β-xylosidase activity and side chain cleavage activity, the T reesei medium additionally contained all of the defined side chain cleavage media.

activities (acetylesterase, α-glucuronidase and α-arabinosidase), whereas R

oxvspomb and S. olivochromogenes medium contained only esterase. Trichoderma is thus an advantageous host because it produces natural broad-spectrum xylanolytic enzymes, the portions of which can be manipulated by genetic engineering for a variety of applications to provide enzymatic preparations tailored to these enzymes. purposes.

In accordance with the present invention, gene constructs encoding homologous enzymes that are desired for pulp and papermaking purposes can be introduced. In the trichome derman genome, and also enhanced expression can be achieved by the use of strong promoters such as cbhl. and, if desired, new and modified control regions, such as amplifier sequences. Such regulatory ranges are preferably homologous to Trichoderma. The regulatory area, and in particular the pro- · ·. 3Q; the engine may be modified to include only the sequence elements that are required for expression and / or to maintain an area corresponding to large expression levels. tymistasoista. Enhancer sequences may be introduced at the same time as the gene of interest, being a separate DNA element, but operably linked to 15 genes of interest, e.g., a DNA sequence that is collinear with a sequence that provides the gene of interest (e.g., 5 '). - or 3'-non-translation sequence or intron).

In a particularly preferred embodiment, the homologous gene introduced into the Trichoderma genome is a gene encoding a homologous hemicellulase, preferably xylanase.

Two major xylanases produced by T. reesei have been purified (Tenkanen, M., Enzyme Microb. Technol. 14: 566-574 (1992)). The enzymes have isoelectric points of 5.5 (XYLI) and 9.0 (HYLII) and have molecular weights of 19 and 20 kDa, respectively.

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The optimum activity of the xylanase I enzyme from Trichoderma Tees is in the acidic pH range, whereas the x optimum for xylanase Π: η is close to the neutral pH range. Because of the different properties of the two xylanases, one of these enzymes, or a mixture thereof, can be used in various applications (WO 92/03541 and Viikari et al., "Im-15 Portant Properties of Xylanases for Pulp and Paper Industry"). , Kuwahara, M. and Shimada, M. editors,

Proc. 5th Int. Conf. Biotechnology in Pulp and Industry, Uni Publishers Co., Ltd., Tokyo, 1992, ss. 101-106).

According to the invention, it is possible to use genetic engineering methods to enrich the culture medium for the desired · · · ”· / xylanolytic activity and to use this culture medium in the desired application. For example, xylanase I (acidic region) may be preferred in some bleaching steps and conditions, xylanase II may be preferred in some other bleaching sequences and conditions. (alkaline or neutral pH range of 25).

• · ·

In some applications, although one cellulolytic activity may be removed, reduced, inactivated, or repressed, it may be desirable to introduce a gene into the host cells which *. encodes a different cellulolytic enzyme so that one specific cellulolytic • · 30. activity is enhanced. For example, in the feed industry, in addition to hemicellulolytic activity, an enzyme preparation containing increased endoglucanase may be used. amounts. Thus, in addition to xylanases and other hemicellulases, added amounts of endoglucanases may be used in preparations where such hydrolysis is desired.

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In another embodiment, the Trichoderma host is transformed. which already expresses a homologous form of the 5 enzymes by a gene construct encoding a heterologous form of the same enzyme. In a further embodiment, a Trichoderma host which does not express a particular enzyme is transformed with one or more gene constructs encoding an enzyme (s) that are heterologous to Trichoderma.

The present invention provides increased amounts of a heterologous enzyme having activity desired for pulp and paper production by introducing into a specific locus a gene producing such a heterologous desired enzyme, or introducing the gene into multiple copies of the Trichoderma genome as described above.

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In a preferred embodiment, the gene encoding the desired enzyme is inserted into the cbh1 locus so that it is operably linked to the strong cbh1 promoter.

As will be described later, enhanced production is achieved by the use of strong promoters such as cbh1. As described later, increased levels of the 20 heterologous enzymes can also be achieved when the capacity of Trichoderman «·:: cellulase is generally reduced, even if the heterologous gene is not inserted into the cbh1 locus.

The gene encoding the desired enzyme, either homologous or heterologous, such as, for example, * 125 kheme hemicellulose hydrolysing enzyme, can be integrated into the Trichoderma genome. , such that the gene is inserted into a general expression vector, for example pAMH11O described in EP 244 234. pAMH11O is derived from pUC19 (Yanish-Perron et al., Gene 33: 103-119 (1985)), and it contains the promoter, · · · ', rb, and termination codon of the cbhl gene, and a bridging fragment, located between the promoter and stop codon, and can be deleted by cleavage with SacII and NdeI. When the ends are truncated, any DNA, cDNA, or chromosomal • · * * · * * DNA can be inserted between the promoter and the stop codon The desired gene can be inserted into the cbh1 expression cassette. a is located in the plasmid between pAMH115, the cbh1 promoter and the stop codon.

17

Transcriptional control elements of other genes can be used where cbh1 elements are not desired. For example, a vector construct containing transcriptional regulatory regions of the 3-phosphoglycerate kinase gene (pgk), which can be used as the 3-phosphoglycerate kinase, a key enzyme for the production of ATP by glycolysis, is expressed in the absence of glucose under which conditions the cellulases are synthesized.

10

Although the inventors do not intend to be bound by any particular theory, the number of copies of the desired gene integrated into the Trichoderma genome and the location of the gene integration in the genome appear to affect the efficiency of expression of the desired gene. In a preferred embodiment, integration of the desired gene is targeted to a specific locus. The use of linear DNA helps in this direction of integration. In a highly preferred embodiment, integration of the desired gene into the Trichoderman cbhl locus is directed.

The DNA constructs of this invention can be used to transform Trichoderma strain 20. Such strains include, for example, T. reesei strains QM9414 • a · (ATCC 26921), RUT-C-30 (ATCC 56765), and highly efficient mutants such as · · · *; for example, VTT-D-79125, a descendant of QM91414 (Nevalainen 1985, Techniques Research Center of Finland Publications 26, (1985), Espoo, Finland). Trichoderma λ · · · · · V can be transformed by any method known in the art, and in particular by the technique disclosed in EP 244 234.

• · ·

Trichoderma host cells can be cultured and the desired enzymes produced by cultivating a host strain having the desired properties under any condition that '. make it possible to express the desired enzymes. For example, Trichoderma- · ^ 30; a host strain having the desired properties can be cultivated in a liquid culture zone, which may contain, for example, 6% Solka Floc cellulose, 3% * ««. 0.5% KH 2 PO 4 used, 0.5% (NH 4) 2 SO 4, 0.1% structo * · · * · '*. Glucose is a sensitive suppressor of cellulase production by Trichoderma strains and requires an inducer such as cellulose, lactose or soforose (Allen et al., Biotechnology and Bioengineering 33: 650-656 (1989). The pH of Trichoderma cultivation should be kept at about 5, by adding phosphoric acid or ammonia and maintaining the temperature at 30 ° C. However, the temperature should be adjusted according to the strain and its enzyme preparation produced (Merivuori et al. Biotechnology Lett.

12: 117-120 (1990)).

DNA Cloning The vector systems can be used in a method of preparing Trichoderma hosts for the production of the enzymes of the invention. One element provided by such a vector construct may encode a sequence of at least one homologous gene whose activity is to be deleted, diminished, inactivated, deleted or repressed. Such a vector construct (a) may further provide 15 separate vector constructs (b) encoding at least one desired gene to be integrated into the Trichoderman genome, and (c) an selectable marker linked to (a) or (b) ) ratio. Alternatively, a separate vector may be used.

£ 0 The cloned DNA used in the hosts of the invention may or may not contain t · t · ψ · CV CV introns. Above all, such genomic DNA can be obtained by fusion with the DNA gene sequence * /, the native 5'-promoter region of the sequence, and / or the 3'-transcriptional end region of the transcript. if such sequences are capable of working in Trichoderma. In addition, * ·! · / 25 such genomic DNA can be obtained by combining with gene sequences encoding a 5 'untranslated region of mRNA and / or by combining with a gene sequence encoding a non-translational 3' region. translate. To the extent that * - · * where the Trichoderma host cell can recognize transcriptional and / or translational regulatory signals related to mRNA and protein expression, the 5'- * · '3ft and / or 3' regions of the organic gene, non-transcriptional, and / or 5 'and / or 3' regions of the mRNA, which are non-translational, may be maintained and used for transcription and translation i «i * · · \. regulation. Genomic DNA can be stored according to known methods of the invention (see, for example, Guide to Molecular Cloning Techniques, S.L. Berger et al.).

19 editors, Academic press 81987). Preferably, the mRNA preparation used should be enriched in the mRNA encoding the desired protein, either naturally or by isolation from cells producing high levels of protein, or by in vitro enrichment of mRNA by methods commonly used in mRNA preparation. 5 enrichment of specific sequences, such as sucrose gradient centrifugation, or mRNA is enriched both naturally and in vitro.

When cloned into a vector, suitable DNA preparations (either genomic DNA or cDNA) for this purpose are randomly cleaved, or enzymatically cleaved, respectively, into appropriate vectors to form a recombinant gene library (either genomic or cDNA).

DNA encoding the desired protein can be inserted into the DNA vector by conventional techniques including blunt or alternating ends, appropriate filling of the cohesive ends, treatment with alkaline phosphatase to avoid unwanted splicing and binding with suitable ligases. Such manipulation techniques disclosed by Maniatis, T., et al., Supra, are known in the art.

# ^ 20 Libraries containing clones encoding a desired protein, identified by any selective DNA of said protein, may be screened. by selective means, such as, for example, by (a) hybridizing to a suitable nucleotide • »* t · j. ·. malic acid probe (s) containing a sequence specific for this IM · protein, or (b) using a hybridization-selected probe

»IM

· * · 2 £ translation analysis in which the organic mRNA which hybridizes to the clone in question is translated in vitro and the translation products are characterized ·: * further, or c) if the clonamt gene sequences themselves are capable of expressing the mRNA, the imi: a protein product obtained by translation with the help of a host containing 9 clones is ammunologically mixed.

• *

-M

# ··· Oligonucleotide probes specific for the desired proteins can be * t * 9, ·. j is used to identify clones corresponding to such proteins which can be constructed based on the amino acid sequence information of the protein.

20

Because the genetic code is degenerate, more than one code may be used to encode a particular amino acid (Watson, JD, Molecular Biology of the Gene, Third Edition, WA Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357 ). Peptide fragments are analyzed to identify the amino acid sequences encoded by the oligonucleotides with the lowest degree of degeneration.

This is preferably done by identifying sequences containing amino acids encoded by a single codon.

Although a single oligonucleotide sequence may encode an amino acid sequence, it may often be encoded by any sequence of similar oligonucleotides. Importantly, when all members of this set contain oligonucleotide sequences capable of encoding the same peptide fragment and thus possibly contain the same nucleotide sequence as the gene encoding the peptide fragment, only one member of the Saija contains a nucleotide sequence identical to the exon encoding the gene . Because this member is within the sequence and is capable of hybridizing to DNA even when the other members of the series are absent, it is possible to use an unfractionated set of oligonucleotides in the same way as using a single oligonucleotide to clone a gene encoding a peptide.

Using the genetic code (Watson, JD, Molecular Biology of the Gen., Third Edition, WA Benjamin, Inc., Menlo Park, CA (1977), the amino • · · * ··; acid sequence to identify one or more oligonucleotides, each capable of encoding a protein. The likelihood that a particular oligonucleotide actually constitutes the actual protein can be estimated by taking into account the abnormalities and bases. the frequency at which a particular codon is actually used in · · · (encoding a particular amino acid) in eukaryotic cells, Lathe, R., et al., published such "codon usage rules" in J. Molec. Biol. 183: 1-12 (1985). · · "Codon usage rules" identify a single oligonucleotide sequence or set of oligonucleotides containing the theoretical, " the most "nucleotide • · 30. otid sequence capable of encoding the identified protein sequence.

21 or oligonucleotide series) can be synthesized by methods well known in the art (see, for example, Synthesis and Application of DNA and RNA, Narang, SA, 1987, Academic Press, San Diego, CA) and used as a probe of the desired , for the identification of a cloned gene when using such types,

5 well known methods. Maniatis, T. et al., (Molecular Cloning, A

Laboratory Manual, Cold Sring Harbor Laboratories, Cold Spring Harbor, NY (1982)) and Hames, B.D., et al. (Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)) have published nucleic acid hybridization and clone identification techniques.

10

Cloning techniques using oligonucleotides and PCR are well known in the art ("PCR Protocols" in A Guide to Methods and Application, Innis et al., Academis Press, San Diego (1990)). Those members of the gene library described above that have been found to be capable of such hybridization are then analyzed to determine the extent and nature of the desired genomic coding sequences they contain.

To facilitate expression of the desired DNA coding sequence, the DNA probe described above is labeled with a moiety which is expressed. Such a detectable group may be any material having detectable physical or chemical properties.

In the field of nucleic acid hybridization, expression materials are well-developed, and most of the labels which are in the present invention can be used. useful in these methods. Particularly useful are non-radioactive labels, such as digoxigenin nucleotides (dUTP) or radioactive labels, Ϊ5. such as 32P, 3H, 14C, 35S, 125J or the like. Any radioactive label that provides a sufficient signal and has a sufficient half-life can be used. The oligonucleotide may be radiolabelled or non-radioactively labeled by a number of methods known in the art and available for these purposes. commercial reagent kits.

jp /: • · • Alternatively, polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as bio-· · · · ♦ 1 phine, digoxigenin, enzyme or fluorinating group, see for example Leary , 22 JJ, et al., Proc. Natl. Acad. Sci. USA 80: 4045 (1983); Renz, M., et al., Nucl. Acids Res. 12: 3435 (1984); and Renz, M., EMBO J. 6: 817 (1983).

Thus, in summary, the actual identification of a protein sequence (or partial 5 protein sequence) makes it possible to identify a theoretically "most likely" DNA sequence or series of sequences capable of encoding the peptide in question. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the "most probable set" of oligonucleotides), a DNA-10 molecule (or series of DNA molecules) capable of acting as a probe is obtained. , by identifying and isolating clones containing the protein gene.

In an alternative way of gene cloning, a library is prepared using 15 expression vectors to clone DNA, or preferably cDNA, made from a cell capable of expressing the desired protein. The kernel is then screened for members expressing the protein, for example, by screening the kernel for antibodies to the protein.

Therefore, the above methods can identify gene sequences which are capable of encoding a desired protein or fragments of that protein. For further characterization of such gene sequences and for the production of recombinant protein, it is desirable to express the proteins encoded by these sequences. Such expression identifies those clones that express proteins having the property of the desired protein. properties. These properties include, inter alia, the ability to specifically bind antibodies directed against the protein, to elicit the production of antibodies capable of binding the protein, and the ability to provide the recipient cell with a function specific for the protein.

39.: The sequences encoding the cloned protein obtained by the methods described above, and preferably in double-stranded form, can be operably linked by sequences. which control transcriptional expression in the expression vector and to export the · · · • · ·

Trichoderma host cell. in order to produce the recombinant protein or functional 23 derivatives thereof. Depending on which strand of the protein coding sequence is operably linked to sequences controlling transcriptional extrusion, it is possible to express antisense RNA or a functional derivative thereof.

A nucleic acid molecule, such as DNA, is said to be "capable of expression" of a polypeptide if it contains expression control sequences that contain transcriptional regulatory information, and if such sequences are "operably linked" to the nucleic acid sequence encoding the polypeptide.

A functional linkage is a linkage by which a sequence is linked to a regulatory sequence (or sequences) in such a way that expression of the sequence is placed under the influence or control of a regulatory sequence. Two DNA sequences (such as a protein coding sequence and a promoter region linked to the 5 'end of the coding sequence) are said to be operably linked if (1) the induction of promoter function does not result in a read-phase mutation, (2) does not interfere with expression control. direct expression of mRNA, antisense RNA, or protein; or (3) not interfere with the ability of the promoter regulatory region to transcribe the template. Thus, the promoter would be operably linked to the DNA sequence if the promoter were able to effect the transcription of that DNA sequence.

