EP0670893A1 - Nouvelles preparations enzymatiques et leurs procedes de production - Google Patents

Nouvelles preparations enzymatiques et leurs procedes de production

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Publication number
EP0670893A1
EP0670893A1 EP93910050A EP93910050A EP0670893A1 EP 0670893 A1 EP0670893 A1 EP 0670893A1 EP 93910050 A EP93910050 A EP 93910050A EP 93910050 A EP93910050 A EP 93910050A EP 0670893 A1 EP0670893 A1 EP 0670893A1
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EP
European Patent Office
Prior art keywords
gene
trichoderma
reesei
host
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP93910050A
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German (de)
English (en)
Inventor
Pirkko Suominen
Helena Nevalainen
Ritva Saarelainen
Marja Paloheimo
Tarja Lahtinen
Richard FAGERSTRÖM
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AB Enzymes Oy
Original Assignee
Alko Oy AB
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Filing date
Publication date
Application filed by Alko Oy AB filed Critical Alko Oy AB
Publication of EP0670893A1 publication Critical patent/EP0670893A1/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes

Definitions

  • 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.
  • Cellulose is a linear polysaccharide of glucose residues connected by ⁇ -1,4 linkages.
  • cellulose is usually associated with lignin together with hemicelluloses such as xylans and glucomannans.
  • hemicelluloses such as xylans and glucomannans.
  • the major part of the lignin is extracted to get acceptable cellulose pulp product.
  • 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.
  • Heroicellulases 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
  • the hemicellulose should be free of cellulases, which would harm the cellulose fibers.
  • 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
  • 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 14:566-514 (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- ⁇ -l,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.
  • Xylans are complex heteropolymers mainly consisting of xylose and arabinose.
  • Xylans have a backbone consisting of ⁇ -l,4-linked xylopyranose units, which may be substituted with acetyl residues and residues of arabinose and methyl glucuronic acid (Timell, T.E., et al, Wood Sci. Technol. 1:45-10 (1967)).
  • Xylans are, after cellulose, the second most abundant carbohydrate fraction of plant biomass.
  • Figure 1 shows the general strategy for deleting a gene.
  • Figure 2 shows the restriction map of the 5.7 kb (Kpn ⁇ ) 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 Sw ⁇ l 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
  • Figure 5 shows the pALK476 fragment used in the transformations.
  • the 1.9 kb cbhl -flanking region (Scal-EcoRl) is from 2.2 kb upstream of the cbhl -coding region, the 1.8 kb 3'-region (BamHL-EcoR ⁇ ) is from 1.4 kb downstream of the end of the cbhl -coding region.
  • the amdS gene was from p3SR2 (3.1 kb Spel-Xba ⁇ fragment) and the xln2 gene was from pALK475 (5.0 kb Smal 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, ALKO2221 16/26 and ALKO2721 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 ressei xlnl gene [S ⁇ Q ID No.:3:]. The coding regions are indicated by upper case letters
  • 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.
  • 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.
  • cellulase enzymes For efficient hydrolysis of cellulose to glucose, at least three cellulase enzymes (three types of cellulase enzyme activity) are needed: randomly cleaving endoglucanases (1,4,- ⁇ -D-glucan glucanohydrolase, EC 3.2.1.4) which usually attack substituted soluble substrates and show no activity to crystalline cellulose; cellobiohydrolase (1,4- ⁇ -D-glucan cellobiohydrolase, EC 3.2.1.91) which is capable of degrading crystalline cellulose * .x has no activity towards derivatized cellulose and ⁇ -glucosidase ( ⁇ -D-glucoside glycohydrolase, EC 3.2.1.21) which degrades cellobiose and cello-oligosaccharides to yield glucose.
  • randomly cleaving endoglucanases (1,4,- ⁇ -D-glucan glucanohydrolase, EC 3.2.1.4
  • cellobiohydrolase (1,4-
  • CBH I and CBH II immunologically distinctive cellobiohydrolases
  • CBH II immunologically distinctive cellobiohydrolases
  • 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.
  • 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 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-l,4- ⁇ -D-xylanase and endo-l,4, ⁇ -D-mannanase.
  • Small oligosaccharides are further hydrolyzed by 1,4- ⁇ -D-xylosidase, 1,4- ⁇ -D- mannosidase and 1,4- ⁇ -D-glucosidase.
  • the side groups are split off by ⁇ -L-arabinosidase, ⁇ -D-glucuronidase and ⁇ -D-galactosidase. Esterified side groups are liberated by various esterases.
  • the definition of hemicellulolytic enzymes is taken from Viikari et al, "Hemicellulases for Industrial Applications,” in Bioconversion of Forest and Agricultural Wastes (1992).
