CA2136350C - Xylanases from trichoderma reesei and methods for their production - Google Patents

Abstract

The structure of the T. reesei xln1 and xln2 genes and the primary structure of proteins are described. Enzyme preparations enriched in hemicellulase enzymes are described. Such enzyme preparations may also be partially or completely deficient in cellulose degrading activity. Such preparations may be utilized in a crude, unpurified form and are especially useful in the production of pulp and paper.

Description

Xylanases From Trichoderma Reesei and Methods for their Production Field of the Invention The present invention is related to enzyme preparations with unique enzyme profiles. Methods for the production of such enzyme preparations by genetically engineering members of the species Trichoderma are disclosed. These preparations contain high levels of xylanase enzymes and are especially useful in the pulp and paper industries.

Background of the Invention r- Cellulose is a linear polysaccharide of glucose residues connected by 0-1,4 linkages. In nature, cellulose is usually associated with lignin together with hemicelluloses such as xylans and glucomannans. In the pulp and paper industry, in chemical pulping (cooking) of the wood, the major part of the lignin is extracted to get acceptable cellulose pulp product. However, the resulting pulp is brown, mainly because of the small portion of the lignin still remaining in the pulp after cooking. This residual lignin is traditionally removed in a multi-stage bleaching procedure using typically a combination of chlorine chemicals and extraction stages.
Peroxide, oxygen and ozone are also used when the use of the chlorine chemicals is wanted to be reduced or avoided totally.
Hemicellulases can be used in enzyme-aided bleaching of pulps to decrease chemical dosage in subsequent bleaching or to increase brightness of the pulp (Kantelinen et al., International Pulp Bleaching Conference, Tappi 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 Centre of Finland, VTT Publications 114, Espoo, 1992). Naturally, in this use, the hemicellulose should be free of cellulases, which would harm the cellulose fibers.
The use of hemicellulose hydrolyzing enzymes in different bleaching sequences is known from WO 89/08738, EP 383,999, WO 91/02791, EP
395,792, EP 386,888 and WO 91/05908.
Other industrial applications for hemicellulolytic enzymes are in the production of thermomechanical pulps, where the aim of the use of hemicellulolytic enzymes is decreased energy consumption.
Hemicellulolytic enzymes can be used to improve drainage of recycled pulp or in the production of dissolving pulps (Viikari et al., "Hemicellulases for Industrial Applications," In: Bioconversion of Forest and Agricultural Wastes, Saddler, J., ed., CAB International, USA (1993)).
The use of hemicellulolytic enzymes for improved water removal from mechanical pulp is known from EP 262,040, EP 334,739 and EP
351,655 and DE 4,000,558).
When the hydrolysis of biomass to liquid fuels or chemicals is considered, the conversion of both cellulose and hemicellulose is essential to obtain a high yield (Viikari et al., "Hemicellulases for Industrial Applications," In: Bioconversion of Forest and Agricultural Wastes, Saddler, J., ed., CAB International, USA (1993)).
Also, in the feed industry, there is a need to use a suitable combination of enzyme activities to degrade the high 0-glucan and hemicellulose containing substrate.

To make the use of hemicellulolytic enzymes an economically realistic possibility in different applications, the production costs of the enzyme must be lowered. This means that the production levels must be high and no expensive purification steps can be included. The most SUBSTITUTE SHEET

"-*'VO 93/24621 2136350 PCT/F193/00221 economical way would be to choose the production strains and conditions in such a way that the culture supematant would be rich in required activities and the side activities would be only minor components.
Consequently, there is a clear demand for enzyme preparations, which contain unique enzyme profiles, taylor-made, that is, designed spesifically for the purposes of the industry in which they are to be used, and which can be obtained in a cost-effective manner, such as, for example, directly from the culture medium of the microorganism which has been modified so that it produces the desired enzymes, but not appreciable quantities of undesired enzymes.
Xylans are complex heteropolymers mainly consisting of xylose and arabinose. Xylans have a backbone consisting of P-1,4-linked xylopyranose units, which may be substituted with acetyl residues and residues of arabinose and niethyl glucuronic acid (Timell, T.E., et al., Wood Sci. Technol. 1:45-70 (1967)). Xylans are, after cellulose, the second most abundant carbohydrate fraction of plant biomass. A number of enzymes are needed for complete hydrolysis of xylan, the most important ones belonging t o-rhe group of endo-j3-xylanases (Biely, P., Trends Biotechno13:286-290 (1985); Dekker, R.F.H., in Hignehi, T., ed., Biosynthesis and biodegradation of wood components (Academic Press Inc., Orlando), pp. 505-533 (1985); Woodward, J., Top Enzyme Ferment.
Biotechnol. 8:9-30 (1984)).
Various microorganisms are capable of degrading xylans, and xylanases have been found in both prokaryotes and eukaryotes (Dekker, RF.H., Richards, G.N., Adv. Carbohydrate Chem. Biochem. 32:277-352 (1976)). Xylanolytic micro-organisms often produce multiple xylanases to attack the different bonds in these molecules. In a recent review, Bastawde (Bastawde, K.B., World J. Microbiol. Biotechnol. 8:353-368 (1992)) presented a compilation of the current knowledge about the properties and modes of action of microbial endoxylanases. There are several reports on the molecular cloning of these enzymes from bacteria SUBSTITUTE SHEET

(e.g. Ghangas, G. S. et al., J. Bacteriol. 171:2963-2969 (1989); Lin, L.-L., Thomson, J. A., Mol. 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, T. R., Lee, D. A., Curr.
Microbiol. 23:15-19 (1991)) but few from fungi (Boucher, F.
et al., Nucleic Acids Res. 16:9874 (1988); Ito, K. et al., Biosci. Biotec. Biochem. 56:906-912 (1992); Maat, J. et al., in Visser, J. et al., eds., Xylans and Xylanases (Elsevier Science, Amsterdam), pp. 349-360 (1992); van den Broeck, H.
et al., "Cloning and expression of xylanase genes from fungal origin", EP 463,706 Al (1992)).

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

Trichoderma reesei also produces all the enzymes needed for complete hydrolysis of native substituted xylans (Poutanen, K., et al., J. Biotechnol. 6:49-60 (1987)).
Multiple endo-R-1,4-xylanases have been purified from culture filtrates of Trichoderma (Baker, C. J., et al., Phytopathology 67:1250-1258 (1977); Hromova, 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 4a endoxylanases of T. reesei with isoelectric points at pH 5.5 (endoxylanase I) and pH 9.0 (endoxylanase II) have been characterized (Tenkanen, M., et al., Enzyme Microb.

Technol. 14:566-574 (1992)).

There is a need for efficient microbial and especially fungal xylanase producers, for example, for producing enzyme mixtures for enzyme-aided pulp bleaching (Kantelinen, A., et al., "Hemicellulases and Their Potential Role in Bleaching," Int. Pulp Bleaching Conference, Tappi Proceedings 1-9 (1988); Lahtinen, T., et al., "Using Selective Trichoderma Enzyme Preparations in Kraft Pulp Bleaching," in Biotechnology in the Pulp and Paper Industry, Kuwahara and Shimada, eds., Uni Publishers Co., Ltd., 5 Tokyo, Japan, pp. 129-137 (1992); Viikari, L., et al., "Bleaching with Enzymes," Biotechnology in the Pulp and Paper Industry, Proc. 3rd Int.
Conf., Stockholm, pp. 67-69 (1986); Viikari, L., et al., Proc. 4th Int.
Symp. Wood and Pulping Chemistry, Paris 1:151-154 (1987)).

Summary of the Invention Recognizing the importance of producing, in an economical way, higher amounts of hemicellulolytic enzymes, and producing these enzymes as an ideal combination with other wood or plant material degrading enzymes, the inventors have investigated the use of recombinant DNA
techniques in the design of hosts which would be useful as a large-scale source of recombinantly produced enzymes of interest to the applications of hemicellulolytic enzymes.
These studies have resulted in the development of fungal hosts which express large amounts of desirable enzymes and preferably large amounts of hemicellulolytic enzymes.
These studies have also resulted in the developmeiit of fungal hosts which not only express large amounts of desirable enzymes, preferably hemicellulolydc enzymes, but also are deficient in at least one enzymatic component of the cellulase degradation system.

The invention relates to fungal hosts belonging to the genera Trichoderma which are capable of expressing high levels of xylanase activity, or, fungal hosts which, in addition, are partially or completely deficient in cellulase activity.

This invention also relates to fungal hosts which produce high levels of xylanolytic enzymes and certain cellulolytic enzymes, preferably endoglucanases, but which, in addition, are partially or completely deficient in other cellulase activity, and preferably deficient in cellobiohydrolases.

This invention also relates to enzyme compositions which are enriched in such hemicellulase activity or which in addition are partially or completely deficient in certain cellulolytic activities.

This invention also relates to isolation and cloning of xylanase genes from Trichoderma.

This invention further relates to new hosts (plant, yeast, fungal or bacterial) which contain and express such Trichoderma xylanases.

According to one aspect of the present invention, there is provided an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequence of T. reesei pI 5.5 xylanase (SEQ ID No. 4) or another nucleic acid molecule encoding a protein having endo-beta-l,4-xylanase activity and which hybridizes to the complement of SEQ ID NO. 3 at 60 to 68 C in the presence of 6 x SSC + 0.1% SDS and washing at 60 to 68 C in the presence of 6 x SSC + 0.1% SDS.

According to another aspect of the present invention, there is provided an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the amino acid sequence of T. reesei pI 5.5 xylanase set forth in SEQ ID No. 4.

6a According to still another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequence of T. reesei pI 9 xylanase set forth in SEQ ID NO. 2.

According to still another aspect, the invention provides an isolated polypeptide encoded by the nucleic acid as described herein.

According to still another aspect of the present invention, there is provided a method for the production of an enzyme preparation enriched in xylanase activity, said method comprising: (1) transforming a host cell with the recombinant vector comprising an isolated nucleic acid molecule as described herein; (2) culturing said host cell in culture medium under conditions wherein a protein having xylanase activity expressed by said recombinant construct is synthesised from said transformed host cell of step (1); (3) and collecting said culture medium.

According to yet another aspect of the present invention, there is provided a recombinant vector comprising an isolated nucleic molecule described herein. In certain embodiments, the nucleic acid molecule in the vector is operably linked to a T. reesei promoter selected from the group consisting of the xlnl promoter, the xln2 promoter, the cbhl promoter, the cbh2 promoter, the egll promoter, and the e912 promoter.

According to a further aspect of the present invention, there is provided a recombinant host cell transformed with the recombinant vector described herein.

According to yet a further aspect of the present invention, there is provided a culture medium obtained after culture of a host cell as described herein.

6b According to still a further aspect of the present invention, there is provided an enzyme preparation having endo-beta-1,4-xylanase activity or pI 9 xylanase activity, obtained by purifying, drying, concentrating or immobilizing the culture medium as described herein.

According to another aspect of the present invention, there is provided a use of the culture medium or the enzyme preparation described herein in the pulp and paper or the feed industries.

Brief Description of the Drawings Figure 1 shows the general strategy for deleting a gene.

Figure 2 shows the restriction map of the 5.7 kb (KpnI) insert of pALK475. The location of xln2 gene is marked with an arrow. The inserts in the subclones pALK573, pALK574, pALK570 and pALK476 are shown by separate lines.
The SmaI site at the 5' end of the fragment is derived from the polylinker of pUC19.

Figure 3 shows the nucleotide sequence of the T. reesei xln2 gene [SEQ ID No.:1:]. The coding regions are expressed in upper case letters [SEQ ID No.:2:1. Peptide sequences obtained from purified protein are indicated by an underline; sequence indicated by a double underline was used for preparing the PCR primer. The TATA box is indicated by dotting. Putative signal cleavage site is marked with an arrow (T) and putative sites for N-glycosylation with a triangle (V). The N-terminal of the mature protein is shown with an arrow (Y_). The two glutamic acids proposed to be involved with an active site are boxed.

~136350 Figure 4 shows plasmid pALK476, a plasmid for targeting the xln2 gene to the cbhl locus. The xln2 gene is under the control of its own promoter.
Figure 5 shows the pALK476 fragment used in the transfonnations.
The 1.9 kb cbhl S'-flanking region (Scal-EcoRI) is from 2.2 kb upstream of the cbhl-coding region, the 1.8 kb 3'-region (BamHI-EcoRI) is from 1.4 kb downstream of the end of the cbhl-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 area of the xln2 gene in the fragment is 2.3 kb in size.

Figure 6 shows the relative xylanase production levels of the transformants of Figure 5 calculated relative to that of T. reesei VTT-D-79125. The columns show the mean values and the range of production levels among the transformants of each host strain. One flask of each transformant was grown. The numbers of transformants tested were (CBHI+'"): T. reesei VTT-D-79125 19/38, ALK02221 16/26 and ALK02721 22/31.

Figure 7 shows the construction of pALK174 (xln2 gene is fused to the cbhl promoter). Only the relevant restriction sites are shown.
Figure 8 shows the relative increase in production of xylanase activity in pALK174 transformants.
Figure 9 shows the restriction map of the 2.3 kb EcoRI insert of pALK572. The location of xlnl gene is marked with an arrow.
Figure 10 shows the nucleotide sequence of the T. ressef xlnl gene [SEQ ID No.:3:]. The coding regions are indicated by upper case letters [SEQ ID No.:4:]. Peptide sequences obtained from purified protein are shown underlined; the sequence with a double underline was used for preparing the PCR primer. The TATA box is indicated by dotting.
Putative signal cleavage site is marked with an arrow (T). The N-terminal of the mature protein is shown with an arrow (-). The two glutamic acids proposed to be involved with an active site are boxed.

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.W

Figure 11 shows the construction of plasmid pALK807 (xlnl gene is fused to the cbhl promoter). Only the relevant restriction sites are shown.
Figure 12 shows an FPLC analysis of the CBHI negative transformant VTT-D-87312; Figure 12A shows its comparison to the untransformed host.
Figure 13 shows a diagram of the plasmid pALK99.
Figure 14 shows a diagram of the replacement of the chromosomal cbh2 gene with the argB gene.
Figure 15 shows the construction of the plasmid pALK412.
Figure 16 shows a diagram of plasmid pPLE3.
Figure 17 shows the construction of a Acbhl acbh2 strain; trpC-mutant of VTT-D-79125 (UV).
Figure 18 shows the enzyme profiles of the hypercellulolytic mutant strain of T reesei VTT-D-79125 (UV) and the genetically engineered derivative of that strain that lacks cellobiohydrolase.
Figure 19 shows modified enzyme profiles of the genetically engineered T. reesei strains #1, #2 and #3.

Detailed Description of the Invention I. Definitions In the description that follows, a number of terms used in recombinant DNA (rDNA) technology are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Cellulase. Cellulase is a collective term which encompasses enzymes which catalyze reactions which participate in the degradation of insoluble cellulose to soluble carbohydrate. The term "cellulase" is known in the art to refer to such a group of enzymes. For efficient hydrolysis of SUBSTITUTE SHEET

U

cellulose to glucose, at least three cellula.se enzymes (three types of cellulase enzyme activity) are needed: randomly cleaving endoglucanases (1,4,-P-D-glucan glucanohydrolase, EC 3.2.1.4) which usually attack substituted soluble substrates and show no activity to crystalline cellulose;
cellobiohydrolase (1,4-A-D-glucan cellobiohydrolase, EC 3.2.1.91) which is capable of degrading crystalline cellulose but has no activity towards derivatized cellulose and P-glucosidase ((3-D-glucoside glycohydrolase, EC
3.2.1.21) which degrades cellobiose and cello-oligosaccharides to yield glucose. Each of the three main types of enzymes listed above occurs in multiple forms. For example, two immunologically distinctive cellobiohydrolases, CBH I and CBH II are known. In addition, 5-8 electrophoretically distinct endoglucanases are known. Synergistic action between some of these enzymes has been demonstrated. Cellulase activity is synonymous with cellulolytic activity.
The biosynthesis of cellulases is provoked or induced by cellulose, cellobiose, sophorose and lactose, and repressed by glucose or other readily utilizable carbon sources.
By a Trichoderma host which is "substantially incapable" of synthesizing one or more cellulase enzymes is meant a Trichoderma host in which the activity of one or more of the cellulase enzymes is depressed, deficient, or absent when compared to the wild-type (untransformed) Trichoderma.
Hemicellulolytic enzymes (hemicellulases). For the enzymatic degradation and modification of hemicelluloses, several different enzymes are needed, each of which are termed "hemicellulase." The two main glycanases depolymerizing the hemicellulose backbone are endo-1,4-(3-D-xylanase and endo-1,4,(3-D-mannanase. Small oligosaccharides are further hydrolyzed by 1,4-(3-D-xylosidase, 1,4-(3-D-mannosidase and 1,4-(3-D-glucosidase. The side groups are split off by a-L-arabinosidase, a-D-glucuronidase and a-D-galactosidase. Esterified side groups are liberated by various esterases.

