HK1156972B - Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials - Google Patents

Description

Compositions and methods comprising cellulase variants having reduced affinity for non-cellulosic materials

I. Statement regarding rights to inventions arising in federally sponsored research and development

The invention is generated under government support, and has conditional grant numbers granted by the energy department: DE-FC36-08GO 18078. The government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No. 61/059,506 filed 6/2008, which is incorporated herein by reference.

Field of the invention

The present invention relates to enzymes, in particular cellulase variants. Nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, methods of identifying other effective cellulase variants, and methods of using the compositions are also described.

IV, background

Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. They can be degraded by a variety of microorganisms (e.g., bacteria, yeast, and fungi) that produce extracellular enzymes capable of hydrolyzing polymeric substrates to monomeric sugars and used as an energy source (Aro et al, J Biol Chem, 276: 24309-. The possibility of cellulose becoming the main renewable energy source is great due to the limitations of the non-renewable source pathway (Krishna et al, Bioresource Tech, 77: 193-. Efficient utilization of cellulose by biological processes is one approach to overcome food, feed and fuel shortages (Ohmiya et al, Biotechnol Gen Engineer Rev, 14: 365-.

Cellulases are enzymes that hydrolyze cellulose (β -1, 4-glucan or β D-glycoside linkages), resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have traditionally been divided into three major classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and beta-glucosidases (beta-D-glucosinolglucohydrolases, EC 3.2.1.21) ("BG") (Knowles et al, TIBTECH 5: 255-. Endoglucanases act mainly on the amorphous part of the cellulose fiber, whereas cellobiohydrolases are also capable of degrading crystalline cellulose (Nevalainen and Penttila, Mycota, 303-319, 1995). Thus, the presence of cellobiohydrolases in cellulase systems is required for efficient dissolution of crystalline Cellulose (Suurnakki et al, Cellulose 7: 189-. The role of β -glucosidase is to release D-glucose units from cellobiose, cellooligosaccharides and other glucosides (Frer, J Biol Chem, 268: 9337-9342, 1993).

Various bacteria, yeasts and fungi are known to produce cellulases. Some fungi produce a complete cellulase system that is capable of degrading cellulose in crystalline form, allowing for convenient mass production of cellulases by fermentation. Filamentous fungi play a particular role due to the lack of cellulose hydrolyzing ability of many yeasts, such as Saccharomyces cerevisiae (see, e.g., Wood et al, Methods in Enzymology, 160: 87-116, 1988).

The fungal cellulase classifications of CBH, EG and BG can be further extended to include multiple components in each classification. For example, a number of CBHs, EGs and BGs have been isolated from a number of fungal sources, including Trichoderma reesei (also known as hypocrea jecorina) which contains the following known genes: two CBHs, CBHI ("CBH 1") and CBHII ("CBH 2"); at least 8 species of EG, i.e., EG I, EG II, EG III, EGIV, EGV, EGVI, EGVII, and EGVIII; and at least 5 BG's, BG1, BG2, BG3, BG4, and BG 5. EGIV, EGVI and EGVIII also have xyloglucanase activity.

In order to efficiently convert crystalline cellulose to glucose, a complete cellulase system is required, which includes components from each of the CBH, EG and BG classifications, as well as separate components that are less effective in hydrolyzing crystalline cellulose (Filho et al, Can J Microbiol, 42: 1-5, 1996). A synergistic relationship has been observed between cellulase components from different classes. Particularly, the EG cellulase and the CBH cellulase synergistically interact to more effectively degrade cellulose.

Cellulases are known in the art to be effective in treating textiles for the purpose of enhancing the cleaning ability of detergent compositions, for use as softeners, for improving the feel and appearance of cotton fabrics, etc. (Kumar et al, Textile Chemist and Colorist, 29: 37-42, 1997). Cellulase-containing detergent compositions have been described which have improved cleaning performance (U.S. Pat. No. 4,435,307; and British application Nos. 2,095,275 and 2,094,826) and are useful for treating fabrics to improve the feel and appearance of textiles (U.S. Pat. Nos. 5,648,263, 5,691,178 and 5,776,757; British application No.1,358,599). Thus, cellulases produced in fungi and bacteria have received great attention. In particular, fermentation of Trichoderma spp (e.g.Trichoderma longibrachiatum) or Trichoderma reesei (Trichoderma longibrachiatum) has been shown to produce an intact cellulase system capable of degrading cellulose in crystalline form.

Although cellulase compositions have been described previously, there remains a need for new and improved cellulase compositions. The improved cellulosic compositions are useful in household detergents, textile processing, biomass conversion, and paper making. Cellulases that exhibit improved performance are of particular interest.

Overview of the invention

The present invention relates to cellulase variants modified to reduce binding to non-cellulosic material. Typically, the cellulase variant has increased cellulolytic activity in the presence of non-cellulosic material compared to a wild-type cellulase. In some embodiments, the cellulase variant has a reduced net charge (i.e., more negative) compared to a wild-type cellulase. In some embodiments, the cellulase variant has a less positive charge than a wild-type cellulase. In some embodiments, the cellulase is modified by removal of one or more positive charges. In some embodiments, the cellulase is modified by the addition of one or more negative charges. In some embodiments, the cellulase is modified by removal of one or more positive charges and addition of one or more negative charges.

In some embodiments, the present invention relates to cellobiohydrolase I (CBH1) or cellobiohydrolase II (CBH2) variants. In some embodiments, the cellulase variant is a mature form having cellulase activity with a substitution at one or more positions selected from the group consisting of 63, 77, 129, 147, 153, 157, 161, 194, 197, 203, 237, 239, 247, 254, 281, 285, 288, 289, 294, 327, 339, 344, 356, 378, and 382, wherein the position is determined by comparison with the amino acid sequence set forth in SEQ ID NO: 3 (e.g., wild-type hypocrea jecorina CBH2), and wherein the substitution at one or more positions results in the cellulase variant having a net charge that is more negative than the reference cellulase. In some embodiments, CBH2 is modified by removal of one or more positive charges, which in some embodiments requires the substitution of lysine or arginine with a neutral amino acid (e.g., by replacing K or R with N or Q or other neutral residue). In some embodiments, CBH2 is modified by the addition of one or more negative charges, which in some embodiments requires the replacement of neutral amino acids with negatively charged amino acids (e.g., by D or E replacing N or Q or other neutral residues). In some embodiments, CBH2 is modified by removing one or more positive charges and adding one or more negative charges, which in some embodiments requires the substitution of lysine or arginine with a negatively charged amino acid (e.g., the substitution of K or R by D or E). In general, the polypeptide having the amino acid sequence of SEQ ID NO: 3 amino acid sequence the CBH2 variant has increased cellulolytic activity in the presence of lignin compared to wild-type hypocrea jecorina CBH 2. The present invention also provides a variant of CBH comprising one or more substitutions in the mature form of CBH, said substitutions being selected from the group consisting of K129, K157, K194, K288, K327, K356, R63, R77, R153, R203, R294, R378, N161, N197, N237, N247, N254, N285, N289, N339, N344, N382, Q147, Q204, Q239, Q281, D151, D189, D211, D277, D405, E146, E208 and E244, wherein said substitutions are according to SEQ ID NO: 3, mature form of hypocrea jecorina CBH 2. In some embodiments, the variant comprises additional substitutions at one or more additional positions selected from the group consisting of 146, 151, 189, 208, 211, 244, 277 and 405, wherein the additional positions are determined by comparison to SEQ ID NO: 3 (CBH2) is numbered with reference to the amino acid sequence of cellobiohydrolase II (CBH 2). In some embodiments, other substitutions at one or more other positions include substitutions of aspartic acid or glutamic acid with a neutral amino acid (e.g., by substituting N or Q or other neutral residues for D or E). In some embodiments, the other substitutions at one or more other positions include one or more of D151N, D189N, D211N, D277N, D405N, E146Q, E208Q, and E244Q, wherein the position is determined by comparison to SEQ ID NO: 3 (CBH2) are numbered corresponding to the amino acid sequence of the reference cellobiohydrolase II. In some preferred embodiments, the substitution at one or more positions is selected from 1,2, 3, 4, 5,6, 7, 8, 9 and 10 positions. In some preferred embodiments, the cellulase variant is derived from a parent cellulase selected from Hypocrea jecorina CBH2, Hypocrea corninis (Hypocrea koningii) CBH2, humicola insolens (humicola insolens) CBH2, vibrio cellulolyticus (Acremonium cellulolyticus) CBH2, Agaricus bisporus (Agaricus bisporus) CBH2, Fusarium oxysporum (Fusarium oxysporum) EG, Phanerochaete chrysosporium (phanerochaetobacter chrysosporium) CBH2, Talaromyces emericus (Talaromyces emersonii) CBH2, trichophyton fuscus (thermobifida) CBH 3 CBH2, thermobifida EG 6A/2, thermobifida EG. In some preferred embodiments, the cellulase variant is derived from a parent cellulase having an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 and SEQ ID NO: 13 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. In some embodiments, the more negative net charge is-1 or-2 compared to reference CBH 2.

The invention also provides cellulase variants, wherein the variants are mature forms having cellulase activity, comprising chemical modification of lysine residues, removing the positive charge of the lysine residues. In some preferred embodiments, the chemical modification comprises treatment with a material selected from the group consisting of succinic anhydride, acetoxysuccinic anhydride, maleic anhydride, tartaric anhydride, phthalic anhydride, trimetallicalhydride, cis-aconitic anhydride, t-nitrophthalic anhydride, acetic anhydride, butyric anhydride, isobutyric anhydride, hexanoic anhydride, valeric anhydride, isovaleric anhydride, and pivalic anhydride. In some preferred embodiments, the cellulase variant is derived from a parent cellulase selected from hypocrea jecorina cellobiohydrolase I, hypocrea jecorina cellobiohydrolase II, hypocrea jecorina endoglucanase I, hypocrea jecorina endoglucanase II, and hypocrea jecorina beta-glucosidase. In some preferred embodiments, the cellulase variant is derived from a parent cellulase selected from the group consisting of hypocrea jecorina CBH2, hypocrea cornininium CBH2, humicola insolens CBH2, vibrio cellulolyticus CBH2, agaricus bisporus CBH2, fusarium oxysporum EG, phanerochaete chrysosporium CBH2, rabdosia emersonii CBH2, thermobifida fusca 6B/E3 CBH2, thermobifida fusca 6A/E2 EG, and cellulomonas fipronii CenA EG. Also provided are cellulase variants derived from a parent cellulase having an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 and SEQ ID NO: 13 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. In some embodiments, the cellulase variant comprises a substitution at one or more positions selected from the group consisting of 63, 77, 129, 147, 153, 157, 161, 194, 197, 203, 237, 239, 247, 254, 281, 285, 288, 289, 294, 327, 339, 344, 356, 378, and 382, wherein the position is determined by a method that is analogous to SEQ ID NO: 3 (CBH2) are numbered in correspondence with the amino acid sequence of the reference cellobiohydrolase II.

The invention also relates to CBH2 variants comprising from 1 to 26 substitutions selected from K129E, K157E, K194E, K288E, K327E, K356E, R63Q, R77Q, R153Q, R203Q, R294Q, R378Q, N161D, N197D, N237D, N247D, N254D, N285D, N289D, N339D, N344D, N382D, Q147E, Q204E, Q239E and Q281E. In some embodiments, CBH2 variants include combinations of substitutions selected from: i) K157E/K129E; ii) K157E/K129E/K288E/K194E; iii) K157E/K129E/K288E/K194E/K356E/K327E; iv) K157E/K129E/K288E/K194E/K356E/K327E/R153Q/R294Q/R203Q/R378Q; v) K157E/K129E/K288E/K194E/K356E/K327E/R153Q/R294Q/R203Q/R378Q/N382D/N344D/N327D/N339D; vi) K157E/K129E/K288E/K194E/K356E/K327E/R153Q/R294Q/R203Q/R378Q/N382D/N344D/N327D/N339D/N289D/N161D/Q204E/Q147E; vii) K157E/K129E/K288E/K194E/K356E/K327E/R153Q/R294Q/R203Q/R378Q N382D/N344D/N327D/N339D/N289D/N161D/Q204E/Q147E/N285D/N197D/N254D/N247D; and viii) K157E/K129E/K288E/K194E/K356E/K327E/R153Q/R294Q/R203Q/R378QN 382D/N344D/N327D/N339D/N289D/N161D/Q204E/Q147E/N285D/N197D/N254D/N247D/Q239E/Q281E/R63Q/R77Q.

In some embodiments, the CBH2 variant comprises 1 to 8 substitutions selected from D151N, D189N, D211N, D277N, D405N, E146Q, E208Q, and E244Q. In some embodiments, CBH2 variants include combinations of substitutions selected from: i) D189N/E208Q/D211N/D405; and ii) D189N/E208Q/D211N/D405/E244Q/D277N/D151/E146Q.

Also described are isolated nucleic acids encoding CBH2 variants having cellobiohydrolase activity as described in the preceding paragraphs. In a first aspect, the invention encompasses an isolated nucleic acid encoding a polypeptide having cellobiohydrolase activity, which polypeptide is a variant of glycosyl hydrolase family 6, and wherein the nucleic acid encodes a substitution at a residue that reduces the net charge as compared to wild-type hypocrea jecorina CBH 2.

In another aspect, the disclosure relates to an isolated nucleic acid encoding a CBH2 variant, wherein the variant comprises a substitution in the mature form of CBH2 at a position selected from the group consisting of: K129E, K157E, K194E, K288E, K327E, K356E, R63Q, R77Q, R153Q, R203Q, R294Q, R378Q, N161D, N197D, N237D, N247D, N254D, N285D, N289D, N339D, N344D, N382D, Q147E, Q204E, Q239E, Q281E, D151N, D189N, D211N, D277N, D405N, E146Q, E208Q and E244Q, wherein the substitutions are according to SEQ ID NO: 3, mature form of hypocrea jecorina CBH 2.