20 • · »• · · I". ' The exact nature of the regulatory regions required for gene expression may vary according to species and cell types, but generally they must necessarily contain 5 · 5 sequences that do not undergo transcription or translation (non-coding sequences). · ·

• ·· I

), and are associated with transcription and translation initiation, respectively.

• · · t .25.

• · ·

Protein expression in Trichoderma hosts requires the use of regulatory regions that operate in such hosts. Many different transcriptional and translation control sequences can be used, since Trichoderma generally recognizes eukaryotic >> t>; host-derived transcriptional controls, such as control sequences derived from fungi with filaments. For eukaryotes that are not · · transcribed not translated, such control regions can provide • · · · ·. starting methionine codon (AUG), or is absent depending on whether the cloned sequence contains the methionine in question. These regions generally contain a promoter region sufficient to direct the initiation of RNA synthesis in the host cell.

As is well known, translation of eukaryotic mRNA begins at the codon encoding the first methionine. For this reason, it is better to ensure that the link between the euka ryotic promoter and the DNA coding for the protein or its functional DNA sequence does not contain any intervening codons capable of coding for methionine. The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same read phase as the DNA encoding protein 10) or in the read phase mutation (if the AUG codon is not in the same read sequence as the protein coding sequence).

Regulatory Zones In a preferred embodiment, the desired protein is secreted into the surrounding culture medium due to the presence of a Trichoderma homologous secretion signal sequence. If the desired protein does not have its own signal sequence, or if such a signal sequence does not function well in Trichoderma. then the protein coding sequence may be operably linked to a signal sequence homologous or heterologous to the Trichoderin. The desired coding sequence can be linked to any signal sequence that allows the protein to be secreted from a Trichoderma host, such as Trichoderma. . ·. man cellobiol hydrolase I protein sequence. Such signal sequences • · • · ·:. can be constructed with or without specific protease sites such that the signal sequence is responsible for subsequent deletion.

·· * · .25- • · ·

Transcription initiation regulatory signals that allow repression or ··· activation may be selected so that expression of functionally linked genes can be altered. Of interest are regulatory signals that are sensitive to temperature such that, by varying the temperature, expression can be repressed or initiated, or .30: expression can be targeted by chemical regulation, e.g., substrate or meta. under the control of the bolite. Also interesting are structures where • · · '·; and (a) the desired protein mRNA and (b) the antisense RNA targeting cellulase enzymes are provided in forms that can be transcribed, such as in a form that is complemented by a single cellulase enzyme in the host protein. expression repression by antisense RNA.

Translational signals are not essential for expression of antisense RNA-5 sequences.

If desired, the sequence encoding the protein can be obtained by the above-described methods without 3'-regions that do not undergo transcription and / or translation. A non-transcriptional 3'region may retain its regulatory sequence structure portion 10, which terminates transcription, or those that terminate translation, or those that direct polyadenylation.

The vectors of the invention may further comprise other functionally linked regulatory moieties, such as enhancer sequences.

15

Detection of Trichoderman Stable Transformants

Genetically stable Trichoderma transformants are constructed in a preferred embodiment by linking the DNA of the desired protein to the host chromosome. The sequence encoding the desired '20a protein can be from any source. The DNA may be fused within a V de Novo cell or, in a most preferred embodiment, by transformation. . ·, With a vector that is functionally inserted into the host chromosome, for example DNA · · • «·: components that promote the integration of DNA sequences into chromosomes.

X35: Selecting cells that have permanently integrated the introduced DNA into their chromosomes, including the introduction of one or more markers that *: 1 allow selection of host cells that on the chromosome include: an expression vector, for example, a marker can confer resistance to a biocide, for example, antibiotic resistance or resistance to heavy metals such as copper • 0 · and the like. The marker gene to be selected is either directly linked to DNA gene sequences. which must be expressed or introduced into the same cell by cotransfection.

• »· · • · · · · · · 26

Important factors in the selection of a particular plasmid or virus-derived vector include: recipient cells containing the vector should be readily identified and selected from those containing no vector; the number of copies of the desired vector desired for a particular host; and to be able to "transport 5 vectors back and forth" between host cells of different species.

Once a vector or DNA sequence containing construct (s) for expression is prepared, the DNA construct (s) is introduced into a suitable host cell by any of several suitable means, including the transformation described above. After vector insertion of the si-10 vector, recipient cells are grown in a selective medium that selects for growth of the transformed cells. Expression of the cloned (s) gene sequence (s) results in the production of the desired protein or fragment of this protein. This protein is expressed in transformed cells continuously or in a regulated manner.

15

The coding DNA sequences obtained by the above methods provide sequences which by definition encode a desired protein and which can then be used to obtain the antisense RNA gene sequences of the desired protein because the antisense RNA sequence is located within its sequence. opposite strand # 20 of the strand where transcription of the peptide core mRNA takes place. The antisense DNA strand »· · •• V may also be operably linked to a promoter in an expression vector such that. transformation with this vector yields a host capable of expressing antisense RNA in the * * ♦ transformed cell. The antisense RNA and its expression can be used to interact with endogenous DNA or RNA in a manner that ·? inhibits transcription or translation of the gene or causes repression of one or both of them. The use of antisense RNA probes to suppress gene expression is discussed in Lichtenstein, C., Nature 333: 801-802 (1988).

• · ♦ ♦ · ·

Trichoderma is a particularly useful and practical host for the enzyme of the invention; for the synthesis of preparations, since Trichoderma is capable of secreting large amounts of protein, for example concentrations of 40 g / l of culture medium have been reported; «· ·. ·. ; the homologous Trichoderman cbhl promoter is a highly suitable promoter for expression of the gene of interest because it is a potent single copy promoter 27 that normally directs up to 60% of the protein secreted from the Trichoderma host; the transformation system is versatile and can be applied to any gene of interest. The Trichoderma host provides an "animal cell type" that strongly glycosylates mannose; and the cultivation of Trichoderma is supported by prior, extensive experience in fermentation techniques on an industrial scale.

Trichoderma hosts lacking at least one cellulase enzyme According to the present invention, it is possible to enrich the Trichoderma hosts for the enzyme whose activity is desired for pulp and papermaking purposes by inactivating or removing at least one cellulase enzyme. In one embodiment, only the cbh1 gene is mutated. Since the majority of Trichoderma secreted proteins may be cellulase activity encoded by the cbhl gene (cellobiohydrolase, CBHI, protein), constructing Trichoderma hosts in which the cbhl gene has been mutated to an inactive form may increase which Trichoderma secretes into the culture medium. In another embodiment, the desired gene is preferably inserted into the cbh1 locus. such that expression of the desired gene is operably linked to the strong cbh1 promoter.

In a highly preferred embodiment, a cassette containing the desired gene already operably linked to the homologous cbh1 promoter is inserted into the cbh1 locus.

In the hosts of the invention, any one cellulolytic enzyme, some. or all of said enzymes may be removed or inactivated, reduced, or repressed by methods known in the art so that * 1 · 25. hosts that are partially or completely unable to degrade cellulose to glucose are obtained. Unwanted cellulolytic enzyme activities ··· can be removed, reduced, inactivated or repressed by a variety of methods, for example r ··· by inactivating the gene (s) encoding the enzyme (e.g. by introducing a read-phase mutation into the gene) , deleting a complete gene or large fragments of it, such that the gene is replaced by another DNA by homologous recombination, gene region compensation, further integration, double-stranded. by yellow crossing over and transforming the host cell with a gene construct which is capable of expressing the antisense RNA targeted to the coding sequence of that gene, etc.

For example, genes encoding cellulolytic activity may be inactivated as disclosed in European Patent Application EP 137 280 EP 244 234.

Trichoderma fungi produce large numbers of identical, predominantly haploid, mononuclear conidia, which provide an excellent material for various mutagenic treatments. However, even a haploid, mutated nucleus can produce a heterokaryotic 10 colony (mycelium) if the mutation initially becomes attached to only one strand of the DNA double helix (mosaicism). The number of mosaic mutants is influenced by both the mutagen and the dose used. Haploid, mononuclear conidia-forming fungi can address the problem of heterokaryotic fungal mycelium by allowing conidia to form and re-isolate colonies derived from a single, single conidia. This cycle can be repeated several times.

Examples of chemical mutagens useful in mutagenizing the Tricho-derma hosts of the invention include alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS) and diethyl sulfate,? (D FLAT). Useful are hydroxylamine and chemicals that remove amido- • v. groups of DNA bases such as nitric acid. Ionizing radiation (r- and X-ray radiation) and ultraviolet radiation (UV) are examples of physical mutagens which. are useful in mutagenesis of the Trichoderma strain.

«1 · · t ··» M »} '£>. Using solid media, it is possible to rapidly screen for colonies * derived from mutagenized colonies by screening for specific *! ' in the presence and absence of enzymes, and with the aid of said substrates, the enzyme produced t? 1, ί: can be quantitatively determined. According to the state of the art, there are several types of solid culture media: ·: 1 ·: which are suitable for use with enzymes, such as, e.g. Racellular Amylolytic Enzymes, Pectinase, Protease, Chitinase, B-galacto ···. expression of tosidase and cellulase, urease, RNAase, and DNAase.

• · 1 · «·>« · p I · 29

Many fungi form large, dispersed colonies as they grow on solid culture media. An increase in colony growth limiting chemicals may therefore be desired to allow the development of more than one (up to 100) colonies per plate. Among the substances used for this purpose are 5 rose Bengal, beef bile and phosphorus D, Triton X-100 and saponin. For some fungi, bacterial replication techniques may be used when examining the properties of fungal colonies on different culture media.

Following screening of the plates, selected colonies are typically cultured in shake flasks in 10 liquid media for measurement of enzyme activity. The best isolates, which express enhanced enzyme production at the scale of shake flasks, may be subjected to a second mutagenic treatment if desired.

Homologous genes which, according to the invention, are desirable to be inactivated or deleted include, for example, the cellulase genes cbh1. cbh2. egll. eg! 2. (formerly egl3;

Saloheimo et al., Gene 63: 11-21 (1988)) (encoding proteins cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II) or combinations of these genes. Deactivation of any of these genes produces a host that is partially or completely lacking in its ability to degrade cellulose to glucose. Sellulo-. Thus, lytic activity can be deleted at the genomic level by deleting the gene * * i t or converting it into a form that cannot be expressed. The activity can also be deleted at the translation level by hybridizing the protein-encoding mRNA to the antisense RNA to such an extent that translation of the hybridized RNA is inhibited.

··· * · · *: In a preferred embodiment, the cellulase activity is selectively inactivated so that some cellulase components are inactivated but not all. For example, if the host's ability to hydrolyze β-glucan is to be maintained, then endoglu- • ♦

Vs canase genes would be inactivated.

ψ 9 * ♦ ·· ♦ • ♦ 'öd Inactivation of one or more cellulase genes may be based on Trichoderma reesei /; *. transformation with a plasmid with a defective gene as described in EP 244 234. Homologous fusion of the plasmid with the chromosomal cellulase gene locus results in insertional inactivation of the endogenous cellulase gene of T. reesei. The plasmid used for transformation contains only a portion of the cellulase coding region and produces an inactive protein. Hedgehogs do not contain 5 'flanking sequences. The read phase mutation may also be provided in the truncated coding region. A selectable marker (e.g., amdS (acetamidase) or argB5 (omitinecarbamoyltransferase) or a screening marker (e.g., IacZ) can be linked to a plasmid used for transformation, or the transformation can be performed as a cotransformation, meaning that the selectable marker and the The gene can be inactivated by homologous recombination using circular DNA that is collinearly linked to Tricho-10 derman chromosomal DNA.

An unwanted gene can be deleted using the operating principle described in Figure 1. The recipient strain is transformed with a linear DNA fragment that contains a selectable marker gene (such as trpC. ArgB or amdS1 and / or an attractive, desired foreign gene that is expressed and flanked by the 5 'and 3' of the gene to be deleted). Homologous linkage at the A locus thus results in the displacement of the A gene by the selectable marker and / or the desired gene B. If the 5 'region in the transforming fragment is upstream of the promoter region, the promoter in the resulting complement strain will also be deleted. The Trichoderma gene, preferably the cellulase gene whose flanking regions can be cloned (isolated), first and foremost, the linear DNA fragment can be ligated to form a circular plasmid, or in addition, the circular form may contain: · · • DNA needed for replication in a bacterium (for example, E. coli) Linear • ·· · * : * fragments used for deletion of unwanted genes can be constructed, for example, from pUC19 (Yanish-Perron et al., Gene 33: 103-119 (1985)).

.. * · * This method is described in more detail in Example IB, which describes a deletion of the cbh2- · · · * gene from the Trichoderma genome using the method ♦: ··.

• 30:. ·: *. Clones of cellulase enzymes which can be used to construct mutant sequences for the inactivation of homologous sequences in a host according to the invention have been described. Any mutant sequence which results in inhibition of enzyme activity can be used. For example, Shoemaker, S., et al. Bio / Technology 1: 691-696 (1983) and Teeri et al. (Teeri, T., et al. Bio / Technology 1: 696-699 (1983)). 5 have cloned the gene for the natural CBHI of cellobiohydrolase, and the entire nucleotide sequence of the gene is known (Shoemaker, S., et al. Bio / Technology 1: 691-696 81983)). The major endoglucanase (EG I) gene from T. reesee has also been cloned and characterized (Penttilä, M., et al. Gene 45: 253-263 (1986); EP 137 280; Van Arstel, JNV, et al.) Bio / Technology 5: 274-278 81987) as well as ee 2 (originally eg! 3 10 (Saloheimo, M., et al. Gene 63: 11-21 81988)).

The enzyme preparation

According to the invention there is provided a process for producing high levels of enzymes, preferably hemicellulases, which are desired in pulp and paper processing. Also provided is a process for preparing an enzyme which is partially or totally lacking in cellulolytic activity (i.e., the ability to completely degrade cellulose to glucose) and enriched with enzymes desired in pulp and paper processing and preferably hemicellulases. By "deficient cellulolytic j20 activity" is meant the reduced, reduced, weakened or suppressed capacity of · · · · to break down cellulose to glucose. Such preparations may be obtained directly from the host of the invention. In addition, if the desired activities are present in more than one recombinant host, such preparations may be isolated from appropriate hosts and combined before being used in the method of the invention.

It is contemplated to provide an enzyme preparation in which the specific enzyme activities are enriched or absent, either partially or completely, so as to satisfy the requirements for specific use in the pulp, paper and feed industry.

* 30: Enzyme activities may be added or removed as described above to provide *: *. reducing, deleting, or retaining or reducing the desired activity. For example, if the preparation is to be used to improve the strength of the mechanical pulp, then the enzyme preparation of the invention may provide enzymes that enhance or facilitate cellulose fiber binding. Similarly, the enzyme preparation of the invention may also provide, for mass production, enzymes that enhance or facilitate such swelling.

In order to obtain the enzyme preparations of the invention, the recombinant hosts described above having the desired properties (i.e., hosts substantially capable of expressing one or more cellulases and capable of expressing the desired enzymes) are cultured under appropriate conditions, wherein the desired enzyme is secreted and from said culture medium for a period of 10 min well known in the art.

The enzyme preparation can be produced by culturing the Trichoderma strain in a fermentation vessel with the desired conditions, for example, in a liquid culture medium containing, for example, six percent Solka Floc cellulose (BW40, James River Corporation, Hackensack, NJ), three percent string. , 0.5% KH 2 PO 4, 0.5

(NH4) 2 SO4 and 0.1% structol as an antifoam agent (struktol SB

2023, Schill & Seilacher, Hamburg, FRG). Cellulase production by Trichoderma strains is highly repressed by glucose and requires induction (cellulose, lactose or

soforosis) (Allen et al. Biotechnology and Bioengineering 33: 650-656 (1989). pH

. .20 should preferably be kept at about 5 by adding phosphoric acid or ammonia and keeping the temperature at 30 ° C during cultivation. However, the temperature can be adjusted • · ·. strain and the enzyme preparation produced (Merivuori et al. Biotechnology • · ·

Letters 12 (2): 117-120 (1990)).