  • Enzyme preparation is meant a 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 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, affinity 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.)
  • conventional bleaching chemicals like chlorine, chlorine dioxide, peroxide, etc.
  • RNA that codes for a protein
  • messenger RNA mRNA
  • RNA polymerase II messenger RNA
  • RNA polymerase II RNA polymerase II
  • antisense RNA gene 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.
  • 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.
  • 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.
  • 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 pi 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
  • 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 ampicillin 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.
  • such expression vehicle provides for an enhanced expression of a gene of interest which has been cloned into it, after transformation into a desired host.
  • 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.
  • genetic sequences are capable of encoding a desired enzymic activity and through the expression of such genetic sequences.
  • 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 T" choderma reesei (formerly T viride) is classified as a member of Fungi imperfecti.
  • Fungi imperfecti 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.
  • Trichoderma has been reported to possess a poorly defined 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.
  • 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).
  • 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.
  • 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.
  • 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, ⁇ - xylosidase, ⁇ -arabinosidase, ⁇ -glucuronidase, and acetyl esterase, the activity of any of which may be a desired enzyme in the enzyme preparations of the invention.
  • 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.
  • any other Trichoderma enzyme which oxidizes cellulose may be utilized in the enzyme preparations of the invention and may be a desired enzyme.
  • the T reesei culture filtrate contained all the side-group cleaving activities assayed (acetyl esterase, ⁇ -glucuronidase and ⁇ - arabinosidase) whereas those from F. oxysporum and S. olivochromogenes only contained esterase.
  • Trichoderma is also advantageous as a host because it naturally produces a wide spectrum of xylanolytic enzymes the proportions of which can oe manipulated by genetic engineering for different applications to provide enzyme preparations tailored for those purposes.
  • 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.
  • promoters such as cbhl
  • additional or modified regulatory regions such as, for example, enhancer sequences.
  • such regulatory sequences are 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).
  • 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. 14:566-514 (1992)).
  • the enzymes have isoelectric points of 5.5 (XYLI) and 9.0 (XYLII) and their molecular masses are 19 and 20 kDa, respectively.
  • 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
  • xylanases for Use in Pulp and Paper industry
  • 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 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.
  • xylanase I more acid pH region
  • xylanase II may be preferably (alkaline or near neutral pH range).
  • 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.
  • an enzyme preparation comprising an elevated amount of endoglucanase may be used.
  • a host which expresses elevated levels of endoglucanases, in addition to xylanases or other hemicellulases may be used.
  • 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.
  • a Trichoderma host which does not express a certain enzyme is transformed with one or more genetic constructs encoding enzyme(s) heterologous to Trichoderma.
  • 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.
  • the gene encoding a desired enzyme is inserted into the cbhl locus such that it is operably linked to the strong cbhl promoter.
  • 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 can be integrated into the genome of Trichoderma by inserting the gene into a general expression vector, for example, pAMHHO, which is described in the patent application EP 244,234.
  • pAMHHO is derived from pUC19 (Yanish-Perron et al, Gene 55:103-119 (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 S ⁇ cII and Nde ⁇ . 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 pAMHl lO 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.
  • a vector construction comprising the 3 -phosphogly cerate kinase gene (pgk) transcriptional regulatory regions may be used as 3 -phosphogly cerate 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.
  • pgk 3 -phosphogly cerate kinase gene
  • 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.
  • 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.
  • 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.
  • Trichoderma 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 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.
  • a Trichoderma host strain having the desired properties may be cultivated in a liquid cultivation medium, which may comprise, for example, 6% Solka Floe cellulose, 3% distiller's spent grain, 0.5% KH 2 PO 4 , 0.5% (NH 4 ) 2 SO 4 , 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 55:650-656 (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. 72:117-120 (1990)).
  • 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 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 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.
  • 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)).
  • mRNA can be 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)).
  • the mRNA preparation used will be enriched in mRNA coding for a desired protein, either naturally, by isolation from a 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.
  • DNA preparations either genomic DNA or cDNA
  • suitable DNA preparations 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, 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.
  • 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
  • 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.
  • 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.
  • amino acid sequence may be encoded by only a single oligonucleotide sequence
  • amino acid sequence may be encoded by any of a set of similar oligonucleotides.
  • 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.
  • 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.
  • 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).
  • 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.
  • the above -described DNA probe is labeled with a detectable group.
  • 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 32 P, 3 H, ,4 C, 35 S, 125 I, 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.
  • 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.
  • a non-radioactive marker such as biotin, digoxigenin, an enzyme or a fluorescent group.
  • the actual identification of protein's sequence permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide.