In some embodiments, the disclosure relates to expression cassettes comprising a nucleic acid encoding a CBH2 variant, constructs comprising a nucleic acid encoding a CBH2 variant operably linked to a regulatory sequence, vectors comprising a nucleic acid encoding a CBH2 variant, and host cells transformed with a vector comprising a nucleic acid encoding a CBH2 variant. The invention also provides methods for producing the CBH2 variant by culturing a host cell that expresses the CBH2 variant in culture under conditions suitable for production of the CBH2 variant.

Also provided are compositions comprising the cellulase variants of the preceding paragraphs. In some preferred embodiments, the composition further comprises at least one additional enzyme selected from the group consisting of subtilisin, neutral metalloprotease, lipase, cutinase, amylase, carbohydrase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, and peroxidase.

Provided herein are methods of converting biomass to sugars comprising contacting the biomass with a cellulase variant. Also provided are methods of producing a fuel by contacting a biomass composition with an enzymatic composition comprising a cellulase variant to produce a sugar solution, and culturing with a fermenting microorganism under conditions sufficient to produce the fuel.

Also provided are compositions comprising cellulase variants, including, for example, detergent compositions, feed supplements; and methods of cleaning or fabric care by contacting a surface and/or article comprising a fabric with a detergent composition. Also provided are methods of fabric care treatment, including depilation and surface finishing (surface finishing), by contacting a surface and/or article comprising a fabric with the cellulase variant.

Other objects, features and advantages of the present invention will become apparent from the detailed description set forth below. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings

FIG. 1 shows the saccharification of APB by cellulase preparations of modified (squares) and unmodified (circles) Trichoderma species in the presence of increased amounts of lignin inhibitor. Fig. 1A and 1B show the results after 24 and 48 hours incubation, respectively.

FIG. 2A shows the saccharification of a comparative modified cellulase and FIG. 2B shows the difference in saccharification using modified and unmodified cellulases.

Figure 3 provides an alignment of the amino acid sequences of the various cellulases in mature form: hypocrea jecorina (also known as Trichoderma reesei) CBH2(SEQ ID NO: 3), Hypocrea cornininum CBH2(SEQ ID NO: 4), Humicola insolens CBH2(SEQ ID NO: 5), Vibrio cellulolyticus CBH2(SEQ ID NO: 6), Agaricus bisporus CBH2(SEQ ID NO: 7), Fusarium oxysporum EG (SEQ ID NO: 8), Phanerochaete chrysosporium CBH2(SEQ ID NO: 9), Talaromyces emersonii CBH2(SEQ ID NO: 10), Thermobifida fusca 6B/E3 CBH2(SEQ ID NO: 11), Thermobifida fusca 6A/E2 EG (SEQ ID NO: 12), and Cellulomonas faecalis CenAEG (SEQ ID NO: 13).

Fig. 4 provides a plot of the observed relative frequency of the CBH2 variant SEL as a charge alteration product of the Pretreated Corn Stover (PCS) determination of winners versus expected winners. The reduced CBH2 charge resulted in a significantly higher PCS winner frequency.

FIG. 5 provides a plasmid map of pTTTpyr-cbh 2.

Detailed description of the various embodiments

The present invention relates to cellulase variants modified to reduce binding to non-cellulosic material. Typically, the cellulase variant has increased cellulolytic activity in the presence of non-cellulosic material compared to a wild-type cellulase. In some embodiments, the cellulase variant has a reduced net charge (i.e., more negative) compared to a wild-type cellulase. In some embodiments, the cellulase variant has a less positive charge than a wild-type cellulase. In some embodiments, the cellulase is modified to remove one or more positive charges. In some embodiments, the cellulase is modified to add one or more negative charges. In some embodiments, the cellulase is modified to remove one or more positive charges and to add one or more negative charges.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions and methods described herein. 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. In this application, the use of the singular includes the plural unless specifically stated otherwise. Unless stated otherwise, the use of "or" means "and/or". Likewise, the terms comprising or including as used herein are not intended to be limiting, as are the various forms of comprising or including ("comprising", "including" and "including"). All patents and publications, including all amino acid and nucleotide sequences disclosed in such patents and publications, referred to herein are expressly incorporated herein by reference. The headings provided 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 herein are more fully defined by reference to the specification as a whole.

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 OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2 nd edition, John Wiley AND Sons, New York (1994); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary 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 include the numbers defining the range. Unless otherwise indicated, nucleic acids are written in a 5 'to 3' direction from left to right, respectively; amino acids are written from left to right in the amino to carboxyl direction. With regard to definitions and terminology in this field, the practitioner may point specifically to Sambrook et al, MOLECULAR CLONING: laboratorymanal (2 nd edition), Cold Spring Harbor Press, Plainview, n.y., 1989; and Ausubel FM et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1993. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

I. Definition of

The following terms may be more fully defined by reference to the specification as a whole.

As used herein, the term "polypeptide" refers to a compound consisting of a single chain of amino acid residues joined by peptide bonds. As used herein, the term "protein" may be synonymous with the term "polypeptide".

"variant" means a protein derived from a precursor protein (e.g., a native protein) by adding one or more amino acids at the C-and/or N-terminus, substituting one or more amino acids at one or more different positions in the amino acid sequence, or deleting one or more amino acids at one or both termini of the protein or at one or more positions in the amino acid sequence, or modifying one or more amino acids by changing charge (i.e., by removing positive charge, adding negative charge, or by removing positive charge and adding negative charge simultaneously). Preparation of cellulase variants may be carried out by any method known in the art, including chemical modification of amino acids by modifying a DNA sequence encoding the native protein, transforming the modified DNA sequence into a suitable host, and expressing the modified DNA sequence to form the variant enzyme. The variant cellulases of the invention include peptides comprising an altered amino acid sequence compared to the amino acid sequence of the precursor enzyme, wherein the variant cellulases retain the characteristic cellulolytic properties of the precursor enzyme but may have altered properties in some particular respect. For example, a variant cellulase may have increased pH optimization, or increased temperature or oxidative stability, or reduced affinity or binding to a non-cellulosic material, but may retain its characteristic cellulolytic activity. It is believed that the variants according to the invention may be derived from a DNA fragment encoding a cellulase variant wherein the functional activity of the expressed cellulase variant is retained. For example, a DNA fragment encoding a cellulase may further comprise a DNA sequence or portion thereof encoding a hinge or linker attached at the 5 'or 3' end to the cellulase DNA sequence, wherein the functional activity of the encoded cellulase domain is retained. The terms variant and derivative are used interchangeably herein.

"equivalent residues" can also be defined by determining homology at the level of tertiary structure to a precursor cellulase whose tertiary structure has been determined by X-ray crystallography. Equivalent residues are defined as the atomic coordinates of two or more backbone atoms (N to N, CA to CA, C to C and O to O) of the specific amino acid residues of cellulase and hypocrea jecorina CBH2 within 0.13nm, preferably within 0.1nm, after alignment. Alignment was achieved after orienting and positioning the best model with respect to hypocrea jecorina CBH2 to produce the maximum overlap of atomic coordinates of the non-hydrogen protein atoms of the cellulase. The best model is the crystallographic model that allows the lowest R-factor to be obtained at the highest resolution of experimental diffraction data, as can be seen for example in US 2006/0205042.

Equivalent residues functionally analogous to the specific residues of hypocrea jecorina CBH2 are defined as amino acids of cellulases that can adopt a conformation such that they alter, modify or promote protein structure, substrate binding or catalysis in a manner that is defined and attributed to the specific residues of hypocrea jecorina CBH 2. Furthermore, they are residues of cellulases (the tertiary structure of which has been obtained by x-ray crystallography) that occupy similar positions to the extent that the atomic coordinates of at least two side chain atoms of a residue are within 0.13nm of the corresponding side chain atoms of hypocrea jecorina CBH2, although the backbone atoms of a given residue may not satisfy the equivalence rule based on occupying homologous positions. Zou et al (1999) (reference 5, supra) showed the crystal structure of hypocrea jecorina CBH 2.

The term "nucleic acid molecule" includes RNA, DNA and cDNA molecules. It will be appreciated that as a result of the degeneracy of the genetic code, a plurality of nucleotide sequences encoding a given protein may be produced, for example CBH2 and/or variants thereof. The present invention contemplates every possible variant nucleotide sequence encoding a variant cellulase (e.g., CBH2), all of which are possible given the degeneracy of the genetic code.

A "heterologous" nucleic acid construct or sequence has a partial sequence that is not native to the cell in which it is expressed. Heterologous in terms of control sequences means that the control sequences (i.e., promoters or enhancers) do not substantially act to regulate the expression of the gene currently being regulated. Typically, the heterologous nucleic acid sequence is not endogenous to the cell or part of the genome in which it is present, and has been added to the cell by infection, transfection, transformation, microinjection, electroporation, and the like. A "heterologous" nucleic acid construct can contain a combination of control sequences/DNA coding sequences that is the same or different from the combination of control sequences/DNA coding sequences found in the native cell.

As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. "expression vector" refers to a vector that has the ability to incorporate and express a heterologous DNA segment in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of a suitable expression vector is within the knowledge of one skilled in the art.

Thus, an "expression cassette" or "expression vector" is a nucleic acid construct, produced recombinantly or synthetically, with a series of specialized nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette may 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, among other sequences, 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 used as a cloning vector, which forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes.

As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence that is capable of being expressed in a cell, and expression of the selectable marker confers upon the cell containing the expressed gene the ability to grow in the presence of a corresponding selective agent or corresponding selective growth conditions.

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 host cells expressing the target gene. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also referred to as "control sequences"), is necessary for the expression of a given gene. Generally, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

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) that is composed of portions of different genes, including regulatory elements. Chimeric gene constructs for host cell transformation typically include a transcriptional regulatory region (promoter) operably linked to a heterologous protein coding sequence, or in a selectable marker chimeric gene, a selectable marker gene encoding a protein that confers resistance to a transformed cell, e.g., an antibiotic. Exemplary chimeric genes for transforming host cells of the invention include transcriptional regulatory regions (constitutive or inducible), protein coding sequences and terminator sequences. The chimeric gene construct may also include a second DNA sequence encoding a signal peptide if secretion of the target protein is desired.

Nucleic acids are operably linked when they are placed in a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or 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 linkage of DNA sequences is contiguous and, in the case of a secretory leader, contiguous and in frame. However, enhancers need not be contiguous. Ligation is achieved by ligation at conventional restriction sites. If such sites are not present, synthetic oligonucleotide adaptors (adaptors), linkers or primers for PCR are used according to conventional practice.

As used herein, the term "gene" means a segment of DNA involved in producing a polypeptide chain, which may or may not include regions preceding and following the coding region, such as 5 ' untranslated (5 ' UTR) or "leader" sequences, and 3 ' UTR or "untranslated tail" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

Typically, a nucleic acid molecule encoding a variant cellulase (e.g., CBH2) can hybridize to a wild-type sequence as provided herein under moderate to high stringency conditions, such as the nucleic acid sequences set forth in SEQ ID NOs: 1. however, in some cases, a CBH 2-encoding nucleotide sequence having a substantially different codon usage is used, whereas the protein encoded by the CBH 2-encoding nucleotide sequence has the same or substantially the same amino acid sequence as the native protein. For example, the coding sequence may be modified to facilitate faster expression of CBH2 in a particular prokaryotic or eukaryotic expression system, depending on the frequency of specific codons used by the host (Te' o et al, FEMS Microbiology Letters, 190: 13-19, 2000; for example, gene optimization for expression in filamentous fungi is described).

A nucleic acid sequence is considered "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize to each other under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "highest 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; whereas "low stringency" occurs at about 20-25 ℃ below the Tm. Functionally, conditions of highest stringency can be used to identify sequences that have stringent or near stringent identity to the hybridization probe, while conditions of high stringency can be used to identify sequences that have about 80% or greater sequence identity to the probe.

Moderate or high stringency hybridization conditions are generally known in the art (see, e.g., Sambrook et al, 1989, chapters 9 and 11; and Ausubel, F.M. et al, 1993; expressly incorporated herein by reference). Examples of high stringency conditions include hybridization in 50% formamide, 5XSSC, 5 fold Denhardt's solution, 0.5% SDS, and 100. mu.g/ml denatured vector DNA at about 42 ℃ followed by 2 washes in 2 fold SSC and 0.5% SDS at room temperature, and 2 additional washes in 0.1 XSSC and 0.5% SDS at 42 ℃.

The term "recombinant" when used with respect to, for example, a cell or nucleic acid, protein or vector, means 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, recombinant cells express genes that are not visible in the native (non-recombinant) form of the cell, or express otherwise abnormally expressed, under-expressed, or not expressed at all, native genes.

As used herein, the terms "transformed," "stably transformed," or "transgenic" in reference to a cell mean that the cell has a non-native (heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained over multiple generations.

As used herein, the term "expression" refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene. The process includes 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 reference to the incorporation 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).

Thus the term "CBH 2 expression" refers to the transcription and translation of the CBH2 gene or variants thereof, the products of which include precursor RNA, mRNA, polypeptides, post-translationally processed polypeptides and derivatives thereof, including CBH2 from related species such as Trichoderma koningii (Trichoderma koningii), Hypocrea (also known as Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride (Trichoderma virride)) and Hypocrea schweinitizi. By way of example, assays for CBH2 expression include Western blots for CBH2 protein, Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) assays for CBH2 mRNA, and phospho-swollen Cellulose (PASC) and p-hydroxybenzoyl hydrazine (PAHBAH) assays, as described in: (a) PASC: (Karlsson, J. et al, (2001), Eur. J. biochem, 268, 6498-: (Lever, M. (1972) Analytical Biochemistry, 47, 273; Blakeney, A.B. & Mutton, L.L, (1980) Journal of Science of Food and agriculture, 31, 889; Henry, R.J. (1984) Journal of the Institute of Brewing, 90, 37).

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

The term "signal sequence" refers to an amino acid sequence at the N-terminal portion of a protein that facilitates secretion of the mature form of the protein outside the cell. The mature form of the extracellular protein lacks a signal sequence that is cleaved off during secretion.