The enzyme preparation is recovered from the culture medium by methods known in the art. Since the hosts of the invention may be partially or completely lacking in cellulase activity, the advantage of the invention is that the enzyme preparation of the invention can be used directly from the culture medium without further purification. Such preparations may be *: · *: lyophilized if desired, or otherwise concentrated and / or • SO *. stabilize for storage. The enzyme preparations of the invention are very economically feasible and usable because (1) the enzymes can be used in; ·, · crude form; isolation of a specific enzyme from the culture medium is not necessary; and · and (2) since the enzymes are secreted into the culture medium, only the culture medium needs to be recovered 33 to obtain the desired enzyme preparation; the enzyme does not need to be extracted from the Trichoderma host.

If desired, the expressed protein may be further purified under conventional conditions such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, and the like.

The Trichoderma and enzyme preparations of the invention have further applications in the manufacture of feed. For example, feed treated with the enzyme preparations of the invention would have a high nutritional value for domestic animals because they can digest such feed more easily.

The invention will be described in more detail in the following examples. These examples illustrate only a few concrete embodiments of the invention. It goes without saying that a person skilled in the art will create several similar applications. For this reason, it should not be interpreted that the examples limit the invention, but rather illustrate it.

EXAMPLES

Materials and Methods. .20 Transformation of T. reesei: T. reesei was transformed and AmdS + and ArgB + transformants were selected as described by Penttilä et al. in Gene 61: 155-164 (1987).

• · ·: .25

Phleomycin resistant transformants were screened as described by Durand et al. in Biochemistry and Genetics of Cellulose Degradation, pp. 135-151, 1987, J.-P. Aubert, P. Beguin, and J. Millet (editors) Academic Press, New York.

• · * 30: Equimolar amounts of plasmid DNA (5-10 μg) were used in the co-transformation performed with p3SR2 and pAMH111. For transformations that gave: * ·. · Phleomycin resistance, relative amounts of plasmid DNA were used which were 1: 1 or 2: 1 for pAMH • · 111 and pAN8-1, respectively. When 34 p3SR2 and pMS4 were used in cotransformation, plasmid pMS4 was added in a 3 to 4-fold molar excess. The transformants were purified via conidia, that is, the conidia suspension was again inoculated onto plates with selective media, so that each colony originated from a single conidia.

5

In transformations performed with a linear DNA fragment, the amount of DNA used ranged from two to five micrograms. The selectable marker (amdS (acetamidase) or areB (omitinecarbamoyltransferase, OTCase, E.C. 2.1.3.3) or trpC (tryptophan) was within the transforming fragment.

Isolation and analysis of DNA from E. coli Plasmid DNA was isolated by standard methods (Maniatis et al. 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold 15 Spring Harbor, N.Y.). Chromosomal DNA was isolated from T. Theses using the method of Raeder and Broda, Lett. Appl. Microbiol. 1: 17-20 (1985)). Southern and Northem hybridizations were performed using standard techniques (Maniatis et al., Supra. 1982). Westem (blotting) technique was according to Maniatis et al., Supra. 1982).

:: 20 Liquid Cereal Trays and Conditions for Trichoderma • · · · · · ·

All liquid cultures of Trichoderma were started with conidiospores grown on · · ·: potato dextrose agar as described by Bailey and Nevalainen in Enzym.

Microb. Technol. 3: 153-157 (1981)). Liquid cultures in shake flasks were performed by · · ·: .25 according to Bailey and Nevalainen, Enzyme Microb. Technol. 3: 153-157 (1981) except that

Finnfloc was replaced by Solca Floc cellulose. The medium used in fermenter cultures contained six percent Solka Floc cellulose, 3 percent tough, 0.5 percent KH2PO4, 0.5 percent (NH4) 2 SO4, and 0.1 percent structure. . The pH was maintained between 4.0 and 4.8 with the addition of phosphoric acid or ammonia. Ferments - *: 3O? at 30 ° C. Maximum enzyme yields were obtained in five days in laboratory fermentations and in four days using a 100 liter fermentation vessel.

• · · • · «• · 35

enzyme assays

All enzyme activities were determined from the culture supernatant fractions after centrifugation for 20 minutes at 3000 rpm for 5 minutes. Endoglucanase activity was measured using hydroxyethyl cellulose as a substrate (HEC, non-aqueous, Fluka AG 54290, pract. Grade) and was performed as described by Bailey and Nevalainen in Enzyme Microbiol. Technol. 3: 153-157 (1981) and Comission on Biotechnology, International Union of Pure and Applied Chemistry; Measurement of Cellulase Activities, Center for Biochemical Engineering Research, Indian Institute of Technology, Delhi, New Delhi - 10016 (1984)), and xylanase activity was measured as a substrate for birch xylanase (Roth No. 7500) and by the method of Bailey et al. in J. Bact. 23: 257-270 (1992). TCA precipitated proteins were determined by the method of Lowry et al., J. Biol. Chem. 193: 265-275 (1951), and bovine serum albumin was the substrate.

Cellobiohydrolase activity against filter paper (Filter Paper Unit) (FPU) was measured as determined by the Comission on Biotechnology, International Union of Pure and Applied Chemistry, Measurement of Cellulase Activities, Indian Institute of Technology, Delhi, New Delhi - 10016 ( 1984)).

:: * 20: ·: · Determination of Endoglucanase I by ELISA: Concentration of endoglucanase I protein in culture supernatant fractions was determined by sandwich ELISA using a double antibody. Assays were performed on 96-chamber * · · 25-fold flat bottom microtiter plates at 37 ° C (unless otherwise stated). Each step was terminated by a triple wash using phosphate buffered saline pH 7.2 containing 0.05% Tween 20N and 0.02% • · · '·. sodium azide (PBS / Tween).

The plates were coated overnight at 4 ° C with mouse monoclonal antibodies: T: directed against endoglucanase I (anti-EGI antibody-EI2). At the unused points on the plastic surface, 1% BSA in PBS / Tween was placed for one hour. Appropriate dilutions of culture supernatant fractions were then added and incubated for 36 hours for two hours, followed by incubation for two hours with rabbit polyclonal antibodies raised against endoglucanase I. Bound rabbit antibodies were detected by incubation for two hours with porcine polyclonal antibodies raised against rabbit IgG and conjugated to alkaline phosphatase (Orion Diagnostica, Espoo, Finland). In the final step, p-nitrophenyl phosphate (1 mg / ml) was added and the reaction was quenched after 30 minutes at room temperature with 2N NaOH. The developed yellow color was measured on a photometer at 405 nm. The concentration of endoglucanase I in the culture supernatant fractions was then calculated by comparing the OD 50 values of the fractions with a standard dilution curve prepared with purified endoglucanase I and simultaneously on the same plates.

Fractionation of the culture supernatant fraction by chromatofocusing A chromatographic system consisting of a Pharmacia FPLC equipped with a Mono P HR 5/20 column was used for chromatofocusing. The resin was stabilized in 25 mM bitrate-HCl buffer, pH 6.5. The crude enzyme mixture produced by T. reesei in shake flasks was diluted with the same buffer to a protein concentration of 1 mg / ml.

500 µl samples of enzyme were injected onto the column and eluted with Pharmaly.

; : ¾) te / Polybuffer (Pharmacia, 1 mL PharmalyteR 2.5-5 and 5 mL Polybuffer ™ PB

• · ·: * · *: 54 in 100 mL total, pH adjusted to 3.0 with HCl) to form a pH gradient from 6.5 to 3.0. The flow rate was 30 ml / h. The column effluents were collected in 600 · · ·, ·: μ1: η fractions and the pH and EGI activity were determined.

I · »• · · * • ·· v2 $ Example 1

Cloning, Sequencing, and Enhanced Expression of xln2 of Trichoderma reesei Endoxylanase pl (pl 9) Gene As a result of this example, QM6 Trichoderma *: 5 'reesei was isolated from organic strain xln2 gene encoding endoxylanase pl 9.0. The gene contains a single intron of 108 nucleotides and encodes a 223 amino acid protein where two putative N-glycosylation targets were found. Three different strains of T. reesei were transformed with the aim of obtaining an endogenous cbn1 locus with a 37 construct consisting of the xln2 gene and its own promoter. The highest levels of xylanase production were achieved by using T. reesei strain AL-K02721, a strain constructed by genetic engineering. There was no need to integrate into the cbhl locus to achieve enhanced expression under the xln2 promoter.

5

Materials and Methods

Organisms, plasmids and growth conditions. Plasmids were added in Escherichia coli strain XL1-Blue (Bullock, W.O. et al. Bio / Techniques 5: 376-378 (1987)) or in strain INV-10aF '(Invitrogen, San Diego, CA, USA). The recipient organisms for the xln2 gene were T. reesei mutants which were good cellulase producers, namely strains VTT-D-79125 (Bailey and Nevalainen, Enzyme Microb. Technol. 3: 153-157 (1981)), ALKO-2721 and ALK02221. T. reesei ALK02221 is a mutant of strain VTT-D-79125 that produces a small amount of protease. T. reesei ALKO 2721 is a mutant trpC minus (trpC) of strain VTT-D-79125. obtained by UV light treatment and in which the cbh2 locus has been replaced by the Aspergillus nidulans gene trpC (Yelton, M.M., et al., Proc. Natl. Acad. Sci. USA 81: 1470-1474 (1984)). The strain also contains several other integrated copies of the trpC gene.

:: * 20 The plasmids used in this study include pBluescript (Stratagene, San: ***: Diego, CA, USA), pCRIOOO (Invitrogen) and pUC19 (Yanish-Perron, C., et al., Gene *, · / 33 : 103-119 (1985). The selection marker amdS came from plasmid p3SR2 (Kelly and Hynes, EMBO J. 4: 475-479 (1985)), which was kindly donated by Dr. M. Hynes. from cbhl .. !! flanking regions were isolated from T. reesei strain ALK02466 (Harkki, A., et al. Enzy-: 25 Me Microb. Technol. 13: 227-233 (1991)) using plasmid release.

Cultures of E. coli were grown in L-broth supplemented with ampicillin ♦ ♦ * * \ * (50 μg / ml), if necessary, at 37 ° C overnight (Maniatis, T., et al. Molecular Cloning: A Laboratory

Manual, Cold Spring Harbor, NY (1982)). Trichoderma strains were cultivated with *: * 30 agar plates of PD (Potato Dexstrose Broth, Difco, Detroit, USA). For production of xylnase «: T: strains of T. reesei were grown for 7 days in shake flasks (30 ° C, 250 rpm) in a medium (pH 5.5) that induced 38 cellulases and xylanase and contained 4 % whey, 1.5% nitrogen source from various components, 1.5% KH2PO4, and 0.5% (NH4) 2SO3.

Peptide cleavage and amino acid sequencing. Purified xylanase II (Tenka-5en, M., et al., Enzyme Microb. Technol. 14: 566-574 (1992)) from T. Theses (VTT-D-79125) was digested with 2% (w / w) w) trypsin (TPCK-treated, Sigma Chemical Company, St. Louis, MO, USA), which was yksiprosenttisessa am-bicarbonate at 37 ° C. for two and a half hours Three percent (w / w) trypsin was added to incubation was continued overnight (13 h). Peptides 10 were separated by high performance liquid chromatography (HPLC) using

Rexchrom Prep-5/300 ODS Reverse Phase Column. The column was eluted at a rate of one ml / minute in a linear solvent gradient running from 5% (v / v) acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) in 60 minutes at 24 ° C. To 60% (v / v) 15 ACN containing 0.06% TFA. The absorbance was measured at 218 nm. Prior to amino terminal sequencing, 10 µg of xylanase II was treated with 1 U pyroglutamate thiaminopeptidase (Boehringer Mannheim, Mannheim, Germany) at 37 ° C overnight in 100 mM potassium phosphate buffer, pH 8.0 containing 10 mM EDTA and 5 mmol of DTT. The amino terminals of the proteins and peptides were sequenced so that they: were digested in a gas-liquid pulse sequencer (Kalkkinen and Tilgmann, J. Protein Chem.

7: 242-243 (1988)). The released PTH amino acids were analyzed by sequential coupled thin-column reverse phase HPLC.

• DNA processing and transformations. Standard constructs were used for DNA constructs.

I f I

* '* 25 reported by Maniatis et al. in Molecular Cloning: A Laboratory Manual,

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). Plasmid and cosmic ··· di-DNAs were isolated on Quiagen columns (Diagen GmbH, Diisseldorf, Germany) according to manufacturer S · · \.

.1 * ·· • · * ** 30 E. coli were transformed according to D. Hanahan, J. Mol. Biol. 166: 557-580 (1983), and I I reesei strains according to the method of M. Penttilä et al., Gene 61: 155-164 (1987) with the following variants used: before transformation protoplasts was subjected to heat shock (Berges and Barreau, J. Gen. Microbiol. 135: 601-504 (1987)), 25% PEG 90 was replaced by 60% PEG4000 and transformed at room temperature rather than transformed on ice. Transformants were purified via conidia on selective acetamide-CsCl plates (Penttilä, M., et al. Gene 61: 155-164 (1987) before being transferred to PD inclined surfaces.

5 Isolation of RNA. T. reesei. VTT-D-79125 was cultured in a fermentor, xylanase-inducing medium, for three days. The mycelia were collected, frozen and ground to a fine powder under liquid nitrogen. Total RNA and subsequently mRNA were isolated by digestion with proteinase K, phenol extracts, and oligo-dT-10 cellulose purification as described by Bartels and Thompson in Nucleic Acid

Res. 11: 2961-2978 (1983): The resulting mRNA was fractionated by size using DMSO sucrose gradient centrifugation (Boedtker, H., et al. Biochemistry 15: 4765-4770 (1976)).

15 Cloning of xylanase II cDNA. The first strand of cDNA was prepared from one µg of mRNA using a cDNA synthesis kit (Boehringer Mannheim), the oligo-dT primer was replaced with the hybrid dT17 adapter primer (5'-GAC-TCG-AGA-ATT). -CAT-CGA-dT17 3 ') [SEQ ID NO: 5]. This cDNA was used as a template for polymerase chain reaction (PCR) amplification (Frohman, MA, Race: Rapid Amplification of cDNA Ends, by PCR Protocols, Innis, MA, et al., Editors, Academic Press Inc., San Diego, CA, pp. 28-38 (1990), directed by a '.0' gene-specific primer (sense 5 'GG (A / C / G) -TGG-CA (A / G) -CCN -GGN-ACN-% (ii: AA 3 ') [SEQ ID NO: 6] deduced from peptide sequence plus' ··· adapter primer (5 'GAC-TCG-AGA-ATT-CAT-CGA 3') [SEQ ID NO: 100 µl of the PCR reaction mixture contained 5 µl of 1:25 diluted cDNA, 100 pmoles of each primer, 5 µmoles of dNTP, 1x PCR buffer and 1.5 units of Taq. «·· *: *. ** DNA polymerase (Boehringer Mannheim) Amplification using a programmable temperature control (MJ: Research Inc) comprised 30 cycles, amplified at 95 ° C. ··· * * for one minute, 55 At 1 ° C for one minute and 72 ° C for two minutes. After the last episode, the period was extended by 10 minutes. The resulting PCR fragments were cloned using TA-cloning reagents (Invitrogen) manufactured by the manufacturer • · * ♦ * ·; and sequencing was confirmed.

Isolation of 40 xln2 gene. The genomic cosmid library of T. reesei QM6a (Mandels and Reese, J. Bacteriol. 73: 269-278 (1957)) was screened by hybridization with a xylanase II PCR probe according to the manufacturer's instructions (Boehringer Mannheim, Germany).

5 Nucleotide sequencing. Templates for nucleotide sequencing were generated by single deletions according to the manufacturer's instructions for the pBluescript Exo / Mung DNA sequencing system (Stratagene). DNA was sequenced in both directions using ABI reagents (Applied Biosystems, Foster City, USA) based on fluorescence labeled T7 and T3 primers and a Taq cycle sequencing method according to the supplier's instructions. The sequencing reactions were analyzed with an ABI 373A sequencer.

Enzyme and Protein Assays. Enzymes were determined from culture supernatants after mycelial removal. Xylanase activity was measured with birch xylan 15 (Roth 7500) as a substrate and the method of Bailey, M.J. in J. Biotechnol. 23: 257-270 (1992). Cellobiohydrolase I (CBHI) production was detected by Wester blot or dot blot methods using CBHL specific monoclonal antibodies CI-89 or CI-261 (Aho, S., et al. Eur. J. Biochem. 200 : 643-649 (1991).

