HK1098784B - Cbh1 homologs and variant cbh1 cellulases - Google Patents

Description

CBH1 homologue and CBH1 cellulase variant

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to the following U.S. provisional applications: 60/456,368, filed on 21/3/2003 (attorney docket number GC793P), and 60/458,696, filed on 27/3/2003 (attorney docket number GC793-2P), all of which are incorporated herein by reference.

Statement regarding rights to inventions made in federally sponsored research and development project

Part of this work was funded by a subcontract ZCO-0-30017-01 with the U.S. national renewable energy laboratory, which is subject to the basic contract DE-AC36-99GO10337 with the U.S. department of energy. Accordingly, the U.S. government may have certain rights in this invention.

Technical Field

The present invention relates to homologues and variants of Hypocrea jecorina (trichoderma reesei) CBH 1. The present invention relates to isolated nucleic acid sequences encoding polypeptides having cellobiohydrolase (cellobiohydase) activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the recombinant variant CBH polypeptides and novel homologs of hypocrea jecorina CBH 1.

Reference to the literature

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Background

Cellulose (Cellulose) and hemicellulose (hemicelllose) are the most abundant plant materials produced by photosynthesis. They are degraded by many microorganisms and are used as a source of energy, including bacteria, yeasts and fungi, which produce extracellular enzymes capable of hydrolyzing polymeric substrates to monomeric sugars (Aro et al, 2001). The potential of cellulose as the main renewable energy source is enormous due to the limitations of non-renewable resource pathways (Krishna et al, 2001). The efficient use of cellulose by biological methods is one approach to address the shortage of food, feed and fuel (Ohmiya et al, 1997).

Cellulases (celluloases) are enzymes that hydrolyze cellulose (β -1, 4 glucans or β D-glycosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides (cellooligosaccharides) and similar substances. Cellulases have traditionally been divided into three main types: endoglucanases (EC3.2.1.4) ("EG"), exoglucanases (Exoglucanases) or cellobiohydrolases (EC3.2.1.91) ("CBH") and beta-glucosidases ([ beta ] -D-glucoside glucohydrolases; EC 3.2.1.21) ("BG"). (Knowles et al, 1987; Shulein, 1988). Endoglucanases act mainly on the amorphous part of the cellulose fiber, whereas cellobiohydrolases also degrade crystalline cellulose (Nevalainen and Penttila, 1995). Thus, the presence of cellobiohydrolases in cellulase systems is essential for efficient solubilization of crystalline cellulose (Suurnakki, et al 2000). The role of β -glucosidase is to release D-glucose units from cellobiose, cellooligosaccharides and other glycosides (Freer, 1993).

Cellulases are known to be produced by a wide variety of bacteria, yeasts and fungi. Some fungi produce a complete cellulase system capable of degrading cellulose in crystalline form, so that cellulase is readily produced in large quantities by fermentation. Filamentous fungi play a particular role because many yeasts, such as Saccharomyces cerevisiae, lack the ability to hydrolyze cellulose. See, e.g., Aro et al, 2001; aubert et al, 1988; wood et al, 1988, and Coughlan, et al.

The CBH, EG and BG classifications of fungal cellulases can be further refined, including multiple components in each class. For example, a variety of CBHs, EGs and BGs have been isolated from various fungal sources, including trichoderma reesei, which contains the following known genes: 2 CBHs, CBH1 and CBHII, at least 8 EGs, EG I, EG II, EG III, EG IV, EGV, EGVI, EGVII and EGVIII, and at least 5 BGs, BG1, BG2, BG3, BG4 and BG 5.

In order to efficiently convert crystalline cellulose to glucose, a complete cellulase system is necessary, which includes components from each of the CBH, EG and BG classes, whereas isolated components are less effective at hydrolyzing crystalline cellulose (Filho et al, 1996). It has been observed that there is a synergistic relationship between different classes of cellulose components. In particular, EG-type cellulases and CBH-type cellulases synergistically interact to more efficiently degrade cellulose. See, for example, Wood, 1985.

Cellulases are known in the art to be useful in the treatment of fabrics, for enhancing the cleaning ability of detergent compositions, as softeners, for improving the feel and appearance of cotton fabrics, and the like (Kumaret al, 1997).

Cellulase-containing detergent compositions (U.S. Pat. Nos. 4,435,307; British applications Nos. 2,095,275 and 2,094,826) with improved cleaning properties have been described, as well as cellulase-containing detergent components for fabric treatment to improve The feel and appearance of fabrics (U.S. Pat. Nos. 5,648,263, 5,691,178 and 5,776,757; British application No. 1,358,599; The Shizuoka prefecture Hammmamatsu textile Industrial Research Report, Vol.24, pp.54-61, 1986).

Thus, cellulases produced in fungi and bacteria have received attention. In particular, Trichoderma spp (e.g. Trichoderma longibrachiatum) or Trichoderma reesei (Trichoderma reesei) has been shown to ferment to produce complete cellulase systems capable of degrading crystalline forms of cellulose.

Although cellulosic compositions have been described previously, there remains a need for new and improved cellulosic compositions which will be useful in household detergents, stonewashing compositions or laundry detergents, among others. Cellulases that exhibit improved performance are particularly valuable.

Summary of The Invention

The present invention provides isolated cellulase proteins, referred to herein as desired cellulases, and nucleic acids encoding the desired cellulases. The desired cellulase may be selected from the group consisting of variant CBH1 from Hypocrea jecorina and novel CBH1 from Hypocrea schwerinae (Hypochrea schweinitzii), Hypocrea orientalis (Hypochea orientalis), Trichoderma pseudokoningii (Trichoderma pseudokoningii) or Trichoderma viride (Trichoderma konilangbra).

Provided are variant CBH1 cellulases (CBH1 variants), wherein the variants comprise substitutions or deletions at positions corresponding to one or more of the following residues: l6, S8, P13, Q17, G22, T24, Q27, T41, S47, N49, T59, T66, A68, C71, A77, G88, N89, A100, N103, A112, S113, L125, T160, Y171, Q186, E193, S195, C210, M213, L225, T226, P227, T232, E236, E239, G242, T246, D249, N250, R251, Y252, D257, D259, S278, T281, L288, E354, T296, S297, A299, N301, F311, L318, E325, N336, D394, T336, A394, S341, S352, F356, K354, T356, T295, T359, N368, T368, P277, P384, P277, N384, P277, P18, P384, and P384 from hypocrea CBH 1.

In another aspect, the variant CBH1 comprises a substitution at a position corresponding to one or more of the following residues: q186(E), S195(A/F), E239S, G242(H/Y/N/S/T/D/A), D249(K/L/Y/C/I/V/W/T/N/M), E325(S/T), T332(A/H/Y/L/K), and P412 (T/S/A).

In a second embodiment, the present invention provides hypocrea orientalis CBH 1.

In a third embodiment, the present invention provides hypocrea schweinez CBH 1.

In a fourth embodiment, trichoderma koningii CBH1 is provided.

In a fifth embodiment, Trichoderma pseudokoningii CBH1 is provided.

In another embodiment of the invention, nucleic acids encoding the desired cellulases of the invention are provided. In another embodiment, the DNA is in a vector. In a further embodiment, the vector is used to transform a host cell.

In another embodiment of the invention, a method for producing a desired cellulase of the invention is provided. The method comprises the steps of culturing a host cell transformed with a nucleic acid encoding a desired cellulase in a suitable culture medium under conditions suitable for the production of the desired cellulase and obtaining the desired cellulase so produced.

In yet another embodiment of the invention, a detergent is provided comprising a surfactant and a desired cellulase. In one aspect of the invention, the detergent is a laundry detergent or a dish detergent. In another aspect of the invention, the desired CBH1 cellulase is used in the treatment of cellulose containing fabrics, in particular, for stonewashing or indigo dyed denim. Alternatively, the cellulase of the invention may be used as a feed additive for wood pulp treatment and for the reduction of biological material (bioglass) to glucose.

Brief Description of Drawings

FIG. 1 shows the nucleic acid sequence (lower row) (SEQ ID NO: 1) and the amino acid sequence (upper row) (SEQ ID NO: 2) of wild-type Cel7A (CBH1) of Hypocrea jecorina.

FIGS. 2A and 2B show the amino acid alignment of hypocrea jecorina (also known as Trichoderma reesei) (SEQ ID NO: 2), hypocrea orientalis (SEQ ID NO: 5), Sarcodon schweinezii (SEQ ID NO: 8), Trichoderma koningii (SEQ ID NO: 11), and Trichoderma pseudokoningii (SEQ ID NO: 14) members of the Cel7A family. Consensus sequences are also shown.

FIG. 3 is the genomic DNA sequence of hypocrea orientalis CBH1 (SEQ ID NO: 3). Introns are in bold and underlined.

FIG. 4 is the signal sequence (4A) (SEQ ID NO: 4) and the mature amino acid sequence (4B) (SEQ ID NO: 5) of Hypocrea orientalis CBH 1.

FIG. 5 is the genomic DNA sequence of hypocrea schwernicki CBH1 (SEQ ID NO: 6). Introns are in bold and underlined.

FIG. 6 is the signal sequence (6A) (SEQ ID NO: 7) and the mature amino acid sequence (6B) (SEQ ID NO: 8) of hypocrea schweinez CBH 1.

FIG. 7 is the genomic DNA sequence of Trichoderma koningii CBH1 (SEQ ID NO: 9). Introns are in bold and underlined.

FIG. 8 is the signal sequence (8A) (SEQ ID NO: 10) and the mature amino acid sequence (8B) (SEQ ID NO: 11) of Trichoderma reesei CBH 1.

FIG. 9 is the genomic DNA sequence of Trichoderma pseudokoningii CBH1 (SEQ ID NO: 12). Introns are in bold and underlined.

FIG. 10 is the signal sequence (10A) (SEQ ID NO: 13) and the mature amino acid sequence (10B) (SEQ ID NO: 14) of Trichoderma pseudokoningii CBH 1.

FIG. 11 is pRAX1 vector. The vector was based on the plasmid pGAPT2, except that a 5259bp HindIII fragment (Molecular Microbiology 199619: 565-574) of the sequence of the genomic DNA fragment AMA1 of Aspergillus nidulans (Aspergillus nidulans) was inserted. Bases 1-1134 comprise the Aspergillus niger glucoamylase gene promoter. Bases 3098 to 3356 and 4950 to 4971 comprise the A.niger glucoamylase terminator. The A.nidulans pyrG gene was inserted in 3357 to 4949 as a marker (marker) for fungal transformation. There is a Multiple Cloning Site (MCS) into which a gene can be inserted.

FIG. 12 is the pRAXdes2 vector backbone. This vector was based on the plasmid vector pRAX 1. The entry cassette has been inserted into pRAX1 vector (indicated by the arrow inside the circular plasmid). This cassette contains the recombination sequences attR1 and attR2 and the selectable markers (marker) catH and ccdB. The vector is according to Gateway^TMThe guidelines in the first edition of Cloning Technology, pages 34-38, are prepared and can only be replicated in Escherichia coli DB3.1 of invitrogen; in other E.coli hosts, the ccdB gene is lethal. First, PCR fragments were obtained using primers containing attB1/2 recombination sequences. This fragment was recombined with pDONR201 (available from Invitrogen corporation); this vector contains attP1/2 recombination sequences, with catH and ccdB located between the recombination sites. The PCR fragment was recombined into a vector called ENTRY using the BP clonase enzyme of invitrogen, and clones with the inserted PCR fragment were treated with 50. mu.g `Kanamycin was selected out because ccdB expressing clones did not survive. Now, att sequences are changed and are referred to as attL1 and attL 2. The second step is to recombine the clone with pRAXdes2 vector (containing attR1 and attR2catH and ccdB between recombination sites). The insert from the ENTRY vector was recombined into a target vector (destination vstor) using LP clonase enzyme of invitrogen. Using 100. mu.g/ml ampicillin, only pRAXCBH1 vector was selected because ccdB was lethal and the ENTRY vector was sensitive to ampicillin. By this method, expression vectors are now prepared and can be used to transform A.niger.

FIG. 13 provides an illustration of the pRAXdes2CBH1 vector used to express nucleic acid encoding a CBH1 homologue or a CBH1 variant in Aspergillus. Nucleic acids encoding homologues or variants of the CBH1 enzyme are cloned into the vector by homologous recombination of att sequences.

Detailed Description

The invention will now be described in detail by reference to the following definitions and examples. All patents and publications, including sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al, DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley AND Sons, New York (1994) AND Hale & Marham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, NY (1991) provide the skilled artisan with a general explanation of many of the terms used in the present invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numerical ranges are inclusive of the endpoints used to define the range. Unless otherwise indicated, nucleic acids are written from left to right in the 5 '-3' direction; amino acid sequences are written from left to right in the direction from amino terminus to carboxy terminus. With regard to definitions and terminology in this field, the practitioner may refer in particular to Sambrook et al, 1989 and Ausubel FM et al, 1993. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

The headings set forth herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below may be more fully defined by reference to the specification as a whole.

All publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing the compositions and methodologies that might be used in connection with the invention.

1. Definition of

"cellulases" (cellulases), "cellulolytic enzymes" (cellulolytic enzymes) or "cellulases" (cellulases) refer to exoglucanases (exoglucanases) or exocellobiohydrolases (exocellobiohydrolases) and/or endoglucanases (endoglucanases) and/or beta-glucosidases of bacteria or fungi. These three different forms of cellulase enzymes act synergistically to convert cellulose and its derivatives to glucose.