SUBSTITUTE SHEET

WO 93/24621 2136350 P(.T/F193/00271 ,...

The defnzition of hemicellulolytic enzymes is taken from Viikari et al., "Hemicellulases for Industrial Applications," in Bioconversion of Forest and Agricultural Wastes (1992).
Enqme preparation. By "enzyme preparation" is meant a 5 composition containing enzymes which have been extracted (either partially or completely purified) from fungi or the medium used to grow such fungi. Therefore, the term "enzyme preparation" is meant to include a composition comprising medium previously used to culture such fungi and any enzymes which the fungi have secreted into such medium during 10 the culture.
Culture medium. By culture medium is meant a medium previously used to culture a fungi ("spent" culture medium), such culture medium containing enzymes which the fungi have secreted into the medium during the culture. The culture medium is usable as such or as partially or completely purified, concentrated, dryed or immobilized. If desired, the expressed endoglucanase protein may be further purified in accordance with conventional conditiosn, such as extraction, precipitation, chromatography, affmity chromatography, electrophoresis, or the like.
Bio-bleaching. By "bio-bleaching" is meant the extraction of lignin from cellulose pulp after the action of hemicellulose degrading enzymes with or without lignin degrading enzymes. Removal of the lignin may be restricted by hemicelluloses either physically (through reprecipitation onto the fiber surface during cooking) or chemically (through lignin-carbohydrate complexes). The hemicellulase activity partially degrades the hemicellulose, which enhances the extractability of lignins by conventional bleaching chemicals (like chlorine, chlorine dioxide, peroxide, etc.) (Viikari et al., "Bleaching with Enzymes" in Biotechnology in the Pulp and Paper Industry, Proc. 3rd Int. Conf., Stoclholm, pp. 67-69 (1986); Viikari et al., "Applications of Enzymes in Bleaching" in Proc. 4th Int. Symp. Wood and Pulping Chemistry, Paris, Vol. 1, pp. 151-154 (1987); Kantelinen et al., "Hemicellulases and their SUBSTITUTE SHEET

WO 93/24621 2136350 PC'T/F193/00221 Potential Role in Bleaching" in International Pulp Bleachrng Conference, Tappi Proceedings, pp. 1-9 (1988)). The advantage of this improved , bleachability is a lower consumption of bleaching chemicals and lower environmental loads or higher final brightness values. Hemicellulolytic enzymes improve bleachability by making the lignin removal with conventional bleaching chemicals easier.
Gene. A DNA sequence containing a template for a RNA
polymerase. RNA that codes for a protein is termed messenger RNA
(mRNA) and, in eukaryotes, is transcribed by RNA polymerase II.
However, it is also known to construct a gene containing a RNA
polymerase II template wherein a RNA sequence is transcribed which has a sequence complementary to that of a specific mRNA but is not normally translated. Such a gene construct is herein termed an "antisense RNA
gene" and such a RNA transcript is termed an "antisense RNA." Antisense RNAs are not normally translatable due to the presence of translational stop codons in the antisense RNA sequence.
A"complementary DNA" or "cDNA" gene includes recombinant genes synthesized by reverse transcription of mRNA and from which intervening sequences (introns) have been removed.
By an enzyme homologous to a Trichoderma host of the invention is meant that an untransformed Trichoderma of the same species as the host species naturally produces some amount of the native protein; by a gene homologous to a Trichoderma host of the invention is meant a gene found in the genome of an untransformed Trichoderma of the same species as the host species.
By an enzyme heterologous to a Trichoderma host of the invention is meant that an untransformed Trichoderma of the same species as the host species does not naturally produce some amount of the native protein;
by a gene heterologous to a Trichoderma host of the invention is meant a gene not found in the genome of an untransformed Trichoderma of the same species as the host species.

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HybridiZation. By hybridization are meant conditions, under which all Trichoderma xylanase genes hybridize to the nucleic acid sequence encoding the amino acid sequence of T. reesei p1 5.5 xylanase or to the nucleic acid sequence encoding the amino acid sequence of T. reesei pI 9 xylanase. These conditions are characterized by hybridization preferably in 60 to 68 C in the presence of 6 x SSC + 0.1% SDS and washing in in 60 to 68 C in the presence of 6 x SSC + 0.1% SDS.
Cloning vehicle. A plasmid or phage DNA or other DNA sequence (such as a linear DNA) which provides an appropriate nucleic acid environment for the transfer of a gene of interest into a host cell. The cloning vehicles of the invention may be designed to replicate autonomously in prokaryotic and eukaryotic hosts. In Trichoderma, the cloning vehicles generally do not autonomously replicate and instead, merely provide a vehicle for the transport of the gene of interest into the Trichoderma host for subsequent insertion into the Trichoderma genome.
The cloning vehicle may be further characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about replication and cloning of such DNA. The cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicilltin resistance. The word "vector" is sometimes used for "cloning vehicle." Alternatively, such markers may be provided on a cloning vehicle which is separate from that supplying the gene of interest.
Expression vehicle. A vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene of interest which has been cloned into it, after transformation into a desired host. In a preferred embodiment, such expression vehicle provides for an enhanced expression SUBSTITUTE SHEET

of a gene of interest which has been cloned into it, after transformation into a desired host.
In a preferred embodiment, the gene of interest which is provided to a fungal host as part of a cloning or expression vehicle integrates into the fungal chromosome. Sequences which derive from the cloning vehicle or expression vehicle may also be integrated with the gene of interest during the integration process.
The gene of interest may preferably be placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences provided by the vector (which integrate with the gene of interest). If desired, such control sequences may be provided by the fungal host's chromosome as a result of the locus of insertion.
Expression control sequences on an expression vector will vary depending on whether the vector is designed to express a certain gene in a prokaryotic or eukaryotic host (for example, a shuttle vector may provide a gene for selection in bacterial hosts) and may additionally contain transcriptional elements such as, enhancer elements, termination sequences, and/or translational initiation and termination sites.

II. Genetic Engineering of the Trichoderma Hosts The process for genetically engineering the hosts of the invention is facilitated through the cloning of genetic sequences which are capable of encoding a desired enzymic activity and through the expression of such genetic sequences. As used herein, the term "genetic sequences" is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences which are capable of encoding a desired enzyme are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.
The mesophilic imperfect fungus Trichoderma reesei (formerly T. viride) is classified as a member of Fungi imperfecti. Fungi imperfecti SUBSTITUTE SHEET

...

is a catch-all category of fungi which have no sexual reproduction or obvious affinities with sexually reproducing genera, such as the highly characteristic Aspergillus. Although Trichoderma has been reported to possess a poorly defmed sexual stage being an imperfect state of the perfect ascomycete species Hypocrea, the genera Aspergillus and Trichoderma are clearly to be considered taxonomically very different.
The improved enzyme preparations according to this invention are produced by the fungus Trichoderma which has been modified by recombinant DNA techniques. The Trichoderma hosts of the invention are modified so as to be able to produce high levels of enzymes, preferably hemicellulases. In addition, these hosts may be modified so as to be totally deficient in at least one cellulase enzyme (whose activity is undesirable during pulp and paper processing). Thus, although the remaining cellulase activities may be unaffected, the Trichoderma hosts of the invention are partially or completely deficient in the necessary complement of enzymes which will fully degrade cellulose to glucose, and, as a result, such degradation is greatly lowered or completely blocked.

Ila. Transformation of Trichoderma with a New Genetic Construct In one embodiment, the Trichoderma hosts of the invention which may be partially or completely deficient in at least one cellulase activity are transformed with a genetic construct capable of expressing at least one desired pulp and paper processing enzyme which is homologous to Trichoderma, so as to provide for increased amounts of this enzyme in the Trichoderma host. Examples of desired pulp and paper processing homologous enzymes include, for example, hemicellulases and pectin-degrading enzymes. Trichoderma is inherently capable of producing a variety of hemicellulases including endoxylanases, mannanases, 0-xylosidase, a-arabinosidase, a-glucuronidase, and acetyl esterase, the activity of any of which may be a desired enzyme in the enzyme SUBSTITUTE SHEET

preparations of the invention. Also, native Trichoderma produces minor amounts of pectin degrading enzymes like polygalacturonase which may be classified as a desired enzyme in the enzyme preparations of the invention.
Further, any other Trichoderma enzyme which oxidizes cellulose may be 5 utilized in the enzyme preparations of the invention and may be a desired enzyme.
Comparison with xylanolytic enzymes produced by Trichoderma reesei QM 9414, Aspergillus awamori VTT-D-75028, Fusarium oxysporum VTT-D-80134, Bacillus subtilis ATCC 12711 and 10 Streptomyces olivochromogenes ATCC 21713 has shown that the highest xylanase activity was produced by T. reesei. Therefore, under conditions where it is desired to retain xylanase activity, T. reesei is an advantageous host. (Poutanen, K., "Characterization of Xylanolytic Enzymes for Potential Applications" in Disssertation for the degree of Doctor of 15 Technology, Technical Research Centre of Finland, Publications 47).
Further, although the above ireparations from the different microbial origins differed with respect to (3-xylosidase activity and side-group cleaving activities, the T. reesei culture filtrate contained all the side-group cleaving activities assayed (acetyl esterase, a-glucuronidase and a-arabinosidase) whereas those from F. oxysporum and S. olivochromogenes only contained esterase. Thus Trichoderma is also advantageous as a host because it naturally produces a wide spectrum of xylanolytic enzymes the proportions of which can be manipulated by genetic engineering for different applications to provide enzyme preparations tailored for those purposes.
According to this invention, the genetic constructs which encode homologous enzymes which are desirable for pulp and paper processing purposes may be introduced into the genome of Trichoderma and enhanced expression can also be achieved by using strong promoters such as cbhl and, if desired, additional or modified regulatory regions, such as, for example, enhancer sequences. Preferably, such regulatory sequences are SUBSTITUTE SHEET

WO 93/24621 PCT/F193/0027' ,....

homologous to Trichoderma. A regulatory region, and especially a promoter, may be modified to contain only those sequence elements needed for expression and/or to retain a region which is responsible for high expression levels. Enhancer sequences may be introduced concurrently with the gene of interest as a separate DNA element but operably-linked to such gene of interest, for example, as a DNA sequence which is colinear with that providing the gene of interest (for example, in a 5' or 3' non-translating sequence, or in an intron).
In a highly preferred embodiment, the homologous gene introduced to the genome of Trichoderma is a gene encoding a homologous hemicellulase, preferably xylanase. The two main 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 (XYLII) and their molecular masses are 19 and 20 kDa, respectively.
The xylanase I enzyme from Trichoderma reesei has optimal activity in the more acid pH region, whereas xylanase II has its pH optimum in the near-neutral pH range. Because of the different properties of the two xylanases, either one or a mixture of these enzymes can be used in different applications (WO 92/03541 and Viikari et al., "Important Properties of Xylanases for Use in Pulp and Paper industry" In:
Biotechnology in Pulp and Paper Industry, Kuwahara, M. and Shimada, M., eds., Proc. 5th Int. Conf. Biotechnology in Pulp and Industry, Uni Publishers Co., Ltd., Tokyo, 1992, pp. 101-106).
According to the invention, it is possible by using genetic engineering methods to enrich the desired xylanolytic activity to the culture medium and use this culture medium in a desired application. For exarnple, in some bleaching sequences and bleaching conditions it may be advantageous to use xylanase I (more acid pH region), in some other bleaching sequences and bleaching conditions xylanase II may be preferably (alkaline or near neutral pH range).

WO 93/24621 PC.'I'/FI93/00221 In some applications, although one cellulolytic activity may be eliminated, reduced, inactivated, or repressed, it may be desirable to introduce a gene encoding a different cellulolytic enzyme into the host cells so as to enhance one specific cellulolytic activity. In addition to hemicellulolytic activity, for example, in feed industry, an enzyme preparation comprising an elevated amount of endoglucanase may be used.
Thus, in those preparations in which such hydrolysis is desired, a host which expresses elevated levels of endoglucanases, in addition to xylanases or other hemicellulases may be used.
In another embodiment, the Trichoderma host which already expresses a homologous form of an enzyme is transformed with a genetic construct encoding a heterologous form of the same enzyme. In a further embodiment, a Trichoderma host which does not express a certain enzyme is transformed with one or more genetic constructs encoding enzyme(s) heterologous to Trichoderma.
According to this invention increased amounts of a heterologous enzyme whose activity is desired for pulp and paper processing purposes are achieved by introducing the gene producing such heterologous desired enzyme into a specific locus and/or introducing the gene in multicopies into the genome of Trichoderma as described above.
In a preferred embodiment, the gene encoding a desired enzyme is inserted into the cbhl locus such that it is operably linked to the strong cbhl promoter. As described below, enhanced production is achieved by using strong promoters such as cbhl. Increased amounts of the desired heterologous enzyme are also achieved when Trichoderma's cellulase producing capacity is lowered in general, even if the heterologous gene is not inserted into the cbhl locus.
A gene encoding a desired enzyme, either homologous or heterologous, such as a hemicellulose hydrolyzing enzyme, can be integrated into the genome of Trichoderma by inserting the gene into a general expression vector, for example, pAMH110, which is described in SUBSTITUTE SHEET

WO 93/24621 P(.'I'/FI93/00221 e3 J !~ ~, the patent application EP 244,234. pAMH 110 is derived from pUC 19 (Yanish-Perron et al., Gene 33:103-119 (1985)) and includes the promoter and terminator of the cbhl gene and a stuffer fragment between the promoter and terminator sequences which can be removed by digestion with SacII and Ndel. After the ends are made blunt, any DNA, cDNA or chromosomal DNA can be inserted between the promoter and terminator.
The desired gene can be inserted to the cbhl expression cassette in the plasmid pAMH110 between the cbhl promoter and terminator.
Transcriptional regulatory elements of other genes may be used where it is desired not to use the cbhl elements. For example a vector construction comprising the 3-phosphoglycerate kinase gene (pgk) transcriptional regulatory regions may be used as 3-phosphoglycerate kinase, a key enzyme for ATP generation by glycolysis, is expressed in the presence of glucose under which conditions the synthesis of cellulases is repressed.
While the inventors do not intend to be bound by any particular theory, the effectiveness of the expression of the desired gene seems to be dependent both on the number of copies of the desired gene integrated to the genome of Trichoderma and on the location of integration of the gene in the genome. In a preferred embodiment, the integration of a desired gene is directed into a specific locus. The use of a linear DNA helps in directing the integration into a homologous locus. In a highly preferred embodiment, the integration of a desired gene is directed into the Trichoderma cbhl locus.
The DNA constructions prepared according to this invention can be used to transform any Trichoderma strain. Such strains include, for example, T. reesei strains QM9414 (ATCC 26921), RUT-C-30 (ATCC
56765), and highly productive mutants like VTT-D-79125, which is a descendant of QM9414 (Nevalainen 1985, Technical Research Centre of Finland Publications 26, (1985), Espoo, Finland). The transformation of SUBSTITUTE SHEET

WO 93/24621 P(,'I'/FI93/0022]

Trichoderma may be performed by any technique known in the art and especially by the technique taught in EP 244,234.
The Trichoderma host cells may be cultivated and the desired enzymes produced by cultivating the host strain having the desired properties under any conditions which allow expressing of the desired enzymes. For example, a Trichoderma host strain having the desired properties may be cultivated in a liquid cultivation medium, which may comprise, for example, 6% Solka Floc cellulose, 3% distiller's spent grain, 0.5% KH2PO4, 0.5% (NH4)ZSO4, 0.1% struktol. The cellulase production by Trichoderma strains is sensitive to glucose repression and require an inducer such as, for example, cellulose, lactose or sophorose (Allen et al., Biotechnology and Bioengineering 33:650-656 (1989)). The pH in Trichoderma cultivation should be kept at approximately pH 5 by the addition of phosphoric acid or ammonia and the temperature may be kept at 30 C during the cultivation. However, the temperature should be adjusted according to the strain and according to the enzyme preparation to be produced (Merivuori et al., Biotechnology Lett. 12:117-120 (1990)).