20 •:: Results •

Peptide Sequences Several tryptic peptides were obtained from purified endoxylanase II and directly sequenced (see Figure 3 for peptide sequences). However, no amino terminal sequence was found for the organic peptide, suggesting that it was • ♦ · · ;; blocked. Amino-terminal sequence (Q) -X-Ile-Gln-Pro-Gly-Thr-Gly-Tyr-Asn [sec- ♦ ♦ · • · · *. SEQ ID NO: 8] was obtained after treatment with pyroglutamate 30 * thiaminopeptidase. The first amino acid (X) of the sample treated with pyroglutamate aminopeptidase could not be clearly identified due to the confusing peaks, • · · _ v; which were obtained in an HPLC chromatogram.

Cloning of T. reesei xln2 cDNA for mRNA-specific xylanase II accumulation in T. reesei cultures was determined by Nothem hybridization using the nucleotide-5-derived oligonucleotide deduced twice from the peptide sequence (shown below). 3). The results indicate that the xylanase Π: η mRNA was most abundant in the mycelium grown for three days and that the size of the xylanase II specific mRNA was about 0.7 kb (data not shown).

An oligomer primer specific for xylanase II (see above) and a non-specific dT adapter primer were used to amplify the xylanase sequence from the first strand of the cDNA. An amino acid sequence deduced from the nucleotide sequence of the subclone pALK564, which contains the longest fragment synthesized in the PCR reaction, contained several xylanase II peptide sequences obtained by direct sequencing.

This confirmed that my PCD clone was coding for xylanase H.

Isolation of the T. reesei xln2 gene and nucleotide sequence Four clones were isolated from the T. reesei genomic cosmid library by hybridization with the pALK564 insert (insert = PCR-synthesized xln2 cDNAl. Subclone:. · · PALK-475, contained a hybridizing 5.7 kb Kpn1 fragment from the cosmid clone and contained in the pUC19 vector was analyzed by restriction mapping (Fig. 2). Hybridizing fragments from pALK475, 2, A 3 kb HindIII / 1.5 kb HindIII / Xho I and a 1 kb Xho I was subcloned and <RTIgt; · 26 </RTI> was partially sequenced to express the xIII gene. .

The nucleotide and deduced amino acid sequences of the xln2 gene are shown in Figure 3. From DNA ·· · *** I a sequence similar to TATA box was found which was -140 nucleotides apart. from ATG (Figure 3). Primary translation initiation site (ATG) flanked by a 30 * highly conserved 5 'CA C / AA / C ATG 3' consensus sequence found in ♦ filamentous fungi and other eukaryotes (Ballance, JD, "Transformation • · Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi, Leon 42 and Berka, eds., Marcel Dekker Inc., New York, p. -29 (1991)), is 99 nucleotides upstream of the codon assigned to the N-terminal amino acid of xylanase II. The xln2 gene encodes a 223 amino acid protein. In this protein, 33 amino acids precede the N-terminus obtained by direct peptide sequencing. This pre-5 prosequence contains a putative signal sequence between Alal9 and Ala20 which is cleaved by peptidase (von Heijne, G., Nucl. Acid Res. 14: 4683-4690 (1986). Comparison of the genomic sequence with cDNA sequences revealed a single intron of 108 nucleotides, indicating that the mature xylanase II protein consists of 190 amino acids and has a calculated molecular weight of 20.8 kDa, which is well compatible with the 20 x 10 kDa obtained for purified xylanase II (Tenkanen, M ., et al .. Enzyme

Microb. Technol. 14: 566-574 (1992)). Sequence analysis also revealed two putative (Asn-X-Ser / Thr, where X is not Pro; Gavel and von Heijne, Protein Engineering 3: 433-442 (1990) target of glycosylation (mature protein Asn38 and Asn61)).

15 Construction of pAlk476

The construction of plasmid pALK476 is shown in Figure 4. This plasmid is useful when the xln2 gene is targeted to the cbh1 locus. The xln2 gene is expressed under the control of its natural promoter. 5.0 kb Smal fragment. derived from 20 plasmids pALK475 (Figure 2), containing the xln2 gene and a promoter (2.3 kb upstream of the gene), was ligated to the SpeI site of pALK425 (supplemented with Kleno · · ·: Willa).

• xyln2 Enhanced Expression in T. Theses 25: To enhance xylanase expression in T. Theses, three strains of VTT-D-79125 ,. ALK02221 and ALK02721, which differed in their enzyme production profiles, were transformed with the EcoRI fragment derived from plasmid pALK476 (Figure 4).

• · ·

An expression cassette containing the xln2 gene and its own promoter (Figure 30 '5) was targeted to the cbh1 locus. such that cbhl flanking regions were used.

Transformants were purified, 57 T. reesei strain VTT-D-79125, 42 ALK02221. *: and 53 transformations of ALK02721 and were grown in shake flasks in 43 cultures where conditions induced xylanase. Xylanase production was measured as birch xylan-degrading activity from culture supernatants. The transformants were tested for CBHI production to determine the frequency of targeting to the cbh1 locus and to isolate the transformants in which the xln2 expression cassette was inserted into the cbh1 locus.

Of the transformants where it has been combined elsewhere, replacement of the cbh1 locus with the xln2 expression cassette produces a CBHI negative (CBHI) phenotype expressed by Westem blots and dot motes using a CBHI specific monoclonal antibody. Effectiveness of targeting to the cbhl locus. as determined by the CBHI phenotype was good in each case: 67, 62 and 58%, respectively, in the VTT-D-10 79125, ALK02221 and ALK02721 transformants.

Figure 6 shows the relative xylanase activity of transformants grouped according to their CBHI + / "phenotype. The increase in the number of copies of xln2 increased the production of xylanase activity in both strains of CBHI 'and CBHI + transformants. (T. reesei ALK02221 and ALK02721) approximately 4.5-fold (T. reesei VTT-D-79125) to the corresponding host strains .The highest activities (nkat / ml) were obtained when T. reesei ALK02721 was used as the host. the best transformants of reesei ALK02221 produced less than half of the activity obtained with the best transformants of the other 20 strains. There was little difference in xylanase activity between the CBHI + and CBHI phenotypes of T1: reesei ALK02221 and ALK02721 transformants (Figs. 6). Among the transformants of VTT-D-79125 T. reesei which are trans- *. type CBHI +, were on average superior to xylanase-producing M · J.

To evaluate the effect of the genetic background on the expression of the xln2 gene in three hosts, the relative increase in xylanase activity in CBHI transformants was calculated. CBHI transformants were used to eliminate all effects. which the differences in the integration site had on gene expression. The majority of the 30 * CBHI transformants in each host strain (42-86%, Table 1) showed the same increase in xylanase. Most transformants that were removed from this comparison are · · ·; produced higher xylanase activities. From this it can be inferred that the transformants shown in Table 1 are likely to have one extra copy of xln2 at cbhl-44 in addition to organic xln2. The mean xylanase activity of these transformants and its mean increase relative to the host strain are shown in Table 1. The mean increase by strain was in the range of 600 nkat / ml (ALK02221) to 27000 nkat / ml (ALK02721).

5

table 1

Relative increase in xylanase production in selected series of CBHr transformants

Stock Avg. activity Avg.

(nkat / ml) increase (nkat / ml) VTT-D-79125 3700 (+/- 20%) 1400 10 transformants (50%) 5100 (+/- 20%) ALK02221 3800 (+/- 10%) 600 transformants (85%) 4400 (+/- 15%) ALK02721 10200 (+/- 15%) 2700 Transformants (42%) 12900 (+/- 10%) 15 1 2 The percentage (%) of CBHr transformants with apparently is an extra copy of the xln2 gene.

·. · I View results •

Several reports have been presented concerning the molecular cloning of bacterial xylanases (e.g., Ghangas, GS et al., J. Bacteriol. 171: 2963-2969 (1989); line ···· Thomson, Mol. Mol. Gen. Genet. 228: 55-61 (1991); Sharek, F., et al., Gene 107: 75-82 (1991); Whitehead and Lee, Curr. Microbiol., 23:15. -19 (1991). There have also been recent reports on the cloning of filamentous fungal xylanases, according to • · · 25; including reports on Aspergillus tubieens (van den Broeck, N., et al.,

"Cloning and Expression of Xylanase Genes from Fungal Origin," EP 0 463 706 AI

* 1 (1992)), A. nigerin var. awamorita (Maat, J., et al. "Xylanases and their Application

In Bakery "in Xylans and Xylanases, Visser, J., et al., Editors, Elsevier 2

Science, Amsterdam, ss. 349-360 (1992)), A. kawachiita (Ito, K., et al., Biosci. Biosci. Biol.: Technol. Biochem. 56: 906-912 (1992), and T. reesei (Suominen, P., et al .. "Genetic 45

Engineering of Trichoderma reesei to Produce Suitable Enzyme Combinations for Applications in the Pulp and Paper Industry, "in Biotechnology in the Pulp and Paper Industry, Kuwahara and Shimada, editors, Uni Publishers Co., Ltd., Tokyo, Japan, pp. 439-445. (1992); Törrönen, A., et al., Bio / Technology 10: 1461-1465 5 (1992)) Genes for the T. reesei organic strain QM6a xylanase II which have been cloned by the inventors (see also Suominen, P ., et al., "Genetic Engineering of Trichoderma Reesei to Produce Suitable Enzyme Combinations for Applications in the Pulp and Paper Industry," in Biotechnology in the Pulp and Paper Industry, Kuwahara and Shimada, editors, Uni Publishers Co., Ltd., Tokyo, Japan, pp. 439-445 (1992)) and the xylanase II genes of the RUTC-30 mutant strain, which were cloned by Törrönen et al., Bio / Technology 10: 1461-1465 (1992), were not completely identical. The main differences were between the pre-propeptide sequences, which gives no cause This indicates that there were differences in signal processing.

According to the inventors' results, the T. reesei gene xln2 contains one long intron of 10S base pairs. Filamentous fungal introns are usually smaller, about 50 base pairs in length, but they can also be considerably longer, such as Vanhanen et al. have shown T. reesei for 3-phosphoglycerate kinase, Curr. Genet. 12: 181-186 (1989). A suitably long pre-sequence (33 to 20 amino acids) of the xln2 gene contains one major cleavage site for signal peptidase. This • · · ···: is consistent with a signal sequence containing 19 amino acids followed by a · · · • 14 amino acid propeptide, which, after translation, can be cleaved from the mature protein. Törrönen et al., Bio / Technology 10: 1461-1465 (1992) have reported that the signal pre-peptide of V T. reesei strain RutC-30 xylanase II (p1 9) comprises 32 amino acids. ·· Ϊ3! acid and has two putative signal peptidase cleavage sites that are very close to the translation start site (5 and 11 amino acids, respectively). This would result in somewhat short signal sequences. Biphasic protein treatment similar to that proposed for xylanase II herein has been shown to occur in A. nieer. glucoamylase (Innis, MA, et al. Science 228: 21-26 (1985)) and it is reported that it occurs by T. reesei cellobiohydrolase II (Teeri, T., et al., Gene 51:43 -52 (1987).

• 1

Total structure occurring on T. reesei xn! 2. is also present in the A. niger 1 · 1 xylanase gene (van den Broek, N., et al., "Cloning and Expression of Xylanase" · Genes from Fungal Origin, EP 0 463 706 A1 (1992), but does not occur, for example, in 46 A. kawachi with a xylanase gene containing nine introns and encoding a larger protein (32.7 kDa) (Ito, K., et al. Biosci. Biotechnol. Biochem. 56: 906-912 (1992)).

Xylanase production in three strains of T. reesei could be increased by increasing the number of copies of the xln2-5 gene. Of these strains, T. reesei VTT-D-79125 is a major cellulase-producing mutant, and ALK02721 was used as a host because it already pre-produced large amounts of xylanase. Xylanase was also desired to be produced by T. reesei ALK02221, a low protease producer, both because of low xylanase production and concomitant low protease background and the desire to increase the relative amount of xylanase in the culture medium.

The greatest increase in xylanase activity was from about two to about four times that of the corresponding host strain. Transformants of the low-protease-producing strain ALK02221 and the xylanase-15 activities of the host engineered ALKO-2721 were unaffected by whether the xln2 gene was integrated into the cbh1 locus or elsewhere in the genome (CBHI + / 'phenotype). For T. reesei VTT-D-79125 transformants, integration of the xln2 gene elsewhere (CBHI + phenotype) than the cbh1 locus appeared to produce a higher enzyme yield. These findings are somewhat inconsistent with the findings of Harkki et al. (Enzyme Microb. Technol. 13: 227-233 (1991)) suggesting that an optimal expression of the expression cassette would require its integration into the cbh1- locus. It should be noted, however, that in the present study, the x1n2 promoter was used in place of the cbh1 promoter, expressed by Harkki et al., Enzyme Microb. TECNOL. 13: 227-233 (1991), used egll with cDNA. This • 1 · ** '/ indicates that the cbhl locus may not be the best environment for expression under the xln2 promoter.

• · · »« ·. The genetic background appeared to have a significant effect on xln2 expression, despite the fact that T. reesei strains ALK02221 and ALK02721 are derived from VTT-D 9 79125. Transformants of T.reese strain ALK02721 30], which also had the highest xylanase activity, showed the greatest increase in xylanase production (2700 nkat / ml). xln2 one. additional copy expression more than quadrupled according to host (Table 1).

11 · • · · · · · · · · · · · 47

Example 2 Increased Production of XYL2: Construction of the xln2 gene Associated with the cbh1 Promoter and Transformation of Three Trichoderma reesei Strains 5 Construction of pALK174 is shown in Figure 7. A first PCR fragment containing 674 bp and cbh1 was constructed. the exact linker site of the promoter to the xln2 signal sequence (and the xln2-ene Xhol site). using plasmid pALK475 as a template (Figure 2). The oligonucleotides used were the following: 5 'to 10 primer containing the end of the cbh1 promoter and the start of the putative xln2 signal sequence, the 3' primer having the sequence derived from the xln2 gene and containing the intracellular Xhol site (see map of pALK476). , Figure 5, or x1 n2 sequence).

5'-primer (39-mer) (SEQ ID NO: 11]: 15 5'-C AACCGCGGA CTG CGC ATC ATG GTC TCC TTC ACC TCC CT Sac End of the putative xln2-sig-cbhl promoter sequence 20 m

»« I

3'-primer (26-mer) [SEQ ID NO: 12]: • t «• · •» r

I * I

. '* 5'-GGG AGC CGC TCG AGC GGT GGT TGC GG

! ·· ν _Xhsi * ·· $$ xln2 sequence »t« «100 µl PCR reaction mixture contained 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 0.01% gelatin, w / v) 50 pmoles of each e, primer, 10 ng of template DNA, 0.2 mmol of dNTP and 2 U of Taq polymerase * · 30 (Boehringer Mannheim). Reaction conditions were: denaturation for one minute at 95 ° C, primer attachment at 60 ° C, extension (copy of DNA sequences at 72 ° C for 30 cycles), and final extension, 9 minutes.

et al. ♦ ·· t · 48 PCR fragment was purified using Mermaid® reagents and equipment (Bio 101 Inc., La Jolla, California, USA). After being treated with T4 DNA polymerase, the fragment was cut with Sach. to be linked to the cbhl promoter.

Plasmid pAMH11O containing the cbh1 promoter was cut with NdeL (supplemented

Klenow) and SacIL. The above described PCR fragment was bound to digested pAMH110 (pALK174X). The fusions and the sequence synthesized by PCR were confirmed by sequencing.

Plasmid pAKJ174 was constructed such that the xln2 promoter. located in pALK476 was replaced with the cbh1 promoter: a 2.9 bp Kpn1-XhoI fragment from pALK174X containing the cbh1 promoter fused to the xln2 gene. and the xln2 sequence. which was connected to the internal XhoLote. was bound to the isolated vector containing the pALK476 fragment after pALK476 was cleaved with KpnL and XhoI.

Plasmid pALK174 contains, in addition to the 3 'regions of amdS and cbh1, as in pAJLK476 (Figure 4), the xln2 gene. linked to the cbh1 promoter and a 1.9 base pair end codon (3 'region) of the xln2 gene.