  • a DNA molecule or set of DNA molecules, capable of functioning as a probe(s) for the identification and isolation of clones containing the protein's 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.
  • 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.
  • sequences controlling transcriptional expression e.g., a Trichoderma host cell
  • 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 “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 of the sequence under the influence or control of the regulatory sequence.
  • Two DNA sequences 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 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.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • 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- coding) sequences involved with initiation of transcription and translation respectively.
  • Trichoderma requires the use of regulatory regions functional in such hosts.
  • a wide variety of transcriptional and translational regulatory sequences can be employed, since Trichoderma generally recognize eukaryotic host transcriptional controls, such as, for example, those of other filamentous fungi.
  • control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such a methionine.
  • a promoter region sufficient to direct the initiation of RNA synthesis in the host cell.
  • - translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine.
  • the linkage between a eukaryotic promoter and a DNA sequence which encodes the protein, or a functional derivative thereof does not contain any i-ntervening 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).
  • 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
  • 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.
  • 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.
  • 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.
  • constructs wherein both (a) a desired protein's mRNA and (b) antisense RNA directed to a 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.
  • 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.
  • 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 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • a casette comprising a desired gene already operably linked to the homologous cbhl promoter is inserted into the cbhl locus.
  • any one, some, or all of the cellulolytic enzymes may be eliminated, reduced, inactivated, or repressed by methods 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 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 antisense RNA directed against the coding sequence for that gene, etc.
  • 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, predominantly haploid uninucleate conidia which constitute excellent material for various mutagenic treatments.
  • 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 on both the mutagen and dose used.
  • 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 Trichoderma hosts of the invention include alkylating agents, such as, for example, N-methyl-N-nitro-N-nitrosoguanidine ( ⁇ TG), ethylmethanesulphonate (EMS) and diethylsulphate (DES). Hydroxylamine and chemicals deaminating D ⁇ A bases such as nitrous acid are also useful. Ionizing radiation ( ⁇ - and X-rays) as well as ultraviolet irradiation (UV) are examples of physical mutagens useful in Trichoderma strain mutagenesis. 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.
  • alkylating agents such as, for example, N-methyl-N-nitro-N-nitrosoguanidine ( ⁇ TG), ethylmethanesulphonate (EMS) and diethylsulphate (DES). Hydroxylamine and chemicals de
  • 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.
  • agents used for the purpose are rose bengal, oxgall and phosphon D, Triton X-100 and saponin.
  • replica plating technique analogous to that developed for bacteria can, in certain cases, be used to test the properties ol 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 egl3; Saloheimo et al, Gene 65:11-21 (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 an antisense RNA to a degree which prevents the translation of the hybridized RNA.
  • 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 ⁇ -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)
  • a marker for screening for example, lacZ
  • 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 from the promoter area, the promoter will also be removed in the resulting replacement strain.
  • a selectable marker gene like trpC, argB or amdS
  • Gene A can be any Trichoderma gene, preferably a cellulase gene, the flanking regions of which can be cloned/isolated.
  • 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 pUC19 plasmids (Yanish-Perron et al, Gene 55:103-119 (1985)).
  • 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.
  • the gene for the native cellobiohydrolase CBH I sequence has been cloned by Shoemaker et al (Shoemaker, S., et al, Bio/Technology 7:691-696 (1983)) and Teeri et al.
  • a method for producing high levels of enzymes preferabaly hemicellulases, desirable for pulp and paper processing.
  • 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.
  • 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.
  • 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.
  • the recombinant hosts described above having the desired properties 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 example in a liquid cultivation medium which may comprise for example 6% Solka Floe cellulose (BW40, James River Corporation, Ralphensack, NJ), 3% distiller's spent grain, 0.5% KH 2 PO 4 , 0.5% (NH 4 ) 2 SO 4 , 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 55:650-656 (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.
  • the temperature may be adjusted according to the strain and according to the enzyme preparation to be produced (Merivuori et al, Biotechnology Letters 72(2):117-120 (1990)).
  • the enzyme preparation is recovered from the culture medium by using methods well known in the art.
  • 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.
  • 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.
  • fodder treated with the enzyme preparations of the invention would be of great food benefit to farm animals because it would be easier for them to digest.
  • Phleomycin resistant transformants were screened as described by Durand et al. in: Biochemistry and Genetics of Cellulose Degradation, p.
  • the amount of DNA used varied from 2 to 5 ⁇ g.
  • the selection marker (amdS (acetamidase) or argB (ornithine carbamoyl transferase, OTCase, E.C. 2.1.3.3)or trpC (tryptophane) was within the transforming fragment.