IIb DNA Cloning Vector systems may be used in the method of producing Trichoderma hosts for the production of the enzyme preparations of the invention. One element provided by such vector construction may encode the sequence of at least one homologous gene the activity of which it is desired to eliminate, reduce, inactivate, delete or repress. Such vector construction (a) may further provide a separate vector construction (b) which encodes at least one desired gene to be integrated to the genome of Trichoderma and (c) a selectable marker coupled to (a) or (b).
Alternatively, a separate vector may be used.
The cloned DNA which is used in the hosts of the invention may or may not include naturally occurring introns. Moreover, such genomic SUBSTITUTE SHEET

.....

DNA may be obtained in association with the native 5' promoter region of the DNA genetic sequences and/or with the 3' transcriptional termination region if such sequences are capable of functioning in Trichoderma.
Further, such genomic DNA may be obtained in association with the 5 genetic sequences which encode the 5' non-translated region of the mRNA
and/or with the genetic sequences which encode the 3' non-translated region. To the extent that the Trichoderma host cell can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then the 5' and/or 3' non-transcribed 10 regions of the native gene, and/or, the 5' and/or 3' non-translated regions of the mRNA, may be retained and employed for transcriptional and trans-lational regulation. Genomic DNA can be extracted by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L.
Berger et al., eds., Academic Press (1987)). Alternatively, mRNA can be 15 isolated from any cell which produces or expresses the desired protein, and used to produce cDNA by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al., eds., Academic Press (1987)). Preferably, the mRNA preparation used will be enriched in mRNA coding for a desired protein, either naturally, by isolation from a 20 cells which are producing large amounts of the protein, or in vitro, by techniques commonly used to enrich mRNA preparations for specific sequences, such as sucrose gradient centrifugation, or both.
For cloning into a vector, such suitable DNA preparations (either genomic DNA or cDNA) are randomly sheared or enzymatically cleaved, respectively, and ligated into appropriate vectors to form a recombinant gene (either genomic or cDNA) library.
A DNA sequence encoding a desired protein may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, SUBSTITUTE SHEET

and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., et al., supra, and are well known in the art.
Libraries containing clones encoding a desired protein may be screened and a clone to the desired protein identified by any means which specifically selects for that protein's DNA such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated protein product produced by the host containing the clone.
Oligonucleotide probes specific for the proteins desired in this invention which can be used to identify clones to such protein can be designed from knowledge of the amino acid sequence of the protein.
Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA
(1977), pp. 356-357). The peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.
Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide sequence, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, poten-tially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene.
SUSSTiTUTE SHEET

WO 93/24621 36350 PCT/F193/0027' Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
Using the genetic code (Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977)), one or more different oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding the protein. The probability that a particular oligonucleotide will, in fact, constitute the actual protein's sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe, R., et al., J. Molec. Biol. 183:1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide sequence, or a set of oligonucleotide sequences, that contain a theoretical "most probable" nucleotide sequence capable of encoding the protein's sequence is identified.
The suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the protein's gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) may be synthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed., 1987, Academic Press, San Diego, CA) and employed as a probe to identify and isolate the desired cloned gene by techniques known in the art. Techniques of nucleic acid hybridization and clone identification are disclosed by Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), and by Hames, B.D., et al.
(In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)).

SUBSTITUTE SHEET

Cloning techniques using oligonucleotides and PCR are also well known in the art ("PCR Protocols" in A Guide to Methods and Application, Innis et al., eds., Academic Press, San Diego (1990)). Those members of the above-described gene library which are found to be capable of such hybridization are then analyzed to determine the extent and nature of the desired genomic coding sequences which they contain.
To facilitate the detection of the desired DNA encoding sequence, the above -described DNA probe is labeled with a detectable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are nonradioactive labels such as digoxigenin-nucleotides (dUTP) or radio-active labels, such as 3zP, sH, 14C, 35S, tzsl, or the like. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life. The oligonucleotide may be radioactively or nonradioactively labeled by several methods known in the art and commercial kits for these purposes are available.
Alternatively, polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, digoxigenin, an enzyme or a fluorescent group. See, for example, Leary, J.J., 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 protein's sequence (or a partial sequence of the protein) permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide comple-mentary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable" oligo-nucleotides), one obtains a DNA molecule (or set of DNA molecules), SUBSTITUTE SHEET

WO 93/24621 2136350 PC.'I'/FI93/0022' capable of functioning as a probe(s) for the identification and isolation of clones containing the protein's gene.
In an alternative way of cloning a gene, a library is prepared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing a desired protein, into an expression vector. The library is then screened for members which express the protein, for example, by screening the library with antibodies to the protein.
The above discussed methods are, therefore, capable of identifying genetic sequences which are capable of encoding a desired protein or fragments of this protein. In order to further characterize such genetic sequences, and, in order to produce the recombinant protein, it is desirable to express the proteins which these sequences encode. Such expression identifies those clones which express proteins possessing characteristics of the desired protein. Such characteristics may include the ability to specifically bind antibodies directed against the protein, the ability to elicit the production of antibody which are capable of binding the protein, and the ability to provide a protein specific function to a recipient cell, among others.
The cloned protein encoding sequences, obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a Trichoderma host cell to produce recombinant protein or a functional derivative thereof. Depending upon which strand of the protein encoding sequence is operably linked to the sequences controlling transcriptional expression, it is also possible to express an antisense RNA or a functional derivative thereof.
A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are SUBSTITUTE SHEET

"operably linked" to the nucleotide sequence which encodes the polypeptide.
An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression 5 of the sequence under the influence or control of the regulatory sequence.
Two DNA sequences (such as a protein encoding sequence and a promoter region sequence linked to the 5' end of the encoding sequence) are said to be operably linked if induction of promoter function results in the transcription of the protein encoding sequence mRNA and if the nature of 10 the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the mRNA, antisense RNA, or protein, or (3) interfere with the ability of the template to be transcribed by the promoter region sequence. Thus, a promoter 15 region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
The precise nature of the regulatory regions needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-20 coding) sequences involved with initiation of transcription and translation respectively.
Expression of the protein in the Trichoderma hosts requires the use of regulatory regions functional in such hosts. A wide variety of transcriptional and translational regulatory sequences can be employed, 25 since Trichoderma generally recognize eukaryotic host transcriptional controls, such as, for example, those of other filamentous fungi. In eukaryotes, where transcription is not linked to translation, such , -itrol regions may or may not provide an initiator methionine (AUG) Ion, depending on whether the cloned sequence contains such a methionine.
Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell.

SUBSTITUTE SHEET

WO 93/24621 PCT/FI93/0027' 213~`~5~

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the protein, or a functional derivative thereof, does not contain any intervening codons which are capable of encoding a methionine. The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the protein encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the protein encoding sequence).

Ilc Regulatory Regions In a preferred embodiment, a desired protein is secreted into the surrounding medium due to the presence of a homologous Trichoderma secretion signal sequence. If a desired protein does not possess its own signal sequence, or if such signal sequence does not function well in Trichoderma, then the protein's coding sequence may be operably linked to a signal sequence homologous or heterologous to Trichoderma. The desired coding sequence may be linked to any signal sequence which will allow secretion of the protein from a Trichoderma host, for example, the signal sequence of the Trichoderma cellobiohydrolase I protein. Such signal sequences may be designed with or without specific protease sites such that the signal peptide sequence is amenable to subsequent removal.
Transcriptional initiation regulatory signals can be selected which allow for repression or activation, so that expression of the operably linked genes can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., substrate or metabolite regulation. Also of interest are constructs wherein both (a) a desired protein's mRNA and (b) antisense RNA directed to a SUBSTITUTE SHEET

w0 93/24621 PCT/F193/00221 cellulase enzyme are provided in a transcribable forms such that expression of the desired protein's mRNA is accompanied by antisense RNA
repression of the expression of one of the host's cellulase enzymes.
Translational signals are not necessary when it is desired to express antisense RNA sequences.
If desired, the non-transcribed and/or non-translated regions 3' to the sequence coding for a protein can be obtained by the above-described cloning methods. The 3'-non-transcribed region may be retained for its transcriptional termination regulatory sequence elements; the 3-non-translated region may be retained for its translational termination regulatory sequence elements, or for those elements which direct polyadenylation in eukaryotic cells.
The vectors of the invention may further comprise other operably linked regulatory elements such as enhancer sequences.

IId Detecting Stable Transformants of Trichoderma In a preferred embodiment, genetically stable transformants of Trichoderma are constructed whereby a desired protein's DNA is integrated into the host chromosome. The coding sequence for the desired protein may be from any source. Such integration may occur de novo within the cell or, in a most preferred embodiment, be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, DNA elements which promote integration of DNA sequences in chromosomes.
Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene SUSSTITUTE SHEET

WO 93/24621 PC.'1'/F193/0022' 64. ,~~63.,o sequences to be expressed, or introduced into the same cell by co-transfection.
Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
Once the vector or DNA sequence containing the construct(s) is prepared for expression, the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transformation as described above. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of transformed cells. Expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner.
The DNA encoding sequences, obtained through the methods above, will provide sequences which by definition, encode a desired protein and which may then be used to obtain a desired protein's antisense RNA
genetic sequences as the antisense RNA sequence will be that sequence found on the opposite strand of the strand transcribing the peptide core's mRNA. The antisense DNA strand may also be operably linked to a promoter in an expression vector such that transformation with this vector results in a host capable of expression of an antisense RNA in the transformed cell. Antisense RNA and its expression may be used to interact with an endogenous DNA or RNA in a manner which inhibits or represses transcription or translation of the gene in a highly specific manner. Use of antisense RNA probes to block gene expression is discussed in Lichtenstein, C., Nature 333:801-802 (1988).

SUBSTITUTE SHEET

w0 93/24621 2~ 36350 PCT/F193/00221 .,,,.

Trichoderma is an especially useful and practical host for the synthesis of the enzyme preparations of the invention because Trichoderma is capable of secreting protein at large amounts, for example, concentrations as much as 40 g/L culture fluid have been reported; the homologous Trichoderma cbhl promoter provides a very convenient promoter for expression of genes-of-interest because it is a strong, single copy promoter which normally directs the synthesis of up to 60% of the secreted protein from the Trichoderma host; the transformation system is highly versatile and can be adapted for any gene of interest; the Trichoderma host provides an "animal cell type" high mannose glycosylation pattern; and culture of Trichoderma is supported by previous extensive experience in industrial scale fermentation techniques.

M. Trichoderma Hosts Deficient in At Least One Cellulase En2yme According to this invention, it is also possible to enrich Trichoderma hosts for an enzyme whose activity is desirable for pulp and paper processing purposes by inactivating or eliminating at least one cellulase enzyme. In one embodiment, the cbhl gene is merely mutated. Since the majority of the secreted proteins of Trichoderma may be the cellulase activity encoded by the gene cbhl, (the cellobiohydrolase, CBHI, protein), by constructing Trichoderma hosts in which the cbhl gene is mutated to an inactive form, the relative percent of the remaining proteins secreted by Trichoderma in the culture medium may be increased. In another embodiment, a desired gene is inserted preferably into the cbhl locus such that expression of the desired gene is operably linked to the strong cbhl promoter. In a highly preferred embodiment, a casette comprising a desired gene already operably linked to the homologous cbhl promoter is inserted into the cbhl locus.
In the hosts of the invention, any one, some, or all of the cellulolytic enzymes may be eliminated, reduced, inactivated, or repressed by methods SUBSTITUTE SHEET

WO 93/24621 PCT/F193/0027' known in the art so as to result in the host's partial or complete inability to degrade cellulose to glucose. Undesired cellulolytic enzyme activities can be eliminated, reduced, inactivated, or repressed by several methods, e.g., by inactivating the gene(s) encoding such enzyme (for example, by 5 introducing a frame-shift mutation to the gene), by deleting the entire whole gene or large segments of the gene, by replacing the gene with another DNA via homologous recombination, by compensation of the gene region, by additional integration, by double crossing-over, and by transforming the host cell with a genetic construct capable of expressing an 10 antisense RNA directed against the coding sequence for that gene, etc.
For example, inactivation of genes coding for cellulolytic activities may be performed as described in European Patent Applications EP
137,280 and EP 244,234.
Trichoderma fungi produce large amounts of identical, 15 predominantly haploid uninucleate conidia which constitute excellent material for various mutagenic treatments. However, even a haploid mutated nucleus can produce a heterokaryotic colony (mycelium) if a mutation becomes initially fixed only in one of the two strands of the DNA double helix (mosaicism). The amount of mosaic mutants depends 20 on both the mutagen and dose used. In fungi forming haploid uninucleate conidia, the problem of heterokaryotic mycelium can be handled by allowing conidiation and by reisolation of colonies originating from single separate conidia. This cycle can be repeated several times.
Examples of chemical mutagens useful for mutengenizing the 25 Trichoderma hosts of the invention include alkylating agents, such as, for example, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethylmethanesulphonate (EMS) and diethylsulphate (DES). Hydroxylamine and chemicals deaminating DNA bases such as nitrous acid are also useful.
Ionizing radiation (y- and X-rays) as well as ultraviolet irradiation (UV) 30 are examples of physical mutagens useful in Trichoderma strain mutagenesis.

SUBSTITUTE SHEET

The use of solid media permits rapid screening of thousands of colonies arising from mutagenized conidia for the presence or absence of specific enzymes and allows quantitative estimation of the amount of enzyme produced. Several types of solid media for detection of enzymes, for example, extracellular amylolytic enzymes, pectinase, protease, chitinase, 0-galactosidase and cellulase, lipase, urease, RNAase and DNAase are known in the art.
Many fungi form large diffuse colonies when grown on solid media.
Addition of chemical agents restrictive to colony growth may therefore be desired to allow development of more than one (up to 100) colony per one plate. Among agents used for the purpose are rose bengal, oxgall and TM
phosphon D, Triton X-100 and saponin. With some fungi, replica plating technique analogous to that developed for bacteria can, iri certain cases, be used to test the properties of fungal colonies on different growth media.
Screening on plates is usually followed by cultivation of the selected colonies in shake flasks in a liquid production medium for measurement of enzyme activity. The best isolates showing enhanced enzyme production in shake flask scale may be in a second round of mutagen treatment if desired.
Homologous genes which it is desirable to inactivate or delete according to this invention include, for example, the cellulase genes cbhl, cbh2, egll, egl2 (formerly eg13; Saloheimo et al., Gene 63:11-21 (1988)) (which encode the proteins cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II) or combinations of these genes.
Eliminating the activity of any of these genes will result in a host which is partially or completely deficient in its ability to degrade cellulose to glucose. Such elimination of cellulolytic activity may be achieved at the genomic level, by eliminating the gene or modifying it into a form which is incapable of expression. Such elimination may also be achieved at the translational level, by hybridizing the mRNA which encodes the protein to WO 93/24621 PCT/FI93/002'' an antisense RNA to a degree which prevents the translation of the hybridized RNA.
In a preferred embodiment, a cellulase activity is selectively inactivated so that some, but not all of the cellulase components are inactivated. For example, if it is desired to maintain the host's ability to hydrolyze R-glucan, then the endoglucanase genes would not be inactivated.
The inactivation of, e.g., one of the cellulase genes can be based on transformation of Trichoderma reesei with a plasmid carrying a defected gene as described in patent application EP 244,234. Homologous recombination of the plasmid at the chromosomal cellulase gene locus causes insertional inactivation of the endogenous T. reesei cellulase gene.
The plasmid used for transformation contains only part of the cellulase coding region and produces inactive protein. No 5' flanking sequences are included. A frameshift mutation can also be introduced to the truncated coding region. A selection marker, (for example amdS (acetamidase) or argB (ornithine carbamoyl transferase)) or a marker for screening (for example, lacZ) can be coupled to the plasmid used for the transformation or the transformation can be done as a cotransformation, which means that the selectable marker and the defected gene are on different plasmids (EP
244,234). Inactivation of a gene with homologous recombination may be done with a circular DNA, which integrates in a colinear manner into the Trichoderma chromosomal DNA.
The deletion of an undesired gene can be done by using a strategy the principle of which is described in Figure 1. The recipient strain is transformed with a linear DNA fragment containing a selectable marker gene (like trpC, argB or amdS) and/or a foreign desired gene of interest which is to be expressed, flanked by the 5' and 3' flanking regions of the gene to be deleted. Homologous recombination at the A locus will thus lead to replacement of the A gene with the selection marker and/or desired gene B. If the 5' region in the transforming fragment is taken upstream SUBSTITUTE SHEET

WO 93/24621 213 6 ~ ~ ~_, PCT/FI93/00221 from the promoter area, the promoter will also be removed in the resulting replacement strain. Gene A can be any Trichoderma gene, preferably a cellulase gene, the flanking regions of which can be cloned/isolated.
Moreover 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 bacteria (e.g., in E. coli). The linear DNA fragments used in deletion of undesired genes can be constructed for example from pUC 19 plasmids (Yanish-Perron et al., Gene 33:103-119 (1985)).
This method is described in more detail in the Example 1 B which describes the deletion of cbh2 gene from the genome of Trichoderma by said method.
Clones of the cellulase enzymes have been described which may be used to design mutant sequences for inactivation of homologous sequences in the hosts of the invention. Any mutant sequence which results in the inactivation of the enzyme's activity may be used. For example, the gene for the native cellobiohydrolase CBH I sequence has been cloned by Shoemaker et al. (Shoemaker, S., et al., BiolTechnology 1:691-696 (1983)) and Teeri et al. (Teeri, T., et al., Bio/Technology 1:696-699 (1983)) and the entire nucleotide sequence of the gene is known (Shoemaker, S., et al., Bio/Technology 1:691-696 (1983)). From T. reesei, the gene for the major endoglucanase (EG I) has also been cloned and characterized (Penttilii, M., et al., Gene 45:253-263 (1986); EP 137,280; Van Arstel, J.N.V., et al., BiolTechnology 5:60-64). Other isolated cellulase genes are cbh2 (Patent Application WO 85/04672; Chen, C.M., et al., BiolTechnology 5:274-278 (1987)) and eg12 (originally eg13 (Saloheimo, M., et al., Gene 63:11-21 (1988)).