Many microorganisms produce enzymes that hydrolyze cellulose, including Trichoderma saprophyticus (Trichoderma), the composting bacteria Thermomonospora (Thermomonospora), Bacillus (Bacillus), and Cellulomonas (Cellulomonas); streptomyces (Streptomyces); and the fungi Humicola (Humicola), Aspergillus (Aspergillus) and Fusarium (Fusarium). The enzymes produced by these microorganisms are a mixture of proteins with three types of functions: endoglucanases (EG), Cellobiohydrolases (CBH), and beta-glucosidases, which are useful in the conversion of cellulose to glucose.

As used herein, "desired cellulase" refers to any of the following:

a) CBH1 variants derived from hypocrea jecorina according to the invention;

b) CBH1 homolog from hypocrea orientalis;

c) a CBH1 homolog derived from hypocrea schwerinae;

d) CBH1 homolog from trichoderma koningii;

e) CBH1 homolog from trichoderma pseudokoningii; and

f) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid encoding any one of a-e.

As used herein, "nucleic acid encoding a desired cellulase" ("desired cellulose-encoding nucleic acid") refers to any one of the following:

a) a nucleic acid encoding a CBH1 variant derived from hypocrea jecorina according to the invention;

b) a nucleic acid encoding a CBH1 homologue derived from hypocrea orientalis having the sequence shown in figure 3;

c) a nucleic acid encoding a CBH1 homologue derived from hypocrea schwerinae having the sequence shown in figure 5;

d) a nucleic acid encoding a CBH1 homologue derived from trichoderma koningii having the sequence shown in figure 7;

e) nucleic acid encoding a CBH1 homologue from trichoderma pseudokoningii, having the sequence shown in figure 9and

f) a nucleic acid that hybridizes under stringent conditions to any of the nucleic acids described in a-e above.

"variant" refers to a protein derived from a precursor protein (e.g., a native protein) that is produced by substitution of one or more amino acids at one or a number of different positions in the amino acid sequence. Preparation of enzyme variants is preferably accomplished by modifying a DNA sequence encoding the native protein, transforming the DNA sequence into a suitable host, and expressing the modified DNA sequence to form the derivative enzyme or enzyme variant. The CBH1 enzyme variants of the invention include peptides that contain an altered amino acid sequence compared to the amino acid sequence of the precursor enzyme, wherein the variant CBH enzyme retains the characteristic fiber hydrolyzing properties of the precursor enzyme, but may have altered properties in some particular aspects. For example, a variant CBH enzyme may have an increased pH optimum, or have increased temperature or oxidative stability, but still retain its characteristic cellulolytic activity.

As used herein, the term "gene" refers to a segment of DNA involved in the production of a polypeptide chain, which may or may not include regions preceding and following the coding region, e.g., the 5 ' untranslated region (5 ' UTR) or "leader" sequence and the 3 ' UTR or "trailer" sequence, and may or may not include intervening sequences (introns) between individual coding segments (exons).

The "filamentous fungi" of the invention are eukaryotic microorganisms, including all filamentous forms of the subdivision Eumycotina (see Alexopodos, C.J. (1962), Introductry Mycology, New York: Wiley). These fungi are characterized by vegetative hyphae with a cell wall composed of chitin, cellulose and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon metabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular explant and carbon metabolism may be fermentative. Saccharomyces cerevisiae has a pronounced, very stable diploid phase, whereas in filamentous fungi, such as Aspergillus and Neurospora, diploids only occur shortly before meiosis. Although in some cases yeast may show pseudohyphal growth (pseudohyphal growth), it should be understood that this does not make yeast fall under the definition of filamentous fungi. Saccharomyces cerevisiae has 17 chromosomes, while aspergillus nidulans (a. nidulans) and neurospora crassa (n. crassa) have 8 and 7, respectively. Further examples of differences between s.cerevisiae and filamentous fungi include the inability of s.cerevisiae to process Aspergillus and Trichoderma introns and the inability to recognize many transcriptional regulators of filamentous fungi (Innis, M.A. et al (1985) Science, 228, 21-26).

The term "heterologous" when used in reference to a nucleic acid moiety means that the nucleic acid contains 2 or more subsequences that are not normally found in the same relationship to each other in nature. For example, the nucleic acid is typically produced recombinantly, with, for example, 2 or more sequences derived from unrelated genes, arranged to form a nucleic acid with novel functions, e.g., the sequences can be a promoter from one source and a coding region from another source. Similarly, a heterologous protein often involves two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

A "heterologous" nucleic acid construct or sequence has a portion of a sequence not naturally associated with the cell in which it is expressed. Heterologous sequence with respect to regulatory sequences refers to a control sequence (i.e., promoter or enhancer) that regulates the expression of a gene, but in nature it does not regulate the expression of the gene it now regulates. Typically, the heterologous nucleic acid sequence is not endogenous to the cell or genomic portion in which it is deposited, but is added to the cell by infection, transfection, transformation, microinjection, electroporation, or the like. A "heterologous" nucleic acid construct can contain a regulatory sequence/DNA coding sequence combination that can be the same as or different from the regulatory sequence/DNA coding sequence combination found in the native cell.

As used herein, the term "isolated" or "purified" refers to a nucleic acid or amino acid that is separated from at least one component with which it is naturally associated.

As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. Promoters are generally suitable for use in host cells expressing a target gene. Promoters and other transcription regulating nucleic acid sequences and translation regulating nucleic acid sequences (also referred to as "regulatory sequences") are necessary for the expression of a particular gene. Generally, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancer or activator sequences.

Generally, a "promoter sequence" is a DNA sequence that is recognized by a particular filamentous fungus for expression purposes. A "constitutive" promoter is a promoter that is active under most environmental or developmental conditions. An "inducible" promoter is a promoter that is activated under environmental or developmental regulation. An example of an inducible promoter useful in the present invention is the Trichoderma reesei (hypocrea jecorina) cbh1 promoter, whose Accession Number in GenBank is D86235. In another aspect, the promoter is cbh II from hypocrea jecorina or a xylanase promoter.

Representative promoters include genes derived from Aspergillus awamori (A.awamori) or Aspergillus niger glucoamylase (Nunberg, J.H.et al (1984) mol.cell.biol.4, 2306-2315; Boel, E.et al (1984) EMBO J.3, 1581-1585), Mucor miehei (Mucor miehei) carboxyprotease, Rhodosarcobacter xylinum cellobiohydrolase I (Shoemaker, S.P.et al (1984) European patent application EPO0137280A1), Aspergillus nidulans trp Gene (Yelton, M.et al (1984) Proc.Natl.Acad.Sci.USA 81, 1470-1474; Mullaney, E.J.al (1985) mol.Genlton.199, 37-45), Aspergillus nidulans.149, Aspergillus niger Gene (1985) Gene J.137-137, cell.31, 1984), Saccharomyces cerevisiae (1984) Gene K.31, Aspergillus nidulans.31, Cell K.31, C.31, Na-11, Na-32, K.16, K.32, E.35, E.A.A.35, E.E.E.E.E.E.E.35, E.E.E.35, E.A.E.E.E.E.E.E.E.E.E.E.E.E.E.E., Hypocrea jecorina cbh2 gene, hypocrea jecorina egl gene, hypocrea jecorina eg2 gene, hypocrea jecorina eg3 gene, and higher eukaryotic promoters such as SV40 early promoter (Barclay, s.l. and e.meller (1983) Molecular and Cellular Biology 3.2117-2130).

A nucleic acid is "operably linked" when it is placed in functional communication with another nucleic acid sequence. For example, if a polypeptide is expressed as a preprotein that participates in the secretion of the polypeptide, then DNA encoding a secretagogue leader sequence, i.e., a signal peptide, is operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; alternatively, a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Ligation may be accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide linkers or linkers can be used in accordance with conventional practice. Thus, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, or a string of transcription factor binding sites) and another nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the other sequence.

As defined herein, a "chimeric gene" or "heterologous nucleic acid construct" refers to a non-native gene (i.e., a gene that has been introduced into a host), which may be composed of different gene portions, including regulatory elements. Typically, the chimeric gene construct used to transform the host cell includes a transcriptional regulatory region (promoter) operably linked to a heterologous protein coding sequence, or, in the case of a selectable marker chimeric gene, to a selectable marker gene that encodes a protein that confers antibiotic resistance to the transformed cell. Exemplary chimeric genes for transformation into a host cell of the invention include, constitutive or inducible transcriptional regulatory regions, protein coding sequences, and terminator sequences. The chimeric gene construct may also include another DNA sequence encoding a signal peptide if secretion of the target protein is desired.

The term "recombinant", when used in reference to, for example, a cell or a nucleic acid, protein, vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene that is not found in the native (non-recombinant) form of the cell, or expresses a native gene, but otherwise is abnormally expressed, under expressed, or not expressed at all.

The term "secretagogue signal sequence" refers to a DNA sequence encoding a polypeptide ("secretagogue peptide") that is part of a larger polypeptide that is directed through the secretory pathway of the cell in which it is synthesized. During passage through the secretory pathway, the larger peptide is typically cleaved to remove the secretagogue peptide.

As used herein, the phrases "holocellulase preparation" and "holocellulase composition" are used interchangeably and refer to both naturally occurring and non-naturally occurring compositions. A "naturally occurring" composition is produced by a naturally occurring source that contains one or more cellobiohydrolase types, one or more endoglucanase types, and one or more beta-glucosidase components, wherein each of these components is found in the ratios produced by the source. Naturally occurring compositions are those produced by organisms that are unmodified with respect to the cellulolytic enzymes, and therefore the ratio of enzyme components is not altered from that produced by the natural organism.

"non-naturally occurring" compositions include those produced by: (1) combining the fiber hydrolyzing enzyme components at a naturally occurring rate or at a non-naturally occurring rate, i.e., a modified rate; or (2) modifying the organism to overexpress or inhibit the expression of one or more cellulolytic enzymes; or (3) modifying the organism such that at least one cellulolytic enzyme is knocked out.

"equivalent residues" can also be defined by determining homology at the level of the tertiary structure of the precursor cellulase, which has been determined by X-ray crystallography. Equivalent residues are defined as residues for which the atomic coordinates of 2 or more of the backbone atoms (N to N, CA to CA, C to C and O to O) of a particular amino acid residue are within 0.13nm, preferably within 0.1nm, after alignment (alignment) of cellulase and hypocrea jecorina CBH. In discussing hypocrea jecorina CBH1, the association was achieved after the best model had been oriented and positioned to obtain the maximum overlap of atomic coordinates of the non-hydrogen protein atoms of the cellulase. The best mode is the crystallographic model that gives the lowest R-factor for the diffraction experimental data at the highest resolution available.

Equivalent residues functionally similar to the specific residues of hypocrea jecorina CBH1 are defined as those amino acids of the cellulase which may adopt a conformation such that they may alter, modify or influence the structure, substrate binding or catalysis of the protein in a defined manner which contributes to the specific residues of hypocrea jecorina CBH 1. Further, they are those residues of cellulases (the tertiary structures of which have been obtained by X-ray crystallography) which occupy similar positions to the extent that, although the main chain atoms of a given residue may not meet the equivalence criterion based on occupying homologous positions, the atomic coordinates of at least 2 of the side chain atoms of the residue lie within 0.13nm of the corresponding side chain atoms of hypocrea jecorina CBH.

The term "nucleic acid molecule" includes RNA, DNA and cDNA molecules. It will be appreciated that, due to the degeneracy of the genetic code, multiple nucleotide sequences encoding a given protein, such as CBH1, may be produced. The present invention encompasses every possible variant nucleotide sequence that encodes CBH1, all of which may be given the degeneracy of the genetic code.

As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector having the ability to integrate a heterologous DNA segment and express the heterologous DNA segment in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The choice of an appropriate vector is within the knowledge of the person skilled in the art.

Thus, an "expression cassette" or "expression vector" is a nucleic acid construct, produced recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter.

As used herein, the term "plasmid" refers to a circular double-stranded (ds) DNA construct that is used as a cloning vector and forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotic cells.

As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleic acid sequence that is capable of being expressed in a cell, and expression of the selectable marker enables the cell containing the expressed gene to be grown in the presence of a corresponding selective agent or under corresponding selective growth conditions.

Generally, under conditions of moderate to high stringency (stringency), a nucleic acid molecule encoding variant CBH1 will be as provided herein as SEQ ID NO: 1 (native hypocrea jecorina CBH 1). However, in some cases, a nucleotide sequence encoding CBH1 with substantially different codon usage is used, while the protein encoded by the nucleotide sequence encoding CBH1 has the same or substantially the same amino acid sequence as the native protein. For example, depending on the frequency with which particular codons are used by the host, the coding sequence may be modified to facilitate faster expression of CBH1 in particular prokaryotic or eukaryotic expression systems, e.g., Te' o, et al (2000) describes an optimized way to express genes in filamentous fungi.

One nucleic acid is considered "selectively hybridizable" to another, reference nucleic acid if the two sequences specifically hybridize to each other under moderate to high stringency hybridization and elution conditions. The hybridization temperature is based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5 ℃ (5 ℃ below the Tm of the probe); "high stringency" occurs at about 5-10 ℃ below Tm; "moderate" or "intermediate stringency" occurs at about 10-20 ℃ below the Tm of the probe; "Low stringency" occurs at about 20-25 ℃ below the Tm. Functionally, maximum stringency conditions can be used to identify sequences that have stringent or near stringent identity to the hybridizing probe, while high stringency conditions are used to identify sequences that have about 80% or more sequence identity to the probe.