  • Plasmid DNA from E. coli was isolated using standard methods (Maniatis et al. 1982, Molecular Cloning: A Laboratory Manual, Cold
  • Endoglucanase I protein concentration in the culture supernatant 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 phosphate buffered saline pH 7.2 containing 0.05% Tween 20 and 0.02% sodium azide (PBS/Tween).
  • mice 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 PBS/Tween for 1 hr. Appropriate dilutions of culture supernatant 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.
  • endoglucanase I anti-EGI antibody EI-2
  • j-nitrophenylphosphate (1 mg/ml) 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 supernatant fractions was then calculated by comparing their 0D 405 values with a standard dilution curve prepared using purified endolgucanase I and performed at the same time on the same plate.
  • 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. reesei 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
  • 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 by targeting a construct composed of the xlnl gene with its own promoter to the endogenous cbhl locus. Highest overall production levels for xylanase were obtained using the T. reesei ALKO2721, a genetically engineered strain, as a host. Integration into the cbhl locus was not required for enhanced expression under xln2 promoter.
  • Plasmids were propagated in Escherichia coli strain XL 1 -Blue (Bullock, W.O., et al, Bio/Techniques 5:376-378 (1987)) or in E. coli INVl ⁇ F' (Invitrogen, San Diego, CA, USA).
  • the recipient organisms for the xln2 gene were the high cellulase-producing mutants T. reesei VTT-D-79125 (Bailey and
  • T. reesei ALKO2221 is a low protease mutant of the strain VTT-D-79125.
  • T reesei ALKO2721 is a trpC minus (trpC ' ) UV mutant of VTT-D-79125, in which the cbh.2 locus has been replaced with the trpC gene of Aspergillus nidulans (Yelton, M.M., et al, Proc. Natl. Acad. Sci.
  • the strain also carries several other integrated copies of the trpC gene.
  • the plasmids used in this study included pBluescript (Stratagene, San Diego, CA, USA), pCRlOOO (Invitrogen) and pUC19 (Yanish-Perron,
  • 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 Pantotato Dextrose Broth, Difco, Detroit, USA
  • agar slants were used for growing the Trichoderma strains.
  • T 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% KH 2 PO 4 and 0.5% (NH 4 ) 2 SO 3 . Peptide digestion and amino acid sequencing. Purified xylanase II
  • ⁇ lution 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.
  • ACN acetonitrile
  • TFA trifluoroacetic acid
  • Plasmid and cosmid DNAs were isolated using Qiagen columns (Diagen GmbH, D ⁇ sseldorf, Germany) according to the manufacturer's instructions. E. coli was transformed according to Hanahan, D., J. Mol. Biol
  • T. reesei strains by the method of Penttila, M., et al, Gene 67:155-164 (1987) with the following modifications: the protoplasts were treated by heat shock (Berges and Barreau, J. Gen. Microbiol. 755:601-604 (1989)) before transformation, 25% P ⁇ G 6000 was replaced with 60% PEG 4000 and the transformation was performed at room temperature instead of that on ice. The transformants were purified through conidia on selective acetamide-CsCl plates (Penttila, M., et al, Gene 67:155-164 (1987) before transferring them to PD slants. RNA isolation, T.
  • 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. 77:2961-2978 (1983).
  • the mRNA obtained was size-fractionated using DMSO-sucrose gradient centrifugation (Boedtker, H., et al, Biochemistry 75:4765-4770 (1976)).
  • 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 dT ]7 -adapter primer (5' GAC-TCG-AGA-ATT-CAT-CGA-dT 17 3') [SEQ ID No.:5:].
  • 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-
  • PCR polymerase chain reaction
  • 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 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.
  • 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 373 A 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 described by Bailey, M.J., et al, J. Biotechnol. 23:251-210 (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)).
  • CBHI cellobiohydrolase I
  • 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 mRNA 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 nucleotide and deduced amino acid sequences of the xlnl 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 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.
  • 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, M., et al, Enzyme Microb. Technol 14:566-514 (1992)). Sequence analysis also revealed two putative targets (Asn-X-Ser/Thr where X is not Pro; Gavel and von Heijne, Protein Engineering 5:433-442 (1990)) for N- glycosylation (Asn38 and Asn ⁇ l of the mature protein).
  • Plasmid pALK476 Construction of the plasmid pALK476 is shown in Figure 4. This plasmid is useful for targetting the xlnl gene to the cbhl locus. Expression of the xlnl gene is under the control of its native promoter.
  • Transformants 57 from T. reesei VTT-D-79125, 42 from ALKO2221 and 53 from ALKO2721 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 supematants. 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 xlnl expression cassette was located at the cbhl locus and those in which it was integrated elsewhere.
  • CBHI- CBHI negative 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, ALKO2221 and ALKO2721, respectively.