IV. The Engyme Preparation According to the invention, there is provided a method for producing high levels of enzymes, preferabaly hemicellulases, desirable for pulp and SUBSTITUTE SHEET

WO 93/24621 2 11~~~~ O PCT/F193/0027' ,.~

paper processing. There is also provided a method for producing an enzyme preparation partially or completely deficient in cellulolytic activity (that is, in the ability to completely degrade cellulose to glucose) and enriched in enzymes desirable for pulp and paper processing, preferably hemicellulases. By "deficient in cellulolytic activity" is meant a reduced, lowered, depressed, or repressed capacity to degrade cellulose to glucose.
Such preparations may be obtained directly from the hosts of the invention.
Further, if desired activities are present in more than one recombinant host, such preparations may be isolated from the appropriate hosts and combined prior to use in the method of the invention.
It is envisioned that enzyme preparation which are enriched or partially or completely deficient in specific enzymatic activities will be provided so as to satisfy the requirements of a specific utility in various applications in the pulp and paper industry and in fodder production.
Enzyme activities may be added or deleted as described above to provide, remove or retain or lower a desired activity. For example, if the intended application is improvement of the strength of the mechanical mass of the pulp, then the enzyme preparation of the invention may provide enzymes which enhance or facilitate the ability of cellulose fibers to bind together.
In a similar manner, in the application of pulp milling, the enzyme preparation of the invention may provide enzymes which enhance or facilitate such swelling.
To obtain the enzyme preparations of the invention, the recombinant hosts described above having the desired properties (that is, hosts substantially incapable of expressing one or more cellulase enzymes and capable of expressing the desired enzymes) are cultivated under suitable conditions, the desired enzymes are secreted from the Trichoderma hosts into the culture medium, and the enzyme preparation is recovered from said culture medium by methods known in the art.
The enzyme preparation can be produced by cultivating the Trichoderma strain in a fermentor having the desired properties for SUBSTITUTE SHEET

~.~ 35 example in a liquid cultivation medium, which may comprise for example 6% Solka Floc cellulose (BW40, James River Corporation, Hackensack, NJ), 3% distiller's spent grain, 0.5% KH2PO4, 0.5% (NH4)2SO4, and 0.1%
struktol as an antifoaming agent (struktol SB 2023, Schill & Seilacher, Hamburg, FRG). The cellulase production of Trichoderma strains is sensitive to glucose repression and require an inducer (cellulose, lactose or sophorose) (Allen et al., Biotechnology and Bioengineering 33:650-656 (1989)). The pH should preferably be kept at approximately pH 5 by the addition of phosphoric acid or ammonia and the temperature at 30 C
during the cultivation. However, the temperature may be adjusted according to the strain and according to the enzyme preparation to be produced (Merivuori et al., Biotechnology Letters 12(2):117-120 (1990)).
The enzyme preparation is recovered from the culture medium by using methods well known in the art. However, because the hosts of the invention may be partially or completely deficient in cellulase activity, it is an advantage of the invention that the enzyme preparations of the invention may be utilized directly from the culture medium with no further purification. If desired, such preparations may be lyophilized or the enzymatic activity otherwise concentrated and/or stabilized for storage.
The enzyme preparations of the invention are very economical to provide and use because (1) the enzymes may be used in a crude form; isolation of a specific enzyme from the culture fluid is unnecessary and (2) because the enzymes are secreted into the culture medium, only the culture medium need be recovered to obtain the desired enzyme preparation; there is no need to extract an enzyme from the Trichoderma hosts.
If desired, an expressed protein may be further purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
The Trichoderma and enzyme preparations of the invention have further application in fodder production. For example, fodder treated with SUBSTITUTE SHEET

the enzyme preparations of the invention would be of great food benefit to farm animals because it would be easier for them to digest.
The invention is described in more detail in the following examples, These examples show only a few concrete applications of the invention. It is self evident for one skilled in the art to create several similar applications. Hence the examples should not be interpreted to narrow the scope of the invention only to clarify the use of the invention.
Examples Materials and Methods Transformation of T. reesei Transformation of T. reesei and selection of AmdS+ and ArgB+
transformants were carried out as described by Penttila et al., Gene 61:155-164 (1987).
Phleomycin resistant transformants were screened as described by Durand et al. in: Biochemistry and Genetics of Cellulose Degradation, p.
135-151, 1987, J.-P. Aubert, P. Beguin and J. Millet (eds.), Academic Press, New York.
In cotransformation with p3SR2 and pAMH111, equal molar amounts of plasmid DNA (5-10 g) were used. In transformations conferring phleomycin resistance, the relative amounts of plasmid DNA
used were 1:1 or 2:1 for pAMHl l l and pAN8-1 respectively. When cotransformation was carried out using p3SR2 and pMS4, the plasmid pMS4 was added in 3-4 times molar excess. Transformants were purified through conidia; that is, the conidial suspension was plated again on the selective medium so that every colony started from a single conidia.
In transformations with a linear DNA fragment, the amount of DNA
used varied from 2 to 5 g. The selection marker (amdS (acetamidase) or SUBSTITUTE SHEET

argB (ornithine carbamoyl transferase, OTCase, E.C. 2.1.3.3)or trpC
(tryptophane)) was within the transforming fragment.

Isolation and Analysis of DNA

Plasmid DNA from E. coli was isolated using standard methods (Maniatis et al. 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Chromosomal DNA was isolated from T. reesei using the method of Raeder and Broda, Lett. Appl. Microbiot :l.47-20 (1985)). Southern and Northern hybridizations were performed by standard techniques (Maniatis et al.
supra, 1982). Western blotting was carried out according to Maniatis et al., supra, 1982).

Liquid Cultivation Media and Conditions for Trichoderma All Trichoderma liquid cultures were started from conidiospores grown on potato dextrose agar as described by Bailey and Nevalainen, Enzyme Microb. Technol. 3:153-157 (1981)). Liquid cultivations in shake flasks were performed according to Bailey and Nevalainen, Enzyme Microb. Technol. 3:153-157 (1981), except that Finnfloc was replaced with Solca Floc cellulose. Medium used in fermenter cultivations contained 6%
Solka Floc cellulose, 3% distiller's spent grain, 0.5% KH2PO41 0.5%
(NH4)2SO4 and 0.1% struktol. The pH was kept between 4.0 and 4.8 by addition of phosphoric acid or ammonia. Fermentations were carried out at C. Maximum yield of enzymes was obtained in 5 days in laboratory fermentations and in 4 days in 100 liter fermenter scale.

SUBSTITUTE SHEET

EnUme Assays All assays for enzyme activity were carried out from culture supernatant fractions after removing the mycelia by centrifugation for 20 min at 3000 rpm. Endoglucanase activity using hydroxyethylcellulose as substrate (IEC, mittelviskos, Fluka AG 54290, pract. grade) was measured as described in Bailey and Nevalainen, Enzyme Microb. Technol. 3:153-157 (1981) and Commission on Biotechnology, International Union of Pure and Applied Chemistry; Measurement of Cellulase Activities, Biochemical Engineering Research Centre, Indian Institute of Technology, Delhi, New Delhi - 10016 (1984)) and xylanase activity using birch xylan (Roth No.
7500) as substrate, were measured as described by Bailey et al., J. Bact.
23:257-270 (1992). The TCA precipitated proteins were assayed with the method of Lowry et al., J. Biol. Chem. 193: 2-65-275 (1951) using bovine serum albumin as standard. Cellobiohydrolase activity against filter paper (filter paper unit, FPU) was measured as described in Commission on Biotechnology, International Union of Pure and Applied Chemistry, Measurement of Cellulase Activities, Biochemical Engineering Research Centre, Indian Institute of Technology, Delhi, New Delhi -10016 (1984)).
ELISA Assay for Endoglucanase I

Endoglucanase I protein concentration in the culture supennatant fractions was determined by a double antibody sandwich ELISA. The assays were performed in 96-well flat bottomed microtiter plates at 37 C
(except were noted). Each step was terminated by washing 3 times with TM
phosphate buffered saline pH 7.2 containing 0.05% Tween 20 and 0.02%
sodium azide (PBS/Tween).
The plates were coated with mouse monoclonal antibodies directed against endoglucanase I (anti-EGI antibody EI-2) overnight at 4 C.
Unoccupied sites on the plastic surface were blocked with 1% BSA in WO 93/24621 213t7 350 PCT/F193/00221 PBS/Tween for 1 hr. Appropriate dilutions of culture supematant fractions and purified endoglucanase I were then added and incubated for 2 hrs followed by an incubation with rabbit polyclonal antibodies against endoglucanase I for 2 hrs. Bound rabbit antibodies were detected by incubation with swine polyclonal antibodies against rabbit IgG conjugated to alkaline phosphatase (Orion Diagnostica, Espoo, Finland) for 2 hrs. In an end step p-nitrophenylphosphate (1 mg/nil) was added and the reaction stopped after 30 min at room temperature with 2 N NaOH. The developed yellow color was measured photometrically at 405 nm. The concentration of endoglucanase I in culture supematant fractions was then calculated by comparing their 0D405 values with a standard dilution curve prepared using purified endolgucanase I and performed at the same time on the same plate.

Fractionation of the Culture Supernatant Fraction by Chromatofocusing The chromatographic system consisted of a Pharmacia FPLC
apparatus equipped with a Mono P HR 5/20 column for chromatofocusing.
The resin was stabilized in 25 mM Bistris-HCl buffer, pH 6.5. The crude enzyme mixture produced by T. reesef in shake flask culture was diluted with the same buffer to 1 mg/ml protein content. 500 l enzyme samples were injected into the column and eluted with Pharmalyte/Polybuffer (Pharmacia, 1 ml PharmalyteR 2.5 - 5 and 5 ml PolybufferT' PB 74 in a total 100 ml, adjusted pH to 3.0 with HCl) forming a pH gradient from 6.5 to 3Ø The flow rate was 30 ml/h. Column effluents were collected in 600 l fractions and the pH and EGI activity were assayed.

SUBSTITUTE SHEET

Example Cloning, Sequencing and Enhanced Expression of the Trichoderma reesei Endoxylanase II (pl 9) Gene, xln2 5 In the results presented in this example, the Trichoderma reesei xln2 gene coding for the pI 9.0 endoxylanase was isolated from the wild-type strain QM6a. The gene contains one intron of 108 nucleotides and codes for a protein of 223 amino acids in which two putative N-glycosylation target sites were found. Three different T. reesei strains were transformed 10 by targeting a construct composed of the xln2 gene with its own promoter to the endogenous cbhl locus. Highest overall production levels for xylanase were obtained using the T. reesei ALK02721, a genetically engineered strain, as a host. Integration into the cbhl locus was not required for enhanced expression under xln2 promoter.

15 Materials and Methods Organisms, plasmids and growth conditions. Plasmids were propagated in Escherichia coli strain XL1-Blue (Bullock, W.O., et al., BiolTechniques 5:376-378 (1987)) or in E. coli INV 1 aF' (Invitrogen, San Diego, CA, USA). The recipient organisms for the x1n2 gene were the 20 high cellulase-producing mutants T. reesei VTT-D-79125 (Bailey and Nevalainen, Enzyme Microb. Technol. 3:153-157 (1981)), ALK02721 and ALK02221. T. reesei ALK02221 is a low protease mutant of the strain VTT-D-79125. T. reesei ALK02721 is a trpC minus (trpC ') UV mutant of VTT-D-79125, in which the cbh2 locus has been replaced with the trpC

25 gene of Aspergillus nidulans (Yelton, M.M., et al., Proc. Natl. Acad. Sci.
USA 81:1470-1474 (1984)). The strain also carries several other integrated copies of the trpC gene.
TM
The plasmids used in this study included pBluescript (Stratagene, San Diego, CA, USA), pCR1000 (Invitrogen) and pUC19 (Yanish-Perron, C., et al., Gene 33:103-119 (1985)). The selectable marker, amdS, came from the plasmid p3SR2 (Kelly and Hynes, EMBO J. 4:475-479 (1985)) kindly donated by Dr. M. Hynes. The cbhl flanking regions were isolated from the T. reesei strain ALK02466 (Harkki, A., et al., Enzyme Microb.
Technol. 13:227-233 (1991)), using plasmid rescue.
E. coli cultures were grown at 37 C overnight in L-broth (Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)) supplemented with ampicillin (50 g/ml) as needed. PD (Potato Dextrose Broth, Difco, Detroit, USA) agar slants were used for growing the Trichoderma strains. For xylanase production, the T. reesei strains were grown for seven days in shaker flasks (30 C, 250 rpm) in a cellulase- and xylanase-inducing medium (pH 5.5) containing of 4% whey, 1.5% complex nitrogen source, 1.5% KH2PO4 and 0.5% (NH4)2SO3.
Peptide digestion and amino acid sequencing. Purified xylanase II
(Tenkanen, M., et al., Enzynie Microb. Technol. 14:566-574 (1992)) from T. reesei (VTT-D-79125) was digested with 2% (w/w) trypsin (TPCK-treated, Sigma Chemical Company, St. Louis, MO, USA) in 1% (w/v) ammonium bicarbonate at 37 C for 2.5 h. After addition of 3% (w/w) trypsin, incubation was continued overnight (13 h). The peptides were separated using high-performance liquid chromatography (HPLC) with a Rexchrom Prep-5/300 ODS reverse-phase column. Elution was performed at the rate of 1 ml/min in a linear solvent gradient running from 5% (v/v) acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) to 60% (v/v) ACN containing 0.06% TFA in 60 min at 24 C. Absorbance at 218 nm was measured. Prior to amino-terminal sequencing, 10 g of xylanase II was treated with 1 U of pyroglutamate aminopeptidase (Boehringer Mannheim, Mannheim, Germany) at 37 C overnight in 100 mM potassium phosphate buffer, pH 8.0, containing 10 mM EDTA
and 5mM DTT. Amino-terminals of the protein and the peptides were sequenced by degrading them in a gas-pulsed-liquid-phase sequencer SUBSTITUTE SHEET