The term "host cell" means a cell which contains a vector and supports the replication and/or transcription and translation (expression) of an expression construct. The host cell for use 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 "filamentous fungus" means any and all filamentous fungi recognized by those skilled in the art. Preferred fungi are selected from the group consisting of Aspergillus (Aspergillus), Trichoderma, Fusarium (Fusarium), Chrysosporium (Chrysosporium), Penicillium (Penicillium), Humicola (Humicola), Neurospora (Neurospora), or an alternative sexual type thereof such as Sporotrichum (Emericella), Hypocrea (Hypocrea). The asexual industrial fungus Trichoderma reesei has been shown to be a cloned derivative of hypocrea jecorina of the Ascomycete class (ascomycetes) (see Kuhls et al, PNAS, 93: 7755-7760, 1996).

The term "cellooligosaccharide" refers to an oligosaccharide group containing 2-8 glucose units and having beta-1, 4 linkages, such as cellobiose.

The term "cellulase", "cellulolytic enzyme" or "cellulase" refers to a class of enzymes capable of hydrolyzing a cellulose multimer to shorter cellooligosaccharide oligomers, cellobiose, and/or glucose. Various examples of cellulases, such as exoglucanases, exocellobiohydrolases, endoglucanases and glucosidases, have been obtained from cellulolytic organisms, including in particular fungi, plants and bacteria. Enzymes produced by such microorganisms are mixtures of proteins that have three types of roles that are effective in converting cellulose to glucose: endoglucanases (EG), Cellobiohydrolases (CBH) and beta-glucosidases (BGL or Bglu). These three different types of cellulases act synergistically to convert cellulose and its derivatives to glucose.

Enzymes produced by many microorganisms to hydrolyze cellulose, including trichoderma, the composting bacteria Thermomonospora (Thermomonospora), Bacillus (Bacillus), and Cellulomonas (Cellulomonas); streptomyces (Streptomyces); and the fungi Humicola, Aspergillus and Fusarium.

CBH2 from hypocrea jecorina is a member of glycosyl hydrolase family 6 (hence Cel6), particularly the first member of this family identified in hypocrea jecorina (hence Cel 6). Glycosyl hydrolase family 6 contains both endoglucanase and cellobiohydrolase/exoglucanase, the latter being CBH 2. Thus, the phrases CBH2, CBH2 type protein, and Cel6 cellobiohydrolase are used interchangeably herein.

As used herein, the term "cellulose binding domain" refers to a region of an enzyme or a partial amino acid sequence of a cellulase involved in the cellulose binding activity of the cellulase or a derivative thereof. The cellulose binding domain typically functions by non-covalent binding of cellulase to cellulose, cellulose derivatives or other polysaccharide equivalents thereof. The cellulose binding domain allows or facilitates hydrolysis of the cellulose fibers by a structurally distinct catalytic core region and generally functions independently of the catalytic core. Thus, the cellulose binding domain may not have significant hydrolytic activity due 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 a structural element having catalytic activity. Cellulose binding domains and cellulose binding components are used interchangeably herein.

As used herein, the term "surfactant" refers to any compound having surface active properties conventionally known in the art. 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; an olefinic sulfonate; and alkyl sulfonates. Amphoteric surfactants include quaternary ammonium sulfonates and betaine-type amphoteric surfactants. Such amphoteric surfactants have both positively and negatively charged groups in the same molecule. Nonionic surfactants may include polyoxyalkylene ethers as well as adducts of alkanolamides of higher fatty acids or alkylene oxides 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 mixtures of cotton-or non-cotton containing cellulose, including natural cellulosics and man-made cellulosics (e.g., jute, flax, ramie, rayon, and lyocell).

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

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

As used herein, the term "detergent composition" refers to a mixture intended for use in a laundry substrate for cleaning soiled cellulose-containing fabrics. In the context of the present invention, such compositions may include, in addition to cellulases and surfactants, other hydrolases, builders, bleaches, bleach activators, bluing agents and fluorescent dyes, anti-caking agents, masking agents, cellulase activators, antioxidants and solubilizers.

As used herein, the term "reduces or eliminates expression of the cbh2 gene" means that the cbh2 gene has been deleted from the genome such that the recombinant host microorganism is unable to express the cbh2 gene; or the CBH2 gene or transcript has been modified such that the host microorganism does not produce a functional CBH2 enzyme or produces at a level significantly lower than the unmodified CBH2 gene or transcript.

The term "variant cbh2 gene" means that the nucleic acid sequence of the cbh2 gene from hypocrea jecorina has been altered by removal, addition, and/or manipulation of the coding sequence.

As used herein, the term "purification" generally refers to subjecting cells containing the transgenic nucleic acid or protein to biochemical purification and/or column chromatography.

As used herein, the terms "activity" and "biological activity" refer to biological activity associated with a particular protein and 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 given protein refers to any biological activity that is typically attributed to a protein by one of skill in the art.

As used herein, the term "enriched" means that the concentration of cellulase (e.g., CBH2) is found to be greater relative to the concentration of CBH2 found in a wild-type or naturally occurring fungal cellulase composition. The terms enriched, elevated and enhanced are used interchangeably herein.

Wild type fungal cellulase compositions are compositions produced from naturally occurring fungal sources and include one or more of the BGL, CBH and EG components, each of which is found in proportions produced by the fungal source. Thus, the enriched CBH composition can have an altered ratio of CBH, wherein the ratio of CBH to other cellulase components (e.g., EG, β -glucosidase, and other endoglucanases) is increased. The ratio may be increased by increasing the CBH or decreasing (or eliminating) at least one other component by any method known in the art.

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

Thus, by way of example, naturally occurring cellulase systems may be purified to substantially pure components by known separation techniques well-disclosed in the literature, including ion exchange chromatography at appropriate pH, affinity chromatography, size exclusion, and the like. For example, in ion exchange chromatography (typically anion exchange chromatography), the cellulase components can be separated by elution with a pH gradient or a salt gradient or a pH and salt gradient. The purified CBH may then be added to the enzyme solution to obtain an enriched CBH solution. The amount of CBH produced by the microorganism may also be increased by using molecular genetic methods to overexpress the gene encoding CBH, possibly in combination with deletion of one or more genes encoding other cellulases.

Fungal cellulases may contain more than one CBH component. The different components typically have different isoelectric points so that they can be separated by ion exchange chromatography or the like. A single CBH component, or a combination of CBH components, may be used in the enzyme solution.

When used in an enzyme solution, the homolog or variant CBH2 component is typically added in an amount sufficient to allow the highest rate of release of soluble sugars from the biomass. The amount of homolog or variant CBH2 component added depends on the type of biomass to be saccharified, which can be conveniently determined by one skilled in the art at the time of use, and the weight percent of homolog or variant CBH2 component present in the cellulase composition is preferably between 1 and 100, with illustrative examples being 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, from 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 60 weight percent, from about 15 to about 65 weight percent, from about 15 to about 70 weight percent, about 15 to about 75 weight percent, about 15 to about 80 weight percent, about 15 to about 85 weight percent, about 15 to about 95 weight percent. However, when used, the weight percent of the CBH2 component, homolog or variant, relative to any EG-type component, homolog or variant present in the cellulase composition is preferably from 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, from 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, about 15 to about 35 weight percent, about 15 to about 30 weight percent, about 15 to about 45 weight percent, about 15 to about 50 weight percent.

Cellulase II

Cellulases are enzymes known in the art that hydrolyze cellulose (β -1, 4-glucan or β -D-glycosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. As mentioned above, cellulases are traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) ("EG"), exoglucanases or cellobiohydrolases (EC 3.2.1.91) ("CBH") and beta-glucosidases (EC 3.2.1.21) ("BG").

Some fungi produce complete cellulase systems, including exo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-type cellulases, as well as β -glucosidases or BG-type cellulases. However, sometimes these systems lack both CBH-type cellulases and bacterial cellulases, and also typically include little or no CBH-type cellulases. In addition, the EG component and CBH component have been shown to interact synergistically, degrading cellulose more efficiently. The different components in 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 enzyme mode of action.

It is believed that endoglucanase-type cellulases hydrolyze beta-1, 4-glucosidic bonds within low crystallinity (crystallinity) regions of cellulose, while exocellobiohydrolase-type cellulases hydrolyze cellobiose from either the reducing or non-reducing ends of cellulose. Thus, by creating new strand ends that are recognized by the exo-cellobiohydrolase component, the action of the endoglucanase component may be greatly beneficial to the action of the exo-cellobiohydrolase. Furthermore, cellulases of the beta-glucosidase type have been shown to catalyze the hydrolysis of alkyl and/or aryl beta-D-glucosides, such as methyl beta-D-glucoside and p-nitrophenylglucoside, as well as glycosides containing only carbohydrate residues, such as cellobiose. This produces glucose as a single product of the microorganism and reduces or eliminates cellobiose that inhibits cellobiohydrolases and endoglucanases.

Cellulases also have a variety of applications in detergent compositions, including enhancing cleaning, as softeners and improving the feel of cotton fabrics (Hemmpel, ITB Dyeing/Printing/Finishing 3: 5-14, 1991; Tyndall, Textile Chemist and Colorist 24: 23-26, 1992; and Kumar et al, Textile Chemist and Colorist, 29: 37-42, 1997). Although the mechanism is not part of the present invention, the softening and color repair properties of cellulases have been attributed to the alkaline endoglucanase components in the cellulase composition, as exemplified in U.S. Pat. nos. 5,648,263, 5,691,178 and 5,776,757, which disclose that detergent compositions containing cellulase compositions enriched for a particular alkaline endoglucanase component impart color repair and improved softening to treated garments, as compared to cellulase compositions not enriched for such components. In addition, the use of such alkaline endoglucanase components in detergent compositions has been shown to compensate for the pH requirements of the detergent composition (e.g., by exhibiting maximum activity at alkaline pH of 7.5-10, as described in U.S. Pat. nos. 5,648,263, 5,691,178, and 5,776,757).

Cellulase compositions have also been shown to degrade cotton-containing fabrics, resulting in reduced strength loss of the fabric (U.S. patent No. 4,822,516), causing a barrier to the use of cellulase compositions in commercial detergent applications. It has been suggested that cellulase compositions comprising an endoglucanase component show reduced force loss in cotton containing fabrics compared to compositions comprising an intact cellulase system.

Cellulases have also been shown to be useful In degrading cellulase biomass to ethanol (where cellulases degrade cellulose to glucose and yeast or other microorganisms further ferment glucose to ethanol), both In mechanical pulp processing (Pere et al, In Proc. Tappi Pulping Conf., Nashville, Tenn., 27-31, page 693-696, 1996), as feed additives (WO 91/04673) and In grain wet milling.

Most CBH and EG have a multidomain structure comprising a core domain separated from a Cellulose Binding Domain (CBD) by a linker peptide (Suurnakki 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, FEBS Lett.204: 223-. 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 a CBD is deleted (Linder and Teeri, J.Biotechnol.57: 15-28, 1997). However, the precise action and mechanism of action of CBD remains a matter of thought. CBD has been suggested to increase enzyme activity only by increasing the effective enzyme concentration on the cellulose surface (Stahlberg et al, Bio/technol.9: 286-. Most studies focusing on the effect of cellulase domains on different substrates have been carried out with the core protein of cellobiohydrolases, since the core protein of the enzyme can be conveniently produced by papain-limited proteolysis (Tomme et al, 1988). Various 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 CBH 1; teeri, t, et al, Gene, 51: 43-52, 1987, which discloses CBH 2. Cellulases from species other than Trichoderma are also described, for example Ooi et al, Nucleic Acids Research, Vol.18, No. 19, 1990 disclosing cDNA sequences encoding endoglucanase F1-CMC produced by Aspergillus aculeatus (Aspergillus aculeatus); kawaguchi T et al, Gene 173 (2): 287-8, 1996, which discloses the cloning and sequencing of a cDNA encoding beta-glucosidase 1 from Aspergillus aculeatus; sakamoto et al, curr. genet.27: 435 ℃ 439, 1995 disclosing a cDNA sequence encoding endoglucanase CMC enzyme-1 from Aspergillus kawachii IFO 4308; saarilahti et al, Gene 90: 9-14, 1990, which discloses endoglucanases from Erwinia carotovora (Erwinia carotovora); spilleert R et al, Eur J biochem.224 (3): 923-30, 1994 which discloses the cloning and sequencing of bglA encoding a thermostable β -glucanase from Rhodothermus marinus; and Halldorsdottir S et al, ApplMicrobiol Biotechnol.49 (3): 277-84, 1998, which discloses the cloning, sequencing and overexpression of the Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12. However, there remains a need to identify and characterize new cellulases with improved properties, such as improved performance under conditions of heat stress or in the presence of surfactants, increased specific activity, altered substrate cleavage pattern, and/or high levels of in vitro expression.

The development of new and improved cellulase compositions comprising varying amounts of CBH-type, EG-type and BG-type cellulases is aimed at: such as (1) compositions that degrade wood pulp or other biomass into sugars (e.g., for biochemical production, such as biofuels); (2) detergent compositions exhibiting enhanced cleaning ability; (3) act as softeners and/or improve the feel of cotton fabrics (e.g., "stone washing" or "biopolishing"); and/or (4) a feed composition.

Also provided herein are whole cellulase preparations comprising cellulase variants. The phrase "whole cellulase preparation" as used herein refers to both naturally occurring and non-naturally occurring cellulase-containing compositions. A "naturally-occurring" composition is one that is produced by a naturally-occurring source, the composition containing one or more cellobiohydrolase types, one or more endoglucanase types, and one or more beta-glucosidase components, wherein each of these components is obtained in a proportion produced by the source. A naturally occurring composition is one that is produced from an organism that is unmodified with respect to the cellulolytic enzymes such that the proportions of the component enzymes are not altered from those produced by the natural organism. "non-naturally occurring" compositions include those produced by (1) combining the component cellulolytic enzymes in naturally occurring proportions or in non-naturally occurring (i.e., altered) proportions, or (2) modifying an organism to over-or under-express one or more cellulolytic enzymes, or (3) modifying an organism such that at least one cellulolytic enzyme is deleted. Accordingly, in some embodiments, a whole cellulase preparation may be deficient in one or more of a plurality of EG and/or CBH, and/or β -glucosidase. For example, EG1 may be deleted alone or in combination with other EG and/or CBH.