  • Figure 6 shows the relative xylanase activities of the transformants grouped according to their CBHI + " phenotype. Increase in the xlnl 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 ALKO2221 and ALKO2721) 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 ALKO2721 as a host.
  • the best transformants of 7. reesei ALKO2221 produced less than half the activity obtained with the best transformants of the two other strains.
  • T. reesei VTT-D- 79125 transformants those with the CBHI + phenotype were on average better producers of xylanase.
  • 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.
  • most of the CBHI- transformants (42-86%, Table 1) exhibited similar levels of increase in xylanase activity.
  • the xlnl 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.
  • 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 118:21-26 (1985)) and suggested to occur with T. reesei cellobiohydrolase II (Teeri, T., et al, Gene 57:43-52 (1987)).
  • T. reesei VTT-D-79125 is a high cellulase-producing mutant and ALKO2721 was used as a host because of its pre-existent high xylanase production.
  • pALK174 Construction of pALK174 is shown in Figure 7.
  • a 674 bp PCR fragment containing an exact fusion of the cbhl promoter to the xlnl signal sequence (and xlnl gene to the internal Xh ⁇ site) was synthesized Oby using plasmid pALK475 ( Figure 2) as a template.
  • the oligonucleotides used were as shown below: 5'-primer contained the end of the cbhl promoter and the sequence of the beginning of the putative xlnl signal sequence, 3 '-primer had sequence from ihe xlnl gene including the gene's internal Xh ⁇ l site (see the pALK476 map, Figure 5 or the xlnl sequence).
  • the 100 ⁇ l PCR reaction contained in PCR buffer (10 mM Tris pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 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, final 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 S ⁇ cII for fusion to the cbhl promoter.
  • the plasmid pAMHHO containing the cbhl promoter, was cut with Ndel (filled in by Klenow) and S ⁇ cII.
  • the PCR fragment described above was ligated to the digested pAMHHO (pALK174X). The fusions and the sequence synthesized by PCR were ensured by sequencing.
  • the plasmid pALK174 was constructed by replacing the xlnl promoter in pALK476 by the cbhl promoter: the 2.9 kb Kpnl-Xhol fragment from the plasmid pALK174X, containing the cbhl promoter fused to the xlnl gene and xlnl sequence to the internal - ⁇ T-oI, was ligated to the isolated vector containing fragment of pALK476, after cutting pALK476 with Kp ⁇ l and Xhol.
  • the plasmid pALK174 contains, in addition to the amdS and cbhl 3 '-areas as in pALK476 ( Figure 4), the xlnl gene, fused to the ebb 7 promoter and 1.9 kb terminator (3'-area) of the xlnl gene.
  • the 9.7 kb EcoRI fragment (expression cassette free from the vector sequences) was used to transform three Trichoderma strains, VTT-D- 79125, ALKO2221 and ALKO2721.
  • the strains, transformation method and methods for purification, analyzing and growing the transformants were as described above and in Example 6. 2.2 Production of xylanase by the pALK174 transformants
  • the targeting frequencies to the cbb7 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 supematants as described in Example 3 (cloning of xlnl).
  • the plasmids used in this study were pBluecscript (Stratagene, San Diego, CA, USA) and pCRlOOO (Invitrogen, San Diego, CA, USA).
  • the plasmids were propagated in Escherichia coli strain XLI-Blue (Bullock, W.O. et al, Bio/Techniques 5:376-378 (1987)) or in E. coli INVl ⁇ F
  • 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.
  • 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. Amino terminals of the protein and the peptides were sequenced by degrading them in a gas-pu- .ed-liquid-phase sequencer (Kalkkinen, N., and Tilgmann, C, J. Prot. Chem. 7:242-243 (1988)). The released PTH-amino acids were analysed on-line using narrow-bore reverse-phase HPLC.
  • 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 xlnl gene). The mycelia were ground into a fine powder under liquid nitrogen. Total RNA and mRNA were isolated as described by Barrels and Thompson (Bartels, D., and Thompson, R.D.,
  • the templates for nucleotide sequencing were generated by unidirectional deletions according to the manufacturer's instructions for the pBluescript ⁇ xo/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 373 A Sequencer.
  • 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-Val-Ser-Tyr-(Ser)- Pro-(Ser)-Asn-Thr-Gly-Phe-Ser [S ⁇ Q ID No.: 10:].
  • Five tryptic peptides were also obtained and directly sequenced (see Figure 10 for peptide sequences).
  • 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).
  • the nucleotide sequence of the xlnl gene was determined and is presented in Figure 10 together with th deduced amino acid sequence.
  • a TATA box was found approximately 90 nt upstream of the putative translation start site.