(Kallckinen and Tilgmann, J. Protein Chem. 7:242-243 (1988)). The released PTH-amino acids were analyzed on-line using narrow-bore reverse-phase HPLC.
DNA manipulation and transformations. Standard DNA methods described by Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) were used for the DNA constructions. 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, D., J. Mol. Biol.
166:557-580 (1983), and T. reesei strains by the method of Penttild, M., et al., Gene 61:155-164 (1987) with the following modifications: the protoplasts were treated by heat shock (Berges and Barreau, J. Gen.
Microbiol. 135:601-604 (1989)) before transformation, 25% PEG6. was replaced with 60% PEGWO and the transformation was performed at room temperature instead of that on ice. The transformants were purified through conidia on selective acetamide-CsCl plates (Penttild, M., et al., Gene 61:155-164 (1987) before transferring them to PD slants.
RNA isolation, T. reesei. VTT-D-79125 was cultured in a fermentor in xylanase-inducing medium for three days. The mycelia were harvested, frozen and ground into a fine powder under liquid nitrogen.
Total RNA and, subsequently, mRNA were isolated using Proteinase K
digestion, phenol extractions and oligo dT-cellulose purification, as described by Bartels and Thompson, Nucleic Acid Res. 11:2961-2978 (1983). The mRNA obtained was size-fractionated using DMSO-sucrose gradient centrifugation (Boedtker, H., et al., Biochemistry 15:4765-4770 (1976)).
Cloning of xylanase H cDNA. The first strand of cDNA was prepared from 1 g of mRNA by using the cDNA synthesis kit (Boehringer Mannheim), replacing the oligo dT-primer with a hybrid dTWadapter primer (5' GAC-TCG-AGA-ATT-CAT-CGA-dT17 3') SUBSTtTUTE SHEET

WO 93/24621 2136350 PCr/F193/00221 [SEQ ID No.:5:]. This cDNA was used as a template for polymerase chain reaction (PCR) amplification (Frohman, M.A., "Race: Rapid Amplification of cDNA Ends," in PCR Protocols, Innis, M.A., et al., eds., Academic Press Inc., San Diego, CA, pp. 28-38 (1990)) directed by a gene specific primer (sense 5' GG(A/C/G)-TGG-CA(A/G)-CCN-GGN-ACN-AA 3') [SEQ ID No.:6:] deduced from a peptide sequence and by the adapter primer (5' GAC-TCG-AGA-ATT-CAT-CGA 3') [SEQ ID
No.:7:]. The 100 l PCR reaction mixture contained 5 l of 1:25 diluted cDNA, 100 pmol of each primer, 5 M dNTP, 1 x PCR buffer and 1.5 units of Taq DNA polymerase (Boehringer Mannheim). Amplification in a programmable thermal controller (M.J. Research Inc.) comprised 30 cycles at 95 C for 1 min, at 55 C for 1 min and at 72 C for 2 min. After the last cycle, the elongation period was extended to 10 min. The PCR fragments obtained were cloned using a TA Cloning kit (Invitrogen) according to the manufacturer's instructions and verified by sequencing.
Isolation of the x1n2 gene. A genomic cosmid library (Suominen et al., MGG, In press) 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 supplier's instructions (Boehringer Mannheim, Germany).
Nucleotide sequencing. The templates for nucleotide sequencing were generated by unidirectional deletions according to the manufacturer's instructions for the pBluescript Exo/Mung DNA sequencing system (Stratagene). DNA was sequenced in both directions by using ABI
(Applied Biosystems, Foster City, USA) kits based on fluorescence-labelled T7 and T3 primers and a Taq cycle sequencing method according to the supplier's instructions. Sequencing reactions were analysed on an ABI 373A Sequencer.
Enzyme and protein assays. Enzymes were assayed from the culture supernatants after removing the mycelia. Xylanase activity was measured, using birch xylan (Roth 7500) as substrate, by the method SUBSTITUTE SHEET

described by Bailey, M.J., et al., J. Biotechnol. 23:257-270 (1992).
Production of cellobiohydrolase I(CBHI) protein was detected by Western blot or dot blot methods using the CBHI specific monoclonal antibodies CI-89 or CI-261 (Aho, S., et aL, Eur. J. Biochem.. 200:643-649 (1991)).

Results Peptide sequences Several tryptic peptides from the purified endoxylanase II were obtained and directly sequenced (see Figure 3 for peptide sequences).
However, no amino-terminal sequence for the native protein was detected, suggesting that it was blocked. After treatment with pyroglutamate aminopeptidase, an amino-terminal sequence of (Q)-X-Ile-Gln-Pro-Gly-Thr-Gly-Tyr-Asn [SEQ ID No.:8:] was obtained. The first amino acid (X) of the pyroglutamate aminopeptidase treated sample could not be deterinined without ambiguity because of confounding peaks in the HPLC
chromatogram.

Cloning of T. reesei xln2 cDNA
The accumulation of xylanase II specific mRNA in T. reesei cultures was determined by Northern hybridization using a nucleotide oligomer deduced from the peptide (marked with a double underline in Figure 3) sequence as a probe. The results indicated that the xylanase II nmRNA was most abundant in mycelia grown for three days and that the size of the xylanase II specific mRNA was about 0.7 kb (data not shown).
The xylanase II specific oligomer (see above) primer and an unspecific dT-adapter primer were used to amplify the xylanase sequence from the first strand of cDNA. The amino acid sequence deduced from the nucleotide sequence of the subclone pALK564, containing the longest of the fragments synthesized in a PCR reaction, contained several of the SUBSTITUTE SHEET

xylanase II peptide sequences obtained by direct sequencing. This confumed that the PCR clone coded for xylanase II.

Isolation and nucleotide sequence of the xln2 gene of T. reesei Four clones were isolated from a genomic cosmid library of T. reesei 5 through hybridization with the insert of pALK564 (insert= PCR-synthesized xln2 cDNA). A subclone, pALK475, containing a hybridizing 5.7 kb KpnI fragment from the cosmid clone in the pUC19 vector was analysed by restriction mapping (Figure 2). The hybridizing 2.3 kb HindIII, 1.5 kb HindIIl/XhoI and 1 kb Xhol fragments from pALK475 were 10 subcloned and partially sequenced to reveal the xln2 gene.
The nucleotide and deduced amino acid sequences of the xln2 gene are shown in Figure 3. Sequence resembling the TATA box was found in the DNA at a distance of -140 nucleotides from the ATG, (Figure 3). The primary translation start site (ATG), flanked by the highly conserved 15 consensus sequence 5' CA C/A A/C ATG 3' found in filamentous fungi and other eukaryotes (Ballance, J.D., "Transformation systems for filamentous fungi and an overview of fungal gene structure," in Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi, Leon and Berka, eds., Marcel Dekker Inc., New York, pp. 1-29 (1991)), is 20 99 nucleotides upstream of the codon for the determined N-terminal amino acid of xylanase II. The xln2 gene codes for a protein of 223 amino acids.
In this protein, the N-terminal, obtained by direct peptide sequencing, is preceded by 33 amino acids. This preprosequence contains a putative signal peptidase cleavage site (von Heijne, G., Nucl. Acid Res. 14:4683-25 4690 (1986)) between Alal9 and A1a20. Comparison of the genomic sequence with the cDNA sequences revealed one intron of 108 nucleotides.
This indicates that the mature xylanase II protein consists of 190 amino acids and has a calculated molecular weight of 20.8 kDa. This is in good agreement with the 20 kDa obtained for purified xylanase II (Tenkanen, 30 M., et al., Enzyme Microb. Technol. 14:566-574 (1992)). Sequence SUBSTITUTE SHEET

analysis also revealed two putative targets (Asn-X-Ser/Thr where X is not Pro; Gavel and von Heijne, Protein Engineering 3:433-442 (1990)) for N-glycosylation (Asn38 and Asn6l of the mature protein).

Construction of pAL%476 Construction of the plasmid pALK476 is shown in Figure 4. This plasmid is useful for targetting the x1n2 gene to the cbhl locus.
Expression of the x1n2 gene is under the control of its native promoter.
The 5.0 kb SmaI fragment from the plasmid pALK475 (Figure 2), containing the x1n2 gene and promoter (2.3 kb of the gene's upstream area), was ligated to Spel site (filled in with Klenow) of the plasmid pALK425.

Enhanced expression of xln2 in T. reesei To enhance xylanase expression in T. reesei, three strains, VTT-D-79125, ALK02221 and ALK02721, differing in their enzyme production profiles, were transformed with the EcoRI fragment from the plasmid pALK476 (Figure 4). The expression cassette containing the xln2 gene with its own promoter (Figure 5) was targeted to the cbhl locus, using the cbhl flanking regions.
Transformants, 57 from T. reesei VTT-D-79125, 42 from ALK02221 and 53 from ALK02721 transformations, were purified and grown in shake flask cultures in xylanase-inducing conditions. Xylanase production was measured as birch xylan degrading activity in the culture supernatants. The transformants were tested for CBHI protein production to determine the targeting frequencies to the cbhl locus and to distinguish between transformants in which the x1n2 expression cassette was located at the cbhl locus and those in which it was integrated elsewhere.
Replacement of the cbhl locus by the x1n2 expression cassette results in a SUBSTITUTE SHEET

WO 93/24621 2436350 P(.'I'/F193/00221 CBHI negative (CBHI ) phenotype which was detected in Western blots and dot blots by using a CBHI specific monoclonal antibody. Targeting efficiency to the cbhl locus, determined as the CBHI- phenotype was high in each case: 67%, 62% and 58% in transformants of VTT-D-79125, ALK02221 and ALK02721, respectively.
Figure 6 shows the relative xylanase activities of the transformants grouped according to their CBHI' phenotype. Increase in the xln2 copy number resulted in increased production of xylanase activity in both CBHI-and CBHI+ transformants of each strain. The best transformants yielded about twofold (T. reesei ALK02221 and ALK02721) to 4.5-fold (T. reesei VTT-D-79125) xylanase activity compared with the respective host strains.
The highest activities (nkat/ml) were obtained by using T. reesei ALK02721 as a host. The best transformants of T. reesei ALK02221 produced less than half the activity obtained with the best transformants of the two other strains. There was little difference in xylanase activity between the CBHI+ and CBHI- phenotype in the transformants of T. reesei ALK02221 and ALK02721 (Figure 6). Among the T. reesei VTT-D-79125 transformants, those with the CBHI+ phenotype were on average better producers of xylanase.
To evaluate the effect of the genetic background on the expression of the xln2 gene in the three hosts, the relative increase in xylanase activity in the CBHI- transformants was calculated. The CBHI- transformants were used to eliminate any effects of differences in the site of integration on gene expression. Within each host strain, most of the CBHI- transformants (42-86%, Table 1) exhibited similar levels of increase in xylanase activity.
Most of the transformants excluded from this comparison produced higher xylanase activities. From this we conclude that it is likely that the transformants shown in Table 1 have one extra copy of xln2 at the cbhl locus, in addition to the natural xln2. The average xylanase activity of these transformants and the average increase compared with the host strain are shown in Table 1. The average increase ranged, depending on the host SUBSTITUTE SHEET

strain, from about 600 nkat/ml (ALK02221) to about 2700 nkat/ml (ALK02721).

Table 1 Relative Increase in Xylanase Production in a Chosen Set of CBHI- transformants Average Activity Average Increase in Strain (nkat/ml) activity (nkat/ml) VTT-D-79125 3700 (+/- 20%) transformants (50%) 5100 (+/- 20%) 1400 ALK02221 3800 (+/-10%) transfornnants (85 %) 4400 (+/- 15%) 600 ALK02721 10200(+/- 15%) transforniants (42%) 12900(+/- 10%) 2700 * The proportion (%) of CBHI' transformants apparently having an extra copy of xln2 is shown in the parentheses.

Discussion There are several reports on molecular cloning of bacterial xylanases (e.g., Ghangas, G.S., et al., J. Bacteriol. 171:2963-2969 (1989); Lin and Thomson, Mol. Gen. Genet. 228:55-61 (1991); Shareck, F., et al., Gene 107:75-82 (1991); Whitehead and Lee, Curr. Microbiol. 23:15-19 (1991)).
Recently, reports on the cloning of xylanases of filamentous fungi have also been published, including those of Aspergillus tubigensis (van dert Broeck, N., et al., "Cloning and Expression of Xylanase Genes from Fungal Origin," EP 0 463 706 Al (1992)), A. niger var. awamori (Maat, J., et al., "Xylanases and Their Application in Bakery," in Xylans and Xylanases, Visser, J., et al., eds., Elsevier Science, Amsterdam, pp. 349-360 (1992)), A. kawachii (Ito, K., et al., Biosci. Biotechnol. Biochem.
56:906-912 (1992)) and T. reesei (Suominen, P., et al., "Genetic Engineering of Trichoderma reesei to Produce Suitable Enzyme Combinations for Applications in the Pulp and Paper Industry," in SUB8TITUTE SHEEIr WO 93/24621 2136350 P(.'I'/F193/00221 ~-Biotechnology in Pulp and Paper Industry, Kuwahara and Shimada, eds., Uni Publishers Co., Ltd., Tokyo, Japan, pp. 439-445 (1992); T6rr6nen, A., et al., BiolTechnology 10:1461-1465 (1992)). The genes for T. reesei xylanase II (pI 9) cloned of the wild-type strain QM6a by us (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 Pulp and Paper Industry, Kuwahara and Shimada, eds., Uni Publishers Co., Ltd., Tokyo, Japan, pp. 439-445 (1992)) and of the mutant strain RutC-30 by TarrSnen, A., et al., Bio/Technology 10:1461-1465 (1992) were not completely identical. The main differences were between the sequences of pre-propeptides thus suggesting differences in the signal processing.
According to our results, the x1n2 gene of T. reesei contains one long intron of 108 bp. The introns in filamentous fungi are usually smaller, around 50 bp in length, but, as shown by Vanhanen et al., Curr.
Genet. 12:181-186 (1989) for the gene of 3-phosphoglycerate kinase in T. reesei, they can also be considerably longer. The fairly long pre-prosequence (33 amino acids) of the x1n2 gene contains one primary cleavage site for the signal peptidase. This is compatible with a signal sequence of 19 amino acids followed by a propeptide of 14 amino acids, which may be post-translationally cleaved from the mature protein.
T6rr6nen, A., et al., Bio/Technology 10:1461-1465 (1992) suggested that the signal pre-propeptide of the xylanase II (pI 9) of T. reesei RutC-30 consists of 32 amino acids and has two putative signal peptidase cleavage sites very close to the translation start site (5 and 11 amino acids, respectively). This would result in rather short signal sequences. Two-step protein processing, similar to what is proposed here for the xylanase II, has been shown to occur with A. niger glucoamylase (Innis, M.A., et al., Science 228:21-26 (1985)) and suggested to occur with T. reesei cellobiohydrolase II (Teeri, T., et al., Gene 51:43-52 (1987)).
Overall structure similar to T reesei xln2 is found in the A. niger xylanase SUBSTITUTE SHEET

gene (van den Broeck, N., et al., "Cloning and Expression of Xylanase Genes from Fungal Origin," EP 0 463 706 Al (1992)) but not, for instance, in the A. kawachii xylanase A gene, which contains nine introns and codes for a larger (32.7 kDa) protein (Ito, K., et al., Biosci.
5 Biotechnol. Biochem. 56:906-912 (1992)).
Production of xylanase could be increased in three T. reesei strains by increasing the copy number of the x1n2 gene. Of these strains, T. reesei VTT-D-79125 is a high cellulase-producing mutant and ALK02721 was used as a host because of its pre-existent high xylanase production. We 10 also wanted to produce xylanase in the low-protease T. reesei ALK02221, both to obtain a xylanase product with a low protease background and to increase the relative amount of xylanase in the culture medium.
The highest increase in xylanase activity ranged from about twofold to over fourfold compared with the respective host strain. Xylanase 15 activities of the transformants of the low-protease strain ALK02221 and those of the genetically engineered host ALK02721 were independent of whether the x1n2 gene was integrated into the cbhl locus or elsewhere in the genome (CBHI' phenotype). For T. reesei VTT-D-79125 transformants, integration of the x1n2 gene elsewhere (CBHI+ phenotype) 20 than into the cbhl locus seemed to result in higher enzyme yield. These observations are somewhat contradictory to those of Harkki, A., et al., Enzyme Microb. Technol. 13:227-23 3(1991), who suggested that for optimal expression the expression cassette would require its integration into the cbhl locus. It should be noted, however, that in the present study 25 _the xln2 promoter was used for expression instead of the cbhl promoter as done by Harkki, A., et al., Enzyme Microb. Technol. 13:227-233 (1991) with egll cDNA. This indicates that the cbhl locus may not be the best environment for expression under the xln2 promoter.
The genetic background seemed to have a significant effect on xln2 30 expression, despite the fact that the strains T. reesei ALK02221 and ALK02721 originate from VTT-D-79125. The increase of xylanase SUBSTITUTE SHEET

WO 93/24621 ~13 6 35 0:-? PCT/F193/00221 production was highest in the transformants of the strain T. reesei ALK02721 (2700 nkat/ml) which also had the highest xylanase activity.
The expression of one additional copy of xln2 varied over fourfold depending on the host (Table 1).