Generally, whole cellulase preparations include enzymes including, but not limited to, (i) Endoglucanases (EG) or 1, 4- β -d-glucan-4-glucanohydrolases (EC 3.2.1.4), (ii) exoglucanases, including 1, 4- β -d-glucan glucanohydrolases (also known as cellodextrins (cellodextrins)) (EC 3.2.1.74) and 1, 4- β -d-glucan cellobiohydrolases (exo-cellobiohydrolases, CBH) (EC 3.2.1.91) and (iii) β -glucosidases (BG) or β -glucoside glucohydrolases (EC 3.2.1.21).

In the present disclosure, the whole cellulase preparation may be derived from any microorganism useful for the hydrolysis of cellulosic material. In some embodiments, the whole cellulase preparation is a filamentous fungal whole cellulase. "filamentous fungi" include all filamentous forms of the subgenus fungi (Eumycota) and the genus Phycomyceta (Oomycota). In some embodiments, the whole cellulase preparation is a whole cellulase of a species of Acremonium (Acremonium), aspergillus, gymnosporium (Emericella), fusarium, Humicola (Humicola), Mucor (Mucor), Myceliophthora (Myceliophthora), Neurospora (Neurospora), cylindrospora (Scytalidium), Thielavia (Thielavia), torticollis (Tolypocladium), or trichoderma. In some embodiments, the whole cellulase preparation is a whole cellulase of Aspergillus aculeatus (Aspergillus aculeatus), Aspergillus awamori (Aspergillus awamori), Aspergillus foetidus (Aspergillus foetidus), Aspergillus japonicus (Aspergillus japonicus), Aspergillus nidulans (Aspergillus nidulans), Aspergillus niger or Aspergillus oryzae. In another aspect, the holocellulase preparation is Fusarium bactridioides (Fusarium bactridioides), Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum (Fusarium culmorum), Fusarium graminearum (Fusarium graminearum), Fusarium graminum (Fusarium graminum), Fusarium heterosporum (Fusarium heterosporum), Fusarium negundi, Fusarium oxysporum (Fusarium oxysporum), Fusarium reticulatum (Fusarium reticulatum), Fusarium roseum (Fusarium roseum), Fusarium sambucinum (Fusarium sambucinum), Fusarium sarcochroosum (Fusarium sarcochromyces), Fusarium sporotrichioides (Fusarium sporotrichinosum), Fusarium trichothecium, Fusarium trichothecioides (Fusarium trichothecioides), Fusarium trichothecium trichothecioides (Fusarium trichothecium), or Fusarium trichothecium, Fusarium trichothecium, or Fusarium trichothecium of the Fusarium trichothecium of the genus Fusarium trichothecium (Fusarium trichothecium sp. In another aspect, the whole cellulase preparation is a whole cellulase of Humicola insolens, Humicola lanuginosa, Mucoramiehei, Myceliophthora thermophila, Neurospora crassa, Scytalidium thermophilum, or Thielavia terrestris (Thielavia terrestris). In another aspect, the whole cellulase preparation is a whole cellulase of Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei (Trichoderma reesei), such as RL-P37(Sheir-Neiss et al, appl. Microbiol. Biotechnology, 20(1984) pages 46-53; Montenecort B.S., Can., 1-20, 1987), QM9414(ATCC No.26921), NRRL 15709, ATCC 13631, 56764, 56466, 56767, or Trichoderma viride (Trichoderma viride), such as ATCC 32098 and 32086. In some embodiments, the whole cellulase preparation is whole cellulase of trichoderma reesei RutC30, which is available from the American Type culture collection (American Type culture collection) such as trichoderma reesei ATCC 56765.

Examples of commercially available cellulase preparations suitable for use in the present invention include, for example, CELLUCLAST^TM(available from Novozymes A/S) and LAMINEX^TM、IndiAge^TMAnd Primafast^TMLaminex BG enzyme, ACCELLERASE^TM100 and ACCELLERASE^TM1500 (available from Genencor Division, Danisco us.inc.).

In the present disclosure, the whole cellulase preparation may be derived from any microbial culture method known in the art, resulting in the expression of an enzyme capable of hydrolyzing a cellulosic material. Fermentation may comprise shake flask cultivation, small or large scale fermentation, such as continuous, batch, fed-batch or solid state fermentation in laboratory or industrial fermentors performed in a suitable medium under conditions allowing the cellulase to be expressed or isolated.

Typically, the microorganisms are cultured in a cell culture medium suitable for use in the production of enzymes capable of hydrolyzing cellulosic material. The cultivation takes place in a suitable nutrient medium containing a carbon source, a nitrogen source and inorganic salts, using methods known in the art. Suitable media, temperature ranges and other conditions suitable for growth and cellulase production are known in the art. As a non-limiting example, the normal temperature range for cellulase production by Trichoderma reesei is 24 ℃ to 28 ℃.

Typically, whole cellulase preparations are used as they are produced by fermentation with little or no recovery and/or purification. For example, once cellulase is secreted by the cells into the cell culture medium, a cell culture medium containing cellulase can be used. In some embodiments, the whole cellulase preparation contains unfractionated components of the fermentation material, including cell culture media, extracellular enzymes, and cells. Alternatively, the whole cellulase preparation may be processed by any convenient method, e.g., by precipitation, centrifugation, affinity, filtration, or any other method known in the art. In some embodiments, the whole cellulase preparation may be concentrated, for example, and then used without further purification. In some embodiments, the whole cellulase preparation contains a chemical agent that reduces cell viability or kills cells. In some embodiments, the cells are lysed or permeabilized using methods known in the art.

Molecular biology

In one embodiment, the present invention provides that the variant cbh2 gene is expressed under the control of a promoter functional in filamentous fungi. Thus, the present invention relies on conventional techniques in the field of recombinant genetics (see, e.g., Sambrook et al, Molecular Cloning, A Laboratory Manual, 2 nd edition, 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Ausubel et al, Current PROTOCOLS INMOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, New York, 1994).

Method for mutating cbh2 nucleic acid sequence

Any method known in the art for introducing mutations is contemplated by the present invention.

The present invention relates to the expression, purification and/or isolation, and uses of variant CBH 2. These enzymes are preferably prepared by recombinant methods using the cbh2 gene from hypocrea jecorina. Fermentation media with or without purification may be used.

After isolation and cloning of the CBH2 gene from hypocrea jecorina, substitutions, additions or deletions corresponding to the substituted amino acids in the expressed CBH2 variant were generated using other methods known in the art, such as site-directed mutagenesis. Likewise, site-directed mutagenesis and other methods of incorporating amino acid changes into expressed proteins at the DNA level are known in the art (Sambrook et al, supra; and Ausubel et al, supra).

DNA encoding amino acid sequence variants of hypocrea jecorina CBH2 were prepared by a variety of methods known in the art. Such methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding hypocrea jecorina CBH 2.

Site-directed mutagenesis is a preferred method of making substitution variants. This technique is generally 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 performing site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the mutation of interest to a single strand of such starting DNA. After hybridization, the entire second strand is synthesized using a DNA polymerase, using the hybridized oligonucleotide as a primer, and the single strand of the starting DNA as a template. Thus, an oligonucleotide encoding the mutation of interest is incorporated into the double-stranded DNA obtained.

PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide (i.e., hypocrea jecorina CBH 2). See Higuchi, PCR Protocols, pp.177-183 (Academic Press, 1990); and Vallette et al, Nuc. acids Res.17: 723-733(1989). See also, e.g., Cadwell et al, PCR Methods and Applications, Vol.2, 28-33 (1992). In short, when a small amount of template DNA is used as a starting material for PCR, primers that differ slightly from the sequence of 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 positions where the primers differ from the template.

Another method for making variants, cassette mutagenesis, is based on Wells et al, Gene 34: 315-. The starting material is a plasmid (or other vector) containing the starting polypeptide DNA to be mutated. The codons to be mutated in the starting DNA were identified. Unique restriction endonuclease sites must be present on each side of the identified mutation site. If such restriction sites are not present, they can be generated by introducing them at the appropriate positions in the starting polypeptide DNA using the oligonucleotide-mediated mutagenesis method described above. The plasmid DNA was linearized by cleavage at these sites. Double-stranded oligonucleotides encoding a DNA sequence containing the mutation of interest between the restriction sites are synthesized using standard procedures, wherein the two oligonucleotide strands are synthesized separately and then hybridized together using standard techniques. The double-stranded oligonucleotide is called a cassette. The cassette is designed to have 5 'and 3' ends compatible with the ends of the linearized plasmid, allowing it to be ligated directly into the plasmid. Now, the plasmid contains the mutated DNA sequence.

Alternatively or additionally, the amino acid sequence of interest encoding a variant CBH2 can be determined and a nucleic acid sequence encoding such amino acid sequence variants can be synthetically produced.

The variant CBH2 so prepared may be further modified, typically depending 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.

Cbh2 nucleic acids and CBH2 polypeptides

A. Variant cbh 2-type nucleic acids

SEQ ID NO: 1 shows the nucleic acid sequence of wild type cbh 2. The invention encompasses nucleic acid molecules encoding the variant cellulases described herein. The nucleic acid may be a DNA molecule.

After cloning the DNA sequence encoding the CBH2 variant into a DNA construct, the DNA is used to transform the microorganism. The microorganism to be transformed for the purpose of expressing the variant CBH2 according to the invention may advantageously comprise a strain derived from a trichoderma species. Thus, a preferred mode for preparing variant CBH2 cellulases according to the invention comprises transforming a trichoderma species host cell with a DNA construct comprising at least a DNA fragment encoding part or all of variant CBH 2. The DNA construct may typically be functionally linked to a promoter. The transformed host cell is then grown under conditions to express the protein of interest. The protein product of interest may then be purified to substantial homogeneity.

In practice, however, the optimal expression vector for a given DNA encoding variant CBH2 may differ from hypocrea jecorina. Thus, it may be most advantageous to express the protein in a transformed host that has phylogenetic similarity to the organism from which variant CBH2 is derived. In an alternative embodiment, Aspergillus niger may be used as expression vector. For a description of transformation techniques using A.niger, see WO 98/31821, the disclosure of which is incorporated herein by reference in its entirety.

Thus, the present description of the aspergillus species expression system is provided for exemplary purposes only as an alternative to expressing the variant CBH2 of the present invention. However, one skilled in the art may be inclined to express DNA encoding variant CBH2 in different host cells as desired, it being understood that the source of variant CBH2 should be considered in determining the optimal expression host. Furthermore, the skilled person is able to select the optimal expression system for a particular gene by routine techniques using tools available in the art.

B. Variant CBH2 polypeptides

The variant CBH2 of the invention has an amino acid sequence derived from the amino acid sequence of precursor CBH 2. The amino acid sequence of the CBH2 variant differs from the precursor CBH2 amino acid sequence by substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. In a preferred embodiment, precursor CBH2 is hypocrea jecorina CBH 2. SEQ ID NO: 3 shows the mature amino acid sequence of hypocrea jecorina CBH 2. Thus, the present invention relates to CBH2 variants comprising an amino acid residue at a position equivalent to a specifically identified residue of hypocrea jecorina CBH 2. The residue (amino acid) of the CBH2 homologue is equivalent (i.e., chemically or structurally binds, reacts or interacts with the same or similar functional capability) to the residue of hypocrea jecorina CBH2 if it is homologous (i.e., corresponds in position in the primary or tertiary structure) or functionally similar to a particular residue or portion of that residue in hypocrea jecorina CBH 2. As used herein, the numbering is intended to correspond to that of the mature CBH2 amino acid sequence (SEQ ID NO: 3).

Alignment of amino acid sequences to determine homology is preferably determined using a "sequence comparison algorithm". Can be determined by, for example, Smith & Waterman, adv.appl.math.2: 482(1981) local homology algorithm; needleman & Wunsch, j.mol.biol.48: 443 (1970); pearson & Lipman, proc.nat' l acad.sci.usa 85: 2444(1988), by computerized application of these algorithms (GAP, BESTFIT, FASTA and TFASTA, in Wisconsin Genetics Software Package, Genetics computer Group, 575 Science Dr., Madison, Wis.) or by visual inspection (visual inspection), which may use a mapping Package, such as the MOE of Chemical Computing Group, Montreal Canada.

An example of an algorithm suitable for determining sequence similarity is the BLAST algorithm described in Altschul et al, j.mol.biol.215: 403-. Software for performing BLAST analysis is publicly available through the national center for Biotechnology information (www.ncbi.nlm.nih.gov). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short byte lengths W in the query sequence that match or satisfy some positive threshold score T when aligned with bytes of the same length in a database sequence. These initial adjacent byte hits act as starting points for finding longer HSPs containing them. Extending byte hits in both directions along either of the two sequences being compared is sufficient to increase the cumulative alignment score. When: the cumulative alignment score decreases by an amount X from the maximum value reached; a cumulative score of 0 or less; or at the end of either sequence, the extension of byte hits is stopped. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The default values used by the BLAST program are: byte length (W) is 11, BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915(1989)) is 50 for alignment (B), expectation (E) is 10, M '5, N' 4, and compare both strands.