20 «

III

;; · / 9.7 kb EcoRI fragment (expression cassette without vector sequence · '1') was used for the Trichodenna strains VTT-D-79125, ALK02221 and ALKO-i. 2721 transformation. Strains, transformation and purification, transfer-

i »S

** V piston analysis and growth was described above and described in Example 6.

· »<« I · · «

Production of Xylanase with pALK174 Transformants The VTT-D-79125, ALK02221 and ALK02721 transformants, respectively 39, 41 and 50, were purified and grown to measure the xylanase activities produced. The 30% hairs achieved by targeting to the cbhl locus, measured using a 9 9 dot blot and a CBHL-specific monoclonal antibody (as in Example 4), were 82, 46 and 84% respectively. The yield of xylanase activity of the CBHI + and CBHL-? X * · · · 'transformants, shown as the relative activity 49 compared to the activity produced by VTT-D-79125, is shown in Figure 8. The bars represent the mean levels of production and the range of transformants in each host strain. The xylanase activity was determined from culture supernatants as described in Example 3 (xln2 cloning).

5

All transformants of the Trichoderma strains produced high levels of xylanase. The best VTT-D-79125 and ALK02221 transformants produced approximately 10-fold xylanase activity as compared to their host strains. The best ALK02721 transformants produced approximately the same amount of xylanase activity as the best VTT-D-79125 and 10 ALKO-2221 transformants, and the increase in activity was three times that of the host. CBHr transformants were on average better producers than transformants with CBHI + phenotype. In CBHT transformants, the cellulose-degrading activity naturally decreased due to deletion of the cbh1 gene.

When the pALK174 construct was used, the levels of xylanase production were higher than those obtained with pALK476 (xln2 under the control of its own promoter, see Example 1).

Example 3?: 2P: Structure of Endoxylanase I, xIII. Trichoderma reesei gene This example shows cloning for the T, reesei gene xlnl. encoding endoxyl • ·: lanase I with a low pI (5.5).

* 25 'Materials and Methods • Bacterial Strains and Plasmids The plasmids used in this study were pBluescript (Stratagene, San Diego, CA). , 30 'USA) and pCRIOOO (Invitrogen, San Diego, CA, USA). Plasmids were added to Esche; richia coli strain XLI-Blue (Bullock, WO et al. Bio / Techniques 5: 376-378 (19-87) or E. coli in INVlaF '(Invitrogen). E. coli cultures were grown at 37 ° C. overnight in L-broth (Maniatis, T. et al. Molecular Cloning: A Laboratory Manual (Cold 50

Sring Harbor Laboratory, Cold Sring Harbor, New York (1982) supplemented, if necessary, with ampicillin (50 µg / ml).

Peptide cleavage and amino acid sequencing

Purified Xylase I (Tenkanen, M. et al. Enzyme Microb. Technol. 14: 566-574 81992) from T. Teese (VTT-D-79125; Baily. M.J., Nevalainen, KH-M). ., Enzyme Microb. Technol. 3: 153-157 (1981)) was digested with 2% (w / w) trypsin (TPCK-treated, Sigma Chemical Company, St. Louis, MO; USA) 10. (Pamo / volume) ammonium carbonate at 37 ° C for 2.5 hours. After addition of 3% trypsin (w / w) to the sample, incubation was continued overnight (13 h). The peptides were separated by high performance liquid chromatography (HPLC) with a Rexchrom Prep-5/300 ODS reverse phase column.

Eluted at a rate of 1 mL / minute in a linear solvent gradient from 5% (v / v) acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) to 60% (v / v) ACN. containing 0.06% TFA in 60 minutes at 24 ° C. The absorbance at 218 nm was measured. The amino terminus of the proteins and peptides was sequenced by digestion in a gas-liquid pulse sequencer (Kalkkinen, N. and Tilgmann, C., J. Prot. Chem. 7: 242-243 (1988).). amino acids were analyzed by thin-layer reverse phase liquid chromatography with high resolution.

DNA Manipulations and Transformations 25 'DNA constructs used standard DNA techniques as described by Maniatis et al. . pictures-. vat (Maniatis et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor • · ·

Laboratory, Cold Spring Harbor, New York (1982)). Plasmid and cosmid DNAs were isolated using Qiagen columns (Diagen GmbH, Diisseldorf, Germany) * according to the manufacturer's instructions. E. coli was transformed according to Hanahan (Hanahan, D., J. Mol. Biol. 166: 557-580 (1983)).

51 RNA Isolation for RNA Isolation T. reesei (VTT-D-79125) was grown in a fermentor in a xylanase-inducing medium; At 30 ° C for three days (as described above for the isolation of the xln2 gene, as described hereinabove). The mycelium was ground to a fine powder under liquid nitrogen. Total RNA and mRNA were isolated as described by Bartels and Thompson (Bartels, D., and Thompson, R.D., Nucleic Acid Res. 11: 2961-2978 (1983)) and size-fractionated using a DMSO sucrose gradient (Boedtker, H). et al., Biochemistry 15: 4765-4770 (1976)).

10

Cloning of xylanase I cDNA The first strand of the cDNA was synthesized and subsequently amplified by PCR as described herein for xln2, except that the oligomer (5 'AA (T / C) -TA) was used as the gene-specific primer. (T / C) -GA (T / C) -CA (G / A) -AA (T / C) -TA (T / C) -GA 3 ') [SEQ ID NO: 9] deduced from the xylan I protein N-terminal sequence. The resulting PCR fragment, 0.6 kb, was subcloned into the pCRI0000 vector and sequenced.

• Isolation of 201 xlnl-ene • Genomization of T. reesei (QM6a, Mandels, M., Reese, ET, J. Bacteriol. 73: 269-280 (1957)). the cosmid library was screened with a xylanase I cDNA PCR fragment labeled with digoxigenin (see Example 1) according to the supplier's instructions (Boehringer, Mannheim, Mannheim, Germany). One positive cosmid clone was obtained. A restriction site map of the xIII region of the cosmid was prepared, and the 2.3 kb EcoRI fragment j was subcloned into pBluescript (pALK572) for sequencing.

Nucleotide Sequencing 30 "• · *:. - Templates for nucleotide sequencing were generated by unidirectional deletions according to the instructions provided by the manufacturer for the pBluescript-Exo / Mung DNA sequencing system (Stratagene). The DNA was sequenced. directions using 52 ABI reagents (Applied Biosystems, Foster City, USA) based on fluorescence-labeled T7 and T3 primers and Taq cycle sequencing according to the supplier's instructions The sequencing reactions were analyzed with the ABI 373A sequencer.

Results Cloning of T. reesei xln1 cDNA

The amino-terminal sequence of purified xylanase I was obtained as Ala-Ser-Ile-Asn-Tyr-Asp-Gln-10 Asn-Tyr-Gln-Thr-Gly-Gly-Gln-Val-Ser-Tyr (Ser) -Pro- ( Ser) -Asn-Thr-Gly-Phe-Ser [SEQ ID NO: 10]. Five tryptic peptides were also obtained and directly sequenced (see Figure 10 for peptide sequences).

A Northem hybridization using an oligomer as deduced from the N-terminal peptide sequence of xylanase I (see above) gave x-lanase I mRNA size of about 0.7 kb.

PCR amplification of the first strand of cDNA motifed a fragment of 0.7 kb. Translation of this DNA nucleotide sequence yielded a protein which: 2P: contained all xylanase I peptide sequences, including the N-terminal sequence (see Figure 10).

Isolation and nucleotide sequence of the T. reesei xln2 gene One positive clone was isolated from the T. reesei genomic cosmid library. The xylanase I gene (xIII) was found in the EcoRI fragment of the cosmid. which was 2.3 kilobases • · «* :: (Figure 9).

The nucleotide sequence of the xln1 gene was determined and is shown in Figure 10 together with the deduced amino acid sequence. About 90 nucleotides upstream · · · were found in the TATA box; from the presumed translation origin. Of the three different inferences of translation · · preceding the N-terminus of the mature protein, only one was in an environment that resembled the well-conserved 5 'CA-C / AA / C-ATG 3' that 53 filamentous fungi have for translation initiation (Ballance, JD, Leone, SA, Berka, RM, editors, Molecular Industrial Mycology, System and Application for Filamentous Fing (Marcel Dekker, Inc., New York), pp. 1-29 (1991). Thus, the gene encodes 229 amino acids. In this protein, the N-terminus (obtained by direct peptide sequencing) is preceded by a 51 amino acid signal propeptide containing the major cleavage site of the signal sequence (von Heijne, Nucleic Acids Res. 14: 4683-4690 (1986), which is Alal9 and Met20). (Figure 10) Comparison of the genomic sequence with the cDNA sequence revealed a single intron of 62 base pairs based on these results, concluding that mature xylanase I protein has 178 to 10 amino acids and has a calculated molecular weight of 19.1 kDa. well with the 19 kDa obtained for purified xylanase I isolated from T. reese (Tenkanen, M. et al., Enzyme Microb. Technol. 14: 566-574 (1992)), as distinct from xylanase II. gene (see here) is that there is no sequence for N-glycosylation (Asn-X-Ser / Thr, where X is not Pro; Gavel, Y., von Heijne, G., Protein Engineering 15 3: 433 -442 (1990).

Comparison of T. reesei xylanase I with other xylanases

Both proposed catalytic glutamic acids of xylanases (Katsube, Y. et al.

: 2P: Proc. 2nd Int. Conference Protein Engineering (Japan Scientific Societies Press, Tokyo) • · ·: ss. 91-96 (1990); Wakarchuk, W. et al., Visser et al., Editors, Xylans and Xylanases (Elsevier Science, Amsterdam), p. 439-442 (1991), found in xylanase; · ϊ: I by direct alignment of the sequence and corresponding to the mature enzyme • · ·

Glu75 and Glul64. Table 2 directly compares the aligned sequence, ··· ** I i · 215 ', which shows the percentage similarity between the various xylanases. The matrix. the upper right half represents the results of direct alignment of the complete mature sequences, and the lower left half of the matrix represents the sequence results between the putative glutamic acids of the xylanases.

• · # · «• I · • ·« · • 54 ·

Table 2

% Identity of Alignment of Sequences for Different Xylanases as Matrix. Upper right: Alignment of mature enzyme sequence. Lower left half: sequence comparison between two proposed active sites on glutamic acid TI, T. reesei xylanase I; TII, T. reesei xylanase II; TV, T. viride (Yaguchi, M. et al., In Visser, J. et al., Eds., Xylans and Xylanases (Elsevier Science, Amsterdam), pp -154 (1992a)); Har, T. harzianum (Yaguchi, M. et al., In Visser, J. et al., J. Xylans and Xylanases (Elsevier Science, Amsterdam), 435438 (1992b)); AT, A. tubieensis (van den Broeck, H. et al. "Cloning and expression of xylanase genes from fungal origin," EP 0 463 706 AI (1992)); SC, Schizophyllum commune (Oku, T. et al., Canadian Fed. Biol. Soc. Annu. Meet (Quebec City), Abst. 676 (1988)); AK, A. kawachii (Ito, K. et al. Biosci. Biotec. Biochem. 56: 906-912 (1992), aligned the first 190 amino acids); BC, Bacillus circulans (Yang, R.C. A. et al., Nucleic Acids Res. 16: 7187 (1988)); BP, P. pumilus (Fukasaki, E. et al., FEBS Lett. 171: 197-201 (1984)); BS, B. subtilis (Paice, M. G. et al., Arch. Microbiol 144: 201-206 (1986)); CA, Clostridium acetobutyllicum (Zappe H. et al., Nucleic Acids Res. 18: 2179 (1990)); SS, Streptomycces sp. 36a (Nagashima, M. et al., Trends Actinomycetologia 91-96 (1989)).

_ π ΤΠ TV TH AT SC AK BC BP BS CA SS

Tl 10 · S2 52 51 47 42 16 49 40 49 40 43 ΤΠ 60 100 98 94 43 34 17 52 48 52 46 52 TV «0 99 100 94 43 54 23 52 49 52 46 51

• · · mbb ^ B ^^^^ MB MM MMM1 · ^^^^ B BBBB ^ M ^ MMM

TH 57 98 97 100 43 56 19 52 48 52 44 51

• · · MM ^ M HMMBBM MM BBBBBBBI MM B ^ HBBB MMM · HMM BM ^ M · BBBM ^ B ^ MM ^ M

AT 62 57 57 58 100 38 17 40 34 40 32 33 • · «BMMM ΜΜΜΜΜ M ^ B ^ MB BMB ^ M BMB MH · B ^ MBM BBBB B ^ MBM BBMMB MM ^ HM ^ M ·; 1V SC 52 51 51 52 SO 100 15 52 42 52 44 50

• · · BMi ^^ B MMM MH ^ MM MMMMI B ^ M ^ M MBB, BBBBBB Bi ^^ BM BBM B ^ MMBi BBBBBBa HMBBBB BBBB

· ;;; AK 20 21 20 21 23 21 100 14 18 14 20 19

• · · BBBMB ^ B «B ^ MMB BBMBM MBM MBBBM ^^^ BM MB ^ BB BBBB M ^ MBM BBB ^ B

BC 53 54 54 56 S3 54 16 100 48 100 45 58 BP 52 57 57 58 47 48 17 59 100 48 71 49 • · · HBMBM MMB MB ^^ B BBBBBM ^ B ^ B · BBMBM BBB ^^ B ^^ BBM ^ BBBBB ^ B ^^^ B · 1Β · ν ^ BS 53 54 54 56 53 54 14 99 60 100 45 58

• · · MMMBBM MMiMB MM ^ BBMM BBMBM BMBB ^ BMBBB · ^^^ MB BBMBB ΒΒΒϋ BBBB BMM ^ M

CA 52 51 52 52 42 47 19 57 73 58 100 46

MBB MMMMMi BB ^^^ B ^ BBIBi BB ^ MB ^^ MB BBM BBBBBB BMBBB BBBB B ^^ M ^ B ^ M ^ BMB

SS 50 53 53 53 46 49 18 64 56 64 52 100 • · LbbJLbbbbJmbJLbbb-LbbbLbb ^ m ^^^^^^^ bb ^ imJbbbbJLbbb ··· • · · · · · ··· · • · · · · · · 55

It can be seen that T. reesei xylanase pH (pH 9.0) is almost identical to the small molecule xylanases of T. viride and T. harzianum (Yaguchi, M. et al., Visser, J. et al. , editors, Xylans and Xylanases (Elsevier Science, Amsterdam), pp. 149-154 (1992); Yaguchi, M. et al., in Visser, J. et 5 aL, publishers, Xylans and Xylanases (Elsevier Science, Amsterdam) , pp. 435-438 (1992)). The direct alignment of the region between the two active sites of glutamic acids gave in most cases a greater degree of similarity than the complete, mature sequences which were directly aligned.

Examination of the Results T. reesei produces at least two different xylanases, xylanase I with a pI of 5.5, and xylanase II with a pI of 9, which are small proteins of 19kDa and 20kDa, respectively (Tenkanen, M. et al., Enzyme Microb. Technol. 14: 566-574 (1992)). The corresponding xln1 and xln2 genes have been cloned and described herein, the overall constructs of the xln1 and xln2 genes were very similar; they were the same size, and each contained only one intron and a long prepropeptide. However, only these two xylanases have 54% similarity at the DNA level and 52% at the amino acid level. Thus not only their primary structures are different but also their: 2b! enzymatic properties such as resistance to various pH values or temperatures; and kinetic parameters such as Tenkanen et al. (Tenkanen, M., et al., 9, 9, 9, Enzyme Microb. Technol. 14: 566-574 (1992)). The xylanases are divided into two subclasses F and G. based on hydrophobic group analysis (Henrissat, J.B., Visser, J. et al., Eds., Xylans and Xylanases, Proc. Int. Symp.

::: 25 Wageningen (Elsvier, Amsterdam), ss. 97-110 (1991)) that these two subclasses. xylanases multiply in different ways. The high degree of similarity between T. reesei xylanases I and II compared to other xylanases (Table 2), with the exception of A. kawachi xylanase A, suggests that they, like other low molecular weight xylanases, also belong to the subgroup GA kawachi in the case of xylanase. (Ito, K. et al. Biosci.