Exaynple 2 Increased production of XYLII: x1n2 gene fused to the cbhl promoter Construction of p.ALK174 and transformation of three Trichoderma reesei strains Construction of pALK174 is shown in Figure 7. First, a 674 bp PCR fragment containing an exact fusion of the cbhl promoter to the xln2 signal sequence (and xln2 gene to the internal XhoI site) was synthesizeu Oby using plasmid pALK475 (Figure 2) as a template. The olig-,nucleotides used were as shown below: 5'-primer contained the end of the cbhl promoter and the sequence of the beginning of the putative xln2 signal sequence, 3'-primer had sequence from ihe x1n2 gene including the gene's internal Xhol site (see the pALK476 map, Figure 5 or the x1n2 sequence).

S'-primer (39-mer) [SEQ ID No.:11:J:

5'- C AAC CGC GGA CTG CGC ATC ATG GTC TCC TTC ACC TCC
CT
SacII beginning of the putative end of the cbhl promoter x1n2 signal sequence SUBSTITUTE SHEET

~136370 3'-primer (26-mer) [SEQ ID No.:12:J:

5'-GGG AGC CGC TCG AGC GGT GGT TGC GG
Xhol x1n2 sequence The 100 l PCR reaction contained in PCR buffer (10 mM Tris pH 8.3, 50 mM KCI, 1.5 mM MgC12, 0.01% gelatin, w/v) 50 pmol of each primer, 10 ng of the template DNA, 0.2 mM dNTP and 2U of Taq-polymerase (Boehringer Mannheim). The reaction conditions were: denaturation 1 min. at 95 C, annealing 1 minute at 60 C, extension 2 minutes at 72 C for 30 cycles, fmal extension was 9 minutes.
The PCR fragment was purified by using the Mermaid kit (BIO
101 Inc., La Jolla, California, USA). After treating it with the T4 DNA
polymerase, it was cut with SacII for fusion to the cbhl promoter.
The plasmid pAMH110, containing the cbhl promoter, was cut with NdeI (filled in by Klenow) and SacII. The PCR fragment described above was ligated to the digested pAMH110 (pALK174X). The fusions and the sequence synthesized by PCR were ensured by sequencing. The plasmid pALK174 was constructed by replacing the x1n2 promoter in pALK476 by the cbhl promoter: the 2.9 kb KpnI XhoI fragment from the plasmid pALK174X, containing the cbhl promoter fused to the xln2 gene and x1n2 sequence to the internal XhoI, was ligated to the isolated vector containing fragment of pALK476, after cutting pALK476 with Kpnl and Xhol.
The plasmid pALK174 contains, in addition to the amdS and cbhl 3'-areas as in pALK476 (Figure 4), the x1n2 gene, fused to the cbhl promoter and 1.9 kb terminator (3'-area) of the x1n2 gene.
The 9.7 kb EcoRl fragment (expression cassette free from the vector sequences) was used to transform three Trich'oderma strains, VTT-D-79125, ALK02221 and ALK02721. The strains, transformation method and methods for purification, analyzing and growing the transformants were as described above and in Example 6.

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õ~..

2.2 Production of xylanase by the pAL%174 transformants VTT-D-79125, ALK02221 and ALK02721 transformants, 39, 41 and 50, respectively, were purified and grown to measure the xylanase activities produced. The targeting frequencies to the cbhl locus were, measured by using dot blot method and a CBHI specific monoclonal antibody (as in Example 4) 82, 46 and 84%, respectively. The production of xylanase activity, shown as relative activity compared to the activity produced by VTT-D-79125, of the CBHI+ and CBHI- transformants is shown in the Figure 8. The columns show the mean values and the range of production levels among the transformants of each host strain. One flask of each transformant was grown. The xylanase activity was determined from the culture supernatants as described in Example 3 (cloning of xlnl ).
High production of xylanase was obtained in transformants of all the three Trichoderma strains. Tlie best VTT-D-79125 and ALK02221 transformants produced about 10 times the amount of xylanase activity compared to their host strains. The best ALK02721 transformants produced about the same amount of xylanase activity as the best VTT-D-79125 and ALKO2221 transformants and the increase compared to the host was over three fold. In average, the CBHI- transformants were better producers than those with the CBH+ phenotype. Naturally, in the CBHI-transformants the cellulolytic activity has decreased because of the deletion of the cbhl gene.
The production levels of xylanase, when pALK174 construction was used, were higher compared to those obtained by using pALK476 (xln2 under the control of its own promoter, see Example 1).

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WO 93/24621 PCT/F193/0022' Example 3 Structure of the Endoxylanase I, xlnl, Gene of Trichoderma reesei In this example, the cloning of the T. reesei gene xlnl which codes for the low pl (5.5) endoxylanase I is presented.

Materials and methods Bacterial strains and plasmids The plasmids used in this study were pBluecscript (Stratagene, San Diego, CA, USA) and pCR1000 (Invitrogen, San Diego, CA, USA). The plasmids were propagated in Escherichia coli strain XLI-Blue (Bullock, W.O. et al., Bfo/Techniques 5:376-378 (1987)) or in E. coli INV1aF
(Invitrogen). E. coli cultures were grown at 37 C overnight in L-broth (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) (1982)) supplemented with ampicillin (50 g/ml) as needed.

Peptide digestion and amino acid sequencing Purified xylanase I (Tenkanen, M. et al., Enzyme Microb.
Technol. 14:566-574 (1992)) from T. reesei (VTT-D-79125; Baily, M.J., Nevalainen, K.H.M., Enzyme Microb. Technol. 3:153-157 (1981)) was digested with 2% (w/w) trypsin (TPCK-treated, Sigma Chemical Company, St. Louis, MO, USA) in 1% (w/v) ammonium bicarbonate at 37 C for 2.5 h. After addition of 3% (w/w) trypsin to the sample, incubation was continued over night (13 h). The peptides were separated using high-performance liquid chromatography (HPLC) with a Rexchrom Prep-5/300 ODS reverse-phase column. Elution was performed at the rate of 1 ml/min in a linear solvent gradient running from 5% (v/v) acetonitrile (ACN) containing 0.1 % trifluoroacetic acid (TFA) to 60% (v/v) ACN
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2136j5o containing 0.06% TFA in 60 min at 24 C. Absorbance at 218 nm was measured. Amino terminals of the protein and the peptides were sequenced by degrading them in a gas-pulsed-liquid-phase sequencer (Kalkkinen, N., and Tilgmann, C., J. Prot. Chem. 7:242-243 (1988)). The 5 released PTH-amino acids were analysed on-line using narrow-bore reverse-phase HPLC.

DNA manipulation and transformations Standard DNA methods described by Maniatis et al. (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor 10 Laboratory, Cold Spring Harbor, New York) (1982)) were used for the DNA constructions. 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)).

15 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 hereinfor the isolation of the xln2 gene). The mycelia were ground into a fine powder under liquid nitrogen. Total RNA and mRNA were isolated as 20 described by Bartels and Thompson (Bartels, D., and Thompson, RD., Nucleic Acid Res. 11:2961-2978 (1983)) and size-fractionated using DMSO-sucrose gradient centrifugation (Boedtker, H. et al., Biochemistry 15:4765-4770 (1976)).

Cloning of xylanase I cDNA
25 The synthesis of the first strand of cDNA and the subsequent PCR
amplifications were performed as described hereir. for x1n2 except that here an oligomer (5' AA(T/C)-TA(T/C)-GA(T/C)-CA(G/A)-AA(T/C)-TA(T/C)-GA 3') [SEQ ID No.:9:] deduced from the N-terminal sequence SUBSTITUTE SHEET

of the xylanase I protein was used as the gene-specific primer. The 0.6 kb PCR fragment obtained was subcloned into the pCR1000 vector and sequenced.

Isolation of xlnl gene A T. reesei (QM6a, Mandels, M., Reese, E.T., J. Bacteriol. 73:269-280 (1957)) genomic cosmid library was screened with a digoxigenin-labelled PCR fragment of xylanase I cDNA (see Example 1) according to the supplier's instructions (Boehringer Mannheim, Mannheim, Germany). One positive cosmid clone was obtained. A restriction map of the xlnl area of the cosmid was prepared, and a 2.3 kb EcoR1 fragment was subcloned into pBluescript (pALK572) for sequencing.

Nucleotide sequencing The templates for nucleotide sequencing were generated by unidirectional deletions according to the manufacturer's instructions for the pBluescript Exo/Mung DNA sequencing system (Stratagene). DNA was sequenced in both directions by using ABI (Applied Biosystems, Foster City, USA) kits based on fluoresence-labelled T7 and T3 primers and a Taq cycle sequencing method according to the supplier's instructions. Sequencing reactions were analysed on an ABI 373A Sequencer.

Results Cloning of T. reesei xlnl cDNA
The amino-terminal sequence of purified xylanase I was found to be Ala-Ser-Ile-Asn-Tyr-Asp-Gln-Asn-Tyr-Gln-Thr-Gly-Gly-Gln- V al- 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).

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Northern hybridization using the oligomer deduced from the xylanase I N-terminal peptide sequence (see above) as a probe indicated that xylanase I
mRNA was approximately 0.7 kb in size.
PCR amplification of the first strand of cDNA resulted in a fragment of 0.7 kb. The nucleotide sequence of this DNA when translated into a protein contained all the xylanase I peptide sequences, including the N-terminal (see Figure 10).

Isolation and nucleotide sequence of the T. reesei xlnl gene One positive clone was isolated from a genomic cosmid library of T.
reesei. The gene for xylanase I(xlnl ) was found in a 2.3 kb EcoRI
fragment of the cosmid (Figure 9).
The nucleotide sequence of the xlnl gene was determined and is presented in Figure 10 together with the deduced amino acid sequence. A TATA
box was found approximately 90 nt upstream of the putative translation start site. Of the three different putative translation initiation sites preceding the N-terminus of the mature protein, only one was in an environment resembling the highly conserved consensus sequence 5' CA-C/A-A/C-ATG 3' for the translation start site among filamentous fungi (Ballance, J.D., in Leone, S.A., Berka, RM., eds., Molecular Industrial Mycology. Systems and applications for filamentous fungi (Marcel Dekker, Inc., New York), pp. 1-29 (1991)). Thus the gene codes for a protein of 229 amino acids. In this protein, the N-terminal (obtained by direct peptide sequencing, see above) is preceded by a 51 amino acid long signal propeptide containing a primary signal sequence cleavage site (von Heijne, Nucleic Acids Res. 14:4683-4690 (1986)) between the amino acids Ala19 and Met2O (Figure 10). Comparison of the genomic sequence with the cDNA sequence revealed one intron of 62 bp. On the basis of these data, it can be concluded that the mature xylanase I is a protein of 178 amino acids with a calculated molecular weight of 19.1 kDa. This is in good agreement with the 19 kDa obtained for purified xylanase I from T. reesei SUBSTITUTE SHEET

(Tenkanen, M. et al., Enzyme Microb. Technol. 14:566-574 (1992)).
Unlike the gene of xylanase II (see herein), there is no sequence (Asn-X-Ser/Thr where X is not Pro; Gavel, Y., von Heijne, G., Protein Engineering 3:433-442 (1990)) for N-glycosylation.

Comparison of T. reesei xylanase I with other xylanases Both of the proposed catalytic glutamic acids of xylanases (Katsube, Y.
et al., Proc. 2nd Int. Conference Protein Engineering (Japan Scientific Societies Press, Tokyo), pp. 91-96 (1990); Wakarchuk, W. et al., in Visser J. et al., eds., Xylans and Xylanases (Elsevier Science, Amsterdam), pp.
439-442 (1991)) were found in xylanase I by sequence alignment, corresponding to Glu75 and Glu164 of the mature enzyme (Figure 10).
Table 2 presents a sequence alignment comparison showing the percent identity between different xylanases. The upper right half of the matrix shows the results of an alignment of complete mature sequences, and the lower left half of the sequences between the proposed active site glutamic acids of xylanases.

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WO 93/24621 Z1363j U PCT/F193/00221 Table 2 Identity % of sequence of alignments between different xylanases expressed as a matrix. Upper right half: mature enzyme sequence alignments.
Lower left half: sequence alignments between the two proposed active site glutamic acids.

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. 149-154 (1992a)); TH, T. harzianum (Yaguchi, M.
et al., in Visser, J. et al., eds., Xylans and Xylanases (Elsevier Science, Amsterdam), pp. 435-438 (1992b)); AT, A. tubigensis (van den Broeck, H.
et al., "Cloning and expression of xylanase genes from fungal origin," EP 0 463 706 Al (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), the first 190 amino acids were aligned); BC, Bacillus circulans (Yang, R. C. A. et al., Nucleic Acids Res. 16:7187 (1988)); BP, P. pumilus (Fukasaki, E. et al., FEBSLett. 171:197-201 (1984)); BS, B. subtilis (Paice, M.G. et al., Arch.
Microbiol 144:201-206 (1986)); CA, Clostridium acetobutylicum (Zappe H.
et al., Nucleic Acids Res. 18:2179 (1990)); SS, Streptonryces sp. 36a (Nagashima, M. et al., Trends Actinomycetologia:91-96 (1989)).

TI TII TV TH AT SC AK BC BP BS CA SS

It can be seen that T. reeset xylanase II (pI 9.0) is almost identical with the T. viride and T. harzianum small, high pI xylanases (Yaguchi, M.
et al., in Visser, J. et al., eds., Xylans and Xylanases (Elsevier Science, SUBSTlTUTE SHEET

WO 93/24621 2 1 3 635 ~ PCT/FI93/00221 60 Amsterdam), pp. 149-154 (1992); Yaguchi, M. et al., in Visser, J. et aL, eds., Xylans and Xylanases (Elsevier Science, Amsterdam), pp. 435-438 (1992)). Alignment of the region between the two active site glutamic acids resulted in most cases in a higher identity than when complete mature sequences where aligned.

Discussion 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 19 kDa and 20 kDa, respectively (Tenkanen, M. et al., Enzyme Microb. Technol.
14:566-574 (1992)). The corresponding genes xlnl and xln2 have been cloned and described herein. In overall structure the xlnl and xln2 genes are very similar; they are about the same size and both contain only one intron and a long pre-propeptide. However, these two xylanases only show a 54 % identity at the DNA level and a 52 % identity at the amino acid level. Thus, they are clearly different not only in primary structure but also in enzymatic properties such as tolerance of different pH values or temperatures and kinetic parameters as shown by Tenkanen et al.
(Tenkanen, M. et al., Enzyme Microb. Technol. 14:566-574 (1992)).
Based on hydrophobic cluster analysis, xylanases have been divided into two subfamilies, F and G (Henrissat, B. et aL, Gene 81:83-95 (1989)).
Subfamily F comprises the high-MW xylanases whereas the low-MW
xylanases belong to subfamily G. Henrissat (Henrissat B., in Visser, J.
et al., eds., Xylans and Xylanases, Proc. Int. Symp. Wageningen (Elsevier, Amsterdam), pp. 97-110 (1991)) suggests that the xylanases of these two subfamilies undergo different folding. The high identity of T. reesei xylanases I and II to all other xylanases compared (Table 2), except the A.
kawachii xylase A, suggests that they too, like the other low-MW
xylanases, belong to subfamily G. In the case of the A. kawachii xylanase A (Ito, K. et al., Biosci. Biotec. Biochem. 56:906-912 (1992)), an identity SUBSTITUTE SHEET

WO 93/24621 2136350 PCr/F193/00221 of only about 20% to the T. reesei xylanases was found. This xylanase has a higher MW of 32.7 kDa, and it has been proposed to belong to subfamily F (Ito, K. et al., Biosci. Biotec. Biochem. 56:906-912 (1992)).