Moderately stringent and highly stringent hybridization conditions are well known in the art (see, e.g., Sambrook, et al, 1989, Chapters 9and 11, and Ausubel, f.m., et al, 1993, expressly incorporated herein by reference). Examples of highly stringent conditions include hybridization at about 42 ℃ in 50% formamide, 5 XSSC, 5 XDenhardt's solution, 0.5% SDS and 100. mu.g/ml denatured vector DNA followed by 2 washes with 2 XSSC and 0.5% SDS at room temperature and 2 additional washes with 0.1 XSSC and 0.5% SDS at 42 ℃.

As used herein, the terms "transformed", "stably transformed" or "transgenic" when used to describe a cell means that the cell has a non-native (heterologous) nucleic acid sequence that is integrated into the genome of the cell or acts as an episomal plasmid that is maintained through multiple generations.

As used herein, the term "expression" refers to the process of producing a polypeptide from the nucleic acid sequence of a gene. This process includes both transcription and translation.

The term "introduced" when referring to the insertion of a nucleic acid sequence into a cell, means "transfection" or "transformation" or "transduction" and includes the integration of a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

Next, the term "expression of a desired cellulase" refers to the transcription and translation of a desired cellulase gene, the products of which include precursor RNA, mRNA, polypeptide, post-translationally processed polypeptide. By way of example, analyses of CBH1 expression include Western blot (Western blot) of CBH1 protein, Northern blot (Northern blot) and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of CBH1mRNA, and endoglucanase activity analyses, as described in Shoemaker S.P.and Brown R.D.Jr. (Biochim. Biophys.acta, 1978, 523: 133-146) and Schulen (1988).

The term "alternative splicing" refers to the process of producing multiple polypeptide isoforms from a single gene, which involves splicing together discrete exons, which occurs during the processing of some, but not all, of the gene transcript. Thus a particular exon can be associated with any of several alternative exons to form messenger RNA. Alternatively spliced mrnas produce polypeptides ("splice variants"), some of which are common and others of which are different.

The term "host cell" refers to a cell that contains a vector and supports the replication and/or transcription and translation (expression) of an expression construct. The host cell used in the present invention may be a prokaryotic cell, such as E.coli, or a eukaryotic cell, such as a yeast, plant, insect, amphibian, or mammalian cell. Typically, the host cell is a filamentous fungus.

The term "cellulase" refers to a species of enzyme that is capable of hydrolyzing cellulose polymers to shorter cellooligosaccharide oligomers, cellobiose, and/or glucose. Many examples of cellulases, such as exoglucanases, exocellobiohydrolases, endoglucanases and glucosidases, have been obtained from cellulose-hydrolysable organisms, including fungi, plants and bacteria, among others.

CBH1 from hypocrea jecorina is a member of glycosyl hydrolase family 7(Cel7), specifically, the first member of this family (Cel7) identified in hypocrea jecorina. Glycosyl hydrolases family 7 contains both endoglucanases and cellobiohydrolases/exoglucanases, the latter being CBH 1. Thus, the phrases CBH1, CBH 1-type protein and Cel7 cellobiohydrolase are used interchangeably herein.

As used herein, the term "cellulose binding domain" refers to a portion of the amino acid sequence of a cellulase enzyme, or a region of the enzyme, which is involved in the cellulose binding activity of the cellulase enzyme or a derivative thereof. The cellulose binding domain or module generally functions by non-covalently binding the cellulase to cellulose, cellulose derivatives, or other polysaccharide equivalents thereof. The cellulose binding domains allow or contribute to the hydrolysis of cellulose fibres by structurally distinct catalytic core regions and, typically, they act independently of the catalytic core. Thus, the cellulose binding domain does not have significant hydrolytic activity, which is attributable to the catalytic core. In other words, the cellulose binding domain is a structural element of the tertiary structure of the cellulase protein that is different from the structural element having catalytic activity.

As used herein, the term "surfactant" refers to any compound that is generally recognized in the art as having surface active properties. Thus, for example, surfactants include anionic, cationic and nonionic surfactants such as those commonly found in detergents. Anionic surfactants include linear or branched alkyl benzene sulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl or alkenyl groups; alkyl or alkenyl sulfates; olefin sulfonates and alkyl sulfonates. Amphoteric surfactants include quaternary ammonium sulfonates and betaine-type amphoteric surfactants. Such ampholytic surfactants have both positively and negatively charged groups in the same molecule. Nonionic surfactants can include polyethylene glycols, as well as higher fatty acid alkanolamides or alkylene oxide adducts thereof, fatty acid monoglycerides, and the like.

As used herein, the term "cellulose containing fabric" refers to any sewn or unsewn fabric, yarn or fiber, made from cotton or non-cotton containing cellulose, or from a cotton or non-cotton containing cellulose blend including natural cellulose and man-made cellulose (such as jute, flax, ramie, rayon and lyocell).

As used herein, the term "cotton-containing fabric" refers to sewn or unsewn fabrics, yarns or fibers made from pure cotton or cotton blends including cotton fabrics (cotton woven fabrics), cotton knits (cotton knits), denim fabrics (cotton denims), cotton yarns (cotton yarns), raw cotton, and the like.

As used herein, the term "stonewashing composition" refers to a formulation used for stonewashing cellulose-containing fabrics. Stonewashing compositions are used to modify cellulose-containing fabrics prior to sale, i.e., during the manufacturing process. In contrast, detergent compositions are primarily used for cleaning soiled garments, not in the manufacturing process.

As used herein, the term "detergent composition" refers to a mixture intended for use in a washing medium for washing soiled cellulose-containing fabrics. For purposes of the present invention, such compositions may include, in addition to cellulase and surfactant, additional hydrolytic enzymes, builders, bleaching agents, bleach catalysts, blue bleaching agents (blunting agents) and fluorescent dyes, caking inhibitors, masking agents (masking agents), cellulase activators, antioxidants, and solubilizing agents.

As used herein, the term "reduction or elimination of CBH1 gene expression" means that the CBH1 gene has been deleted from the genome and thus cannot be expressed by a recombinant host microorganism, or that the CBH1 gene has been modified such that a functional CBH1 enzyme cannot be produced by the host microorganism.

The term "variant CBH1 gene", or "variant CBH 1", respectively, means that the nucleic acid sequence of the CBH1 gene from hypocrea jecorina has been altered by removal, addition and/or manipulation of the coding sequence, or that the amino acid sequence of the expressed protein has been modified, in accordance with the invention described herein.

As used herein, the term "active" or "bioactive" refers to a biological activity associated with a particular protein, which are used interchangeably herein. For example, the enzymatic activity associated with a protease is proteolytic, and thus, the active protease has proteolytic activity. Thus, the biological activity of a particular protein refers to any biological activity that one of skill in the art would normally consider the protein to possess.

When applied to an enzyme solution, the homologous or variant CBH1 component is generally added in an amount sufficient to allow the soluble sugars to be released from the biological material at a maximum rate. The amount of CBH1 homologue or variant added depends on the type of biological material to be saccharified, which can be readily determined by the skilled person. However, when applied, the weight percent of CBH1 homolog or variant component relative to the EG-type component present in the cellulase composition is from preferably about 1, preferably about 5, preferably about 10, preferably about 15 or preferably about 20 weight percent to preferably about 25, preferably about 30, preferably about 35, preferably about 40, preferably about 45 or preferably about 50 weight percent. Further, preferred ranges may be from about 0.5 to about 15 weight percent, about 0.5 to about 20 weight percent, from about 1 to about 10 weight percent, from about 1 to about 15 weight percent, from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, From about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 20 weight percent, from about 15 to about 25 weight percent, from about 15 to about 30 weight percent, from about 15 to about 35 weight percent, from about 15 to about 40 weight percent, from about 15 to about 45 weight percent, from about 15 to about 50 weight percent.

Host organism

Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota. Filamentous fungi are characterized by vegetative hyphae, which have cell walls composed of chitin, glucan, chitosan, mannan, and other complex polysaccharides, vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic.

In the present invention, the filamentous fungal parent cell may be a cell of the following species-but not limited to these species-: trichoderma, such as Trichoderma longibrachiatum (Trichoderma longibrachiatum), Trichoderma viride (Trichoderma viride), Trichoderma koningii (Trichoderma koningii), Trichoderma harzianum (Trichoderma harzianum); penicillium sp. (Penicillium sp.); humicola species (Humicola sp.) including Humicola anova (Humicola insolens) and Humicola grisea (Humicola grisea); chrysosporium sp (Chrysosporium sp.) including Chrysosporium lucknowense (c.lucknowense); gliocladium sp.); an Aspergillus species; fusarium sp, Neurospora sp, Hypocrea sp, and Emericella sp. As used herein, the term "trichoderma" or "trichoderma spp" refers to any fungal strain that has previously been classified into the genus trichoderma or any fungal strain that is currently classified into the genus trichoderma.

In a preferred embodiment, the filamentous fungal parent cell is an Aspergillus niger, Aspergillus awamori, Aspergillus aculeatus or Aspergillus nidulans cell.

In another preferred embodiment, the filamentous fungal parent cell is a Trichoderma reesei cell.

Cellulase III

Cellulases are considered in the art to be enzymes that hydrolyze cellulose (β -1, 4 glucan or β D-glucoside linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides and the like. As previously set forth, cellulases have traditionally been divided into three main types: endoglucanases (EC3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC3.2.1.91) ("CBH") and beta-glucosidases (EC 3.2.1.21) ("BG") (Knowles, et al, 1987; Schulen, 1988).

Some fungi produce complete cellulase systems including exo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-type cellulases, beta-glucosidases or BG-type cellulases (Schulein, 1988). However, sometimes these systems lack CBH-type cellulases, and bacterial cellulases generally also contain little or no CBH-type cellulases. Furthermore, it has been shown that the EG component and CBH component interact synergistically to degrade cellulose more efficiently. See, for example, Wood, 1985. The different components of a multi-component or complete cellulase system, i.e. the various endoglucanases and exo-cellobiohydrolases, typically have different properties, such as isoelectric point, molecular weight, degree of glycosylation, substrate specificity and mode of enzyme action.

It has also been shown that cellulase compositions can degrade cotton-containing fabrics, resulting in a diminished strength loss of the fabric (us patent 4,822,516), which has led to a reluctance to use cellulase compositions in commercial detergent applications. It has been suggested that cellulase compositions comprising an endoglucanase component show a reduced strength loss on cotton-containing fabrics compared to compositions comprising an intact cellulase system.

Cellulases have also been shown to be useful in the degradation of cellulosic biomass to ethanol (where cellulases degrade cellulose to glucose and yeast or other microorganisms further glycolyze glucose to ethanol), in mechanical pulp processing (Pere et al, 1996), as feed additives (WO 91/04673) and in grain wet milling (wet milling).

Most CBH and EG have a multidomain structure consisting of a Cellulose Binding Domain (CBD) and a core domain separated by a linker peptide (surnakki et al, 2000). The core domain contains the active site, while the CBD interacts with cellulose by binding the enzyme to cellulose (van Tilbeurgh et al, 1986; Tomme et al, 1988). CBD is particularly important in the hydrolysis of crystalline cellulose. It has been shown that cellobiohydrolases have a significantly reduced ability to degrade crystalline cellulose when CBD is absent (Linder andteeeri, 1997). However, the exact action and mechanism of action of CBD remains a corollary. CBD has been proposed to enhance enzyme activity only by increasing the effective enzyme concentration on the cellulose surface (Stahlberg et al, 1991), and/or by unwinding individual cellulose chains from the cellulose surface (Tormo et al, 1996). Most studies involving the action of cellulase domains on different substrates were conducted using the core protein of cellobiohydrolases, since their core protein can be readily produced by limited proteolysis by papain (Tomme et al, 1988). Many cellulases have been described in the scientific literature, examples of which include: from Trichoderma reesei: shoemaker, s.et al, Bio/Technology, 1: 691-696, 1983, which discloses CBHI; teeri, t.et al, Gene, 51: 43-52, 1987, which discloses CBHII. Cellulases from other species than Trichoderma have also been described, for example Ooi et al, 1990 disclosing cDNA sequences encoding the endoglucanase F1-CMC produced by A.aculeatus; kawaguchi T et al, 1996, which discloses cloning and sequencing of cDNA encoding β -glucosidase from aspergillus aculeatus; sakamoto et al, 1995, which discloses a cDNA sequence encoding endoglucanase CMCase-1 of Aspergillus kawachii (Aspergillus kawachii) IFO 4308; saarilahti et al, 1990, disclosing endoglucanases from Erwinia carotovora; spilleaert R, et al, 1994, which discloses the cloning and sequencing of bglA, which encodes a thermostable β -glucanase of Rhodothermus marinus; halldorsdottir S et al, 1998, discloses the cloning, sequencing and overexpression of the Rhodothermus marinus gene, which encodes a thermostable cellulase of glycosyl hydrolase family 12. However, there is still a need to identify and characterize novel cellulases that should have improved properties, such as improved performance under heat stress or in the presence of surfactants, increased specific activity, altered substrate cleavage pattern and/or high level expression in vitro.