30 Biotec. Biochem. 56: 906-912 (1992)), only about 20% similarity with T1 · · * * * * reesei xylanases was observed. The former xylanase has a higher molecular weight of 32,9 kDa and has been proposed to be in the class F (Ito, K. et al. Bios ci. Biotec. Biochem. 56: 906-912 (1992)).

56

Example 4 Increased yield of XYLI with Trichoderma. transformed with a construct containing the xln1 gene linked to the cbhl promoter (pALK807) Construction of the plasmid pALK807 and transformation of Trichoderma strain ALK0221

Plasmid pALK807 was contraindicated using the same strategy as used in constructing pALK174 (Figure 11). A 98 bp fragment containing the cbh1 promoter junction with the putative xlnl signal sequence and 10 xlnl junction at the internal XhoI site was prepared by PCR (see map of pALK572 in Figure 2 or the xlnl sequence used), and so on. 5'-primer (39-mer) [SEQ ID NO: 13]:

15 5 - C AAC CGC GGA CTG CGC ATC ATG GTT GCC TTT TCC AGC CT

Sac II putative xlnl signal sequence cbhl promoter end 3'-primer (30 mer) [SEQ ID NO: 14]: 2b: · «·

5'-CAG GCT CGA GGC CTG TGG GCA TCG CCA GAG

• i-: XhoI

• · ♦ · · · xlnl Sequence • · · • I ···

• M

::: 25. Plasmid pALK572 containing the xln1 gene was used as a template for the PCR reaction.

• · · '!!! The reaction was allowed to occur and the Mote was purified as used for construction of plasmid pALK174 · · ·. To obtain plasmid pALK805, the purified and SacII-XhoI digested [PCR fragment was ligated into pALK486, which had been cut with SacII-XhoI and contained the cbh1 promoter. To construct plasmid pALK806, the xln1 sequence downstream of the XhoI site was isolated from plasmid pALK572. treated with • · '· "PstK (amplified by T4 DNA polymerase) -XhoI, and the fragment was combined with 57 pALK805 treated with BamHK-supplemented with Klenow) - XhoI. pALK805 was obtained to give the EcoRI-Spel fragment ( supplemented with Klenow) obtained from pALK174 containing the amdS gene and the 3 'flanking region of cbhl (as in pALK174) was combined with Eco RV cut pALK806.

5 T. reesei strain ALK02221 was transformed with an 8.2 Kb Notl phrase from plasmid pALK807.

Production of xylanase by pALK807 transformants 10

A total of 51 pALK807 transformants were purified, analyzed, and grown as described in Example 2. The targeting efficiency to the cbh1 locus was 35%. The xylanase activity was measured as in Example 2 but at pH 4.3 which is within the optimum range for XYLI activity (Tenkanen et al., Enzyme Microb. Technol. 14: 566-574 (1992)). The results are shown in Table 3 as an increase in xylanase activity relative to the host strain ALK02221. Thirty of the best xylanase producers obtained have been included. One transformant was grown for each transformant.

The best transformants produced more than ten times the amount of xylanase activity: 20: as compared to the starting strain. The best xylanase producers were the CBHI + phenotype, so «i ·! · 1 differed from XYLII (pALK174) transformants.

f · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ···

• f I

• · «M ··· • · • • im •% •

III

• · · • · · ft • · · · · · · 58

Table 3

CBHI +/- (dot blot) of transformants X

number compared to ALK02221 5 (control) (+) ALK02221 20 (+) 11.8 5 (+) 11.7 18 (+) 10.4 10 4 (+) 9.8 33 (+) 9.5 32 (-) 9.2 21 (+) 8.9 3 (+) 8.9 15 46 (+) 8.8 40 (+) 8.2 1 (+) 8.2 43 (+) 8.0 10 (+) 8.0 20 19 (+) 8.0 8 (+) 7.9 51 (+) 7.8 39 (+) 7.6 '.: Ί: 12 (_) 7) 5: 2S. 7 (-) 7.2 36 (+) 6.9 41 (-) 6.8 * · **: 35 (+) 6.8 49 (-) 6.6 .30 * 29 (-) 6.6 : T; 24 (+) 6.3 50 (-) 5.9 14 (-) 5'9 26 (+) 5.8: 3S: 15 (-) 5.8 52 (+) 5.1 • · ___ 59

Example 5 A. Inactivation of the master cellulase cbhl gene 5 T. In the theses, the master cellulase-encoding chhl gene was inactivated by homologous recombination with plasmid pMS4 containing a 0.8 kb internal fragment of cbh1 cDNA is a read phase mutation. Plasmid pMS4 was prepared as follows: pTTcO1 (Teeri et al. Anal. Biochem. 164: 60-67 (1987); Penttilä et al. Gene 63: 103-112 (1988)) containing 10 full-length cbhl genes. The cDNA clone contained in the pUC8 vector (Vieira and Messing, Gene 19: 259-268 (1982)) was treated with BglII. which cuts in the signal sequence (Shoemaker et al., Bio / Technology 1: 691-696 (1983)) and with BglII. The resulting 0.8 kb DNA fragment containing the 5 'region of the cbh1 cDNA was blunt ended with Srnuclease and ligated with the EcoRL-cut blunt-ended pUC18 vector (Yanish-15 Perron et al., Gene 33: 103-119 (1985)). The resulting clone was cut in the middle of the cbhI fragment with EcoRL and then complemented with EcoRL. The resulting plasmid thus contains a read-phase mutation centered on the cDNA fragment of truncated cbh1.

i2S0t T.reesei VTT-D-79125 (Bailey and Nevalainen, Enzyme Microb. Technol. 3: 153-157 (1981)) was co-transformed with pMS4 and p3SR2. p3SR2 contains a 5 kb DNA fragment containing the A. nidulans amdS gene. which has been cloned into? · · * · * / pBR322 (Kelly and Hynes, EMBO J. 4: 475-479 (1985)). Transformants were selected on the basis of the · amdS + phenotype, followed by purification from conidia. Approximately 600 x 25 clones from 200 independent transformants were grown on microtiter plates and their cellulase phenotype was analyzed by Ouchterloney immunodiffusion (Ouchterloney,

/ ♦ ·, Allergy 5: 1-78 (1958)) using undiluted medium and CBHL • · · * (specific sheep antiserum).

. · ♦ ·· ft »k) Many of the names did not produce any detectable CBHL. One of these VTT-D-87312- · ··· ft · * · * * strains was confirmed to be CBHI-negative by analysis of growth medium by SDS-PAGE and FPLC with no peak corresponding to CBHL was detected (compare Figure 12 and Figure 12A). The total amount of protein secreted by the 60 CBHI negative strain was about half that of T. reesei VTT-D-79125. Filter paper cleavage activity, FPU activity (Mandels, MH, Reese, ET and Spano, LA (eds.); John Wiley and Sons, New York, 1976) observed in the culture supernatant of strain VTT-D-87312, was significantly reduced, and it was about 20 percent of normal activity. The absence of a major cellular biohydrolase, which normally represents about 60% of the total secreted protein, did not significantly alter the growth characteristics of the strain.

deletion of the cbh2 gene and its promoter 10

The Trichoderman cbh2 gene was replaced by the Aspereillus argB gene as described below. Plasmid pALK99 was constructed as the source of the transformation fragment (Figure 3). Plasmid pALK99 was constructed as follows. The PvuII fragment of plasmid pUC19 (containing the Bubble Buster) was replaced with a new synthetic multilink fragment containing the recognition sites for the following restriction enzymes: XhoI-Stul-SmaI-XbaI-

PvuII-Sall-Xhol. The new plasmid was designated pALK96. This plasmid was cut with XbaI and PvuII, and a 2.1 kb Xba I-PvuII fragment from the 3 'region of the cbh2 gene (see Figure 14) was ligated to it. The resulting plasmid was cut with PvIL and Hindi and ligated with a 3.4 kb PvuII fragment from cbh2-20. 5 'region of the gene (see Figure 14). Both the 3'- and 5'-fragments were derived from the clone cbh21ambda (Teeri et al., Gene 51: 43-52 (1987)). The resulting plasmid was named • ·. . ·. pALK98, respectively. Aspergillus nidulans gene (2.6 kb SalI fragment) • · · • · ·. was ligated between the 3 'and 5' regions of the cbh2 gene, a unique PvuII site of plasmid pALK98. The resulting plasmid was designated pALK99. A transforming fragment that isolates • · ·· 23 *; from pALK99 as the XhoI fragment, thus contains the Aspergillus argB gene as a SaU fragment, which is a 2.6 kb base (Berse et al., Gene 25: 109-117 (1983)) and is located in the cbh2 gene. , Between the 5 ': T: flanking region of the 4 kb (PvuII-PyuII fragment) and the 3' flanking region of 2.1 kb (PyuII-Xball fragment) (see Figure 14). The T. reesei VTT-D-87305 ArgB mutant strain 30 ·: (Penttilä et al. 1987, Gene 61: 155-164) was transformed with this fragment such that. selection of the arginine prototype was used. ArgB + transformants were screened for CBHIT phenotype by Western blotting using a monoclonal antibody directed against CBHII. Replacement of the cbh2 locus with transforming 61 fragments in CBHII transformants was then confirmed by Southern blots. Strain ALKO 2564 is an example of this kind of "replacement strain" and no longer contains the cbh2 gene.

By the method described above, the Trichoderman cbh2 gene was replaced by the Aspergillus trpC gene.

The trpC gene of Aspergillus nidulans (4.2 kb XhoI fragment blunt-ended with Klenow) was ligated between the 3 'and 5' regions of the cbh2 gene into a unique PvuII site of plasmid pALK98. The resulting plasmid was designated pALK402. The transforming fragment isolated from pALK402 as the XhoI fragment contains the trpC gene of A. nidulans as a 4.2kb XhoI fragment (Yelton et al. PNAS, 91: 1470-1474 (1984) located cbh2-). between the 3.4 '5' flanking region of the gene and the 2.1 '3' flanking region 15. The trpC mutant strain of T. reesei ALK02319 was transformed with tryptophan prototroph for selection. For the CBHIL phenotype by Western (blotting) a monoclonal antibody against CBHII, replacement of the cbh2 locus in the CBHIT phenotype by the fragment transformed was confirmed by Southem blots Strain AL-20. K02721 is an example. from this kind of "substitution strain", and it no longer contains the .V cbh2 gene.

• • • • • • • •: ·. Example 6

Construction of a CBH1 negative strain that does not produce EGI • · ··· '; increased amounts ··· CBHI-negative strain VTT-D-87312 described in Example 5A, trans- · · ·: T: was formed with plasmid pAMH 111 to enhance EGI expression on CBHI in a negative background. Plasmid pAMH 111 was constructed using the generic $ 9 expression vector pAMH 110 (these two plasmids are described in EP 244 234).

pAMH 110 was constructed from pUC19 (Yanish Perron et al., Gene 33: 103-119 (1985)).

• · ·: The single NdeI site of pUC19 was destroyed by filling its ends with the well with Klenow polymerase, and then the plasmid was treated with EcoRI and

PstL and bound to the cbh1 promoter and terminal codon fragments to provide an expression cassette. The promoter fragment was a 2.6 kb base EcoRI-PstI fragment from pAMH 102 (Harkki et al., Bio / Technology 7: 596-603 (1989)). The terminal codon was a 0.75 kb AvaH fragment contained in the PstI fragment. which also included an adapter and a TAA stop codon in each of the three read phases. PAMH 110 was then treated with SacIL and NdeI to remove the DNA between the cbh1 promoter and the terminal codon, and the treated ends blunt-ended with S1 nuclease and Klenow polymerase. Expression eg11 cDNA was taken from plasmid pTTc11 (Teeri et al., Anal. Biochem. 164: 60-67 (1987); Penttila et al., Yeast 3: 175-185 (1987) as a 1.6 kb EcoRI-BamHI fragment. which was blunt-ended with Klenow polymerase and ligated into an expression cassette to obtain plasmid pAMH111. Transformation was performed by co-transformation with pAMH111 and plasmid pAN8-1 (Mattem et al., "Transformations of Aspergillus orvzae" in Abstracts of the 19th Edition). Lecmres of Molecular Gene-15 tics of Yeasts and Filamentous Fungi and its Impact on Biotechnology, Lunteren,

34, (Netherlands) carrying the Steptoallotheicus hindustanus phleomycin resistance gene under the gpd promoter of A. nidulans. Another marker must be used if the VTT-D-87312 strain was already AmdS +, as in this example. Transformants were purified and examined for endoglucanase production in shake flask cultures. About 20. 20% of the cultures had hydroxyethylcellulose (HEC) hydrolyzing activity greater than that of the recipient strain. The amount of EGI protein (Table 4) in the supernatant fraction of the shake flask culture was examined from three transformants with HEC active. Southern blot analysis of these transformants showed that in the best · · · · endoglucanase producing clone (ALKO 2466) an expression cassette containing * ··· egll cDNA between the cbh1 promoter and end codon sequences, was integrated into the chromosomal cbhl locus via end codon sequences located on the *; · insert The amount of secreted protein increased in this transformant strain (ALKO 2466): approximately four-fold compared to control (Table 4).

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ‘63

Table 4 Characterization of EGI Production by T. reesei VTT-D-87312 (CBHI Negative VTT-D-79125 Transformant) and ALKO 2493, ALKO 2466 and ALKO 2494- is derived from VTT-D-87312 transformed with plasmid pAMH111.

TT-D-79125 is an untransformed mutant strain of T. reesei that produces high levels of cellulase. All strains were grown in shake flasks as described in Materials and Methods. The total amount of EGI protein and secreted protein was measured after seven days of culture as described in Materials and Methods.

10

EG 1 Protein Total% secreted EGI

female protein 15% secretion (mg / ml) (mg / ml) of total protein VTT-D-87312 0.35 4 ^ 5 7/7 20 ALCO 2493 1.25 5.2 24.0 ALCO 2466 1.90 5.8 32.8 ALKO 2494 1.40 5.9 23.7 VTT-D-79125 0.75 10.6 7.1 25 • · · HY Example 7 Simultaneous inactivation of the cbhl gene and egll multiplication of copy number 3Q:. The cbhl gene of T. reesei VTT-D-79125 was replaced by Trichoderman egll cDNA and lit · amdS gene. The egII cDNA was bound between the promoter of the cbh1 gene and the terminal codon. Plasmid pALK412 was constructed to be the source of the transforming fragment. Plasmid ··· pALK412 was prepared as described in Figure 15.

IMI

35: Plasmid p3SR2 harboring the amdS gene of Aspergillus nidulans was cloned into pBR322 (Kelly and Hynes, EMBO J. 4: 475-479 (1985) and treated with SphI). and Xbal: A 3.2 kb DNA fragment containing the entire amdS gene was obtained.

• · · • · · I. was combined with the Sph I and Xba I cut pUC19 vector (Yanish-Perron et al., Gene · ·· 33: 103-119 (1985)). The resulting plasmid was designated pALK410.

64

A DNA fragment containing the 1.65 kb base cbh1 gene from the Scal site of the 3 'region. which was in the coding region was isolated as a Scal-BamHI fragment and blunt-ended with Klenow. This fragment was ligated to the Xba I site of plasmid pALK410 (made blunt-ended with Klenow). In this case, the 3'fragment was isolated from plasmid pTT11.

The plasmid pTT11 (Teeri et al., Bio / Tech. 1: 696-699 (1983)) contains a 1.8 kb cbhI region. 3 'from the BamHI site in the coding region and was cloned into the BamHI site of pBR322. The gene can be isolated. from other sources, for example -10 clone 44A (Teeri et al. Bio / Tech 1: 696-699).

The resulting plasmid was pALK411. It was treated with ScaL and Sphl. The 5.8 kb fragment was bound to the plasmid pPLE3 cut with Scal and SphL (Nevalainen et al., 1990, Molecular Industrial Mycology: Systems and Applications for 15 Filamentous Fungi, Leong et al., Pp. 129-148). contains the cDNA for the egll gene between the promoter and terminal codon regions of the cbh1 gene cloned into pUC18 (Figure 16) .The promoter and terminal codon regions are derived from the pAMH11O expression vector (Nevalainen et al., Molecular Industrial Myc. Systems and Applications for Filamentous Fungi, Leong et al., Eds., Pp. 129-148 (1990)).