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-. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences can 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 test amino acid sequence to the protease amino acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

For the purposes of the present invention, the degree of identity may suitably be determined by Computer programs known in the art, for example as provided in the GCG Program package (Program Manual for the WisconsinPackage, 8 th edition, month 8 1994, Genetics Computer Group, 575 scientific drive, Madison, Wis., USA 53711) (Needleman, S.B. and Wunsch, C.D. (1970), Journal of Molecular Biology, 48, 443-45), using GAPs with the following settings for polynucleotide sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

Structural alignments between Trichoderma reesei CBH2 and other cellulases can be used to identify equivalent/corresponding positions in other cellulases having moderate to high degrees of homology, e.g., homology of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% to Trichoderma reesei CBH2(SEQ ID NO: 3). One way to obtain the structural alignment is to use the Pile Up program from the GCG package, with a default value for the gap penalty, i.e., a gap creation penalty of 3.0 and a gap extension penalty of 0.1. Other structural alignment methods include hydrophobic cluster analysis (Gaboriaud et al, FEBS Letters, 224: 149-.

An exemplary alignment of the mature forms of the various reference cellulases is provided in figure 3. The reference cellulases include: hypocrea jecorina (also known as Trichoderma reesei) CBH2(SEQ ID NO: 3), Hypocrea cornininum CBH2(SEQ ID NO: 4), Humicola insolens CBH2(SEQ ID NO: 5), Vibrio cellulolyticus CBH2(SEQ ID NO: 6), Agaricus bisporus CBH2(SEQ ID NO: 7), Fusarium oxysporum EG (SEQ ID NO: 8), Phanerochaete chrysosporium CBH2(SEQ ID NO: 9), Talaromyces emersonii CBH2(SEQ ID NO: 10), Thermobifida fusca 6B/E3 CBH2(SEQ ID NO: 11), Thermobifida fusca 6A/E2 EG (SEQ ID NO: 12), and Cellulomonas faecalis CenAEG (SEQ ID NO: 13). The sequences were aligned using the ClustalW and MUSCLE multiple sequence alignment algorithm. Table 1 provides a matrix showing the percent cellulase identity of the sequence alignment of fig. 3.

TABLE 1 cellulase percent identity matrix

*

Percent _ ID	3	4	5	6	7	8	9	10	11	12	13
												3	100	95.5	62.3	64.7	59.6	63.1	55.4	63.4	31.9	13.5	27
4	95.5	100	61.6	64	59.1	63.6	54.7	63	32.9	13.5	26.8
												5	62.3	61.6	100	59.1	57.6	61.3	54	58.8	31.9	15.9	26.6
6	64.7	64	59.1	100	58.6	56.4	54	72.6	32.8	13.5	29.2
												7	59.6	59.1	57.6	58.6	100	55.8	69.1	58.1	34.9	17.5	27.6
8	63.1	63.6	61.3	56.4	55.8	100	48.7	54.8	31.1	13.9	25.2
												9	55.4	54.7	54	54	69.1	48.7	100	52.6	32.4	15.4	25.6
10	63.4	63	58.8	72.6	58.1	54.8	52.6	100	33.9	13.2	27.3
												11	31.9	32.9	31.9	32.8	34.9	31.1	32.4	33.9	100	15.9	36.3
12	13.5	13.5	15.9	13.5	17.5	13.9	15.4	13.2	15.9	100	12.8
												13	27	26.8	26.6	29.2	27.6	25.2	25.6	27.3	36.3	12.8	100

Numbers in the topmost row and left column correspond to SEQ ID NOs of the aligned sequences of fig. 3.

When evaluating a given nucleic acid Sequence against GenBank DNA sequences and other public databases, Sequence searches are typically performed using the BLASTN program. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all open reading frames for amino acid sequences in GenBank protein sequences and other public databases. BLASTN and BLASTX were both run using default parameters: an open gap penalty of 11.0 and an extended gap penalty of 1.0, using the BLOSUM-62 matrix (see, e.g., Altschul et al, 1997).

V. expression of recombinant CBH2 variants

The methods of the invention rely on the use of cells to express variant CBH2, and do not require specific methods of CBH2 expression. The variant CBH2 is preferably secreted from the cell. The present invention provides host cells transduced, transformed or transfected with expression vectors comprising variant CBH 2-encoding nucleic acid sequences. Culture conditions such as temperature, pH, etc., are those previously used in the parent host cell prior to transduction, transformation, or transfection, and 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 that functions in a host cell line operably linked to a DNA fragment encoding variant CBH2, such that variant CBH2 is expressed in the cell line.

A. Nucleic acid construct/expression vector

A natural or synthetic polynucleotide fragment encoding a variant CBH2 ("CBH 2-encoding nucleic acid sequence") may be incorporated into a heterologous nucleic acid construct or vector, which is capable of introduction into and replication in a filamentous fungal or yeast cell. The vectors and methods disclosed herein are suitable for use in host cells for expression of variant CBH 2. Any vector may be used as long as it is replicable and viable in the introduced cells. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available. Cloning and expression vectors are also described in Sambrook et al, 1989; ausubel F M et al, 1989 and Stratel et al, The Molecular Biology of The Yeast Saccharomyces, 1981, all of which are expressly incorporated herein by reference. Suitable expression vectors for fungi are described In van den Hondel, c.a.m.j.j. et al, (1991) In: bennett, J.W. and Lasure, L.L. (eds.) More Gene management functional Press, pages 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. Generally, the DNA sequence is inserted into the appropriate restriction enzyme site by standard methods. It is believed that such methods and related subcloning methods should fall within the knowledge of one skilled in the art.

Recombinant filamentous fungi comprising a variant CBH2 coding sequence may be produced by introducing a heterologous nucleic acid construct comprising the variant CBH2 coding sequence into cells of a selected filamentous fungal strain.

Once the variant cbh2 nucleic acid sequence of the desired morphology is obtained, it can be modified in various ways. Where the sequence relates to a non-coding flanking region, the flanking region may be excised, mutagenized, etc. Thus, transitions, transversions, deletions and insertions may be performed on the naturally occurring sequence.

The selected variant CBH2 coding sequences can be inserted into an appropriate vector according to generally known recombinant techniques and used to transform filamentous fungi capable of expressing CBH 2. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences encoding substantially the same or functionally equivalent amino acid sequences can be used to clone and express variant CBH 2. Thus, it is understood that such substitutions in the coding region fall within the scope of sequence variants encompassed by the present invention. Any and all such sequence variants can be used in the same manner as described herein for the parent CBH 2-encoding nucleic acid sequence.

The invention also includes recombinant nucleic acid constructs comprising one or more variant CBH 2-encoding nucleic acid sequences as described above. The construct comprises a vector, such as a plasmid or viral vector, into which the sequence of the invention has been inserted, in either forward or reverse orientation.

The heterologous nucleic acid construct can include a coding sequence for variant cbh2 that is (i) isolated; (ii) in combination with other coding sequences; for example as a fusion protein or signal peptide coding sequence, wherein the cbh2 coding sequence is the predominant coding sequence; (iii) in combination with non-coding sequences, e.g., introns and control elements, such as promoter and terminator elements or 5 'and/or 3' untranslated regions, which are effective for expression of the coding sequence in a suitable host; and/or (iv) in a vector or host environment, wherein the cbh2 coding sequence is a heterologous gene.

In one aspect of the invention, the variant CBH 2-encoding nucleic acid sequence is transferred into cells in vitro using a heterologous nucleic acid construct, with established filamentous fungal and yeast lines being preferred. For long-term production of variant CBH2, stable expression is preferred. Thus, any method effective to produce stable transformants may be used in the practice of the present invention.

Suitable vectors are generally equipped with selectable marker-encoding nucleic acid sequences, insertion sites and appropriate control elements, such as promoter and terminator sequences. The vector may contain regulatory sequences for efficient expression of the coding sequence in a host cell (and/or in the context of a vector or host cell in which the modified soluble protein antigen coding sequence is not normally expressed), including, for example, non-coding sequences such as introns and control elements, i.e., promoter and terminator elements or 5 'and/or 3' untranslated regions, 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).

Exemplary promoters include constitutive promoters and inducible promoters, examples of which include the CMV promoter, SV40 early promoter, RSV promoter, EF-1 α promoter, promoters containing Tet Response Elements (TRE) in the tet-on or tet-off systems as described (ClonTech and BASF), the β actin promoter, and the metallothionein promoter which can be upregulated by the addition of certain metal salts. Promoter sequences are DNA sequences recognized by the particular filamentous fungus used for expression purposes. It is operably linked to a DNA sequence encoding a variant CBH2 polypeptide. Such linkage includes placing the promoter in the disclosed expression vector against the start codon of the DNA sequence encoding the variant CBH2 polypeptide. The promoter sequence contains transcriptional and translational control sequences that mediate the expression of the variant CBH2 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 aspartic protease encoding gene of Aspergillus niger or Mucor miehei; hypocrea jecorina (trichoderma reesei) cbh1, cbh2, egl1, egl2, or other cellulase encoding genes.

The selection of appropriate selectable markers depends on the host cell, and appropriate markers for different hosts are generally known in the art. Typical selectable marker genes include argB from Aspergillus nidulans or Trichoderma reesei, amdS from Aspergillus nidulans, pyr4 from Neurospora crassa or Trichoderma reesei, pyrG from Aspergillus niger or Aspergillus nidulans. Other exemplary selectable markers include, but are not limited to, trpc, trp1, oliC31, niaD, or leu2, included in heterologous nucleic acid constructs used to transform mutant strains (e.g., trp-, pyr-, leu-, etc.).

Such selectable markers confer upon the transformants the ability to utilize metabolites not normally metabolized by filamentous fungi. For example, the amdS gene from hypocrea jecorina, encoding acetamidase, allows the transformant cells to grow with acetamide as a nitrogen source. The selectable marker (e.g., pyrG) may restore the ability of the auxotrophic mutant strain to grow on selective minimal medium, or the selectable marker (e.g., lic31) may confer the ability of the transformant to grow in the presence of an inhibitory drug or antibiotic.

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

The practice of the present invention may employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, 1989; freshney, Animal CellCulture, 1987; ausubel et al, 1993; and Coligan et al, Current protocol sin Immunology, 1991.

B. Host cells and culture conditions for CBH2 production

(i) Filamentous fungi

Accordingly, the present invention provides filamentous fungi comprising cells that have been modified, selected, and cultured in a manner effective to result in the production or expression of variant CBH2, relative to a corresponding untransformed parent fungus.

Examples of parent filamentous fungal species that may be treated and/or modified for expression of variant CBH2 include, but are not limited to, trichoderma, e.g., trichoderma reesei, trichoderma longibrachiatum, trichoderma viride, trichoderma koningii; penicillium species (Penicillium sp.); humicola species (Humicola sp.), including Humicola insolens; aspergillus species (Aspergillus sp.); microsporum species (Chrysosporiumsp); a Fusarium species; hypocrea species and chaetomium species.

The CBH2 expressing cells were cultured under conditions commonly used for the culture of parental fungal lines. Generally, cells are cultured in standard media containing physiological salts and nutrients, such as Pourquie, J.et al, Biochemistry and Genetics of cell Degradation, Aubert, J.P. et al, Academic Press, pp.71-86, 1988 and Ilmen, M.et al, appl.environ.Microbiol.63: 1298, 1306, 1997. Culture conditions are also standard, e.g., cultures are incubated in shake flask cultures or fermentors at 28 ℃ until the desired expression level of CBH2 is obtained.

Preferred culture conditions for a given filamentous fungus can be found in the scientific literature and/or from fungal sources, such as the American type culture Collection (ATCC; www.atcc.org /). After fungal growth has been established, the cells are exposed to conditions effective to cause or allow expression of variant CBH 2.

In the case where the CBH2 coding sequence is under the control of an inducible promoter, an inducer, such as a sugar, metal salt, or antibiotic, is added to the medium at a concentration effective to induce expression of CBH 2.

In one embodiment, the strain comprises Aspergillus niger, which is an efficient strain for obtaining over-expressed proteins. For example, the A.niger var awamori (A. niger var awamori) dgr246 is known to secrete increased amounts of secreted cellulase (Goedegebauur et al, curr. Genet (2002) 41: 89-98). Other strains of the A.niger P.awamori variety, such as GCDAP3, GCDAP4 and GAP3-4, are known (Ward et al, appl. Microbiol. Biotechnol. 39: 738-743, 1993).

In another embodiment, the strain comprises trichoderma reesei, which is an effective strain for obtaining over-expressed proteins. For example, Sheir-Neiss et al, appl.Microbiol.Biotechnol.20: 46-53(1984) RL-P37 secretes increased amounts of cellulase. Functional equivalents of RL-P37 include Trichoderma reesei strain RUT-C30(ATCC No.56765) and strain QM9414(ATCC No. 26921). These strains are also thought to be effective for over-expressing variant CBH 2.

When it is desired to obtain a variant CBH2 that lacks potentially harmful native cellulolytic activity, it may be beneficial to obtain a Trichoderma host cell strain from which one or more cellulase genes have been deleted prior to introduction of a DNA construct or plasmid containing a DNA fragment encoding the variant CBH 2. Such strains can be prepared by 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 variant CBH2 cellulase in a host microorganism lacking one or more cellulase genes, the identification and subsequent purification steps are simplified. Any genes from Trichoderma species that have been cloned can be deleted, such as cbh1, cbh2, egl1, and egl2 genes, as well as genes encoding EG III and/or EGV proteins (see, e.g., U.S. Pat. No.5,475,101 and WO 94/28117, respectively).

Gene deletion can be accomplished by inserting the morphology of the gene of interest to be deleted or disrupted into a plasmid by methods known in the art. The deletion plasmid is then cut at appropriate restriction sites, which are internal to the coding region of the gene of interest, and the gene coding sequence, or a portion thereof, is replaced with a selectable marker. DNA sequences flanking the locus from which the gene is to be deleted or disrupted, preferably between about 0.5-2.0kb, remain on either side of the selectable marker gene. Suitable deletion plasmids may typically have unique restriction enzyme sites present therein such that the fragment containing the deleted gene (including flanking DNA sequences), and the selectable marker gene, is removed as a single linear fragment.

The selectable marker must be selected so as to enable detection of the transformed microorganism. Any selectable marker gene expressed in the selected microorganism may be suitable. For example, for aspergillus species, the selectable marker is selected such that its presence in the transformant 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 species gene may be used, which if absent from the host strain, results in the host strain exhibiting an auxotrophic phenotype. Similarly, there are selectable markers for trichoderma species.