Example 4 Increased production of XYLI by Trichoderma transformed with the construction containing the xlnl gene fused to the cbhl promoter (pAL%807) Construction of the plasmid pAL%807 and transformation of the Trichoderma strain ALK02221 The plasmid pALK807 was constructed by using the same strategy as in construction pALKl74 (Figure 11). The 98 bp PCR fragment containing the fusion of the cbhl promoter to the putative xlnl signal sequence and xlnl sequence to the internal XhoI site (see the pALK572 map in Figure 2 or the xlnl sequence) was made by PCR using the following oligonucleotides:

5'-primer (39-mer) [SEQ ID No.:13:1:

5'- C AAC CGC GGA CTG CGC ATC ATG GTT GCC TTT TCC AGC CT
SaciI beginning of the putative end of the cbhl promoter xlnl signal sequence 3' primer (30-mer) [SEQ ID No.:14:]:

5'-CAG GCT CGA GGC CTG TGG GCA TCG CCA GAG
XhoI
x1nl. sequence SUBSTITUTE SHEET

WO 93/24621 PCf/F193/00221 Plasmid pALK572 containing the xlnl gene was used as a template for the PCR reaction. The reaction and purification of the product were done like in constructing pALK174. To obtain pALK805, the purified and SacII-XhoI digested PCR fragment was ligated to the SacII-Xhol cut plasmid pALK486 containing the cbhl promoter. To construct pALK806, the xlnl sequence downstream from the XhoI site was isolated from the plasmid pALK572 by PstI (filled by T4 DNA polymerase) -XhoI digestion and the fragment was ligated to the BamHI (filled in with Klenow) - XhoI digested pALK805. pALK805 was obtained by ligating the EcoRI-SpeI fragment (filled in by Klenow) from pALK424, containing the amdS gene and the chbl 3'-flanking region (as in pALK174), into the EcoRV cut pALK806.
T. reesei strain ALK02221 was transformed with the 8.2 kb NotI
fragment from the plasmid pALK807.

Production of xylanase by the pALK807 transformants, Total of 51 pALK807 transformants were purified, analyzed and grown as in Example 2. Targeting efficiency to the cbhl locus was 35%.
The xylanase activity was measured as in Example 2, but at the pH 4.3 which is at the optimum pH range for the XYLI activity (Tenkanen et al., Enzyme Microb. Technol. 14:566-574 (1992)). The results, as an increase in xylanase activity produced compared to that of the host strain ALK02221 are shown in the Table 3. Thirty best xylanase producers obtained are included. One bottle of each transformant was grown.
The best transformants produced over 10 fold the amount of xylanase activity compared to the parent. Differing from the XYLII
(pALK174) transformants, the best xylanase producers were CBHI+.
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Table 3 x transformant CBHI+/- compared to number (dot blot) ALK02221 (control) (+) 20 (+) 11.8 5 (+) 11.7 18 (+) 10.4 4 (+) 9.8 33 (+) 9.5 32 (-) 9.2 21 (+) 8.9 3 (+) 8.9 46 (+) 8.8 40 (+) 8.2 1 (+) 8.2 43 (+) 8.0 10 (+) 8.0 19 (+) 8.0 8 (+) 7.9 51 (+) 7.8 39 (+) 7.6 12 (-) 7.5 7 (-) 7.2 36 (+) 6.9 41 (-) 6.8 (+) 6.8 49 (-) 6.6 29 (-) 6.6 30 24 (+) 6.3 50 (-) 5.9 14 (-) 5.9 26 (+) 5.8 15 (-) 5.8 35 52 (+) 5.1 SUBSTITUTE SHEET

Example 5 A. Inactivation of the Major Cellulase cbhl Gene The cbhl gene which encodes the major cellulase in T. reesei was inactivated by homologous recombination with plasmid pMS4 containing a 0.8 kb internal fragment of the cbhl cDNA bearing a frame shift mutation.
The pMS4 plasmid was prepared on the following way: the plasmid pTTcOl (Teeri et al., Anal. Biochem. 164:60-67 (1987); Penttila et al., Gene 63:103-112 (1988)), which contains the full length cDNA clone of the cbhl gene in the pUC8 vector (Vieira and Messing, Gene 19:259-268 (1982)), was digested with Bgll cutting in the signal sequence (Shoemaker et al., BiolTechnology 1:691-696 (1983)) and with BgIII. The resulting 0.8 kb DNA fragment bearing the 5' region of the cbhl cDNA was made blunt-ended with S, nuclease and was ligated to an EcoRl cut, blunt-ended pUC18 vector (Yanish-Perron et al., Gene 33:103-119 (1985)).
The clone obtained was cut in the middle of the cbhl fragment with EcoRI. The EcoRI generated termini were then filled in and back-ligated.
The resulting plasmid pMS4 thus contains a frameshift mutation in the middle of the truncated cbhl cDNA fragment.
T. reesei VTT-D-79125 (Bailey and Nevalainen, Enzyme Microb.
Technol. 3:153-157 (1981)) was cotransformed with pMS4 and p3SR2.
p3SR2 carries a 5 kb DNA fragment containing the A. nidulans amdS gene cloned into pBR322 (Kelly and Hynes, EMBO J. 4:475-479 (1985)).
Transformants were selected on the basis of the AmdS+ phenotype after which they were purified from conidia. About 600 clones from 200 independent transformants were then grown on microtiter plates and their cellulase phenotype was tested by the Ouchterlony immunodiffusion (Ouchterlony, Progr. Allergy 5:1-78 (1958)) using undiluted growth medium and the CBHI specific sheep antiserum.
A number of strains produced no detectable CBHI. The CBHI
negative character of one of these strains VTT-D-87312 was confirmed by SUBSTaTUTE aHEET

WO 93/24621 213635t1 PCr/FI93/00221 V

analyzing the growth medium in SDS-PAGE and in FPLC, in which no peak corresponding CBHI was seen (compare Figure 12 and Figure 12A).
The amount of total secreted protein of the CBHI negative strain was about half of that secreted by the T. reesei VTT-D-79125. The filter paper 5 degrading activity, FPU (Mandels et al.,"Measurement of Saccharifying Cellulase," in: Biotechnol. Bioeng. Symp. no. 6., p. 21-33, Gaden, E.L., Mandels, M.H., Reese, E.T., and Spano, L.A. (eds.), John Wiley and Sons, New York, 1976) activity detected in the culture supematant fraction of the strain VTT-D-87312 was significantly reduced and was about 20% of 10 normal. The lack of the major cellobiohydrolase which normally represents about 60% of the total secreted protein did not notably change the growth properties of the strain.

B. Deletion of the cbh2 Gen ti Its Promoter The cbh2 gene of Trichoderma replaced with the Aspergillus argB
15 gene as described below. Plasmid pAI.K99 was constructed to be the source of the transforming fragment (Figure 13). Plasmid pALK99 was constructed in the following way. The PvuH fragment (containing the multilinker) of the plasmid pUC19 was replaced by a new synthetic multilinker fragment containing recognition sites for the following 20 restriction enzymes: XhoI-StuI-SmaI XbaI-PvuII-SalI XhoI. The new plasmid was called pALK96. This plasmid was cut with Xbal and PvuH
and a 2.1kb XbaI-PvuII fragment from the 3' region of the cbh2 gene (see Figure 14) was ligated into it. The resulting plasmid was cut with PvuII
and HincII and ligated with the 3.4 kb PvuII-fragment from the 5' area of 25 the cbh2-gene (see Figure 14). Both the 3' and 5' fragments were originally from the X clone cbh2lambdal (Teeri et al., Gene 51:43-52 (1987)). The resulting plasmid was called pALK98. The Aspergillus nidulans argB gene (2.6kb Sali fragment) was then ligated between the 3' and 51 regions of the cbh2 gene into the unique PvuII site of plasmid SUBSTiTUTE SHEET

WO 93/24621 PCf/F793/00227 ...., pALK98. The resulting plasmid was called pALK99. Thus, the transforming fragment which is isolated from pALK99 as a XhoI fragment contains the Aspergillus argB gene as a 2.6 kb Sall fragment (Berse et al., Gene 25:109-117 (1983)) between 3.4kb (PvuII-PvuII fragment) of the 5' flanking region and 2.1kb (PvuII-Xbal fragment) of the 3' flanking region of the cbh2 gene (see Figure 14). T. reesei VTT-D-87305 ArgB- mutant strain (Penttila et al., 1987, Gene 61:155-164) was transformed with this fragment using selection for arginine prototropy. ArgB+ transformants were then screened for CBHII- phenotype by Western blotting using monoclonal antibody against CBHII. Replacement of the cbh2 locus by the transforming fragment in the CBHII-transformants was then confirmed by Southern blots. Strain ALKO 2564 is an example of this kind of "replacement" strain and thus does not contain the cbh2 gene any more.
With the method described above, the cbh2 gene of Trichoderma was replaced with the Aspergillus trpC gene.
The Aspergillus nidulans trpC gene (4.2 kb Xhol fragment blunt-ended with Klenow enzyme) was ligated between the 3'and 5'regions of the cbh2 gene into the unique PvulI site of the plasmid pALK98. The resulting plasmid was called pALK402. The transforming fragment which is isolated from pALK402 as XhoI fragment contains A. nidulans trpC gene as a 4.2 kb XhoI fragment (Yelton et al., PNAS, 91:1470-1474 (1984) between the 3.4 kb of the 5'flanking region and 2.1 kb of the 3'flanking region of the cbh2 gene. T. reesei ALK02319 trpC' mutant strain was transformed with this fragment using selection for tryptophane prototrophy. TrpC+
transformants were screened for CBHII- phenotype by Western blotting using monoclonal antibody against CBHII. Replacement of the cbh2 locus by the transforming fragment in the CBHII- transformants was confirmed by Southern blots. Strain ALK02721 is an example of this kind of "replacement" strain and thus does not contain the cbh2 gene any more.

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WO 93/24621 2136350 PC,'I'/F193/00221 Example 6 Construction of a CBHI Negative Trichoderma Strain Producing Elevated Amounts of EGI

The CBHI negative strain VTT-D-87312 described in Example 5A
was transformed with the plasmid pAMH 111 to enhance EGI expression in a CBHI negative background. The plasmid pAMH 111 was constructed using the general expression vector pAMH 110 (both of these plasmids are described in EP 244,234). pAMH 110 was built from pUC19 (Yanish Perron et al., Gene 33:103-119 (1985)). First the single Ndel site of pUC19 was destroyed by filling in the recessed ends with Kienow polymerase, and then the plasmid was digested with EcoRI and PstI and ligated to cbhl promoter and terminator fragments to make an expression cassette. The promoter fragment was a 2.6 kb EcoRI-PstI fragment from the plasmid pAMH 102 (Harkki et al., BiolTechnology 7:596-603 (1989)).
The terminator was a 0.75 kb AvalI fragment contained in a PstI fragment which also included an adaptor with the TAA stop codon in all three reading frames. pAMH 110 was then digested with SacII and Nde1 to remove a piece of DNA between the cbhl promoter and terminator, and the digested ends were made blunt-ended with S, nuclease and Klenow polymerase. The egll cDNA to be expressed was taken from the plasmid pTTc11 ((Teeri et al., Anal. Biochem. 164:60-67 (1987); Penttilli et al., Yeast 3:175 -185 (1987); as a 1.6 kb EcoRI-BamHI fragment, made blunt-ended with Klenow polymerase, and ligated into the expression cassette to give plasmid pAMH 111. Transformation was carried out as a cotransformation with pAMH111 and the plasmid pAN8-l (Mattern et. al., "Transformations of Aspergillus oryzae," In: Abstracts of the 19th Lunteren Lectures of Molecular Genetics of Yeasts and Filamentous Fungi and its Impact on Biotechnology, Lunteren, the Netherlands, p.34, (1987)) carrying the phleomycin resistance gene of Steptoallotheicus hindustanus under the SUBSTOTUTE SHEET

A. nidulans gpd promoter. Another marker must be used if, as in this example, the strain VTT-D-87312 was already AmdS+. Transformants were purified and tested for endoglucanase production in shake flasks cultures. In about 20 % of the transformants, the level of hydroxyethylcellulose (HEC) hydrolyzing activity was higher than in the recipient strain. The amount of EGI protein (Table 4) in the shake culture supernatant fraction was analyzed from three transformants showing high HEC activity. Southern blot analysis of these transformants showed that in the best endoglucanase producing clone (ALKO 2466) the expression cassette containing the egll cDNA between the cbhl promoter and terminator sequences was integrated in the chromosomal cbhl locus through the terminator sequences on the insert. The amount of secreted EGI protein in this transformant strain (ALKO 2466) was increased about four fold over that of the control (Table 4).

Table 4 Characterization of EGI production in T. reesei VTT-D-87312 (CBHI
negative transformant of VTT-D-79125) and in ALKO 2493, ALKO 2466 and ALKO 2494 which arise from VTT-D-87312 transformed with the plasmid pAMH111. TT-D-79125 is the untransformed high-cellulase producing T.
reesei mutant strain. All strains were grown in shake flasks as described in the Materials and Methods. The amount of EGI protein and total secreted protein were measured after 7 days cultivation as described in the Materials and Methods.
EG1 protein Total secreted protein % EGI
% of the total (mg/ml) (mg/ml) secreted protein VTT-D-87312 0.35 4.5 7.7 ALKO 2493 1.25 5.2 24.0 ALKO 2466 1.90 5.8 32.8 ALKO 2494 1.40 5.9 23.7 VTT-D-79125 0.75 10.6 7.1 SUBSTITUTE SHEET

Example 7 Simultaneous Inactivation of the cbhl Gene and Multiplication of the egll Copy Number The cbhl gene of T. reesei VTT-D-79125 was replaced with the Trichoderma egll cDNA and amdS gene. The egll cDNA was ligated between the promoter and terminator of cbhl gene. Plasmid pALK412 was constructed to be the source of the transforming fragment. The plasmid pALK412 was prepared as in Figure 15.
The plasmid p3SR2 which contains the Aspergillus nidulans amdS gene cloned into pBR322 (Kelly and Hynes, EMBO J. 4:475-479 (1985)) was digested with Sphl and with XbaI. The resulting 3.2 kb DNA fragment bearing the whole amdS gene was ligated to the Sphl and Xbal cut pUC 19 vector (Yanisch-Perron et al., Gene 33:103-119 (1985)). The resulting plasmid was called pALK410.
A DNA fragment containing 1.65 kb of the 3' region of the cbhl gene starting from the ScaI site in the coding region was isolated as a ScaI-BamHI fragment and blunt-ended with Klenow-enzyme. This fragment was ligated to the Xbal site (blunt-ended with Klenow enzyme) of the plasmid pALK410. In this case the 3' fragment was isolated from the plasmid pTTl l.
Plasmid pTTI l(Teeri et al., BiolTech 1:696-699 (1983)) contains 1.8 kb fragment of the cbhl region, 3' from the BamHI site in the coding region, cloned into the BamHI site of pBR322. The gene can also be isolated from other sources, for example, from ak clone 44A (Teeri et al., BiolTech 1:696-699).
The plasmid obtained was pALK41 1. It was digested with Scal and with Sphl. The 5.8 kb fragment was ligated to ScaI and Sphl cut plasmid pPLE3 (Nevalainen, et al., In: Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi, Leong et al., eds., pp. 129-148 (1990)) which contains egll cDNA between the promoter and terminator SUBSTITUTE SHEET

regions of cbhl gene cloned into pUC18 (Figure 16). The promoter and terminator regions are from the expression vector pAMH 110 (Nevalainen et al., In: Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi, Leong et al., eds., pp. 129-148 (1990)).
5 The resulting plasmid pALK412 was cut with EcoRI to remove the bacterial DNA. The 9.3 kb pALK412F fragment was also backligated to form plasmid pALK412L.
T. reesei VTT-D-79125 (Bailey and Nevalainen, Enzyme Microb.
Technol. 3:153-157 (1981)) was transformed with plasmid pALK412, with 10 pALK412F linear fragment, with backligated pALK412L and with pALK412F and pALK412L at the same time with a molar ratio of 5:1 respectively. Transformants were selected on the basis of the amdS+
phenotype and purified from conidia on selective medium containing acetamide as a sole nitrogen source.
15 Purified transformants were grown on microtiter plates and were screened for CBHI- phenotype by Western blotting using polyclonal antibody against CBHI protein. About one third of the pALK412F
transformants produced no detectable CBHI. There was one CBHI"
transformant among forty strains that had been transformed with the 20 plasmid pALK412.
CBHI' transformants were tested for endoglucanase production in shake flasks cultures. In all of these transformants the level of hydroxyethylcellulose (HEC) hydrolyzing activity was higher than in the recipient strain. The best transformants secreted 4-5 times the 25 endoglucanase activity of the recipient strain.
Southern blot analysis of the CBHI' transformants showed that their cbhl locus was replaced by the vector fragment. The chromosomal DNA
of the transformants was digested with Xhol and hybridized with the 0.5 kb fragment of the cbhl coding region probe.
30 The best endoglucanase producing strains had more than one copy of the vector fragment which carries the gene of interest inserted into the SUBSTITUTE SHEET

WO 93/24621 ~ ~ ~ ~ ~ ~ Q PCT/FI93/00221 Trichoderma genome.