The development of new and improved cellulase compositions containing different levels of CBH type cellulases is valuable for the following uses: (1) for use in detergent compositions which exhibit enhanced cleaning ability, act as softeners and/or improve the feel of cotton fabrics (e.g., "stone-washing" or "biopolishing"), (2) for use in compositions for degrading wood pulp or other biological materials to sugars (e.g., for bioethanol production), and/or (3) for use in feed compositions.

IV molecular biology

In one embodiment, the invention provides for expressing a desired cellulase gene under the control of a promoter functional in a filamentous fungus. The present invention thus relies on conventional techniques in the field of recombinant genetics. Basic documents disclosing general methods for the present invention include: sambrook et al, Molecular Cloning, A Laboratory Manual (2nd ed.1989); kriegler, Gene Transfer and Expression: ALaborory Manual (1990), and Ausubel et al, eds., Current Protocols in molecular biology (1994).

A. Method for identifying homologous CBH1 genes

The nucleic acid sequence of wild type hypocrea jecorina CBH1 is shown in fig. 1. The invention, in one aspect, includes nucleic acid molecules encoding a CBH1 homolog as described herein. The nucleic acid may be a DNA molecule.

Techniques that can be used to isolate DNA sequences encoding homologous CBH1 are well known in the art, including, but not limited to, eDNA library screening and/or genomic library screening with homologous DNA probes, expression screening with activity assays or anti-CBH 1 antibodies. Any of these methods can be found IN Sambrook, et al, or Current PROTOCOLS IN MOLECULAR BIOLOGY, F.Ausubel, et al, ed.Greene Publishing and Wiley-Interscience, New York (1987) ("Ausubel").

B. Method for mutating CBH nucleic acid sequence

Any method known in the art that can introduce mutations is contemplated by the present invention.

The present invention relates to the expression, purification and/or isolation and use of CBH1 variants. These enzymes are preferably prepared by a recombinant method using the cbh gene of hypocrea jecorina.

After the CBH1 gene of hypocrea jecorina has been isolated and cloned, other methods known in the art, such as site-directed mutagenesis, are used to substitute, add or delete the amino acid position in the CBH1 variant corresponding to expression. In addition, site-directed mutagenesis and other methods to effect introduction of amino acid changes in expressed proteins at the DNA level can be found in Sambrook, et al and Ausubel, et al.

DNA encoding amino acid sequence variants of hypocrea jecorina CBH1 were prepared by various methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared DNA encoding hypocrea jecorina CBH 1.

Site-directed mutagenesis is a preferred method of making substitutional variants. This technique is well known in the art (see, e.g., Carter et al. nucleic Acids Res.13: 4431-4443(1985) and Kunkel et al, Proc. Natl. Acad. Sci. USA 82: 488 (1987)). Briefly, in the case of site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of the starting DNA. After hybridization, the hybridized oligonucleotides are used as primers, the single strand of the starting DNA is used as template, and a DNA polymerase is used to synthesize the complete second strand. Thus, an oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA.

PCR mutagenesis is also useful for making amino acid sequence variants of the starting polypeptide, hypocrea jecorina CBH 1. See, Higuchi, PCR Protocols, pp.177-183(Academic Press, 1990); and valley et al, nuc. acids res.17: 723-733(1989). In short, when a small amount of a DNA template is used as a starting material in PCR, primers that differ slightly in sequence from the corresponding region of the template DNA can be used to generate a relatively large number of specific DNA fragments that differ from the template sequence only at the positions where the primers differ from the template.

Another method for making variants, cassette mutagenesis, is based on Wells et al, Gene 34: 315-323 (1985). The starting material is a plasmid (or other vector) containing the starting polypeptide DNA to be mutated. The codons in the starting DNA to be mutated are identified. On each side of the identified mutation site, a unique restriction enzyme site must be present. If such restriction sites are not present, they can be generated using the oligonucleotide-mediated mutagenesis method described above to introduce such sites at appropriate locations in the starting polypeptide DNA. Plasmid DNA is cleaved at these sites to linearize it. Double-stranded oligonucleotides encoding DNA sequences between the restriction sites, but containing the desired mutation, are synthesized using standard methods, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard methods. This double-stranded oligonucleotide is called a cassette. This cassette was designed to have 5 'and 3' ends compatible with the ends of the linearized plasmid so that it can be ligated directly into the plasmid. Thus, the plasmid contains a mutated DNA sequence.

Alternatively, or in addition, the desired amino acid sequence encoding the desired cellulase may be determined and the nucleic acid sequence encoding such amino acid sequence variants may be generated by synthetic means.

The desired cellulase so prepared may be further modified, and this will often depend on the intended use of the cellulase. Such modifications may involve further changes in amino acid sequence, fusion with heterologous polypeptides, and/or covalent modifications.

Cbh1 nucleic acids and CBH1 polypeptides

A. Variant cbh-type nucleic acids

After the DNA sequence encoding the CBH1 variant has been cloned into the DNA construct, the DNA is transformed into a microorganism. Advantageously, the microorganism to be transformed for expression of variant CBH1 according to the present invention may comprise a strain derived from Trichoderma. Thus, according to the present invention, a preferred means for preparing a variant CBH1 cellulase comprises transforming a Trichoderma host cell with a DNA construct comprising at least a fragment of DNA encoding part or all of variant CBH 1. The DNA construct is typically functionally linked to a promoter. The transformed host cell is then grown under conditions to express the desired protein. Subsequently, the desired protein product is purified to substantial homogeneity.

However, it is possible that the optimal expression vector for the particular DNA encoding variant CBH1 may differ from hypocrea jecorina. Thus, it may be most advantageous to express the protein in a transformed host that is similar to the phylogenetic origin of the organism from which variant CBH1 is derived. In an alternative embodiment, A.niger may be used as an expression vector. For a description of the transformation techniques of A.niger, see WO 98/31821, the disclosure of which is incorporated by reference in its entirety.

Thus, the description of the Trichoderma spp expression system herein is provided for illustrative purposes only and provides an option for expressing the variant CBH1 of the present invention. However, one skilled in the art can select DNA encoding variant CBH1 for expression in different host cells, if appropriate, and it will be understood that the source of variant CBH1 should be considered in determining the optimal expression host. Alternatively, one skilled in the art would be able to select the optimal expression system for a particular gene by routine techniques using means available in the art.

B. Variant CBH1 polypeptides

FIG. 1 shows the amino acid sequence of wild type hypocrea jecorina CBH 1. Variant CBH1 polypeptides include substitutions or deletions at positions corresponding to one or more of the following residues: l6, S8, P13, Q17, G22, T24, Q27, T41, S47, N49, T59, T66, A68, C71, A77, G88, N89, A100, N103, A112, S113, L125, T160, Y171, Q186, E193, S195, C210, M213, L225, T226, P227, T232, E236, E239, G242, T246, D249, N250, R251, Y394, D257, D259, S278, T281, L288, E295, T296, S297, A299, N301, F311, L325, N327, D329, T332, A318, S341, S352, S341, S354, T296, S297, T359, T368, N368, P384, P466, P384, P443, N380, P384, T414, T371, T373, T440, T62, T414, and T380, T440, T62, T414, T62, T440, T62, T.

The variant CBH1 of the invention has an amino acid sequence derived from the amino acid sequence of the precursor hypocrea jecorina CBH 1. The amino acid sequence of the CBH1 variant differs from the precursor CBH1 amino acid sequence by substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. FIG. 1 shows the mature amino acid sequence of hypocrea jecorina CBH 1. Thus, the CBH1 variants involved in the present invention contain amino acid residues at positions equivalent to those specifically identified in hypocrea jecorina CBH 1. If a residue (amino acid) of the CBH1 variant is homologous (i.e., corresponds in position in the primary or tertiary structure) or functionally similar (i.e., has the same or similar functional capability in chemical or structural combination, reaction, or interaction) to a particular residue or portion of a residue of hypocrea jecorina CBH1, then that residue (amino acid) of the CBH1 variant is equivalent to that residue of hypocrea jecorina CBH 1. As used herein, the numbering is that corresponding to the amino acid sequence of mature CBH1 as shown in figure 1. In addition to positions within precursor CBH1, specific residues in precursor CBH1 corresponding to amino acid positions linked to the instability of the precursor CBH1 under heat stress are identified herein for substitution or deletion. The amino acid position number (e.g., +51) refers to the number designed for the mature hypocrea jecorina CBH1 sequence shown in fig. 1.

Preferably, the alignment of amino acid sequences is performed using a "sequence comparison algorithm" to determine homology. The optimal alignment of sequences for comparison can be performed by the following method or algorithm: for example, Smith & Waterman, adv.appl.math.2: 482(1981), Needleman & Wunsch, j.mol.biol.48: 443(1970), Pearson & Lipman, proc.nat' l acad.sci.usa 85: 2444(1988), Computer implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA, Wisconsin Genetics software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), visual inspection (visual inspection) or MOE of Chemical Computing Group, Montreal Canada.

One example of an algorithm suitable for determining sequence similarity is the BLAST algorithm, described in Altschul, et al, j.mol biol.215: 403-410 (1990). Software for performing BLAST analysis is publicly available through the national center for Biotechnology information (www.ncbi.nim.nih.gov). This algorithm involves first identifying short strings of length W in the query sequence (HSPs) to determine high ranking sequence pairs (high ranking sequences pairs) that match or satisfy some positive-valued threshold T when aligned with strings of the same length in the database sequence. These initial neighborhood word strings are used as starting points to find longer HSPs containing them. The string extends in both directions along each of the two sequences being compared, as long as the cumulative alignment score is increasing. The extension of strings stops when: the cumulative association score decreases by an amount X from the maximum reached; the cumulative fraction reaches 0 or below 0; or extends to the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and rate of the alignment. The BLASTN program defaults are: the string length (W) was 11, BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915(1989)), alignment (B) was 50, expectation (E) was 10, M '5, N' -4, and the two strands were compared.

The BLAST algorithm then performs a statistical analysis of the similarity between the two sequences (see, e.g., Karlin & Altschul, Proc. nat' l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which represents the probability by which a match between two nucleotide or amino acid sequences will occur by chance. For example, an amino acid sequence is considered similar to a protease if the smallest sum probability in a comparison of the amino acid sequence being tested and the amino acid sequence of the protease is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Additional specific strategies for altering the stability of CBHl cellulases are provided below:

(1) reducing the entropy of backbone unfolding can lead to stability of the enzyme. For example, introduction of proline residues can significantly stabilize proteins by reducing the entropy of unfolding (see, e.g., Watanabe, et al, Eur.J.biochem.226: 277-283 (1994)). Similarly, a glycine residue has no β -carbon and therefore has much more backbone conformational freedom than any other residue. Substitution of glycine, preferably with alanine, may reduce the entropy of unfolding and improve stability (see, e.g., Matthews, et al, proc.natl.acad.sci.usa84; 6663-6667 (1987)). Furthermore, by shortening the outer ring structures (loops), it is possible to improve stability. It has been observed that proteins produced by extreme thermophilic bacteria have shorter external loop structures than their mesophilic homologues (see, e.g., Russel, et al, Current Opinions in Biotechnology 6: 370-374 (1995)). The introduction of disulfide bonds can also effectively stabilize different tertiary structures in association with each other. Thus, the stability of CBH1 variants can be altered by introducing cysteines at positions close to residues of existing cysteines, or by introducing pairs of cysteines that can form disulfide bonds.

(2) The stability of the enzyme can be altered by decreasing the internal cavity by increasing the hydrophobicity of the side chains. Enzyme stability can be increased by reducing the number and volume of internal cavities by maximizing hydrophobic interactions and reducing packing defects (see, e.g., Matthews, Ann. Rev. biochem. 62: 139-160 (1993); Burley, et al., Science 229: 23-29 (1985); Zuber, Biophys. chem. 29: 171-179 (1988); Kellis, et al., Nature 333: 784-786 (1988)). It is known that multimeric proteins from thermophilic organisms often have more hydrophobic subunit interfaces, with a greater degree of surface complementarity, than their mesophilic counterparts (Russel, et al, supra). This principle is believed to apply to the domain interface of the unimer protein as well. Specific substitutions may improve stability by increasing hydrophobicity, including lysine to arginine, serine to alanine, threonine to alanine (Russel, et al, supra). Modifications to alanine or proline can increase the size of the side chain, resulting in reduced cavities, better stacking, and increased hydrophobicity. The stability of the enzyme can be improved by reducing the size of the cavity, increasing the hydrophobicity and improving the complementary substitution of the interface between the CBH1 domains. In particular, changing specific residues at these positions to different residues selected from the group consisting of phenylalanine, tryptophan, tyrosine, leucine, and isoleucine can improve performance.

(3) Balancing the charge in the rigid secondary structure, i.e. the alpha-helix and beta-turn, may improve stability. For example, neutralization of a partial positive charge at the N-terminus of an α -helix with a negative charge on aspartic acid can improve the stability of the structure (see, e.g., Eriksson, et al, Science 255: 178-183 (1992)). Similarly, neutralization of a partial negative charge at the C-terminus of the helix with a positive charge may improve stability. Removal of the positive charge interaction with the N-terminus of the β -turn peptide should be effective in imparting tertiary structural stability. Substitution with a non-positively charged residue can remove the interaction of the undesired positive charge with the amide nitrogen in the turn.