In one embodiment, a pyrG-derived strain of Aspergillus is transformed with a functional pyrG gene to provide a selectable marker for transformation. The pyrG-derived strain may be obtained by selecting a strain of Aspergillus species that is resistant to fluoroorotic acid (FOA). The pyrG gene encodes orotidine-5' -monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with the intact pyrG gene were grown in media lacking uridine, but were sensitive to fluoroorotic acid. By selecting for FOA resistance, it is possible to select pyrG-derived strains that lack functional orotidine monophosphate decarboxylase and require uridine for growth. Using the FOA selection technique, uridine requiring strains lacking functional orotate phosphoribosyltransferase can also be obtained. The cells can be transformed with functional copies of the gene encoding the enzyme (Berges & Barreau, Current. Gene.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 the derivative strain is conveniently carried out using the FOA resistance technique described above, and thus the pyrG gene is preferably used as a selectable marker.

In a second embodiment, a pyr 4-derived strain of a hypocrea species (trichoderma species)) is transformed with a functional pyr4 gene, thereby providing a selectable marker for transformation. The pyr4.sup. -derived strain may be obtained by selecting a strain of a Sarcophyton species (Trichoderma species) which is resistant to fluoroorotic acid (FOA). The pyr4 gene encodes orotidine-5' -monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with the intact pyr4 gene were grown in medium lacking uridine, but were sensitive to fluoroorotic acid. By selecting for FOA resistance, it is possible to select pyr4.sup. -derived strains that lack functional orotidine monophosphate decarboxylase enzyme and require uridine for growth. Using the FOA selection technique, uridine requiring strains lacking functional orotate phosphoribosyltransferase can also be obtained. The cells can be transformed with functional copies of the genes encoding the enzymes (Berges & Barreau, 1991). Selection of the derivative strain is conveniently carried out using the FOA resistance technique described above, and thus the pyr4 gene is preferably used as a selectable marker.

To transform pyrG-Aspergillus species or pyr 4-Hypocrea species (Trichoderma species) such that they lack the ability to express one or more cellulase genes, a single DNA fragment containing a disrupted or deleted cellulase gene is isolated from the deletion plasmid and used to transform a suitable pyr-Aspergillus or pyr-Trichoderma host. Transformants are then identified and selected based on their ability to express pyrG or pyr4, respectively, the gene product, and thus the complementation of the host strain's uridine auxotrophy. The transformants obtained are then subjected to Southern blot analysis to identify and verify double crossover integration events replacing part or all of the coding region of the genomic copy of the gene that is to be deleted with the appropriate pyr selectable marker.

Although the specific plasmid vector described above is directed to the preparation of pyr-transformants, the present invention is not limited to such vectors. Various genes can be deleted and replaced in Aspergillus species or Hypocrea species (Trichoderma species) using the above-described techniques. Furthermore, any available selectable marker may be used, as described above. Indeed, using the above strategy, any cloned and identified gene of any host, such as aspergillus or hypocrea species, can be deleted from the genome.

As mentioned above, the host strain used may be a derivative of a hypocrea species (trichoderma species) which is deleted or has a non-functional gene or a gene corresponding to a selected selectable marker. For example, if the selectable marker pyrG is selected for use in Aspergillus species, a particular pyrG-derived strain is used as recipient for the transformation step. Furthermore, for example, if the selectable marker pyr4 was selected for use in a hypocrea species, a specific pyr 4-derived strain was used as recipient of the transformation step. Similarly, selectable markers that may be used include genes from hypocrea species (trichoderma species) equivalent to the aspergillus nidulans genes amdS, argB, trpC, niaD. Thus, the corresponding recipient strain must be a derivative strain, e.g. argB-, trpC-, niaD-, respectively.

DNA encoding the CBH2 variant is then prepared for insertion into an appropriate microorganism. According to the invention, the DNA encoding the CBH2 variant comprises DNA necessary to encode a protein having functional cellulolytic activity. The DNA fragment encoding the CBH2 variant may be functionally linked to a fungal promoter sequence, for example, the promoter of the glaA gene in aspergillus or the promoter of the CBH1 or egl1 gene in trichoderma.

It is also contemplated that more than one copy of the DNA encoding the CBH2 variant may be recombined into the strain to facilitate overexpression. DNA encoding the CBH2 variant may be prepared by constructing an expression vector carrying DNA encoding the variant. The expression vector carrying the inserted DNA fragment encoding the CBH2 variant may be any vector capable of autonomous replication in a given host organism, or of integration into the DNA of the host, typically a plasmid. In a preferred embodiment, two types of expression vectors are contemplated for obtaining expression of a gene. The first contains DNA sequences in which the promoter, gene coding region and terminator sequences are all derived from the gene to be expressed. If desired, gene truncation may be achieved by deletion of unwanted DNA sequences (e.g., encoding unwanted domains) to retain the domain to be expressed under the control of its own transcriptional and translational regulatory sequences. The vector may also contain a selectable marker to allow selection for integration of multiple copies of the novel gene sequence into the host.

The second type of expression vector is preassembled, containing 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 the universal expression vector under the transcriptional control of the promoter and terminator sequences of the expression cassette.

For example, in aspergillus, pRAX is such a universal expression vector. The gene or part thereof may be inserted downstream of the glaa strong promoter.

For example, in hypocrea, pTEX is such a universal expression vector. The gene or a portion thereof may be inserted downstream of the cbh1 strong promoter.

In the vector, the DNA sequence encoding the CBH2 variant of the invention should be operably linked to transcriptional and translational sequences, i.e., a suitable promoter sequence and a signal sequence in 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 a gene encoding a protein, either homologous or heterologous to the host cell. An optional signal peptide is provided for extracellular production of the CBH2 variant. The DNA encoding the signal sequence is preferably naturally associated with the gene to be expressed, however, the invention contemplates a signal sequence from any suitable source, for example an exo-cellobiohydrolase or an endoglucanase from trichoderma.

Methods for ligating the DNA sequence encoding the variant CBH2 of the present invention with a promoter, and methods for insertion into an appropriate vector are generally known in the art.

The above-described DNA vectors or constructs may be introduced into host cells according to known techniques, such as transformation, transfection, microinjection, microperforation (microperforation), biolistic bombardment, and the like.

In the preferred transformation technique, it must be considered that in hypocrea species (trichoderma species) the permeability of the cell wall to DNA is very low. Thus, the uptake of the desired DNA sequence, gene or gene fragment is at most minimal. There are various methods for increasing the permeability of the cell wall of hypocrea species (trichoderma species) in the derivative strain (i.e. lacking the functional gene corresponding to the selectable marker used) prior to the transformation step.

A preferred method of preparing Aspergillus or Hypocrea species (Trichoderma species) for transformation in the present invention involves the preparation of protoplasts from fungal mycelia. See Campbell et al. Improved transformation efficiency of aspergillus niger the homologous niaD gene of nitrate reductase, curr. genet.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 an osmotic pressure stabilizer present in the suspension medium. These include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Typically, the concentration of these stabilizers ranges between 0.8M and 1.2M. Preferably, about 1.2M sorbitol solution is used in the suspension medium.

The uptake of DNA by the host strain (Aspergillus species or Hypocrea species (Trichoderma species)) is dependent on the calcium ion concentration. Typically about 10mM CaCl is used in the intake solution₂To 50mM CaCl₂. In addition to the requirement for calcium ions, other substances which are usually included in the uptake solution are buffer systems, such as TE buffer (10Mm Tris, pH 7.4; 1mM EDTA) or 10mM MOPS, pH6.0 buffer (morpholine propanesulfonic acid) and polyethylene glycol (PEG). Polyethylene glycol is thought to function by fusing cell membranes, allowing the media contents to be delivered into the cytoplasm of the host cell, and plasmid DNA is transferred into the nucleus, as exemplified by aspergillus or hypocrea strains. Such fusions often result in multiple copies of plasmid DNA being integrated into the host chromosome.

Generally, a density of 10 is used in the conversion^-5To 10^-6mL, preferably 2X10^-5Per mL ofA suspension comprising protoplasts of an Aspergillus species or cells that have been permeabilized. Similarly, a density of 10 was used in the transformation^-8To 10^-9mL, preferably 2X10^-8a/mL suspension containing protoplasts of a hypocrea species (Trichoderma species) or cells that have been subjected to a permeability treatment. A volume of 100. mu.L of an appropriate solution (e.g., 1.2M sorbitol; 50mM CaCl)₂) The protoplast or cell of (1) above, mixed with the target DNA. Typically, a high concentration of PEG is added to the uptake solution. 0.1-1 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, etc. may also be added to the uptake solution to aid in transformation.

Typically, the mixture is then incubated at about 0 ℃ for 10 to 30 minutes. Additional PEG is then added to the mixture to further increase the uptake of the gene or DNA sequence of interest. Typically 25% PEG4000 is added in a volume of 5-15 times the volume of the transformation mixture; however, more or less volume may be suitable. The 25% PEG4000 volume is preferably about 10 times the volume of the transformation mixture. After the addition of PEG, sorbitol and CaCl were added₂Prior to the solution, the transformation mixture is then incubated at room temperature or on ice. The protoplast suspension is then further added to an aliquot of the thawed growth medium. The growth medium only allows growth of the transformants. Any growth medium suitable for growing the transformant of interest may be used in the present invention. However, if Pyr is selected⁺Transformants, preferably using a growth medium without uridine. Subsequent colonies were transferred to growth medium lacking uridine and purified.

At this stage, stable transformants can be distinguished from unstable transformants by faster growth rates and the formation of circular colonies with smooth rather than rough contours on solid media lacking uridine, for example, in Trichoderma. In addition, in some cases, the stability test can be further performed by growing the transformants on a solid non-selective medium (i.e., uridine-containing), harvesting spores from the medium, and determining the percentage of these spores that can subsequently germinate and grow on selective medium lacking uridine.

In a particular embodiment of the above method, the active form of the CBH2 variant is recovered from the host cell after growth in liquid culture medium as a result of correct post-translational processing of the CBH2 variant.

(ii) Yeast

The present invention also contemplates the use of yeast as a host cell for CBH2 production. Several other genes encoding hydrolases have been expressed in different strains of s.cerevisiae. The gene includes sequences encoding the following enzymes: two endoglucanases (Penttila et al, Yeast, Vol.3, pp.175-185, 1987), two cellobiohydrolases (Penttila et al, Gene, 63: 103-112, 1988) and one beta-glucosidase from Trichoderma reesei (Cummings and Fowler, Curr. Genet.29: 227-233, 1996), a xylanase from aureobasidium pullulans (Li and Ljungdahl, appl. environ. Microbiol.62, No.1, pp.209-213, 1996), an alpha-amylase from wheat (Rothstein et al, Gene 55: 353-356, 1987) and the like. Furthermore, cellulase gene cassettes encoding endo- [ beta ] -1, 4-glucanase (END1) of Vibrio cellulolyticus (Butyrivibrio fibrinolvens), Phanerochaete chrysosporium cellobiohydrolase (CBH1), Ruminococcus xanthus (Ruminococcus flavefaciens) dextrinase (CEL1) and Endomyces fibrilizer cellobiohydrolase (Bgl1) have been successfully expressed in laboratory strains of Saccharomyces cerevisiae (Van Rensburg et al, Yeast, Vol.14, pp.67-76, 1998).

C. Introduction of a CBH 2-encoding nucleic acid sequence into a host cell

The invention also provides cells and cellular compositions that are genetically modified to comprise an exogenously provided variant CBH 2-encoding nucleic acid sequence. The parental cell or cell line may be genetically modified (i.e., transduced, transformed or transfected) with a cloning vector or an expression vector. The vector may be in the form of, for example, a plasmid, a viral particle, a phage, or the like, as described above.

The transformation methods of the invention can result in the stable integration of all or part of the transformation vector into the genome of the filamentous fungus. However, transformation of extrachromosomal transformation vectors that result in maintenance of self-replication is also contemplated.

A variety of standard transfection methods can be used to produce Trichoderma reesei cell lines expressing large amounts of heterologous proteins. Some disclosed methods for introducing a DAN construct into a cellulase-producing strain of trichoderma include: lorto, Hayes, DiPietro and Harman, 1993, curr. genet.24: 349-356; goldman, VanMontagu and Herrera-Estrella, 1990, curr. gene.17: 169-; penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6: 155-; for Aspergillus: yelton, Hamer and Timberlake, 1984, proc.natl.acad.sci.usa 81: 1470-; for Fusarium species: bajar, Podila and Kolattukudy, 1991, proc.natl.acad.sci.usa 88: 8202-8212; for Streptomyces species: hopwood et al, 1985, The John Innes Foundation, Norwich, UK; and for the genus bacillus: brigidi, DeRossi, Bertarini, riccaradi and Matteuzzi, 1990, FEMS microbiol.lett.55: 135-138.

Other methods for introducing the heterologous nucleic acid construct (expression vector) into the filamentous fungus (e.g., hypocrea jecorina) include, but are not limited to, using particle gun, permeabilizing the filamentous fungal cell wall prior to the transformation step (e.g., by using a high concentration of a base, such as 0.05M-0.4M CaCl)₂Or lithium acetate), protoplast fusion or agrobacterium-mediated transformation. By using polyethylene glycol and CaCl₂Exemplary methods for treating protoplasts or spheroplasts to transform filamentous fungi are described in Campbell, e.i. et al, curr.genet.16: 53-56, 1989 and Penttila, M. et al, Gene, 63: 11-22, 1988.

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

Furthermore, a heterologous nucleic acid construct comprising a variant CBH 2-encoding nucleic acid sequence may be transcribed in vitro and the resulting RNA introduced into the host cell by generally known methods, e.g. by injection.

The present invention also includes novel and advantageous filamentous fungal transformants, such as hypocrea jecorina and aspergillus niger, for use in the production of fungal cellulase compositions. The invention includes transformants of filamentous fungi, particularly fungi that comprise the coding sequence of variant CBH2, or that have the endogenous CBH coding sequence deleted.