  • 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, R.M., eds., Molecular Industrial Mycology. Systems and applications for filamentous fungi (Marcel Dekker, Inc., New York), pp. 1-29 (1991)).
  • the gene codes for a protein of 229 amino acids.
  • the N-terminal obtained by direct peptide sequencing, see above
  • a 51 amino acid long signal propeptide containing a primary signal sequence cleavage site von Heijne, Nucleic Acids Res. 74:4683-4690 (1986)
  • Figure 10 Comparison of the genomic sequence with the cDNA sequence revealed one intron of 62 bp.
  • the mature xylanase I is a protein of 178 amino acids with a calculated molecular weight of 19.1 kDa.
  • T. reesei 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, 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.
  • 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. 14:566-514 (1992)).
  • the corresponding genes xlnl and xlnl have been cloned and described herein. In overall structure the xlnl and xlnl genes are very similar; they are about the same size and both contain only one intron and a long pre-propeptide.
  • 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. 14:566-514 (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.
  • A. kawachii xylanase A Ito, K. et al, Biosci. Biotec. Biochem. 56:906-912 (1992)
  • T. reesei xylanases an identity 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)).
  • the plasmid pALK807 was constructed by using the same strategy as in construction pALK174 ( 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 Xh ⁇ l site was made by PCR using the following oligonucleotides:
  • Xhol xlnl sequence 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 Sac ⁇ l- Xh ⁇ l digested PCR fragment was ligated to the S ⁇ I--X7zoI cut plasmid pALK486 containing the cbhl promoter.
  • pALK806 To construct pALK806, the xlnl sequence downstream from the Xh ⁇ l site was isolated from the plasmid pALK572 by Pstl (filled by T4 DNA polymerase) -Xh ⁇ l digestion and the fragment was ligated to the Bam ⁇ l (filled in with Klenow) - Xh ⁇ l digested pALK805.
  • pALK805 was obtained by ligating the Eco ⁇ l-Spel 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 ALKO2221 was transformed with the 8.2 kb Notl fragment from the plasmid pALK807.
  • Example 2 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. 14:566-514 (1992)). The results, as an increase in xylanase activity produced compared to that of the host strain
  • ALKO2221 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 + . 10
  • 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.
  • the resulting 0.8 kb DNA fragment bearing the 5' region of the cbhl cDNA was made blunt-ended with Sj nuclease and was ligated to an EcoRI cut, blunt- ended ⁇ UC18 vector (Yanish-Perron et al., Gene 55:103-119 (1985)).
  • the clone obtained was cut in the middle of the cbhl fragment with
  • 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. 5:153-157 (1981)) was cotransformed with ⁇ MS4 and p3SR2.
  • p3SR2 carries a 5 kb DNA fragment containing the A. nidulans amdS gene cloned into pBR322 (Kelly and Hynes, EMBO J. 4:415-419 (1985)). Transformants were selected on the basis of the AmdS + phenotype after which they were purified from conidia.
  • Plasmid pALK99 was constructed to be the source of the transforming fragment ( Figure 13). Plasmid pALK99 was constructed in the following way. The Pvu ⁇ l fragment (containing the multilinker) of the plasmid pUC19 was replaced by a new synthetic multilinker fragment containing recognition sites for the following restriction enzymes: Xhol-Stul-Smal-Xbal-Pvu ⁇ l-Sall-Xhol. The new plasmid was called pALK96.
  • This plasmid was cut with -Xb ⁇ l and -PvwII and a 2.1kb Xb ⁇ l-Pvu ⁇ fragment from the 3' region of the cbhl gene (see Figure 14) was ligated into it.
  • the resulting plasmid was cut with .PvwII and Hindi and ligated with the 3.4 kb Pv ⁇ ll-fragment from the 5' area of the cbb2-gene (see Figure 14). Both the 3' and 5' fragments were originally from the ⁇ clone cbh21ambdal (Teeri et al., Gene 57:43-52 (1987)).
  • the resulting plasmid was called pALK98.
  • the Aspergillus nidulans argB gene (2.6kb Sail fragment) was then ligated between the 3' and 5' regions of the cbhl gene into the unique PVHII site of plasmid pALK98.
  • the resulting plasmid was called pALK99.
  • the transforming fragment which is isolated from pALK99 as a -Xbol fragment contains the Aspergillus argB gene as a 2.6 kb Sail fragment (Berse et al., Gene 25:109-117 (1983)) between 3.4kb (PvuTl-Pvu ⁇ .
  • Strain ALKO 2564 is an example of this kind of "replacement" strain and thus does not contain the cbhl gene any more.
  • the cbhl 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 cbhl gene into the unique PvuII site of the plasmid pALK98.
  • the resulting plasmid was called pALK402.