(4) Stabilization of the tertiary structure by introduction of salt bonds and hydrogen bonds may be effective. For example, ion pair interactions, e.g., between aspartic or glutamic acid and lysine, arginine or histidine, can cause strong stabilizing effects and can be used to adhere different tertiary structural elements, resulting in increased thermal stability. In addition, an increase in the number of hydrogen bonds for charged/uncharged residues, and an increase in the number of hydrogen bonds, can generally improve thermal stability (see, e.g., Tanner, et al, Biochemistry 35: 2597-2609 (1996)). Substitution with aspartic acid, asparagine, glutamic acid, or glutamine can introduce hydrogen bonds to the backbone amide. Substitution with arginine can improve salt bridging and introduce hydrogen bonds into the carbon skeleton carbonyl.

(5) Avoiding susceptibility residues can generally increase thermostability. For example, asparagine and glutamine are susceptible to deamidation and cysteine to oxidation at high temperatures. Reducing the number of these residues at sensitive sites can lead to improved thermostability (Russel, et al, supra). Substitutions or deletions made by any residue other than glutamine or cysteine may increase stability by avoiding thermosensitive residues.

(6) Stabilization and destabilization of ligand binding results in altered stability of CBH1 variants. For example, in a substrate to which the CBH1 variant of the invention is applied, one component may bind to a particular surfactant sensitive/thermosensitive site of the CBH1 variant. By modifying the site by means of substitution, the binding of the component to the variant can be enhanced or reduced. For example, CBH1 binding to nonaromatic residues in the cleft may be substituted with phenylalanine or tyrosine to introduce aromatic side chain stabilization, where the action of the cellulose substrate may occur smoothly with the benzene ring, which increases the stability of CBH1 variants.

(7) Increasing the electronegativity of any surfactant/heat-sensitive ligand can improve stability under surfactant or heat stress conditions. For example, substitution with phenylalanine or tyrosine can increase the electronegativity of the D (aspartic acid) residue by improving shielding from solution, thereby improving stability.

C. Homologous CBH1 nucleic acids and polypeptides

Genomic DNA from a microorganism is immobilized on a membrane. The genomic DNA was hybridized with a gene-specific probe and screened using PCR. The PCR products are isolated and sequenced using techniques well known in the art.

Expression of recombinant CBH1 homologs and variants

The method of the invention relies on the use of cells to express the desired cellulase rather than requiring a specific expression method.

The invention provides host cells transduced, transformed or transfected with expression vectors containing nucleic acid sequences encoding the desired cellulases. Culture conditions, such as temperature, pH and the like, are those used in the parent host cell prior to transduction, transformation or transfection, as will be apparent to those skilled in the art.

In one method, a filamentous fungal cell or yeast cell is transfected with an expression vector having a promoter or biologically active promoter fragment or one or more (e.g., a series) of enhancers, operably linked to a DNA fragment encoding a desired cellulase, that functions in a host cell line such that the desired cellulase is expressed in the cell line.

A. Nucleic acid constructs/expression vectors

A natural or synthetic polynucleotide fragment encoding the desired cellulase (the "nucleic acid sequence encoding the desired cellulase") may be incorporated into a heterologous nucleic acid construct or vector, which is capable of being introduced into and replicated in a filamentous fungal or yeast cell. The vectors and methods disclosed herein are suitable for use in host cells for expression of the desired cellulase. Any vector may be used as long as it can replicate and survive in the introduced cells. A large number of suitable vectors and promoters are known to those of skill in the art and are commercially available. Cloning and expression vectors are also described in Sambrook et al, 1989, Ausubel FM et al, 1989 and Stratanet et al, 1981, each of which is expressly incorporated herein by reference. van den Hondel, c.a.m.j.j.et al (1991) In: suitable expression vectors for fungi are described in Bennett, J.W.and Lasure, L.L, (eds.) More GeneManiporations in fungi.academic Press, pp.396-428. The appropriate DNA sequence may be inserted into a plasmid or vector (collectively referred to herein as a "vector") by a variety of methods. Typically, the DNA sequence is inserted into the appropriate restriction site by standard methods. Such methods and related subcloning methods are believed to be within the knowledge of those skilled in the art.

Recombinant filamentous fungi comprising the coding sequence for the desired cellulase can be produced by introducing a heterologous nucleic acid construct comprising the coding sequence for the desired cellulase into the cells of a selected strain of filamentous fungus.

Once the desired form of the desired cellulase nucleic acid sequence is obtained, it can be modified by various methods. When the sequence includes non-coding flanking regions, the flanking regions may be excised, mutated, and the like. Thus, transitions, transversions, deletions and insertions may be made on the naturally occurring sequence.

The selected sequence encoding the desired cellulase can be inserted into a suitable vector according to known recombinant techniques and used to transform filamentous fungi capable of expressing the cellulase. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences encoding substantially identical or functionally equivalent amino acid sequences can be used to clone and express the desired cellulase. Thus, it will be understood that such substitutions in the coding region are within the scope of sequence variants covered by the present invention.

The invention also includes recombinant nucleic acid constructs comprising one or more nucleic acid sequences encoding a desired cellulase enzyme as described above. The constructs include vectors, such as plasmid vectors or viral vectors, into which the sequences of the invention are inserted, in either a forward or reverse orientation.

The heterologous nucleic acid construct may include a coding sequence for a desired cellulase, which coding sequence may be in the following state: (i) in a detached state; (ii) in combination with another coding sequence, such as a fusion protein or signal peptide coding sequence, wherein the coding sequence for the desired cellulase is the primary coding sequence; (iii) in combination with non-coding sequences, e.g., introns and regulatory elements, such as promoter and terminator elements or 5 'and/or 3' untranslated regions, which have an effect on the expression of the coding sequence in a suitable host; and/or (iv) in a vector or host environment, wherein the coding sequence for the desired cellulase is a heterologous gene.

In one aspect of the invention, established filamentous fungal and yeast lines are preferred for transferring nucleic acid sequences encoding a desired cellulase into cells in vitro using a heterologous nucleic acid construct. For long term production of the desired cellulase, stable expression is preferred. Any method that can efficiently produce stable transformants can be used in the practice of the present invention.

Suitable vectors typically carry a nucleic acid sequence encoding a selectable marker, an insertion site, and suitable control elements, such as promoter and termination sequences. The vector may include regulatory sequences, including, for example, non-coding sequences, such as introns and regulatory elements, i.e., promoter and termination elements or 5 'and/or 3' untranslated regions, which are effective for expression of the coding sequence in a host cell (and/or in a vector or host cell environment in which the modified soluble protein antigen coding sequence is not normally expressed) and are operably linked to the coding sequence. A large number of suitable vectors and promoters are known to those skilled in the art, many of which are commercially available and/or described in Sambrook, et al, (supra).

Representative promoters include constitutive promoters and inducible promoters, examples of which include the CMV promoter, SV-40 early promoter, RSV promoter and EF-1. alpha. promoter, the promoter containing the Tet Response Element (TRE) in the tet-on and tet-off system (Clon Techned BASF), the β -actin promoter and the metallothionein promoter, which can be upregulated by the addition of certain metal salts. The promoter sequence is a DNA sequence recognized by a particular filamentous fungus for expression purposes. It may be operably linked to a DNA sequence encoding a variant CBH1 polypeptide. Such linkage includes positioning the promoter relative to the start codon of the DNA sequence encoding the variant CBH1 polypeptide in the disclosed expression vectors. The promoter sequence contains transcriptional and translational regulatory sequences that mediate the expression of the variant CBH1 polypeptide. Examples include promoters from the following genes: an Aspergillus niger, Aspergillus awamori or Aspergillus oryzae glucoamylase, alpha-amylase or alpha-glucosidase encoding gene; the aspergillus nidulans gpdA or trpC gene; neurospora crassa cbh1 or trp1 gene; an Aspergillus niger or Rhizomucor miehei aspartic protease encoding gene; hypocrea jecorina cbh1, cbh2, egl1, egl2, or other cellulase encoding genes.

The choice of a suitable selectable marker will depend on the host cell, and suitable markers for different host cells are well known in the art. Typical selectable marker genes include argB, from Aspergillus nidulans or Hypocrea jecorina; amdS from Aspergillus nidulans; pyr4 from Neurospora crassa or Hypocrea jecorina; pyrG from Aspergillus niger or Aspergillus nidulans. Other exemplary selectable markers include, but are not limited to, trpc, trp1, oliC31, niaD, or leu2, which may be incorporated into heterologous nucleic acid constructs used to transform mutant strains such as trp-, pyr-, leu-, and the like.

Such selectable markers confer the ability of the transformants to utilize metabolites not normally metabolized by the filamentous fungi. For example, the amdS gene of hypocrea jecorina encoding acetamidase allows transformed cells to grow with acetamide as a nitrogen source. A selectable marker (e.g., pyrG) can restore the ability of an auxotrophic mutant strain to grow in a selective minimal medium, or a selectable marker (e.g., olic31) can confer on a transformant the ability to grow in the presence of an inhibitory drug or antibiotic.

The coding sequence for the selectable marker is cloned into any suitable plasmid using methods commonly used in the art. Exemplary plasmids include pUC18, pBR322, pRAX, and pUC 100. The pRAX plasmid contains the AMA1 sequence from aspergillus nidulans, which allows it to replicate in aspergillus niger.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the knowledge of one of ordinary skill in the art. Such techniques are well described in the literature. See, e.g., Sambrook et al, 1989; freshney, 1987; ausubel, et al, 1993 and Coligan et al, 1991. All patents, patent applications, articles, and publications mentioned herein are expressly incorporated herein by reference.

B. Host cells and culture conditions for CBH1 production

(i) Filamentous fungi

Accordingly, the present invention provides filamentous fungi, comprising cells that have been modified, selected and cultured, which are effective to produce or express a desired cellulase enzyme relative to a corresponding non-transformed parent fungus.

Examples of parental filamentous fungi that may be treated and/or modified to express a desired cellulase include, but are not limited to, Trichoderma, certain Penicillium sp, certain Humicola sp including Humicola insolens; aspergillus species (Aspergillus sp.) include Aspergillus niger, Chrysosporium sp, Fusarium sp, Hypocrea sp, and Chaetomium sp.

The cells expressing the desired cellulase are cultured under conditions typically used to culture parental fungal strains. Typically, cells are cultured in standard media containing physiological salts and nutrients, such as Pourquie, j.et., Biochemistry and Genetics of cell Degradation, eds. aubert, j.p.et., Academic Press, pp.71-86, 1988 and llmen, m.et., appl.environ.microbiol.63: 1298-1306, 1997. Culture conditions are also standard, for example, cultures are incubated at 28 ℃ in a shake incubator or a fermentor until the desired level of the desired cellulase is obtained.

Preferred culture conditions for a given filamentous fungus can be found in the scientific literature and/or obtained from the source of the fungus, e.g., the american type culture collection (ATCC;www.atcc.org) And finding. After growth conditions for the fungus are established, the cells are exposed to an environment effective to cause or allow expression of the desired cellulase.

When the coding sequence for the desired cellulase is under the control of an inducible promoter, an inducing agent, such as a sugar, metal salt or antibiotic, is added to the culture medium at a concentration sufficient to effectively induce the expression of the desired cellulase.

In one embodiment, the strain comprises Aspergillus niger, which is a useful strain for obtaining overexpressed proteins. For example, the A.niger var awamori (A. niger var awamori) dgr246 is known to secrete increased amounts of secreted cellulases (Goedegebauur et al, curr. Genet (2002) 41: 89-98). Other strains of the A.niger theubrious varietal, such as GCDAP3, GCDAP4 and GAP3-4 are also known, see Ward et al (Ward, M, Wilson, L.J.and Kodama, K.H., 1993, appl.Microbiol.Biotechnol.39: 738-743).

In another embodiment, the strain comprises trichoderma reesei, which is a useful strain for obtaining overexpressed proteins. For example, it is known that the ratio of the molecular weight of a compound expressed by Sheir-Neiss, et al, appl.Microbiol.Biotechnol.20: 46-53(1984) to RL-P37. Functional equivalents of RL-P37 include Trichoderma reesei strain RUT-C30(ATCC No. 56765) and strain QM9414(ATCC No. 26921). It is expected that these strains will also be useful in overexpressing variant CBH 1.

When it is desired to obtain a desired cellulase without potentially adverse native cellulolytic activity, it is useful to obtain a host cell in which one or more cellulase genes have been knocked out prior to introduction of a DNA construct or plasmid containing a DNA fragment encoding the desired cellulase. Such strains may be prepared using the methods disclosed in U.S. Pat. No. 5,246,853 and WO 92/06209, the disclosures of which are incorporated herein by reference. By expressing the desired cellulase in a host microorganism lacking one or more cellulase genes, the identification and subsequent purification procedures can be simplified.

The gene knockout can be accomplished by: a form of the gene to be knocked out or disrupted is inserted into the plasmid using methods known in the art. The deletion plasmid (deletion plasmid) is then cleaved at the appropriate restriction site(s) located within the coding region of the gene, and the coding sequence of the gene, or a portion thereof, is replaced with a selectable marker. The flanking DNA sequences of the locus of the gene to be knocked out or disrupted, preferably at about 0.5 to 2.0kb, flank the selectable marker gene. Suitable knock-out plasmids typically have unique restriction sites present so that the knock-out gene, including flanking DNA sequences, and the selectable marker gene can be removed as a single linear fragment.