• • • • • • •; The resulting plasmid pALK412 was cut with EcoRL. to remove bacterial DNA.

The 9.3 kb pALK412F fragment was also re-ligated to form plasmid pALK412L.

25 ': T. reesei VTT-D-79125 (Bailey and Nevalainen, Enzyme Microb. Technol. 3: 153-157 (1981)) was transformed with plasmid pALK412, linear pALK412F fragment, re-bound pALK412 and pALK412F and pALK412L concurrently *: ··· with a molar ratio of the latter to 5: 1. Transformants were selected on the basis of the amdS + - 30 *: phenotype and purified from conidia on a selective medium containing acetamide as the sole nitrogen source.

• · · • · · ·

Purified transformants were grown on microtiter plates and screened for CBHI-65 phenotype by Western blotting using a polyclonal antibody raised against CBHI. About one-third of the pALK412F transformant did not produce any detectable CBHL. Among the forty strains was one CBHr transformant transformed with plasmid pALK412.

5 CBHr transformants were assayed for endoglucanase production in shake flasks. In all these transformants, the hydrolyzing activity of hydroxyethylcellulose (HEC) was higher than in the recipient strain. The best transformants secreted endoglycan activity 4-5 times as much as the recipient strain.

10

Southern blot analysis showed that the vector fragment displaced the cbh1 locus of CBHI 'transformants. Transformant chromosomal DNA was treated with XhoI and hybridized with a 0.5 kb base probe for cbh1.

15

The best endoglucanase producing strains had more than one copy of the vector fragment carrying the gene of interest inserted into the Trichoderma genome.

Example 8 Construction of a T. reesei Strain Negative for 2Q Cellobiohydrolase ························································································

«· · • Technol. 3: 153-157 (1982)) is a good producer of cellulase, it also has improved viability and is better able to produce the secreted protein due to the mutagenesis program by which it was created. Thus, it was desired to utilize the high protein production capacity of this mutant to obtain xylanases without cello biohydrolases for pulp bleaching.

*: **: UV-induced tryptophan-auxotrophic VTT-D-79125 mutant trans was formed by a linear fragment of DNA (Fig. 17) in which cbh2 flanking regions were used to target transforming fragment to the cbh2 locus. Purified TrpC + transformants were grown on microtiter plates and screened for CBHII phenotype by Westem (blotting) technique using monoclonal antibody 66 raised against CBHII. Southem (blotting) technique confirmed that the cbh2 locus was replaced by a transforming fragment. One such cbh2-negative transformant was transformed with another DNA fragment. This time, the cbh1 flanking regions were used to replace the organic-type cbh1 locus with the amdS marker of the set-5 mitase. Purified amdS + transformants were grown on microtiter plates and screened for CBHT phenotype by Westem (blotting) technique using a polyclonal antibody developed against CBHI. Substitution was again ensured by Southem (blotting) technology. The resulting strain does not carry the cbh1 or cbh2 gene and thus is unable to produce cellobiohydrolase under any circumstances.

Fig. 18 illustrates enzyme production profiles obtained with a hypocellulolytic strain and a cellobiohydrolase negative derivative thereof. The production of endoglu-canases is reduced by selecting appropriate fermentation conditions which are well known in the art. The novel strain can produce xylanase as an enzyme composition secreted by the host cell, and such enzyme composition does not contain cellobiohydrolase and thus lacks activity against crystalline cellulose. The very small amount of endoglucanase activity still present does not adversely affect the pulp properties when this kind of preparation is used in pulp enzymatic bleaching.

i · »··« · ·· · • · «• ♦ •» • · *. Example 9: Construction of strains that produce different combinations of cellulases. T. reesei strains producing different combinations of cellulases were constructed using a similar gene deletion strategy as in previous constructs ( Figure 17). In the first series of strains A-D, one cellulase is eliminated at a time. Strain A-D synthesizes everything as a host strain except: ·: ··: CBHI (strain A), CBHIE (strain B), EGI (strain C) or EGIE (strain D).

• 30: The second set, strains E-J, provides mutants that lack all pairs of four cellulases. Strain E lacks CBHI and CBHII. Strain F lacks CBHI and EGI.

•

. ·. : Strain G is missing CBHI and EGII. Strain H lacks CBHII and EGI. From the standpoint I

• · Missing CBHII and EGII. Strain J is missing EGI and EGII.

67

When both the EGI and EGII genes (strain J, Table 5) are deleted, the activity against hydroxyethylcellulose is reduced by more than 10% of the activity produced by the hypercellulolytic host strain VTT-D-79125. Deficiency of CBHI or CBHII proteins (strain E, Table 5) can be determined by assaying for activity against filter-5 paper by a method known in the art. When both CBHI and CBHII are eliminated, no riveting activity is produced. It would be obvious to one skilled in the art that additional modifications such as deleting three or more activities can be constructed using the same strategy.

10

Table 5

Genetically engineered strains for the production of novel cellulase mixtures

Parent CBHI CBHII EGI EGII 15 type A + + + B + - + + C ++ - -ΙΟ + + + - 20 E - - + ΙΕ - + - + G - + + -. H + - - + • ♦ · _ ··· 'I + - + K1 j ++. K + - - l - + - * - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------>; Gene technology has been successfully used to produce derivatives of the hypercellulolytic mutant, · · ·, to produce various cellulase-xylanase mixtures.

Strain 3 replaces the cbhl gene coding sequence with the * 11 coding sequence for strain 3 expressed under cbhl promoter. Thus, the proportion of endoglu-3β * canase produced and secreted by these hosts is increased. Strain 1 produces xylanases with its main activity and under various production conditions, as is known in the art, endoglucanases can be produced without any cellobiohydrolase. In the enzyme mixture produced by strain 3 68, the proportion of endoglucanases increases, and strain 2 produces cello-biohydrolases that are substantially free of endoglucanases.

Example 10 Use of Enzyme Preparations in Bio-Starting Coniferous kraft pulp was decontaminated with oxygen (kappa 16.1, high brightness 35.4%). The pH of the pulp was adjusted to 6 with sulfuric acid. A3273 ("Enzyme A") or A2799 ("Enzyme B") enzyme was used to treat pulp samples containing 250 g of dry matter at a dose of 100 nkat / g of dry matter at pH 6, 55 ° C. for two hours, and washed. The pulp samples were then bleached using a bleaching cycle D „(EO) DED. The control pulp was bleached without enzyme treatment.

The following standard methods were used: brightness (ISO 2470), kappa number (ISO 302), viscosity (ISO 5351/1 and PC yellowing) number (TAPPI 260 pm-81). The pulps were ground in a PFI mill (ISO 5264/2), the test sheets were hand-made according to ISO 5269/1 and the strength properties were examined (ISO 5270).

The enzyme-treated pulps were bleached so that 25% lower C102 doses were used in the first bleaching step than in the control pulp. The pulp pretreated with the enzyme • · · f, * t * achieved complete brightness with approximately 15% lower Cl

»M

(Table 6). This is approximately the same result obtained in earlier experiments with conventionally cooked pulps (Chapter 31-32), 25c. not lignin depleted by oxygen and not treated with elemental chlorine • «·! (Lahtinen, T., et al., Biotechnology in Pulp and Paper Industry, Kuwahara, M. Shimada, M. (eds.) Uni Publishers Co., Tokyo, Japan, ····: pp. 129-137 ( 1992)).

k · »« · ·

• · I

* -SO · Strength properties (viscosity and tear index) correlated with cellulosic side activities: when side activities (enzymes A) were low, 69 similar strength characteristics were obtained as in the control sample. The optical properties were also comparable to the reference material, with the exception of the PC number (yellowing measure), which was even better for pulps treated with enzyme.

• • 4 4 • I · • «f ·« · · 9 9 4 9 4 C t • • m · • · »» · • ♦ * «·« Il »1 · · 4 * ·· * · ·· * · · • ♦ · 9 9 4 4 9 9 • 494 449 9 41 • · 4 4 9 4 • 9 4 i 4414 4 4 * 444 4 4 4 • 49 * · «· · • ·· ♦ 1 70

Table 6. ECF Bleaching Tests

Enzymes Comparison

A

Enzyme Step 5 Dose, nkat / g 100 100 Temperature ° C 55 55 pH 6.0 6.0 Time, Hours 2 2-DO Step 10 CLO 2 Consumption, Activ. Cl 24.2 24.1 31.9 kg / h ____ EO phase

Brightness,% 62.5 62.5 63.1

Kappa 4.6 4.6 4.2 15 D1 Stage C102 Consumption, Act. Cl kg / t 17 17 17

Brightness,% 82.9 82.5 83.5 D2 Stage .20. Consumption of CL02, ”V act · Cl kg / t 6.5 6.3 6.0 • · · | Il ..... —— I ..... M · - ^ —— • · • Final

Brightness,% 90.2 89.5 89.7::: PC Number 0.45 0.54 0.87 25. Viscosity 830 810 840 • • · · ·

Total consumption of · · · CL02, active. Cl kg / t 47.7 47.4 54.9 ··· CL02 consumption, * 3i>. akt. Cl kg / t, ISO 901 * ·] Reduction 46.9 49.4 56.1 16% 12% • ·

Ml • · • 1 71 1 1 71 71 71 71 71 71 71 71 71 71 71 71 71 71 71 71

Properties of pulp after cleaning (tensile index 70 Nm / g) 5 PFI revs

Tear, nNm 2 / g 1123 945 871

Light scattering coefficient m2 / kg 13.8 12.2 14.2 V Alon adsorption coefficient 10 m2 / kg 20.7 20.6 21.6 0.06 __ (^ 08__0.06

Fiber Length mm 2.00 1.99 1.97 <0.20 mm,% 3.23 3.22 3.39 15 1: Assuming 4 kg Cl to increase the brightness per ISO unit Bleaching Conditions: 20

Step DO (E0) D1 E D2. Composition,% 3 10 9 9 9 • · ·!: V Temperature, ° C 55 75 70 70 70 • ·]. ·, Time, min. 60 60 180 60 180 I * · $ 2. End pH 2.9-3.2 10.8 3.7-4.0 10.8 3.5-3.8 • · · · · · · · · · · · · · · C102 dose, kg / t • · · · * : * · Active Cl 24.2 - 17-8 • · · 9 02 pressure, kPa - 200 - - - ··· NaOH dose, kg / t - 17 2.5 9 • · · ·

Having now fully described the invention, it will be apparent to one skilled in the art that the scope of the invention can be achieved over a wide and uniform range of conditions, parameters, etc., without affecting the spirit or scope of the invention or any of its applications; heretofore. All references cited in connection with the invention are hereby incorporated by reference.

• · · • · 72

Sequence Listing

(1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: OY ALKO AB et al.

(B) STREET: Salmisaarenranta 7 H

(C) CITY: Helsinki (E) COUNTRY: Finland (F) POSTAL NUMBER: 00180 (ii) NAME OF THE INVENTION: New Enzyme Preparations and Methods of Preparing them (iii) NUMBER OF SEQUENCES: 14 (v) MACHINE CODE FORM: (A) BETTER TYPE: COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patent Publication Authorization # 1.0, Version # 1.25 (vi) INFORMATION ON CURRENT APPLICATION: (A) APPLICATION NO: PCT / FI93 / 00221 (B) APPLICATION DATE: May-24-1993 (C) CLASSIFICATION: (vii) (A) APPLICATION NO: US 07 / 889,893 (B) APPLICATION DATE: May-29-1992 (2) SEQ ID NO: 1 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 1015 base pairs. .,, (B) TYPE: nucleic acid (C) YARNITY: single strand (D) TOPOLOGY: linear • · • ·. (ix) FEATURES:

(A) NAME / EXPLANATION: CDS

: (B) LOCATION: Range (176..448, 557..952) * · * • · · · (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: • ·· * AAGCTTGATG AGGCCAAATT ATCCGTCAAC TGTCTTATAA AGGAGCCCAT GCCAAACCCC 60 CCC AAACCTGAAC AACCCCAGCA CCTGAACAGT CATACAACCC 120 • · · CTCCAAGCCC AAAAGACACA ACAACTCCTA CTAGCTGAAG CAAGAAGACA TCAAC ATG 178

Met ·; ··· GTC TCC TTC ACC TCC CTC CTC GCC GGC GTC GCC GCC ATC TCG GGC GTC 226. Vai Ser Phe Thr Ser Leu Leu Ala Gly Vai Ala Ala Ile Ser Gly Vai ·: * ·: 5 ίο is TTG GCC GCT CCC GCC GCC GAG GTC GAA TCC GTG GCT GTG GAG AAG CGC 274 'Leu Ala Ala Pro Ala Glu Vai Glu Ser Or Ala Or Glu Lys Arg: 20 25 30 • · · • · CAG ACG ATT CAG CCC GGC ACG GGC TAC AAC AAC GGC TAC TTC TAC TCG 322

Gin Thr Ile Gin Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser 35 40 45 73 TAC TGG AAC GAT GGC CAC GGC GGC GTG ACG TAC ACC AAT GGT CCC GGC 370

Tyr Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Asn Gly Pro Gly 50 55 60 65 GGG CAG TTC TCC GTC AAC TGG TCC AAC TCG GGC AAC TTT GTC GGC GGC 418

Gly Gin Phe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly Gly 70 75 80 AAG GGA TGG CAG CCC GGG ACC AAG AAC AAG TAAGACTACC TACTCTTACC 468

Lys Gly Trp Gin Pro Gly Thr Lys Asn Lys 85 90 CCCTTTGACC AACACAGCAC AACACAATAC AACACATGTG ACTACCAATC ATGGAATCGG 528 ATCTAACAGC TGTGTTTTAA AAAAAAGG GTC ATC AAC TTC TCG GGA AGC TAC 580

Val lie Asn Phe Ser Gly Ser Tyr 95 AAC CCC AAC GGC AAC AGC TAC CTC TCC GTG TAC GGC TGG TCC CGC AAC 628

Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser Arg Asn 100 105 110 115 CCC CTG ATC GAG TAC TAC ATC GTC GAG AAC TTT GGC ACC TAC AAC CCG 676

Pro Leu lie Glu Tyr Tyr lie Val Glu Asn Phe Gly Thr Tyr Asn Pro 120 125 130 TCC ACG GGC GCC ACC AAG CTG GGC GAG GTC ACC TCC GAC GGC AGC GTC 724

Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp Gly Ser Val 135 140 145 TAC GAC ATT TAC CGC ACG CAG CGC GTC AAC CAG CCG TCC ATC ATC GGC 772

Tyr Asp lie Tyr Arg Thr Gin Arg Val Asn Gin Pro Ser lie lie Gly 150 155 160 ACC GCC ACC TTT TAC CAG TAC TGG TCC GTC CGC CGC AAC CAC CGC TCG 820

Thr Ala Thr Phe Tyr Gin Tyr Trp Ser Val Arg Arg Asn His Arg Ser 165 170 175:.: I AGC GGC TCC GTC AAC ACG GCG AAC CAC TTC AAC GCG TGG GCT CAG CAA 868

Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp Ala Gin Gin:. · 180 185 190 195 '·. *. GGC CTG ACG CTC GGG ACG ATG GAT TAC CAG ATT GTT GCC GTG GAG GGT 916:. ·. Gly Leu Thr Leu Gly Thr Met Asp Tyr Gin lie Val Ala Val Glu Gly ί. ·: 200 205 210 • · · ...: TAC TTT AGC TCT GGC TCT GCT TCC ATC ACC GTC AGC TAAAGGGGGC 962. *: *. Tyr Phe Ser Ser Gly Ser Ala Ser lie Thr Val Ser '· *' 215 220 TCTTCTTTTG TGATGTGTGA AAAAAAAAAA AAGGATGGTG GATAAAAGGG GGT 1015 • · · ····. *: *. (2) SEQ ID NO: 2 DETAILS: • · · *: **: (i) SEQUENCE FEATURES:. (A) LENGTH: 223 amino acids *: **: (B) TYPE: amino acid. (D) TOPOLOGY: linear «· · · · *. * * (Ii) MOLECYL-TYPE: protein * · *: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Vai Ser Phe Thr Ser Leu Leu Ala Gly Will Ala Ala Ile Ser Gly 15 10 15 74