Example 8 Construction of a cellobiohydrolase negative T. reesei strain The hypercellulolytic mutant VTT-D-79125 (Bailey, M.J. et al., Enzyme Microb. Technol. 3:153-157 (1982)) is a good cellulase producer, and also, has a markedly better viability and increased capability to produce secreted protein as a result of the mutagenesis program that created it. Thus, we wanted to make use of the high protein production capacity of this mutant to produce xylanases free of cellobiohydrolases to be used in pulp bleaching.
An UV-induced tryptophan auxotrophic mutant of VTT-D-79125 was transformed with a linear fi-< -ient of DNA (Figure 17), on which the cbh2 flanking regions were used to target the transforming fragment into the cbh2 locus. Purified TrpC+ transF rmants were grown on inicrotiter plates and were screened for CBHII" phenotype by Western blotting using monoclonal antibody against CBHII. Replacement of the cbh2 locus by the transforming fragment was confirmed by Southern blotting. One such cbh2 negative transformant was transformed with a second DNA fragment.
This time cbhl flanking regions were used in order to replace the wild type cbhl locus with the amdS marker for acetamidase. Purified amdS+
transformants were grown on microtiter plates and screened for CBHI' phenotype by Western blotting using polyclonal antibody against CBHI
protein. The replacement was again confirmed by Southern blotting. The resulting strain has neither the cbhl nor the cbh2 gene and thus is unable to produce cellobiohydrolases in any conditions.
Figure 18 illustrates the enzyme production profiles of the hypercellulolytic strain and its cellobiohydrolase negative derivative. Production of endoglucanases is lowered by choosing suitable fermentation conditions as well known in the art. With the new strain xylanase can be produced as SUBSTITUTE SHEET

an enzyme composition secreted from the host cell, such enzyme composition not containing cellobiohydrolases, and thus lacking activity against crystalline cellulose. The very small amount of endoglucanase activity still present does not have any harmful effects on the pulp properties when this kind of preparation is used in enzyme aided pulp bleaching.

Example 9 Construction of Strains Producing Different Combinations of Cellulases Using similar gene replacement strategy as in the previous strain constructions (Figure 17), sets of T. reesei strains have been constructed that produce different combinations of cellulases (Table 5). In the first set, strains A-D, one cellulase is eliminated at a time. Strains A-D synthesize everything the parent strain does except for CBHI (strain A), CBHII (strain B), EGI (strain C) or EGII (strain D). The second set, strains E-J, provides mutants missing all pairs of the four cellulases. Strain E lacks CBHI and CBHII. Strain F lacks CBHI and EGI. Strain G lacks CBHI
and EGII. Strain H lacks CBHII and EGI. Strain I lacks CBHII and EGII. Strain J lacks EGI and EGII.
When genes for both EGI and EGII (strain J, Table 5) are deleted, the activity against hydroxyethylcellulose drops to less than 10% of the activity produced by the parent hypercellulolytic strain, VTT-D-79125.
Lack of CBHI or CBHII proteins (strain E, Table 5) can be assayed by examining for activity against filter paper as known in the art. When both CBHI and CBHII are eliminated, no measurable activity is produced. One of skill in the art would recognize that further modifications, such as to eliminate three or more activities may also be constructed by the same strategy.

SUBSTITUTE SHEET

1 .

Table 5 Genetically Engineered Strains to Produce Novel Cellulase Mixtures Strain CBHI CBHII EGI EGII
type A - + + +
B + - + +
C + + - +
D + + + -E - - + +
F - + - +
G - + + -H + - - +
I + - + -J + + - -K + - - -L - - + -Figure 19 illustrate the powerfulness of gene technology to produce the compositions of the invention. Genetic engineering has been successfully used to construct derivatives of the hypercellulolytic mutant to produce different cellulase-xylanase mixtures. In strain 3, the cbhl gene coding sequence is replaced with the egll gene coding sequence, which in strain 3 is expressed under the cbhl promoter. Thus, the proportion of endoglucanases produced and secreted by these hosts is increased.
Strain 1 produces xylanases as the main activity, and in different production conditions, as known in the art, endoglucanases without any cellobiohydrolases can be produced. In the enzyme mixture produced by strain 3, the proportion of endoglucanases is increased, and strain 2 produces cellobiohydrolases essentially free of endoglucanases.

SUBSTITUTE SHEET

WO 93/24621 2136s;? r~ 50 PCT/F193/0027' Example 10 Use of the Enzyme Preparations in Biobleaching Oxygen-delignified softwood kraft mill pulp (kappa 16.1, brightness 35.4%
ISO) was used. The pulp pH was adjusted with sulphuric acid to 6. The pulp samples of 250 g dry matter were treated with enzyme A3273 ("enzyme A") or A2799 ("enzyme B") so that the xylanase dosage was 100 nkat/g pulp dry matter, at pH 6 for 55 C for 2 hours and washed. Then they were bleached using bleaching sequence D. (EO) DED. Reference pulp was bleached without enzyme treatment.
Following standard methods were used: brightness (ISO 2470), kappa number (ISO 302), viscosity (ISO 5351/1) and pc (post-coloring) number (TAPPI 260 pm-81). Pulps were beaten with a PFI mill (ISO 5264/2), handsheets were made according ISO 5269/1 and the strength properties were tested (ISO 5270).
The enzyme treated pulps were bleached using 25% lower C1O2 dosage in the first bleaching stage than in the reference pulp. Enzyme pretreated pulp achieved the full brightness with about 15% less aCl in total (Table 6). This is about the same as achieved in earlier experiments using conventionally cooked pulps (kappa number 31-32) without 02-delignification and bleaching sequences containing elementary chlorine (Lahtinen, T., et al., In: Biotechnology in Pulp and Paper Industry, Kuwahara, M., Shimada, M. (eds.), Uni Publishers Co., Tokyo, Japan, pp.
129-137 (1992)).
The strength properties (viscosity and tear index) correlated with the cellulolytic side activities: with low side activities (enzymes A), strength properties similar to the reference were obtained. Optical properties were also comparable to reference, with the exception of the pc-number (the measure of brightness reversion) which was even better in enzyme treated pulps.

SUBSTi i'fdTE SHEE'f Table 6. ECF bleaching experiments Enzymes Ref A B

Enzyme sta, :
Dose, nkat/g 100 100 -Temp., C 55 55 -5 pH 6.0 6.0 -time, hours 2 2 -DO stage consumption of C102, act.
Cl kg/t 24.2 24.1 31.9 10 EO stage Brightn, % 62.5 62.4 63.1 Kappa 4.6 4.6 4.2 D 1 stage Consumption of C10Z, 15 act. Cl kg/t 17 17 17 Brightn, % 82.9 82.5 83.5 D2 stage Consumption of C102, act. Cl kg/t 6.5 6.3 6.0 20 Final Brightn., % 90.2 89.5 89.7 pc number 0.45 0.54 0.87 Viscosity 830 810 840 Total 25 Consumption of C102, act. Cl kg/t 47.7 47.4 54.9 Consumption of C1021 act. Cl kg/t, at ISO 90' 46.9 49.4 56.1 Reduction 16% 12% --30 Pulp properties after refming (at Tensile Index 70 Nm/g) PFI revs Tear, mNm2/g 1123 945 871 Light scatt. coeff. m2/kg 13.8 2.2 14.2 35 Light abs. coeff. m2/kg 20.7 ". ,6 21.6 0.06 J8 0.06 Fiber length mm 2.00 1.99 1.97 < 0.20 tnm, % 3.23 3.22 3.39 1: assuming that 4 kg aCl increases brightness by 1 ISO unit SUBSTOTUTE SHEET

Bleaching Conditions:
Stage DO (EO) D 1 E D2 Consistency, % 3 10 9 9 9 Temperature, C 55 75 70 70 70 Time, min. 60 60 180 60 180 End pH 2.9-3.2 10.8 3.7-4.0 10.8 3.5-3.8 C102 dose, kg/t 24.2 - 17 - 8 active Cl OZ pressure, bar - 2 - - -NaOH dose, kg/t - 17 2.5 9 -Having now fully described the invention, it will be understood by those with skill in the art that the scope may be performed within a wide and equivalent range of conditions, parameters and the like, without - affecting the spirit or scope of the invention or any embodiment thereof.

The Swedish Patent O{fice PCT/ Fl 9 3 / 0 0 2 2 1 PCT International Application SEQUENCE LISTING
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(i) APPLICANT:
(A) NAME: OY ALKO AB et al.
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(C) CITY: Helsinki (D) COUNTRY: Finland (E) POSTAL CODE: 00180 (ii) TITLE OF INVENTION: Novel Enzyme Preparations and Methods for Their Production (iii) NUMBER OF SEQUENCES: 14 (v) COMPUTER READABLE FORM:
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Met Val Ser Phe Thr Ser Leu Leu Ala Gly Val Ala Ala Ile Ser Gly Val Leu Ala Ala Pro Ala Ala Glu Val Glu Ser Val Ala Val Glu Lys Arg Gln Thr Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser SUBSTITlJTi E SHEET

rl.l/ rl y-) / UU127 The Swedish Pa'.ent Office 02 -08- 1993 pCT Interna-ionat Application 2136350 .,. _ 78 Tyr Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Asn Gly Pro Gly Gly Gin Phe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly Gly Lys Gly Trp Gln Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser Gly Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp Gly Ser Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn His Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp Ala Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val Ser (2) INFORMATION FOR SEQ ID NO:2:

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The Swedish Pa vnt O`:`ice PCT/ FI 9 3/ 0 C 2 2 1 PCT Interna-ional AaplicGtion 0 2-08- 1993 Val Leu Ala Ala Pro Ala Ala Glu Val Glu Ser Val Ala Val Glu Lys Arg Gln Thr Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser Tyr Trp Asn Asp Gly His Gly Gly Val Thr Tyr Thr Asn Gly Pro Giy Gly Gln Phe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly Gly Lys Gly Trp Gln Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser Gly Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp Gly Ser Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn His Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp Ala Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val Ser (2) INFORMATION FOR SEQ ID NO:3:
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The Swedish Patent Office r~ ~~ r I y 3/ 0 0 2 2 1 PCT International AFplication 02_0$_ 1993 Met Tyr Asp Phe Val Leu Gly Ala His Asn Asp His Arg Arg Arg Ala Ser Ile Asn Tyr Asp Gln Asn Tyr Gln Thr Gly Gly Gln Val Ser Tyr Ser Pro Ser Asn Thr Gly Phe Ser Val Asn Trp Asn Thr Gln Asp Asp Phe Val Val Gly Val Gly Trp Thr Thr Gly Ser Ser Ala Pro Ile Asn Phe Gly Gly Ser Phe Ser Val Asn Ser Gly Thr Gly Leu Leu Ser Val Tyr Gly Trp Ser Thr Asn Pro Leu Val Glu Tyr Tyr Ile Met Glu Asp Asn His Asn Tyr Pro Ala Gln Gly Thr Val Lys Gly Thr Val Thr Ser Asp Gly Ala Thr Tyr Thr Ile Trp Glu Asn Thr Arg Val Asn Glu Pro Ser Ile Gln Gly Thr Ala Thr Phe Asn Gln Tyr Ile Ser Val Arg Asn Ser Pro Arg Thr Ser Gly Thr Val Thr Val Gln Asn His Phe Asn Ala Trp Ala Ser Leu Gly Leu His Leu Gly Gln Met Asn Tyr Gln Val Val Ala Val Glu G1y,Trp Gly Gly Ser Gly Ser Ala Ser Gln Ser Val Ser Asn (2) INFORMATION FOR SEQ ID NO:4:

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"rBELT

Claims (25)

CLAIMS:

1. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequence of T. reesei pI 5.5 xylanase (SEQ ID No. 4) or another nucleic acid molecule encoding a protein having endo-beta-1,4-xylanase activity and which hybridizes to the complement of SEQ ID NO. 3 at 60 to 68°C in the presence of 6 × SSC + 0.1%
SDS and washing at 60 to 68°C in the presence of 6 × SSC +
0.1% SDS.

2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence is that of the DNA
sequence of SEQ ID No. 3.

3. An isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the amino acid sequence of T. reesei pI 5.5 xylanase set forth in SEQ ID No. 4.

4. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequence of T.
reesei pI 9 xylanase set forth in SEQ ID NO. 2.

5. The isolated nucleic acid molecule of claim 4, wherein said nucleic acid sequence is that of SEQ ID No. 1.

6. An isolated polypeptide encoded by the nucleic acid of any one of claims 1 to 5.

7. A recombinant vector comprising the isolated nucleic acid molecule of any one of claims 1 to 5.

8. The recombinant vector of claim 7, wherein said vector is selected from the group consisting of a plasmid and a linear DNA.

9. A recombinant host cell transformed with the recombinant vector of claim 7 or 8.

10. The recombinant host cell of claim 9, wherein said host is Trichoderma.

11. The recombinant host cell of claim 10, wherein said Trichoderma is T. reesei.

12. A recombinant vector comprising the isolated nucleic acid molecule of any one of claims 1 to 5, wherein said nucleic acid molecule is operably linked to a T. reesei promoter selected from the group consisting of the xln1 promoter, the xln2 promoter, the cbh1 promoter, the cbh2 promoter, the egl1 promoter, and the egl2 promoter.

13. A recombinant host cell transformed with the recombinant vector of claim 12.

14. The recombinant host cell of claim 13, wherein said host is Trichoderma.

15. The recombinant host cell of claim 14, wherein said Trichoderma is T. reesei.

16. A method for the production of an enzyme preparation enriched in xylanase activity, said method comprising:

(1) transforming a host cell with the recombinant vector of any one of claims 7, 8 and 12;

(2) culturing said host cell in culture medium under conditions wherein a protein having xylanase activity expressed by said recombinant construct is synthesised from said transformed host cell of step (1);

(3) and collecting said culture medium.

17. The method of claim 16, wherein said host cell is genetically incapable of expressing one or more endogenous cellulolytic enzymes.

18. The method of claim 17, wherein said cellulolytic enzyme is selected from the group consisting of CBHI, CBHII, EGI and EGII.

19. The method of claim 18, wherein said cellulolytic enzyme is CBHI.

20. The method of any one of claims 16 to 19, wherein said host is Trichoderma.

21. The method of claim 20, wherein said Trichoderma is T. reesei.

22. Culture medium obtained after culture of the host cell of any one of claims 9 to 11 and 13 to 15.

23. An enzyme preparation having endo-beta-1,4-xylanase activity or pI 9 xylanase activity, obtained by purifying, drying, concentrating or immobilizing the culture medium of claim 22.

24. Use of the culture medium of claim 22 or the enzyme preparation of claim 23 in pulp and paper industries.

25. Use of the culture medium of claim 22 or the enzyme preparation of claim 23 in feed industry.