  • the transforming fragment which is isolated from pALK402 as Xhol fragment contains A.
  • nidulans trpC gene as a 4.2 kb Xhol 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 cbhl gene.
  • T. reesei ALKO2319 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.
  • 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 constmcted 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 55:103-119 (1985)).
  • the single Ndel site of pUC19 was destroyed by filling in the recessed ends with Klenow polymerase, and then the plasmid was digested with EcoRI and Rstl and ligated to cbb7 promoter and terminator fragments to make an expression cassette.
  • the promoter fragment was a 2.6 kb EcoRI-P-ytl fragment from the plasmid pAMH 102 (Harkki et al, Bio/Technology 7:596-603 (1989)).
  • the terminator was a 0.75 kb Avail fragment contained in a Pstl fragment which also included an adaptor with the TAA stop codon in all three reading frames.
  • pAMH 110 was then digested with S ⁇ cII and Ndel to remove a piece of DNA between the cbb7 promoter and terminator, and the digested ends were made blunt-ended with S, nuclease and Klenow polymerase.
  • the eg/7 cDNA to be expressed was taken from the plasmid pTTcl l ((Teeri et al, Anal. Biochem. 164:60-61 (1987); Penttila et al, Yeast 5:175 -185 (1987); as a 1.6 kb EcoRI-R ⁇ HI 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 pAMHl l l and the plasmid pAN8-l (Mattern et. al,
  • 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 cbb7 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).
  • 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. 4:415-419 (1985)) was digested with Sphl and with Xbal.
  • the resulting 3.2 kb DNA fragment bearing the whole amdS gene was ligated to the Sphl and Xbal cut pUC19 vector (Yanisch-Perron et al, Gene 55:103-119 (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 Seal site in the coding region was isolated as a Sc ⁇ l- BamHl fragment and blunt-ended with Klenow-enzyme. This fragment was ligated to the - ⁇ 7j> ⁇ l site (blunt-ended with -Klenow enzyme) of the plasmid pALK410. In this case the 3' fragment was isolated from the plasmid pTTl l.
  • Plasmid pTTl l (Teeri et al, Bio/Tech 7:696-699 (1983)) contains 1.8 kb fragment of the ebb 7 region, 3' from the BamUl site in the coding region, cloned into the Bam ⁇ i site of pBR322.
  • the gene can also be isolated from other sources, for example, from a ⁇ clone 44 A (Teeri et al, Bio/Tech 7:696-699).
  • the plasmid obtained was pALK411. It was digested with Seal and with Sphl. The 5.8 kb fragment was ligated to Seal 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 eg/7 cDNA between the promoter and terminator regions of cbb7 gene cloned into pUC18 ( Figure 16).
  • the promoter and terminator regions are from the expression vector pAMHHO (Nevalainen et al, In: Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi, Leong et al, eds., pp. 129-148 (1990)).
  • 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. 5:153-157 (1981)) was transformed with plasmid ⁇ ALK412, with 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.
  • the hypercellulolytic mutant VTT-D-79125 (Bailey, M.J. et al, Enzyme Microb. Technol. 5:153-157 (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.
  • 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.
  • xylanase can be produced as 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.
  • 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.
  • 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.
  • strain 3 the cbb7 gene coding sequence is replaced with the eg/7 gene coding sequence, which in strain 3 is expressed under the cbb7 promoter.
  • 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.
  • the proportion of endoglucanases is increased, and strain 2 produces cellobiohydrolases essentially free of endoglucanases.
  • Example 10 Example 10
  • 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 0 (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 ⁇ m-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 ClO 2 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 O 2 - 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)).
  • ATC AAC TTT GGC GGC TCT TTT AGT GTC AAC AGC GGA ACT GGC CTG CTT 505 lie Asn Phe Gly Gly Ser Phe Ser Val Asn Ser Gly Thr Gly Leu Leu 100 105 110
  • Arg Arg Arg Ala Ser lie Asn Tyr Asp Gin Asn Tyr Gin Thr Gly Gly 50 55 60 Gin Val Ser Tyr Ser Pro Ser Asn Thr Gly Phe Ser Val Asn Trp Asn 65 70 75 80
  • Xaa lie Gin Pro Gly Thr Gly Tyr Asn 1 5

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Abstract

L'invention concerne la structure des gènes xln1 et xln2 de T. reesei et la structure primaire des protéines correspondantes, ainsi que des préparations enzymatiques enrichies en hemicellulase. De telles préparations enzymatiques peuvent être partiellement ou totalement exemptes d'une activité de décomposition de cellulose. De telles préparations peuvent être utilisées sous une forme brute non purifiée et elles sont particulièrement utiles dans la production de pâte à papier et de papier.