After introduction of the heterologous nucleic acid construct comprising the variant CBH2 coding sequence, the genetically modified cells can be cultured in conventional nutrient media modified as desired for activating promoters, selecting transformants, or amplifying expression of the variant CBH 2-encoding nucleic acid sequence. Culture conditions such as temperature, pH, etc., are previously used for the host cell selected for expression and will be apparent to those skilled in the art.

It is generally believed that progeny of a cell into which such a heterologous nucleic acid construct has been introduced comprise the variant CBH 2-encoding nucleic acid sequence found in the heterologous nucleic acid construct.

The present invention also includes novel and advantageous filamentous fungal transformants, such as hypocrea jecorina, for use in the production of fungal cellulase compositions. Aspergillus niger can also be used to produce variant CBH 2. The invention includes transformants of filamentous fungi, particularly fungi comprising the coding sequence of variant cbh2, or fungi lacking the endogenous cbh2 coding sequence.

Stable filamentous fungal transformants can generally be distinguished from unstable transformants by faster growth rates and the formation of circular colonies with smooth, rather than rough, contours on solid media, for example in trichoderma. In addition, in some cases, stability testing can be further performed by growing the transformants on solid non-selective media, harvesting spores from the media, and determining the percentage of these spores that can subsequently germinate and grow on selective media.

Isolation and purification of recombinant CBH2 protein

In general, variant CBH2 proteins produced in cell culture are secreted into the culture medium and can be purified or isolated, for example, by removing unwanted components from the cell culture medium. However, in some cases, variant CBH2 proteins may be produced in cellular form and must be recovered from cell lysates. In this case, the variant CBH2 protein was purified from the producer cells using techniques routinely used by those skilled in the art. Examples include, but are not limited to, affinity chromatography (Tilbeurgh et al, FEBS Lett.16: 215, 1984), ion exchange chromatography (Goyal et al, Biorese Technol.36: 37-50, 1991; Fliess et al, Eur.J.Appl.Microbiol.Biotechnol.17: 314-318, 1983; Bhikhabhai et al, J.Appl.biochem.6: 336-345, 1984; Ellouz et al, J.Chromatology 396: 307-317, 1987), including ion exchange using materials with high resolution (resolution power) (Medve et al, J.nematology A808: 153-165, 1998), hydrophobic interaction chromatography (Tomaz and Queeiz, J.chromy A123: 123, 1999, Biochur et al: 287, 1999: 295, 1999: 1999-295).

Typically, variant CBH2 proteins are fractionated to isolate proteins with selective properties (e.g., binding affinity to a particular binding agent, such as an antibody or receptor); or proteins having a selected molecular weight range, or isoelectric point range.

Once expression of a given variant CBH2 protein is achieved, the thus produced CBH2 protein is purified from the cell or cell culture. Exemplary methods suitable for such purification include the following: antibody-affinity column chromatography, ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on silica gel or on cation-exchange resins (e.g., DEAE); carrying out chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; and gel filtration using, for example, Sephadex G-75. Various protein purification Methods can be used, and such Methods are known in the art and are described, for example, in Deutscher, Methods in enzymology, volume 182, phase 57, page 779, 1990; scopes, Methods enzymol.90: 479-91, 1982. The purification step chosen depends, for example, on the production method used and the nature of the particular protein produced.

Utilization of Cbh2 and CBH2

It will be appreciated that variant CBH nucleic acids, variant CBH2 proteins, and compositions comprising the activity of variant CBH2 proteins are useful for a wide range of applications, some of which are described below.

The new and improved cellulase compositions comprising varying amounts of BG-type, EG-type, and variant CBH-type cellulases are useful in detergent compositions exhibiting enhanced cleaning ability, function as softeners and/or improve the feel of cotton fabrics (e.g., "stone-washing" or "biopolishing"), in compositions for degrading wood pulp to sugar (e.g., for bioethanol production), and/or in feed compositions. The isolation and characterization of each type of cellulase provides the ability to control various aspects of such compositions.

In one approach, the cellulases of the invention are useful in detergent compositions or in the treatment of fabrics to improve feel and appearance.

Since the hydrolysis rate of cellulose products can be increased by using transformants having at least one additional copy of the cbh gene inserted into the genome, cellulose or heteropolysaccharide-containing products can be degraded at a faster rate and to a greater extent. Products made from cellulose, such as paper, cotton, cellulosic cotton fabrics, and the like, can be more effectively degraded in landfills. Thus, fermentation products obtainable from the transformants, or the transformants themselves, may be used in compositions to help degrade various cellulose products added to overcrowded landfills by liquefaction.

Step saccharification and fermentation are processes in which cellulose present in biomass (e.g., corn stover) is converted to glucose, and then yeast strains convert the glucose to ethanol. Simultaneous saccharification and fermentation is a process in which cellulose present in biomass (e.g., corn stover) is converted to glucose, and simultaneously and in the same reactor, a yeast strain converts glucose to ethanol. Thus, in another approach, the variant CBH-type cellulases of the invention can be used to degrade biomass to ethanol. The production of ethanol from readily available sources of cellulose provides a stable, renewable source of fuel.

Cellulose-based feedstocks include agricultural waste, grass and wood, as well as other low value biomass, such as municipal waste (e.g., recycled paper, yard clippings, etc.). Ethanol may be produced from the fermentation of any of the above cellulosic feedstocks. However, before it can be converted to ethanol, the cellulose must first be converted to sugars.

The variant CBH of the invention can be used with a variety of feedstocks, the choice of which depends on the region where the transformation is carried out. For example, in the midwestern united states, agricultural wastes such as wheat straw, corn stover, and bagasse predominate, while rice straw predominates in california. However, it should be understood that any available cellulosic biomass may be used in any region.

The process of the invention is useful for the production of monosaccharides, disaccharides and polysaccharides as chemical or fermentation feedstocks for microorganisms used for the production of organic products, chemicals and fuels, plastics and other products or intermediates. In particular, the value of the processing residues (dried distillers grains), parent grains from brewing, bagasse, etc.) can be increased by partial or complete dissolution of cellulose or hemicellulose. In addition to ethanol, some chemicals that can be produced from cellulose and hemicellulose include acetone, acetate, glycine, lysine, organic acids (e.g., lactic acid), 1, 3-propanediol, butanediol, glycerol, ethylene glycol, furfural, polyhydroxyalkanoates, cis-muconic acid, animal feed, and xylose.

Cellulase enzyme compositions containing increased amounts of cellobiohydrolases may be used for ethanol production. The ethanol from the process can be further used as octane booster or directly as a gasoline replacement fuel (fuel oil of gasoline), the latter being advantageous because ethanol is environmentally better as a fuel source than petroleum derived products. The use of ethanol is known to improve air quality and may reduce local ozone levels and smoke. Furthermore, the use of ethanol in gasoline substitution is strategically important to buffer the impact of sudden diversion of non-renewable energy and petrochemical supplies.

Ethanol can be produced by saccharification and fermentation processes of cellulosic biomass (e.g., woody, herbaceous, municipal solid waste, and agricultural and forestry residues). However, the ratio of individual cellulases in a mixture of naturally occurring cellulases produced by a microorganism may not be the most efficient for the rapid conversion of cellulose in biomass to glucose. Endoglucanase action is known to produce new cellulose chain ends which are themselves substrates for cellobiohydrolases, thereby improving the hydrolysis efficiency of the entire cellulase system. Thus, the use of increased or optimized cellobiohydrolase activity can greatly enhance ethanol production.

Thus, the cellobiohydrolases of the present invention may be used to hydrolyze cellulose to its sugar components. In one embodiment, the variant cellobiohydrolase is added to the biomass prior to addition of the fermenting organism. In a second embodiment, the variant cellobiohydrolase is added to the biomass simultaneously with the fermenting organism. Optionally, other cellulase components may be present in either embodiment.

In another embodiment, the cellulosic feedstock may be pretreated. The pretreatment may be by increasing the temperature, adding dilute acid, concentrated acid or dilute base solution. The pretreatment solution is added for a time sufficient to at least partially hydrolyze the hemicellulose component, and then neutralized.

The main product of CBH2 action on cellulose is cellobiose, which can be converted to glucose by BG activity (e.g., in fungal cellulase products). Sugars other than glucose and cellobiose may be obtained from biomass by pretreatment of the cellulosic biomass, or by enzymatic action on the biomass. The hemicellulose content of biomass can be converted (by hemicellulases) to sugars such as xylose, galactose, mannose and arabinose. Thus, in biomass conversion processes, enzymatic saccharification can produce sugars that can be used for biological or chemical conversion to other intermediates or end products. Thus, sugars produced from biomass can be used in a variety of processes other than ethanol production. Examples of such conversions are the fermentation of glucose to ethanol (as reviewed in M.E.Himmel et al, pages 2-45, "Fuels and Chemicals from Biomass", ACS symposium series 666, eds. B.C.Saha and J.Woodward, 1997) and other bioconversion of glucose to 2, 5-diketo-D-gluconic acid (U.S. Pat. No. 6,599,722), lactic acid (R.Datta and S-P.Tsai, page 224, above), succinic acid (R.R.Gokarn, M.A.Eiteman and J.Sridhar, page 237-. See also, for example, WO 98/21339.

In addition to cellulase compositions (independent of the level of cellobiohydrolase, i.e., free of cellobiohydrolase, substantially free of cellobiohydrolase, or enhanced cellobiohydrolase), the detergent compositions of the present invention may employ surfactants (including anionic, nonionic, and amphoteric surfactants), hydrolytic enzymes, builders, bleaches, bluing agents, and fluorescent dyes, anti-caking agents, solubilizers, cationic surfactants, and the like. All such components are known in the detergent art. The cellulase composition described above may be added to the detergent composition in the form of a liquid diluent, granules, emulsion, gel, paste (paste), etc. 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 granulate. Preferably, the granules may be formulated to contain a cellulase protecting agent. For a more thorough discussion, see U.S. Pat. No. 6,162,782 entitled "reagent compositions relating cell compositions and determining in CBH2 type compositions," which is incorporated herein by reference.

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

In addition, variant CBH2 nucleic acid sequences can be used to identify and characterize related nucleic acid sequences. A variety of techniques that can be used to determine (predict or verify) the function of a relevant gene or gene product include, but are not limited to: (A) DNA/RNA analysis, such as (1) overexpression, ectopic expression, and expression in other species; (2) gene knockout (reverse genetics, targeted knockout, virus-induced gene silencing (VIGS, see Baulcombe, 100 Yeast of Virology, Calisher and Horzinek eds., Springer-Verlag, New York, N.Y. 15: 189-201, 1999)); (3) methylation status analysis of genes, in particular flanking regulatory sequences; and (4) in situ hybridization; (B) gene product analysis, such as (1) recombinant protein expression; (2) antiserum production; (3) immune localization; (4) biochemical assays for catalytic or other activity; (5) (ii) a phosphorylation state; and (6) interaction with other proteins by yeast double-hybrid analysis; (C) pathway analysis, e.g., based on an over-expressed phenotype or by sequence homology to a related gene, to place the gene or gene product into a particular biochemical or signaling pathway; and (D) other assays that may also be performed to determine or verify the involvement of the isolated gene or its product in a particular metabolic or signaling pathway and to help determine gene function.

Experiment of

The invention is described in more detail in the following examples, which are not intended to limit the scope of the invention as claimed in any way. The accompanying drawings are intended to be considered an integral part of the description and illustration of the present invention. The following examples are provided to illustrate, but not to limit the claimed invention.

In the following experimental publications, the following abbreviations are used: m (mole); mM (millimolar); μ M (micromolar); nM (nanomolar); mol (mole); mmol (millimole); μ mol (micromolar); nmol (nanomole); gm (gram); mg (milligrams); μ g (μ g); pg (picogram); l (liter); mL and mL (milliliters); μ L and μ L (microliters); cm (centimeters); mm (millimeters); μ m (micrometers); nm (nanometers); u (unit); v (volts); MW (molecular weight); sec (seconds); min (minutes); h and hr (hours); deg.C (degrees Celsius); qs (proper amount); ND (not performed); NA (not applicable); rpm (revolutions per minute); h₂O (water); dH₂O (deionized water); HCl (hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodalton); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single-stranded DNA); dsDNA (double stranded DNA); dntps (deoxyribonucleotide triphosphates); RNA (ribonucleic acid); MgCl₂(magnesium chloride); NaCl (sodium chloride); w/v (weight/volume); v/v (volume/volume); g (gravity); OD (optical density); CNPG (chloro-nitro-phenyl- β -D-glucoside); CNP (2-chloro-4-nitrobenzene); APB (acid pretreated bagasse); PASC (phosphoric acid swollen cellulose); PCS (acid pretreated corn stover); pi or Pi (performance index); PAGE (polyacrylamide gel electrophoresis); PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR) and HPLC (high pressure liquid chromatography).

Example 1

Chemical modification of cellulase preparations from Trichoderma species and assays for testing CBH2 variants

This example describes the treatment of a commercial Trichoderma species cellulose preparation LAMINEX BG enzyme complex (Genencor Division, Danisco US, Inc.) with succinic anhydride to acetylate lysine residues. Acetylation of lysine residues of the laminenx BG enzyme complex alters the net charge (e.g., increased negative charge) of the protein. Other similar chemical modifications can also be used to convert the positive charge of lysine to a negatively charged group (e.g., treatment with acetoxysuccinic anhydride, maleic anhydride, tartaric anhydride, and phthalic anhydride) or even to two negative charges (e.g., treatment with 1,2, 4-trimellitic anhydride, cis-aconitic anhydride, and 4-nitrophthalic anhydride). Other chemical modifications can be used to remove the positive charge of lysine residues, resulting in uncharged residues (e.g., acetic anhydride, butyric anhydride, isobutyric anhydride, hexanoic anhydride, valeric anhydride, isovaleric anhydride, and pivalic anhydride treatments).