The selection of the selectable marker must be such that detection of the transformed microorganism is facilitated. Any selectable marker gene that is expressed in the selected microorganism is suitable. For example, for Aspergillus, the selection marker is chosen such that its presence in the transformants does not significantly affect its properties. Such a selectable marker may be a gene encoding a detectable product. For example, a functional copy of an Aspergillus gene may be used, which if absent in the host strain, results in the host strain exhibiting an auxotrophic phenotype.

In a preferred embodiment, the Aspergillus pyrG gene is used to transform the Aspergillus pyrG^-Derivatives, which provide useful selectable markers in transformation. PyrG^-The derivative strain may be obtained by selecting a strain of Aspergillus resistant to fluoroorotic acid (FOA). The pyrG gene encodes orotidine-5' -monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with the entire pyrG gene were grown in medium lacking uridine, but were sensitive to fluoroorotic acid. By selecting for FOA resistance, it is possible to select pyrG lacking a functional orotidine monophosphate decarboxylase^-Derivative strains, such derivative strains requiring uridine for growth. It is also possible to obtain strains that require uridine, lack functional ribose pyrophosphate orotate transferase, using FOA selection techniques. By function of the gene encoding the enzymeIt is possible to transform these cells in sexual copies (Berges)&Barreau, curr. genet.19: 359-365(1991) and van Hartingsveldt et al, (1986) Development of a homogous transformation system for Aspergillus niger based on the pyrG gene. mol. Gen. Gene.206: 71-75). Selection of derivative strains is readily performed using the FOA resistance technique mentioned above, and therefore, the pyrG gene is preferably used as a selectable marker.

To transform pyrG^-Aspergillus so that it is incapable of expressing one or more cellulase genes, a single DNA fragment comprising a disrupted or knocked-out cellulase gene is isolated from the knock-out plasmid and used to transform an appropriate pyr^-An Aspergillus host. Transformants are then identified and selected based on their ability to express the pyrG gene product and to alter the uridine auxotrophy of the host strain. The transformants obtained were then subjected to southern analysis to identify and confirm the occurrence of a double crossover integration (double cross integration) event in which the pyr4 selectable marker replaced part or all of the coding region of the genomic copy of the gene to be knocked out.

Although the specific plasmid vector described above is related to pyr^-Preparation of transformants, but the present invention is not limited to these vectors. Using the above techniques, various genes in Aspergillus strains can be knocked out and replaced. Furthermore, as discussed above, any available selectable marker may be used. In fact, any Aspergillus gene that has been cloned and identified can be deleted from the genome using the strategy described above.

As mentioned above, the host strain used is a derivative of Aspergillus which lacks or has one or more non-functional genes corresponding to the selectable marker of choice. For example, if a pyrG selectable marker is selected, then the particular pyrG^-The derivative strain was used as an acceptor in the transformation procedure. Similarly, a selectable marker comprising an Aspergillus gene equivalent to the Aspergillus nidulans genes amdS, argB, trpC, niaD may also be used. Corresponding acceptor strainsIt must therefore be a derivative strain, such as argB respectively^-、trpC^-、niaD^-。

Then, a DNA encoding the desired cellulase is prepared for insertion into an appropriate microorganism. According to the present invention, the DNA encoding the desired cellulase includes DNA necessary for encoding a protein having functional cellulolytic activity. The DNA fragment encoding the desired cellulase may be functionally linked to a fungal promoter sequence, e.g.the promoter of the glaA gene.

It is also contemplated that more than one copy of the DNA encoding the desired cellulase may be recombined into the strain to aid in over-expression. The DNA encoding the desired cellulase can be prepared by construction of an expression vector carrying the DNA encoding the cellulase. The expression vector carrying the inserted DNA fragment encoding the desired cellulase may be any vector, typically a plasmid, capable of autonomous replication in the particular host organism or capable of integration into the host DNA. In preferred embodiments, two types of expression vectors for obtaining expression of a gene are contemplated. The first type contains DNA sequences in which the promoter, gene coding region and terminator sequences all originate from the gene to be expressed. If desired, a truncated gene can be obtained by deleting an undesired DNA sequence (e.g., a DNA sequence encoding an undesired domain), leaving the domain to be expressed under the control of its own transcriptional and translational regulatory sequences. Selectable markers are also included in the vector so that multiple copies of the novel gene sequence integrated into the host can be selected.

The second type of expression vector is pre-assembled and contains the sequences required for high level transcription and a selectable marker. It is contemplated that the coding region of a gene, or a portion thereof, may be inserted into such a universal expression vector such that it is under the transcriptional control of the promoter and terminator sequences of the expression cassette. For example, pRAX is such a universal expression vector. The gene or a part thereof may be inserted downstream of the glaA strong promoter.

In this vector, the DNA sequence encoding the desired cellulase of the present invention may be operably linked to transcriptional and translational sequences, i.e., a suitable promoter sequence and a signal sequence in reading frame with the structural gene. The promoter may be any DNA sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. An optional signal peptide can be used to allow the desired cellulase to be present extracellularly. The DNA encoding the signal peptide is preferably DNA naturally associated with the gene to be expressed, however, signal sequences from any suitable source are contemplated by the present invention.

Methods for fusing a DNA sequence encoding the desired cellulase of the present invention and a promoter into an appropriate vector are known in the art.

The DNA vector or construct described above may be introduced into a host cell according to known techniques such as transformation, transfection, microinjection, electroporation, biobalistic bombardment (Biolisicbombedment) and the like.

A preferred method of the invention for preparing Aspergillus for transformation comprises preparing protoplasts from fungal mycelia. See, Campbell et al, improved transformation efficiency of A.niger using homologus niaD gene for nitrate reduction enzyme, Current, Gene.16: 53-56; 1989. the mycelium can be obtained from germinated vegetative spores. The mycelium is treated with an enzyme that digests the cell wall to obtain protoplasts. The protoplasts are then protected by adding a osmo-stabilizer to the suspension medium. These include sorbitol, mannitol, potassium chloride, magnesium sulfate and the like. Typically, the concentration of these stabilizers varies between 0.8M and 1.2M. Preferably, a sorbitol solution of about 1.2M is used in the suspension culture.

The uptake of DNA by the host Aspergillus strain is dependent on calcium ion concentration. Usually about 10mM CaCl₂To 50mM CaCl₂In between CaCl₂Is applied in the uptake solution. In the intake solutionIn addition to the requirement for calcium ions, other substances which are usually incorporated are buffer systems such as TE buffer (10Mm Tris, pH 7.4; 1mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinopropanesulfonic acid) and polyethylene glycol (PEG). The effect of polyethylene glycol is believed to be to fuse the cell membrane, thereby allowing the components of the culture medium to be transported to the cytoplasm of Aspergillus and the plasmid DNA to be transported to the nucleus. This fusion often allows multiple copies of the plasmid DNA to be integrated into the host chromosome mildly.

Generally, a suspension containing Aspergillus protoplasts or cells which have been subjected to an osmotic treatment and have a density of 10 is used in the transformation⁵To 10⁶Per mL, preferably 2X 10⁵and/mL. A volume of 100. mu.l of a suitable solution containing these protoplasts or cells (e.g., 1.2M sorbitol; 50mM CaCl₂) Mixing with the desired DNA. Typically, a high concentration of PEG is added to the uptake solution. 0.1 to 1 volume of 25% PEG4000 may be added to the protoplast suspension. However, it is preferred to add about 0.25 volumes to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like may also be added to the uptake solution and aid in the transformation.

Typically, the mixture is then incubated at about 0 ℃ for 10 to 30 minutes. Thereafter, additional PEG is added to the mixture to further enhance uptake of the desired gene or DNA sequence. Typically, 25% PEG4000 is added in a volume equivalent to 5 to 15 times the volume of the transformation mixture; however, larger or smaller volumes may be suitable. The volume of 25% PEG4000 is preferably about 10 times the volume of the transformation mixture. After adding PEG, sorbitol and CaCl were added₂Prior to the solution, the transformation mixture is incubated at room temperature or on ice. The protoplast suspension is then further added to the melted aliquot of growth medium. This growth medium only allows the transformants to grow. Any growth medium suitable for culturing the desired transformants may be used in the present invention. However, if Pyr is being selected⁺Transformants, then preferablyGrowth medium without uridine was used. Subsequently the obtained clones were transferred to uridine-free growth medium and purified.

At this stage, stable transformants, which have a faster growth rate, can be distinguished from unstable transformants, which form circular clones with a smooth, rather than rough, profile on solid media lacking uridine. In addition, in some cases, stability can be further tested by culturing the transformants on non-selective solid medium (i.e., containing uridine), harvesting spores from the medium, and determining the percentage of these spores that subsequently bud and grow in selective medium lacking uridine.

In a specific embodiment of the above process, after cultivation in liquid medium, the desired cellulase is recovered from the host cells in active form, which may be subjected to appropriate post-translational processing.

(ii) Yeast

The present invention also contemplates the use of yeast cells as host cells for the production of the desired cellulase. Several other genes encoding hydrolases have been expressed in various strains of s.cerevisiae. These include sequences encoding two endoglucanases from Trichoderma reesei (Penttila et al, 1987), two cellobiohydrolases (Penttila et al, 1988) and one beta-glucosidase (Cummings and Fowler, 1996), a xylanase from Aureobasidium pullulans (Li and Ljungdahl, 1996), an alpha-amylase from wheat (Rothstein et al, 1987), etc. Furthermore, cellulase gene cassettes encoding Vibrio cellulolyticus (Butyrivibrio fibrinolves) [ β ] -1, 4-endoglucanase (END1), Phanerochaete chrysosporium (CBH1), Ruminococcus flavacinus (Ruminococcus flavefaciens) cellodextrin enzyme (CEL1) and Endomyces fibrilizer cellobiose (Bgl1) were successfully expressed in a laboratory strain of Saccharomyces cerevisiae (Van Rensburg et al, 1998).

C. Introducing a nucleic acid sequence encoding a desired cellulase into a host cell

The invention further provides cells and cellular compositions that have been genetically modified to contain an exogenously supplied nucleic acid sequence encoding a desired cellulase. The parent cell or cell line may be genetically modified (i.e., transduced, transformed or transfected) with a cloning or expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, or the like, as described above.

The transformation method of the invention may result in the stable integration of all or part of the transformation vector into the genome of the filamentous fungus. However, transformation may also result in the maintenance of self-replicating extrachromosomal transformation vectors, which are also contemplated by the present invention.

A number of standard transfection methods can be used to generate Trichoderma reesei cell lines expressing large amounts of heterologous proteins. Some disclosed methods for introducing DNA constructs into cellulase-producing strains of trichoderma include, lorto, Hayes, DiPietro and Harman, 1993, curr. genet.24: 349 to 356; goldman, VanMontagu and Herrera-Estrella, 1990, curr. gene.17: 169 to 174; penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6: 155-164, for Aspergillus, Yelton, Hamerand Timberlake, 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474 for Fusarium, Bajar, Podila and Kolattukudy, 1991, Proc.Natl.Acad.Sci.USA 88: 8202-8212, for Streptomyces, Hopwood et al, 1985, The John Innes Foundation, Norwich, UK, and for Bacillus, Brigidi, DeRossi, Bertarini, Riccaradi and Matteuzzi, 1990, FEMS Microbiol. Lett.55: 135-138.

Any known method for introducing an exogenous nucleotide sequence into a host cell may be used. These methods include the use of calcium phosphate transfection, polybrene (polybrene), protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors, and any other known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). Agrobacterium-mediated transfection methods described in U.S. Pat. No. 6,255,115 may also be used. The only requirement here is that the particular genetic engineering method used is capable of successfully introducing at least 1 gene into a host cell capable of expressing the heterologous gene.

In addition, a heterologous nucleic acid construct containing a nucleic acid sequence encoding the desired cellulase can be transcribed in vitro, and the resulting RNA introduced into the host cell by known methods, e.g., by injection.

The invention further includes novel and useful filamentous fungal transformants, such as hypocrea jecorina and aspergillus niger transformants, which are useful for the production of fungal cellulase compositions. The invention includes transformants of filamentous fungi, particularly fungi that contain sequences encoding the desired cellulase or that have the endogenous cbh coding sequence deleted.

Analysis of CBH1 nucleic acid coding sequences and/or protein expression

After transformation of the cell line with a nucleic acid construct encoding the desired cellulase enzyme, the cell line may be assayed at the protein level, RNA level, or using a functional biological assay specific for cellobiohydrolase activity and/or expression, in order to assess the expression of the desired cellulase enzyme by the cell line.

In one representative application of the desired cellulase nucleic acid and protein sequences described herein, genetically modified strains of filamentous fungi, such as Trichoderma reesei, are engineered to produce increased amounts of the desired cellulase. Such genetically modified filamentous fungi are useful for producing cellulase products with greatly enhanced fiber hydrolyzing ability. In one method, this is accomplished by introducing the coding sequence for the desired cellulase into a suitable host, such as a filamentous fungus, e.g., Aspergillus niger.

Thus, the present invention includes methods for expressing a desired cellulase in a filamentous fungus or other suitable host by introducing an expression vector comprising a DNA sequence encoding the desired cellulase into the cells of the filamentous fungus or other suitable host.

In another aspect, the invention includes a method for modifying the expression of a desired cellulase in a filamentous fungus or other suitable host. Such modifications include, reduction or elimination of endogenous CBH expression.

In general, assays for analyzing the expression of the desired cellulase include northern blotting (northern blotting), dot blotting (DNA or RNA analysis), RT-PCR (reverse transcriptase polymerase chain reaction) or in situ hybridization, the use of appropriately labeled probes (based on the nucleic acid coding sequence) and conventional Southern blotting (Southern blotting) and autoradiography.