EP93910050A 1992-05-29 1993-05-24 Nouvelles preparations enzymatiques et leurs procedes de production Ceased EP0670893A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US88989392A 1992-05-29 1992-05-29
US889893 1992-05-29
PCT/FI1993/000221 WO1993024621A1 (fr) 1992-05-29 1993-05-24 Nouvelles preparations enzymatiques et leurs procedes de production

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EP0670893A1 true EP0670893A1 (fr) 1995-09-13

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EP (1) EP0670893A1 (fr)
JP (1) JPH08500727A (fr)
KR (1) KR950701974A (fr)
AU (1) AU4071893A (fr)
BR (1) BR9306451A (fr)
CA (1) CA2136350C (fr)
WO (1) WO1993024621A1 (fr)

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US7816129B2 (en) 1994-07-29 2010-10-19 Ab Enzymes Gmbh Production and secretion of proteins of bacterial origin in filamentous fungi
CA2209617C (fr) * 1995-01-26 2002-05-28 Novo Nordisk A/S Additifs pour l'alimentation animale, comportant de la xylanase
US6723549B2 (en) 1995-10-17 2004-04-20 Ab Enzymes Oy Cellulases, the genes encoding them and uses thereof
US6184019B1 (en) 1995-10-17 2001-02-06 Röhm Enzyme Finland OY Cellulases, the genes encoding them and uses thereof
ES2525677T3 (es) 1995-10-17 2014-12-29 Ab Enzymes Oy Celulasas, genes que las codifican y usos de las mismas
EP1433843A3 (fr) * 1995-12-18 2005-04-13 AB Enzymes Oy Nouvelles xylanases, gènes les codant, et leurs utilisation
WO1997022691A1 (fr) * 1995-12-18 1997-06-26 Röhm Enzyme Finland OY Nouvelles xylanases et leurs utilisations
US6228629B1 (en) 1995-12-18 2001-05-08 Röhn Enzyme Finland OY Xylanases, genes encoding them, and uses thereof
US6635464B1 (en) 1995-12-18 2003-10-21 Rohm Enzyme Finland Oy Xylanases, genes encoding them, and uses thereof
US6001595A (en) * 1996-11-29 1999-12-14 Rohm Enzyme GmbH Promoters and uses thereof
US6015703A (en) * 1998-03-10 2000-01-18 Iogen Corporation Genetic constructs and genetically modified microbes for enhanced production of beta-glucosidase
AU1783901A (en) * 1999-11-30 2001-06-12 Novo Nordisk Biotech, Inc. Methods for producing a polypeptide using a consensus translational initiator sequence
WO2006016596A1 (fr) * 2004-08-12 2006-02-16 Oji Paper Co., Ltd. Production d'ingrédient fibreux à partir de matériau lignocellulosique et utilisation de ce matériau
DE102004050410A1 (de) 2004-10-15 2006-06-08 Ab Enzymes Gmbh Polypeptid mit Phytaseaktivität und dieses codierende Nucleotidsequenz
DE102006053059A1 (de) 2006-11-10 2008-05-15 Ab Enzymes Gmbh Polypeptid mit Phytaseaktivität und erhöhter Temperaturstabilität der Enzymaktivität sowie dieses codierende Nukleotidsequenz
WO2013176033A1 (fr) * 2012-05-21 2013-11-28 王子ホールディングス株式会社 Fibres fines ainsi que procédé de fabrication de celles-ci, tissu non tissé, et cellulose sous forme de fibres fines
US20200002695A1 (en) * 2017-02-23 2020-01-02 Toray Industries, Inc. Xylanase variant and enzyme composition for decomposing biomass
US20210076704A1 (en) 2017-12-20 2021-03-18 Dsm Ip Assets B.V. Animal feed compositions and uses thereof
BR112021004833A2 (pt) 2018-09-17 2021-06-08 Dsm Ip Assets B.V. composições de ração animal e usos das mesmas

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Publication number Priority date Publication date Assignee Title
FI904214A (fi) * 1990-08-27 1992-02-28 Valtion Teknillinen Hydrolyseringsfoerfarande foer hemicellulosa.
EP1416045A1 (fr) * 1990-10-05 2004-05-06 Genencor International, Inc. Composition contenant de la cellulase fongique pour le traitement de tissus à base de coton

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9324621A1 *

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CA2136350A1 (fr) 1993-12-09
JPH08500727A (ja) 1996-01-30
CA2136350C (fr) 2009-04-07
BR9306451A (pt) 1998-06-30
AU4071893A (en) 1993-12-30
WO1993024621A1 (fr) 1993-12-09
KR950701974A (ko) 1995-05-17

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