Vai Leu Ala Ala Pro Ala Ala Glu Or A Glu Ser Ala Ala A Glu Lys 20 25 30

Arg Gin Thr Ile Gin Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr 35 40 45

Ser Tyr Trp Asn Asp Gly His Gly Gly Vai Thr Tyr Thr Asn Gly Pro 50 55 60

Gly Gly Gin Phe Ser Or Asn Trp Ser Asn Ser Gly Asn Phe Or Gly 65 70 75 80

Gly Lys Gly Trp Gin Pro Gly Thr Lys Asn Lys Vai Ile Asn Phe Ser 85 90 95

Gly Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Vai Tyr Gly Trp 100 105 110

Ser Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Or Glu Asn Phe Gly Thr 115 120 125

Tyr Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Vai Thr Ser Asp 130 135 140

Gly Ser Vai Tyr Asp Ile Tyr Arg Thr Gin Arg Vai Asn Gin Pro Ser 145 150 155 160

Ile Ile Gly Thr Ala Thr Phe Tyr Gin Tyr Trp Ser Vai Arg Arg Asn 165 170 175

His Arg Ser Ser Gly Ser Vai Asn Thr Ala Asn His Phe Asn Ala Trp 180 185 190

Ala Gin Gin Gly Leu Thr Leu Gly Thr Met Asp Tyr Gin Ile Vai Ala 195 200 205

Vai Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Vai Ser. 210 215 220 • · · · · · · · · ·

Iti • · * (2) SEQ ID NO: 3 DETAILS: «* * • ♦ · •« ♦: (i) SEQUENCE FEATURES: ··· ♦ (A) LENGTH: 941 bp ... (B) TYPE: Nucleic Acid * · ♦ · (C) STRIPE: simple strand (D) TOPOLOGY: linear • (ix) FEATURES:

(A) NAME / EXPLANATION: CDS

(B) LOCATION: space (102.392, 455..850) • · · • · · · · (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ____. GAATTCTGCA TATATAAAGC CATGGAAGAA GACGTAAAAC TGAGACAGCA AGCTCAACTG 60 • · • CATAGTATCG ACTTCAAGGA AAACACGCAC AAATAATCAT C ATG GTT GCC TTT 113

Met Vai Ala Phe • m 4 • 1 «*» ·. *: TCC AGC CTC ATC TGC GOT CTC ACC AGC ATC GCC AGT ACT CTG GCG ATG 161

Ser Ser Leu Ile Cys Ala Leu Thr Ser Ile Ala Ser Thr Leu Ala Met 5 10 15 20 75 CCC ACA GGC CTC GAG CCT GAG AGC AGT GTC AAC GTC ACA GAG CGT GGC 209

Pro Thr Gly Leu Glu Pro Glu Ser Ser Or Asn Or Thr Glu Arg Gly 25 30 35 ATG TAC GAC TTT GTT CTT GGA GCT CAC AAT GAT CAT CGC CGT CGT GCT 257

Met Tyr Asp Phe Vai Leu Gly Ala His Asn Asp His Arg Arg Arg Ala 40 45 50 AGC ATC AAC TAC GAC CAA AAC TAC CAA ACT GGC GGA CAA GTC AGC TAT 305

Ser Ile Asn Tyr Asp Gin Asn Tyr Gin Thr Gly Gly Gin Vai Ser Tyr 55 60 65 TCG CCT TCC AAC ACT GGC TTC TCA GTG AAC TGG AAC ACT CAA GAT GAC 353

Ser Pro Ser Asn Thr Gly Phe Ser Vai Asn Trp Asn Thr Gin Asp Asp 70 75 80 TTT GTT GTG GGC GTT GGT TGG ACG ACT GGA TCT TCT GCG TCGGAGGATT 402

Phe Vai Or Gly Or Gly Trp Thr Thr Gly Ser Ser Ala 85 90 95 CTCATCATTC TGCACTTTGA AAGCATCTTC TGACCAACAA GCTTCTCTTA GT CCC 457

Pro ATC AAC TTT GGC GGC TCT TTT AGT GTC AAC AGC GGA ACT GGC CTG CTT 505

Ile Asn Phe Gly Gly Ser Phe Ser Vai Asn Ser Gly Thr Gly Leu Leu 100 105 110 TCC GTC TAT GGC TGG AGC ACC AAC CCA CTG GTT GAG TAC TAC ATC ATG 553

Ser Vai Tyr Gly Trp Ser Thr Asn Pro Leu Vai Glu Tyr Tyr Ile Met 115 120 125 130 GAG GAC AAC CAC AAC TAC CCA GCA CAG GGT ACC GTC AAG GGA ACC GTC 601

Glu Asp Asn His Asn Tyr Pro Ala Gin Gly Thr Vai Lys Gly Thr Vai 135 140 145 ACC AGC GAC GGA GCC ACT TAC ACC ATC TGG GAG AAT ACC CGT GTC AAC 649. Thr Ser Asp Gly Ala Thr Tyr Thr Ile Trp Glu Asn Thr Arg Vai Asn * ··· · 150 155 160 • · · · · · · GAG CCT TCC ATC CAG GGC ACA GCG ACC TTC AAC CAG TAC ATT TCC GTG 697 . Glu Pro Ser Ile Gin Gly Thr Ala Thr Phe Asn Gin Tyr Ile Ser Vai 165 170 175 • · • · ·: Cgg aac tcg ccc agg acc agc gga act gtt act gtg cag aac cac ttc 745 ··· Arg Asn Ser Pro Arg Thr Ser Gly Thr Vai Thr Vai Gin Asn His Phe ··· 180 185 190 • · t • · f ’AAT GCT TGG GCC TCG CTT GGC CTG CAC CTT GGG CAG ATG AAC TAC CAG 793

Asn Ala Trp Ala Ser Leu Gly Leu His Leu Gly Gin Met Asn Tyr Gin 195 200 205 210 • · · ** ·· GTT GTC GCT GTC GAA GGC TGG GGT GGT AGT GGT TCT GCC TCA CAG AGT 841 · * · *; Vai Vai Ala Vai Glu Gly Trp Gly Gly Ser Gly Ser Ala Ser Gin Ser \ 215 220 225 GTC AGC AAC TAGGTTCTGT TGATGTTGAC TTGGAGTGGA TGAGGGGTTT 890. Vai Ser Asn • · GAGCTGGTAT GTAGTATTGG GGTGGTTAGT GAGTTAACTT GACAGACTGC A 941 • »• ♦ · * · (2) SEQ ID NO: 4 DETAILS: 1 SEQUENCE FEATURES: 76 (A) LENGTH (D): amino acids 229 amino acids TOPOLOGY: linear (ii) MOLECYL TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Vai Lower Phe Ser Ser Leu Ile Cys Lower Le Thr Ser Ile Lower Ser 15 10 15

Thr Leu Ala Met Pro Thr Gly Leu Glu Pro Glu Ser Ser Or Asn Vai 20 25 30

Thr Glu Arg Gly Met Tyr Asp Phe Vai Leu Gly Ala His Asn Asp His 35 40 45

Arg Arg Arg Ala Ser Ile Asn Tyr Asp Gin Asn Tyr Gin Thr Gly Gly 50 55 60

Gin Vai Ser Tyr Ser Pro Ser Asn Thr Gly Phe Ser Vai Asn Trp Asn 65 70 75 80

Thr Gin Asp Asp Phe Or Or Gly Or Gly Trp Thr Thr Gly Ser Ser 85 90 95

Ala Pro Ile Asn Phe Gly Gly Ser Phe Ser Or Asn Ser Gly Thr Gly 100 105 110

Leu Leu Ser Vai Tyr Gly Trp Ser Thr Asn Pro Leu Vai Glu Tyr Tyr 115 120 125

Ile Met Glu Asp Asn His Asn Tyr Pro Lower Gin Gly Thr Vai Lys Gly 130 135 140

Thr Vai Thr Ser Asp Gly Ala Thr Tyr Thr Ile Trp Glu Asn Thr Arg 145 150 155 160 • · · ··· · Vai Asn Glu Pro Ser Ile Gin Gly Thr Ala Thr Phe Asn Gin Tyr Ile I '· *. 165 170 175 • · • ·. Ser Vai Arg Asn Ser Pro Arg Thr Ser Gly Thr Vai Thr Vai Gin Asn * ··· 180 185 190 • · • · · * ·· His Phe Asn Ala Trp Ala Ser Leu Gly Leu His Leu Gly Gin Met Asn ·· · 195 200 205 • · ··

Tyr Gin Vai Or Area Or Glu Gly Trp Gly Gly Ser Gly Ser Ala Ser * 210 215 220

Gin Ser Vai Ser Asn ··· 225 ···· · · · · »(2) SEQ ID NO: 5 DETAILS: 1 SEQUENCE FEATURES: • · (A) LENGTH: 18 bp (B) TYPE: Nucleic Acid · * · *; (C) SURFACE: Simple strand • (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GACTCGAGAA TTCATCGA 18 77 (2) SEQUENCE ID NO: 6 DETAILS: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) SURFACE: single strand (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6G: GNG: 6G: SEQ ID NO: 7 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 18 base pairs (B) TYPE: Nucleic acid (C) JURITY: single strand (D) TOPOLOGY: linear (xi) SEQ ID NO: 7 : GACTCGAGAA TTCATCGA 18 (2) SEQ ID NO: 8 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURES: (A) : Peptide. (B) LOCATION: 1 · (D) OTHER INFORMATION: / Product = Peptide / Note = "The amino acid at position 2 with • · * marked" Xaa "is unknown." • · · ··· * (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: * ·· · Xaa Ile Gin Pro Gly Thr Gly Tyr Asn ls ·· * · · · * · ♦ * '' (2) SEQ ID NO: 9 DETAILS: • j. (i) SEQUENCE FEATURES: ···· (A) LENGTH: 20 base pairs · * · *; (B) TYPE: Nucleic Acid • * (C) DRAINAGE: Single strand. (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: • · • AAYTAY GAY C ARAAYTAY GA 20 •: (2) SEQUENCE ID NO: 10 DETAILS: • · · • · (i) SEQUENCE FEATURES: (A) LENGTH: 2 to 5 amino acids (B) TYPE: amino acid 78 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Ala Ser Ile Asn Tyr Asp Gin Asn Tyr Gin Thr Gly Gly Gin Vai Ser 15 10 15

Tyr Ser Pro Ser Asn Thr Gly Phe Ser 20 25 (2) SEQ ID NO: 11 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 39 base pairs (B) TYPE: Nucleic Acid (C) SURFACE: single strand (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CAACCGCGGA CTGCGCATCA TGGTCTCCTT CACCTCCCT 39 (2) SEQ ID NO: 12 DETAILED DESCRIPTION: (i) SEQUENCE FEATURES: (A): C) SURFACE: Simple strand (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGGAGCCGCT CGAGCGGTGG TTGCGG 26 9 • · · ['/' (2) SEQ ID NO: 13 • · 9 9 • (i) SEQUENCE FEATURES:. *** (A) LENGTH: 20 base pairs::: (B) TYPE: nucleic acid * · 1. · (C) DRAINAGE: single strand ·; 1 (D) TOPOLOGY: linear • · · ·: T: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GACTGGCATC ATGGCGCCCT 2 0 9 9 9 9 9 ”” (2) SEQUENCE ID NO: 14 DETAILS: ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) JURITY: single strand (D) TOPOLOGY: linear • · · • · · *, (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: · · «· · 'CAGGCTCGAG GCCTGTGGGC ATCGCCAGAG 30

Claims (27)

An isolated nucleic acid molecule, characterized in that it contains a nucleic acid sequence encoding the 5.5 amino acid sequence of T. reesei xylanase p1 (SEQ ID NO: 4) or another nucleic acid molecule encoding a protein having endo-β- 1,4-xylanase activity and having more than 49% amino acid sequence identity to the 5.5 mature portion of T. reesei xylanase. 2. An isolated nucleic acid molecule, characterized in that it contains a nucleic acid sequence encoding a protein having endo-β-1,4-xylanase activity and having an amino acid sequence more than 62% identical to that portion of T. reesei xylanase p 5.5. which is an active center with a sequence identification number between the glutamic acids 15 at positions 126 and 215 of position 4, including said amino acids. Nucleic acid molecule according to claim 1 or 2, characterized in that it contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 4. 20 Nucleic acid molecule according to any one of claims 1 to 3, characterized in that said nucleic acid sequence is a nucleic acid sequence of DNA having the sequence identification number 3. ♦ • · #]]!!!!!! 5. An isolated nucleic acid molecule, characterized in that it contains a nucleic acid sequence encoding the amino acid sequence of T. reesei xylanase p1 9 (SEQ ID NO: 2) or another nucleic acid molecule which: · · · · · '' Encodes a protein having endo-β-1,4-xylanase activity and having a · · · V · amino acid sequence more than 94% identical to the mature 30 portion of T. reesei xylanase p1. 1 An isolated nucleic acid molecule, characterized in that it contains • · ·. *. a nucleic acid sequence encoding a protein having endo-β-1,4- • · ·; .. * xylanase activity with an amino acid sequence greater than 98% identical to T. • · **; · '35 reesei xylanase pl 9 (SEQ ID NO: 2) with the moiety which is: ***: Glutamic acids at position 119 and 210: * · · in the active center including the amino acids mentioned. • · A nucleic acid molecule according to claim 5 or 6, characterized in that it contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2. Nucleic acid molecule according to any one of claims 5 to 7, characterized in that said nucleic acid sequence is a sequence according to SEQ ID NO: 1. A recombinant vector, characterized in that it contains a nucleic acid molecule according to any one of claims 1-8. A recombinant vector according to claim 9, characterized in that said vector is selected from the group consisting of plasmid and linear DNA. A recombinant host, characterized in that it is transformed with a recombinant vector according to claim 9 or 10. The recombinant host according to claim 11, characterized in that said host is Trichoderma. 20 The recombinant host of claim 12, wherein said Trichoderma is T. reesei. •% • · · • 1 ♦ A recombinant vector comprising an isolated nucleic acid molecule according to any one of claims 1 to 8, characterized in that the nucleic acid molecule is operably linked to a T. reesei promoter selected from the group consisting of the nucleic acid molecule. comprising the xlnl, xln2, cbhl, cbh2, precursor, and egl2 promoter · 15. ♦ · • · · V1 15. Recombinant host transformed with the recombinant vector of claim 14. ··· • · · • · · • A combination host according to claim 15, characterized in that said .1. the host is Trichoderma. A combination host according to claim 16, characterized in that said Trichoderma is T. reesei. ··· - 1 ·• · · · · · · A method for producing an enzyme preparation having an enriched enzyme activity, characterized in that (1) the host is transformed with a nucleic acid molecule according to any one of claims 1-8 or with a recombinant vector according to any one of claims 9,10 or 14; (2) cultivating said host cell under conditions wherein said nucleic acid molecule or recombinant vector is expressed in said (1) to 10 step host cell to produce xylanase; (3) recovering said culture medium. The method of claim 18, wherein said IS host is genetically unable to express one or more endogenous cellulolytic enzymes. A method according to claim 18 or 19, characterized in that said cellulolytic enzyme is selected from the group consisting of CBHI, 20 from CBHII, EGI and EGII. A method according to claim 20, characterized in that said β-cellulolytic enzyme is CBHI. A method according to any one of claims 18 to 21, characterized in that:. The host mentioned in 1 I is Trichoderma. • · · • · · ··· · · · The method of claim 22, wherein said Trichoderma is T. reesei. A culture medium obtained after culturing a host according to any one of claims 11 to 13 or 15 to 17. A product obtained from a culture medium according to claim 24 containing enzymes secreted by the fungi during cultivation by purification. drying, concentrating or immobilizing said culture medium. • · • · · · · · The use of a culture medium as claimed in claim 24 containing enzymes secreted by the fungi during culture or a product derived from the culture medium as claimed in claim 25 in the pulp and paper industry. Use of a culture medium according to claim 24 containing enzymes secreted by the fungi during culture or a product derived from culture medium according to claim 25 for use in the feed industry. · · · ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··· · · • i • · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·