In addition, the production and/or expression of the desired cellulase can be determined directly in the sample, e.g., analyzed for cellobiohydrolase activity, expression and/or production. For example, in Becker et al, Biochem j. (2001) 356: 19-30 and Mitsuishi et al, FEBS (1990) 275: 135-138, each of which is expressly incorporated herein by reference. The ability of CBH1 to hydrolyze isolated soluble and insoluble substrates can be determined using Srisodsuk et al, j.biotech, (1997) 57: 49-57 and Nidetzky and Claeyssensens Biotech.Bioeng (1994) 44: 961-966, by the analytical method described therein. Useful substrates for analyzing cellobiohydrolase, endoglucanase or beta-glucosidase activity include crystalline cellulose, cellophane, phosphoswollen cellulose (phospholic acid), cellooligosaccharide, methylumbelliferyl lactoside (methylumbelliferyl lactoside), methylumbelliferyl cellobioside (celloidosyllactoside), o-nitrophenyl lactoside (ortho-nitrophenyl lactoside), p-nitrophenyl lactoside (para-nitrophenyl lactoside), o-nitrophenyl cellobioside (ortho-nitrophenyl celloside), p-nitrophenyl celloside (para-glucosidase).

In addition, protein expression can be assessed by immunological methods, such as immunohistochemical staining of cells, tissue sections, or immunoassay analysis of tissue culture media, such as western blot (western blot) or ELISA. Such an immunoassay can be used to assess qualitatively and quantitatively the expression of the desired cellulase. The details of these methods are known to those skilled in the art and many reagents for practicing these methods are commercially available.

Purified forms of the desired cellulase can be used to produce monoclonal or polyclonal antibodies specific for the expressed protein, which can be used in various immunoassays. (see, e.g., Hu et al, 1991). Representative assays include ELISA, competitive immunoassays, radioimmunoassays, western blots, indirect immunofluorescence assays, and the like. In summary, commercially available antibodies and/or kits can be used for quantitative immunoassay of expression levels of cellobiohydrolase proteins.

Isolation and purification of recombinant CBH1 protein

Generally, the desired cellulase proteins produced in cell culture are secreted into the culture medium and may be purified or isolated, e.g., to remove undesired components of the cell culture medium. However, in some cases, the desired cellulase protein may be produced intracellularly, which necessitates recovery from the cell lysate. In such a case, the desired cellulase protein is purified from the cells producing it using techniques routinely employed by those skilled in the art. Examples include, but are not limited to, affinity chromatography (Tilbeurgh et al, 1984), ion exchange chromatography methods (Goyal et al, 1991; Fliess et al, 1983; Bhikhabhai et al, 1984; Ellouz et al, 1987), including ion exchange chromatography using materials with high resolution (Medve et al, 1998), hydrophobic interaction chromatography (Tomaz and Queiroz, 1999), and two-phase partitioning (Brumbauer, et al, 1999).

Typically, the desired cellulase proteins are fractionated to isolate the protein with selected properties, such as binding affinity for a particular binding agent, such as an antibody or receptor; or have a selected molecular weight range or isoelectric point range.

Once expression of the particular desired cellulase protein is achieved, the desired cellulase protein thus produced is purified from the cell or cell culture. Representative methods suitable for such purification include: antibody-affinity column chromatography, ion exchange chromatography; ethanol precipitation; reversed phase HPLC; chromatography on silicon or on a cation exchange resin such as DEAE; carrying out chromatographic focusing; SDS-PAGE; precipitating ammonium sulfate; and gel filtration using, for example, Sephadex G-75. Various protein purification methods may be employed, which are known in the art, for example, in Deutscher, 1990; scopes, 1982. The purification step chosen will depend, for example, on the nature of the production process used and the particular protein produced.

Effectiveness of cbh1 and CBH1

It will be appreciated that the desired cellulase nucleic acids, the desired cellulase proteins and compositions containing the desired cellulase protein activity have utility in a wide variety of applications, some of which are described below.

New and improved cellulase compositions containing varying amounts of a desired cellulase are useful in detergent compositions that exhibit enhanced cleaning ability, act as softeners, and/or improve the feel of cotton fabrics (e.g., "stone-washing" or "biopolishing"), they may also be used in compositions that degrade wood pulp to sugar (e.g., for bioethanol production), and/or in feed compositions. The isolation and characterization of each type of cellulase enables control of various aspects of such compositions.

The desired cellulases having reduced thermostability are useful in certain situations, for example, where it is desirable that the activity of the enzyme be neutralized at lower temperatures so that other enzymes that may be present are not affected. In addition, enzymes may play a role in the restricted transition of cellulose, for example, may be used to control the degree of crystallinity and cellulose chain length. After the desired degree of conversion is achieved, the temperature of the saccharification process may be raised above the survival temperature of the destabilized CBH 1. Since CBH1 activity is essential for the hydrolysis of crystalline cellulose, the transition of crystalline cellulose will stop at elevated temperatures.

In one method, the cellulase of the invention is used in the treatment of detergent compositions or fabrics to improve hand and appearance.

Since the rate of hydrolysis of the cellulose product can be achieved by using a transformant having at least one additional copy of the desired cellulase gene, either as a replicable plasmid or inserted into the genome, the cellulose-or heteropolysaccharide-containing product can be degraded at a faster rate and to a greater extent. Products made from cellulose in waste, such as paper, cotton, cellulosic diapers and the like, can be more effectively degraded. Thus, fermentation products obtained from multiple transformants or individual transformants can be used in compositions to help degrade various cellulose products in overcrowded landfills by means of liquefaction.

Separate saccharification and fermentation processes can be used to convert cellulose present in the biological material, such as corn stover, to glucose, followed by conversion of the glucose to ethanol by the yeast strain. Simultaneous saccharification and fermentation processes can convert cellulose present in biological materials such as corn stalks to glucose while in the same reactor, the yeast strain converts glucose to ethanol. Thus, in another method, the desired cellulase of the present invention may function in the degradation of biological material to ethanol. Ethanol production using readily available cellulose sources provides a stable, renewable fuel resource.

Cellulose-based feeds consist of agricultural waste, grass and wood, and other low value biological materials such as municipal waste (e.g., recycled paper, yard trimmings, etc.). Ethanol can be produced by fermenting any of these cellulosic feedstocks. However, before conversion to ethanol, cellulose must first be converted to sugars.

Various different feeds may be used in combination with one or more of the desired cellulases of the invention, the particular choice of which may depend on the region in which the conversion process is carried out. For example, in the midwestern united states, agricultural wastes such as wheat straw, corn stover, and bagasse are predominant, while in california, rice straw is predominant. However, it should be understood that any available cellulosic biomaterial may be used in any location.

Cellulase compositions containing increased amounts of cellobiohydrolases are useful in the production of ethanol. The ethanol obtained by the method can be further used as an octane auxiliary agent or directly used as a fuel for replacing gasoline, and has the advantage that the ethanol is more environment-friendly as a fuel source than petroleum-derived products. It is known that the use of ethanol improves air quality and may reduce local ozone levels and smoke. Moreover, the use of ethanol instead of gasoline is of strategic importance in buffering the effects caused by sudden changes in the supply of non-renewable energy and petrochemicals.

Saccharification and fermentation processes utilizing cellulosic biomass materials such as trees, herbaceous plants, municipal solid waste and agricultural residues, and forestry residues can produce ethanol. However, the ratio of individual cellulases in a naturally occurring cellulase mixture produced by a microorganism may not be most effective for the rapid conversion of cellulose to glucose in a biological material. It is known that endoglucanases act to form new cellulose chain ends, which are themselves substrates for cellobiohydrolases, thereby improving the hydrolysis efficiency of the entire cellulase system. Thus, using increased and optimized cellobiohydrolase activity, ethanol production can be greatly increased.

Thus, one or more cellobiohydrolases of the present invention may be used to hydrolyze cellulose to its sugar components. In one embodiment, the desired cellulase is added to the biological material (bioglass) prior to addition to the fermenting organism. In another embodiment, the desired cellulase is added to the biological material simultaneously with the fermenting organism. Optionally, in each embodiment, additional cellulase components may be present.

In another embodiment, the cellulosic feedstock may be pretreated. The pretreatment may be by raising the temperature and adding any of dilute acid, concentrated acid or dilute base solution. The pretreatment solution is added for a period of time sufficient to at least partially hydrolyze the hemicellulose components prior to neutralization.

In addition to cellulase compositions (i.e., without cellobiohydrolase, substantially without cellobiohydrolase, or with increased cellobiohydrolase), the detergent compositions of the present invention may also employ surfactants including anionic, nonionic and amphoteric surfactants, hydrolytic enzymes, builders, bleaches, blue bleaches and fluorescent dyes, anti-caking agents, solubilizing agents, cationic surfactants and the like. These ingredients are therefore known in the detergent art. The cellulase composition described above may be incorporated into a detergent composition in any form of liquid diluent, granule, emulsion, gel, paste or the like. These forms are known to those skilled in the art. When a solid detergent composition is used, the cellulase composition is preferably formulated as a granule. Preferably, the formulated granules may contain a cellulase protecting agent. For a more complete discussion, see U.S. patent 6,162,782 entitled "reagent compositions relating to cellular compositions in CBH1 type compositions", which is incorporated herein by reference.

Preferably, the cellulase composition is used in a weight percentage of about 0.00005 to about 5 relative to the total detergent composition. More preferably, the cellulase composition is used in a weight percentage of about 0.0002 to about 2 relative to the total detergent composition.

In addition, the desired cellulase nucleic acid sequences can be used to identify and characterize related nucleic acid sequences. Numerous techniques that can be used to determine (predict or confirm) the function of a relevant gene or gene product include, but are not limited to, (a) DNA/RNA analysis, such as (1) overexpression, aberrant expression, and expression in other species; (2) gene knockouts (reverse genetics, targeted knock-outs, virus-induced gene silencing (VIGS, see Baulcombe, 1999), (3) analysis of the methylation status of genes, particularly flanking regulatory regions, and (4) in situ hybridization, (B) analysis of gene products, such as (1) recombinant protein expression, (2) antisera production, (3) immunolocalization, (4) biochemical assays for catalytic or other activity, (5) phosphorylation status, and (6) analysis of interactions with other proteins by yeast two-hybrid analysis, (C) pathway analysis, such as placement of genes or gene products in specific biochemical or signaling pathways based on the phenotype of gene overexpression or sequence homology to related genes, and (D) other analyses, which can also be performed to determine or confirm that isolated genes and their products are involved in specific metabolic or signaling pathways, and help to determine gene function.

Examples

The invention is further described in detail in the following examples, which are not intended to limit the scope of the invention in any way. The accompanying drawings are considered to be part of the specification and description of the invention. All cited references are expressly incorporated herein by reference for all that is described herein.

Example 1

Identification of CBH1 homolog

This example illustrates a novel homologue of CBH1 found in various fungi. Genomic DNA from several different microorganisms was prepared and used to perform PCR reactions to determine whether the DNA of a particular organism encodes a homologous CBH1 cellulase.

Isolation of genomic DNA

Genomic DNA can be isolated using any method known in the art. In this set of experiments we received 48 genomic DNA solutions from different hypocrea and Trichoderma species, hypocrea schwerinae (CBS 243.63), hypocrea orientalis (PPRI3894), Trichoderma pseudokoningii (CBS 408.91) and Trichoderma koningii (isolate 1) from a collaborative Technical University of Vienna (TUV). However, the following scheme may be applied:

cells were grown at 30 ℃ for 24 hours in 20ml Potato Dextrose Broth (PDB). Cells were diluted 1: 20 with fresh PDB medium and grown overnight. 2 ml of cells were centrifuged and the pellet washed with 1ml KC (60g KCl, 2g citric acid per liter, pH adjusted to 6.2 with 1M KOH). The cell pellet was resuspended in 900. mu.l KC. Add 100. mu.l (20mg/ml)Gently mix and manipulate protoplast formation (proplastation) under a microscope at 37 ℃ until more than 95% of the protoplasts are formed, for a maximum period of 2 hours. Cells were centrifuged at 1500rpm (460g) for 10 minutes. Add 200. mu.l TES/SDS (10mM Tris, 50mM EDTA, 150mM NaCl, 1% SDS), mix and incubate for 5 minutes at room temperature. DNA was isolated using Qiagen miniprep isolation kit (Qiagen). The column was eluted with 100. mu.l of milli-Q water and the DNA was collected.

It may be desirable to apply other methods in which it may be used. The system consists of a device for isolating nucleic acidsApparatus andand (4) forming a kit. Can be obtained from Qbiogene

Construction of primers

The PCR reaction is carried out using a standard PCR instrument such as PCT-200Peltier thermal cycler from MJ Research Inc., under the following conditions:

1) 1 minute at 96 ℃ for 1 cycle

2)94 ℃ for 30 seconds

45 ℃ for 90 seconds (1 ℃ per cycle)

72 ℃ for 2 minutes

3) Repeat the second step for 10 cycles

4)94 ℃ for 30 seconds

55 ℃ for 90 seconds

72 ℃ for 2 minutes

5) Repeating the fourth step for 20 cycles

6)7 minutes at 72 ℃,1 cycle, and

7) the temperature was lowered to 15 ℃ for storage and further analysis.

The following DNA primers were constructed for amplifying the homologous CBH1 gene from genomic DNA isolated from various microorganisms. All symbols used herein for proteins and DNA correspond to the IUPAC IUB Biochemical Nomenclature Commission codes (Biochemical Nomenclature communication codes).

Homologous 5 '(FRG 192) and 3' (FRG193) primersThe substance was developed based on the sequence of CBH1 from Trichoderma reesei. Both primers contain at their 5' ends a primer derived fromThe entry (Gateway) cloning sequence of (c). Primer FRG192 contains the attB1 sequence and primer FRG193 contains the attB2 sequence.

FRG192 sequence, aatB1 not shown:

ATGTATCGGAAGTTGGCCG (CBH1 Signal sequence of hypocrea jecorina)

FRG193 sequence, aatB2 not shown:

TTACAGGCACTGAGAGTAG (CBH1 hypocrea jecorina cellulose binding module)

The PCR conditions were as follows: 10 μ L of 10 × reaction buffer (10 × reaction buffer comprises 100mM Tris HCl, pH 8-8.5; 250mM KCl; 50mM (NH)₄)₂SO₄；20mM MgSO₄) (ii) a 0.2mM each of dATP, dTTP, dGTP, dCTP (final concentration), 1. mu.L of 100 ng/. mu.L of genomic DNA, 0.5. mu.L of 1 unit/. mu.L of PWO polymerase (Boehringer Mannheim, Cat #1644-947), FRG192 and FRG 1930.2. mu.M (final concentration) of each primer, 4. mu.L of LDMSO, and water was added to 100. mu.L.

According to these conditions, 4 genes were finally obtained from different species:

1. schweiziz Hypocrea (CBS 243.63)

2. Hypocrea orientalis (PPRI3894)

3. Trichoderma pseudokoningii (CBS 408.91)

4. Trichoderma koningii

Isolation of the Cel7A Gene sequence

The full-length sequence was directly obtained by using an N-terminal primer (FRG192) and a C-terminal primer (FRG 193). The full-length DNA sequence was translated according to 3 open reading frames using Vector NTI software. DNA and protein sequence comparisons with hypocrea jecorina Cel7A were performed to identify putative intron sequences. Translation of the genomic DNA sequence without intron sequences revealed a protein sequence of homologous CBH 1. Full-length genes have been obtained and are shown in FIGS. 3, 5,7 and 9.

Example 2

Expression and thermostability of Cbh1 homologs

The full-length gene of example 1 was transferred to an Aspergillus niger portal compatible destination vector (gateway) developed by Genencor. According to Gateway^TMCloning technology: version 1page 34-38, using pRAX1 as a scaffold, as shown in FIG. 11, to construct the vector.

FIG. 12 shows a newly developed expression vector; this is the product of the transfer of the new gene into the target vector pRAXdes 2. The result was a final expression vector designated pRAXdesCBH1 (specified with germline name).

The constructs were transformed into the A.niger vesiculosis variety (Cao Q-N, Stubbs M, Ngo KQP, Ward M, Cunningham A, Pai EF, Tu G-C and Hofmann T (2000) Penicilliopepsin-JT 2a recombinant enzyme from Penicillium japonicum and ligation of a hydrogen bond in subunit S3 kcat Protein Science 9: 991-1001) according to the method described by Cao et al.

Transformants were streaked onto minimal medium plates (Ballance DJ, Buxton FP, and Turner G (1983) Transformation of Aspergillus nidulans by the organotide-5' -phosphatedicarboxylase gene of Neurospora crassa Biochem Biophys Commun 112: 284-289), and grown at 30 ℃ for 4 days. Spores were collected using methods known in the art (seewww.fgsc.net/fgn48/Kaminskyj.htm). The aspergillus nidulans conidia (surface of conidia-producing culture was scraped with a sterile bent glass rod to scrape off the conidia) were harvested in water and these conidia could be preserved at 4 ℃ for several weeks to several months without severe loss of viability. However, it is freshThe obtained spores germinate with a stronger reproductive capacity. For long term storage, spores can be stored in-20 deg.C, 50% glycerol, or-80 deg.C, 15-20% glycerol. In the case of an 80% aqueous solution, the glycerol can be more easily pipetted. Adding 800 μ l conidia aqueous suspension (which can be stored at 4 deg.C after preparation) into 200 μ l 80% glycerol for storage at-80 deg.C; mu.l of the suspension was added to 600. mu.l of 80% glycerol for storage at-20 ℃. Vortex mixing was performed prior to freezing. To harvest the mutants, a small portion of conidiogenic culture can be removed, placed in 20% glycerol, vortexed, and stored frozen at-80 ℃. In our practice, we preserved them at-80 ℃, in 50% glycerol.

Transformants of the A.niger vesiculosa variety were grown on minimal medium without uridine (Ballance et al 1983). By applying 1cm of a spore-forming growth agar plate (sporulated grown agar plate)²The spore suspension of (2) was inoculated into a100 ml shake flask and incubated at 37 ℃ for 3 days, and transformants having cellulase activity were selected as described in Cao et al (2000).

The CBH1 activity assay is based on the hydrolysis of non-fluorescent 4-methylumbelliferyl- β -lactoside to the products lactose and 7-hydroxy-4-methylcoumarin, which results in the generation of a fluorescent signal. 170 μ l of 50mM NaAc buffer, pH4.5, was removed and added to a 96-well microtiter plate (MTP) (Greiner, Fluotrac 200, art. nr.655076) suitable for fluorescence analysis. Mu.l of the supernatant was added followed by 10. mu.l of MUL (1 mM 4-methylumbelliferyl-. beta. -lactoside (MUL) in milli Q water), and the MTP was placed in FluostarGalaxy (BMG Labtechnologies; D-77656 Offenburg). At 50 ℃ using lambda_320nm(excitation light) and lambda_460nm(light emission), kinetic data were measured, and the measurement was performed for 16 minutes (8 cycles, 120 seconds per cycle). The supernatant with CBH activity was then subjected to hydrophobic interaction chromatography, as described in example 5 below.

The amino acid sequence was deduced as described in example 1. The amino acid sequences of the CBH1 homologues are shown in FIG. 4 (Hypocrea orientalis), FIG. 6 (Hypocrea schweini), FIG. 8 (Trichoderma reesei) and FIG. 10 (Trichoderma pseudokoningii).

The thermal stability of the above homologues was determined as described in example 5 below.

CBH1 homologue	% identity	Tm	ΔTm
				Hypocrea jecorina		62.5
Schweiziz Hypocrea (CBS 243.63)	96.5	61.4	-1.1
				Hypocrea orientalis (PPRI3894)	97.1	62.8	0.3
Trichoderma pseudokoningii (CBS 408.91)	94.9	57.5	-5.0
				Trichoderma koningii	93.0	59.4	-3.1

Table 1: comparison between Tm determination and different CBH1 homologous sequences

It can be seen that the CBH1 cellulase homologue has a slight or negative effect on the thermostability of the CBH1 cellulase variant compared to the wild type. The homologue is closely related to hypocrea jecorina CBH 1; the difference in thermostability between hypocrea jecorina and homologues may suggest that a site with residues other than the amino acid residues found in hypocrea jecorina CBH1 may be involved in thermostability.

Example 3

Identification of sites of importance for stability

The amino Acid sequence of the CBH1 homologue, characterized in example 2 above, was aligned with the hypocrea jecorina sequence using the Clustal W algorithm using vector NTI software (Nucleic Acid Research, 22 (22): 4673-4680, 1994). Fig. 2 shows this association.

Based on the sequence alignment of the homologue with CBH1, three different approaches were used to determine possible sites involved in the stability of the CBH1 enzyme. In the first approach, the site that differs between the catalytic domain of hypocrea jecorina CBH1 and the catalytic domain of at least one homolog with lower stability (i.e., excluding hypocrea orientalis only) was identified as a possible site involved in the thermostability of CBH 1. The identified sites are L6, P13, T24, Q27, S47, T59, T66, G88, N89, T160, Q186, S195, T232, E236, E239, G242, D249, N250, T281, E295, F311, E325, N327, D329, T332, a336, K354, V407, P412, T417 and/or F418 of hypocrea jecorina CBH 1.

In the second approach, sites where residues in hypocrea jecorina or hypocrea orientalis are identical to those found in all homologous enzymes of reduced stability reveal sites that are not linked to Tm deficiency. The sites thus identified as being relevant for stability are L6, T24, Q27, S47, T59, T66, T160, Q186, S195, T232, E236, G242, D249, T281, E295, E325, N327, D329, T332, K354 and/or P412 of hypocrea jecorina CBH 1.

In the final method, the sites that are the same in hypocrea jecorina and hypocrea orientalis, the corresponding residues in hypocrea schweritz are the same or different from either of the two, and the sites having different amino acids in the corresponding sites of trichoderma koningii or trichoderma pseudokoningii are considered as possible sites involved in the thermostability of the enzyme. These sites, empirically shown to be most relevant to Tm stability, were identified as Q186, S195, E325, T332, and P412.

Sites carrying residues other than those found in hypocrea jecorina CBH1 were identified and subjected to site saturated mutagenesis (site saturated mutagenesis).

Example 4

Expression of CBH1 variants

PCR fragments were obtained using the primers and protocol described in example 1. The fragments were purified on agarose gels using Qiagen gel purification kits. According toThe Manual of Portal cloning technology (Gateway)^TMTechnology instruction manual) (version C), utilizingpDONR of^TM201 vector, the purified fragment was subjected to the clonase reaction, the above references being incorporated herein by reference. Then theThe gene was transferred from the ENTRY vector to a target vector (pRAXdes2) to obtain an expression vector pRAXCBH 1.

The cells are transformed with an expression vector containing a nucleic acid encoding the desired cellulase. The host cell A.niger is then grown under conditions that allow the desired cellulase to be expressed, as described in example 2.

Sites other than hypocrea jecorina CBH1 may be involved in the thermostability of the variants for site-saturation mutagenesis as described in example 3.

Example 5

Thermostability of CBH1 variants

The CBH1 cellulase variant was cloned and expressed as described above (see example 4). Then, Cel7A wild type and variants were purified from cell-free supernatants of these cultures by column chromatography. The protein was purified using Hydrophobic Interaction Chromatography (HIC). Post is arranged onSpring fusion chromatography System, applications thereof20HP2 resin, both manufactured by applied biosystems.

The HIC column was equilibrated with 5 column volumes of 0.020M sodium phosphate, 0.5M ammonium sulfate, pH 6.8. Ammonium sulfate was added to the supernatant to a final concentration of about 0.5M, and the pH was adjusted to 6.8. After filtration, the supernatant was applied to the column. After loading, the column was washed with 10 column volumes of equilibration buffer and then eluted with 10 column volumes of 0.02M sodium phosphate pH6.8 using an eluent having a 0.5M to 0M ammonium sulphate gradient. Cel7A eluted at approximately the middle gradient. Fractions were collected and pooled together based on reducing SDS-PAGE gel analysis.

According to Luo, et al, Biochemistry 34: 10669 and Gloss, et al, Biochemistry 36: 5612, melting point.

Data were collected using an Aviv 215 circular dichroism spectrophotometer. At 25 ℃, spectra of variants between 210 and 260 nanometers were acquired. The buffer conditions were 50mM Bis Tris propane/50 mM ammonium acetate/glacial acetic acid, pH 5.5. The protein concentration is maintained between 0.25 and 0.5 mgs/mL. After determining the optimal wavelength to monitor unfolding, the sample was thermally denatured by raising the temperature from 25 ℃ to 75 ℃ under the same buffer conditions. Data was collected every 2 degrees for 5 seconds. Partially reversible unfolding was monitored at a wavelength of 230 nm in a sample cell with a path length of 0.1 cm.

Mutations introduced into the CBH1 cellulase variant had a positive effect on the thermostability of the CBH1 cellulase variant compared to the wild type.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (15)

1. An isolated cellulase protein which is CBH1 from Hypocrea orientalis (Hypocrea orientalis) having the amino acid sequence of SEQ ID NO: 5.

2. an isolated nucleic acid whose sequence encodes or is complementary to a sequence encoding a CBH1 polypeptide selected from the group consisting of SEQ ID NOs: 5.

3. An isolated nucleic acid selected from the group consisting of SEQ ID NOs: 3.

4. a vector comprising the nucleic acid of claim 2.

5. A vector comprising the nucleic acid of claim 3.

6. A host cell transformed with the vector of claim 4.

7. A host cell transformed with the vector of claim 5.

8. A method of producing a CBH1 variant, comprising the steps of:

(a) culturing the host cell of claim 6 in a suitable medium under suitable conditions to produce a CBH1 variant;

(b) obtaining said generated CBH1 variant.

9. A method of producing a CBH1 variant, comprising the steps of

(a) Culturing the host cell of claim 7 in a suitable medium under suitable conditions to produce a CBH1 variant;

(b) obtaining said generated CBH1 variant.

10. A detergent composition comprising a surfactant and CBH1, wherein the CBH1 comprises the isolated cellulase protein of claim 1.

11. The detergent composition of claim 10, wherein the detergent is a laundry detergent.

12. The detergent composition of claim 10, wherein the detergent is a dishwashing detergent.

13. A feed additive comprising the isolated cellulase protein of claim 1.

14. A method of treating wood pulp comprising contacting said wood pulp with the isolated cellulase protein of claim 1.

15. A method for converting a biological material to sugars comprising contacting said biological material with the isolated cellulase protein of claim 1.