Succinic anhydride is used to modify lysine residues on cellulase preparations using a variation of the disclosed method (Lundblad, Chemical Reagents for protein Modification, eds.: R.Lundblad, 3 rd edition, CRC press, 1984). For this reaction, a sample of 236mg of LAMINEX BG enzyme complex was prepared in 1mL of 500mM HEPES buffer pH 8. Before addition of the enzyme complex, a succinic anhydride (Aldrich) solution was prepared by dissolving the powder in DMSO to a final concentration of 500 mg/mL. Aliquots of succinic anhydride were added so that a lysine to succinic acid ratio of > 1: 100 was obtained in the reaction tubes. The other reaction tube was filled with a similar volume of DMSO and enzyme only and used as an unmodified protein control. The tube was vortexed and left overnight at room temperature. The next day, 1: 10 volumes of 1M glycine (pH 3) were added to each tube and the succinic anhydride reaction was quenched.

Chemical modification was verified by comparing the modified and unmodified proteins on a native gel. Aliquots (both chemically modified and unmodified) from each reaction were analyzed by running a native gel (Phast System gels, GE Healthcare) on a gradient of 8-25% at pH 8.8 and 100 volts. After staining the gel with coomassie blue, the protein was observed to verify successful modification. Staining revealed changes in protein band migration, validating changes in charge of various protein components of the cellulase preparation. The modified sample of the cellulase preparation of Trichoderma species is more negatively charged than the unmodified sample.

To separate the modified and unmodified (control) proteins, an aliquot of 80 μ l of each sample was desalted using a rotary desalting column (Pierce). Samples were diluted 1: 10 and NanoDrop was used^TMThe absorbance at 280nm of the desalted sample (including the unmodified control) was measured in duplicate by a spectrophotometer (Thermo) to determine the total protein concentration of the sample.

Zeta potential measurement

This example describes the determination of zeta potential of enzymes and substrates. The presence of particle surface charges affects the distribution of ions in the surrounding interface region. The result is an increase in the concentration of counter ions that are opposite to the particle charge near the particle surface. The non-uniform distribution of ions eventually becomes uniform as they are removed from the surface of the particles. The distance at which uniform distribution is obtained is called the Debye length (1/κ) or screening distance, which depends on the ionic strength shown in the following expression, where ε₀Is the dielectric constant of free space (8.854x 10)^-12F m^-1)，ε_rIs the dielectric constant of the liquid, and k is the Boltzmann constant (1.38X 10)^-23JK^-1) T is the temperature in Kelvin and e is the electronic charge (1.6022x 10)^-19C) I is the molar ionic strength and NA is the Avogadro's constant (6.022x 10)²³mol^-1)。

The molar ionic strength can be calculated from the following equationIn which C is_iIs the concentration of an ionic species, Z_iIs a valence.

For 298K of water, the Debye length expression is reduced to the form described below.

k^-1＝0.304(I^-0.5) (0.3)

The liquid layer around the particles exists as two parts; an inner region (Stern layer) where ions are strongly bound and an outer (diffusion) region where they will not be firmly bound. There is a boundary in the diffusion layer where the ions and particles form a stable body. As the particles move, the ions in the boundary move with them. Those ions on the boundary (beyond the boundary) do not move with the particle. The potential at this boundary (also known as the hydrodynamic shear surface) is defined as the zeta potential.

In electrophoretic light scattering, zeta potential z is calculated from the measured electrophoretic mobility u using the Henry formula shown below, where epsilon is the permittivity, h is the solution viscosity, kappa is the reciprocal Debye length, a is the particle radius, and f (ka) is the Henry function.

κ is in reciprocal length and 1/κ is the "thickness" of the electrical bilayer (Debye length). The parameter a represents the radius of the particle, so κ a is the particleThe ratio of the grain radius to the electrical bilayer thickness. The Henry function f (ka) depends on the particle shape, but it is known to be spherical. In the above expression, it is given in f (0) ═ 1 (Huckel limit) and(Smoluchowski limit). In low dielectric media (or low ionic strength media), huckel limitation of f (ka) ═ 1 is a more suitable model for small particles (e.g., proteins).

Zetasizer NS (Malvern Instruments, UK) was used to measure the zeta potential of the protein according to the principles given above. The zeta potential of fabrics soiled with BMI was measured with SurPass (Anton-Paar, Austria) using the streaming potential tool of the above principle. Definition of surface charge, usually expressed in coulombs:

q_S＝4pe_oe_ra(1+ka)z (0.5)

this can also be expressed as the net charge z multiplied by the basic charge e1.6 x 10-19C:

q_S＝ze (0.6)

thus, the expected zeta potential change due to the net charge increase is given below:

zeta-potential can also be measured using the native gel technique described in example 1 (Sparks et al, Journal of LipidResearch, 33: 123-. Electrophoretic mobility measured with native gels is generally less than in solution because the gel matrix causes blocking. The zeta potential calculated by this method is generally lower than that calculated by the solution-based method. We therefore refer to the zeta potential obtained by the native gel technique as the apparent zeta potential.

The effective charge in a given formulation is expressed as its zeta potential. The use of zeta potential as a common charge scale allows comparison of enzyme variants with different folds (e.g., serine proteases, metallo proteases, etc.) and interactions with different substrates (e.g., BMI microbubs) under conditions of interest (e.g., AATCC HDL detergents). Although zeta potentials are preferred for comparing different protein folds, electrophoretic mobility or measured charge also provides an absolute scale and is sufficient for comparison. BMI performance as a function of enzyme zeta potential is well described by a standard normal distribution, shown by the solid line, with an average μ equal to-9.68 mV, a standard deviation σ of 11.39mV, and a peak at 0.4056[ A600-background ]. The distribution is expressed in normalized reduced coordinates as the BMI activity divided by the peak on the right Y-axis (as a function of the Z-score on the top X-axis). The Z-fraction is defined as (X- μ)/σ as usual, where X in this case is the zeta potential.

Mean μ ═ 9.68mV, standard deviation σ ═ 11.39mV, ζ -potential ζ ═ Z × + μ ═ σ

Reference buffer: 5mM HEPES pH 8.0, 2.5mM NaCl

The normal distribution of each substrate stain is unique under given reaction conditions (pH, conductivity, type of salt, detergent chelant, etc.). The different benefits or advantageous results follow a normal distribution of physical properties (e.g., expression levels and zeta-potentials for ASP and NprE charge step variants) maintained among enzymes from different folds. In a normal distribution, the peak occurs at the mean. Comparison of the enzyme and substrate charges on a common zeta potential scale reveals that optimal BMI performance occurs when the average enzyme zeta potential in this case is-9.68 mV, which-9.68 mV substantially matches the substrate stain zeta potential (measured at-8.97 mV under the same conditions).

The performance levels of a standard normal distribution are conveniently described for their z-scores as shown in Table 1-1 (see Abramowitz and Stegun, Handbook of chemical Functions with formulas, Graphs, and chemical Tables, Dover, New York, 9 th edition, 1964). The conversion to zeta potential is given directly by the mean and standard deviation (defining the distribution for a given application). In this example, the cleaning performance measured for protein folding was limited to zeta potential values between-40 mV and +20 mV. Variants with cleaning performance above 80% of their fold optimum (i.e. z ═ 0.65) are limited to zeta potential values between-17.08 mV to-2.28 mV. Variants with cleaning performance above 90% of their fold optimum (i.e. z ═ 0.46) are limited to zeta potential values between-14.92 mV to-4.44 mV.

Different substrate stains (e.g., grass, body soils, tomatoes) have different zeta potentials with the same formulation, while the same substrate stain also has different zeta potentials with different formulations (e.g., north american HDL, european powder dishwashing detergent). Regardless, the standard deviation of the normal distribution is expected to remain constant despite variations in substrate stain charge. Information on the zeta potential of the enzyme and substrate in a given detergent formulation allows the rapid identification of the predicted performance level of the variant, as well as the direction and magnitude of the charge change required to achieve the optimum performance level. Measuring the zeta potential of a substrate in a desired reaction medium allows optimization of the enzymatic reaction on a particular substrate in that medium. Similar methods can be used to optimize any enzymatic reaction in any medium.

Optimizing reactions on substrates exhibiting variable charge

Cellulose conversion is assessed by techniques known in the art (see, e.g., Baker et al, ApplBiochem Biotechnol, 70-72: 395-. Standard cellulose conversion assays were used in the experiments. In this assay, the enzyme and buffered substrate are placed in a vessel and incubated for a certain period of time at a certain temperature. The reaction is quenched with sufficient 100mM glycine (pH 11) to bring the pH of the reaction mixture to at least pH 10. Once the reaction was quenched, an aliquot of the reaction mixture was filtered through a 0.2 micron membrane to remove solids. The filtered solution was then assayed for soluble sugars by HPLC, following the method described by Baker et al, supra.

Determination of zeta potential of Pretreated Corn Stover (PCS)

According to Schell et al, J Appl Biochem Biotechnol, 105: 69-86[2003]The use amount of the catalyst is 2% w/w H₂SO₄Corn stover was pretreated, followed by multiple washes with deionized water to achieve a pH of 4.5. Sodium acetate was added to give a final concentration of 50mM and the solution was titrated to pH 5.0. The cellulose concentration in the reaction mixture was about 7%.

PCS aliquots, either before or after saccharification by commercial cellulase enzyme mixtures (Spezyme CP and Indiane 44L), were loaded into 1.5mL Eppendorf centrifuge tubes to a volume of about 1/3. The samples were centrifuged at 6,000rpm for 5 minutes and Milli-Q^TMThe supernatant was exchanged with water and the process was repeated 5 times. Preparation of MIlli-Q from rinsed corn stover^TM100mg/mL stock in water. The stock was diluted to 1mg/mL in 50mM sodium acetate buffer (pH5.0) for zeta potential measurement. A1 mL aliquot of each substrate sample was transferred to a clean Malvern instruments (UK) disposable Zetasizer NS^TMIn a transparent tube.

Tables 1-2 show that during the saccharification reaction, the PCS substrate charge, expressed as zeta potential, is nearly twice negative. Without being bound by theory, there are many explanations regarding the increase in net negative charge, including but not limited to: enrichment for lignin, the non-reactive part of the substrate, and non-productive binding or fouling (fouling) of whole cellulases and other proteins. There is an optimal zeta potential of the enzyme for performance (e.g., extent of reaction and rate of reaction) that matches the zeta potential of the substrate under the conditions of the reaction medium. Different biomass pretreatments can greatly affect the initial substrate charge. If the zeta potential of the enzyme or substrate does not match during the reaction, the enzyme-substrate interaction will no longer be optimal. This effect will be significant for variations close to 10mV, which is the case for biomass conversion.

Strategies for remedying such conditions include, but are not limited to: providing an enzyme blend spanning a plurality of charges; fed-batch process means, wherein enzymes with different charges at new optimal values are provided at different reaction times and/or degrees of conversion; control of substrate surface charge by addition of formulation active agents, particularly surfactants (ionic and non-ionic) or other proteins; controlling the surface charge of the substrate by pH adjustment; adjusting the ionic strength by reaction to change the optimum value of the enzyme charge; membrane filtration, in particular reverse osmosis and nanofiltration, to control the ionic strength during the reaction; adding a chelating agent to control the ionic strength by removing salt; and controlling the biomass substrate charge by a pretreatment process.

The following assays were used in the examples below. Any deviations from the procedures provided below are noted in the examples. In these experiments, the absorbance of the product formed after the reaction was completed was measured using a spectrophotometer.

Hexokinase assay for measuring residual glucose

Residual glucose in hypocrea jecorina culture supernatants expressing the CBH2 variants was measured using a hexokinase assay. In a 96-well microtiter plate (Costar Flat Bottom PS), a volume of 5. mu.l of the supernatant was added to 195. mu.l of a glucose hexokinase assay (Instrument laboratory, Breda, Netherlands). Incubate the plate at room temperature for 15 minutes. After incubation, absorbance was measured at 340nm OD. Culture supernatants exhibiting residual glucose were excluded from the mix for further study.

HPLC assay for determining protein content

The concentration of CBH2 variant proteins from mixed culture supernatants was determined using Agilent 1100 (hewlett packard) HPLC equipped with a prostift RP 2H column (Dionex). After equilibration of the HPLC column with 10% acetonitrile containing 0.01% trifluoroacetic acid, 10 microliters of a sample mixed with 10% acetonitrile in 50. mu.l of filtered demineralized water was injected. Elution of compound was performed using a 10% to 30% acetonitrile gradient for 0.3-1 min followed by a 30% to 65% gradient for 1-4 min. Protein concentrations of CBH2 variants were determined from calibration curves generated using purified wild-type CBH2(6.25, 12.5, 25, 50 μ g/ml). To calculate the performance index (Pi or Pi), the ratio of (average) total protein produced by the variants to (average) total protein produced by the same dose of wild type was averaged.

Determination of specific activity by phosphoswelling cellulose (PASC) hydrolysis assay

Cellulose hydrolysis: phosphoric acid expanded cellulose (PASC) was prepared from Avicel according to a known method (Walset, Tappi 35: 228, 1971; and Wood, Biochem J, 121: 353-. The above material was diluted with buffer and water to obtain a 1% w/v mixture to give a final concentration of 50mM sodium acetate, pH 5.0. 100 microliters of a 1% suspension of PASCs in 50mM sodium acetate buffer (pH5.0) was dispersed in a 96-well microtiter plate (Costar Flat Bottom PS). 10 microliters of 5mg/ml culture supernatant from the CBH 2-deleted strain was added to the PASCs, and 5,10, 15, or 20 μ l of mixed culture supernatant from hypocrea jecorina cells expressing the wild-type CBH2 or CBH2 variant was added thereto. Deletion of the CBH2 gene from hypocrea jecorina (also known as trichoderma reesei) is described in U.S. Pat. nos. 5,861,271 and 5,650,322. Make up volumes of acetate buffer were added to make up for the difference in total volume. The microtiter plates were sealed, incubated in a thermostatted incubator at 50 ℃ and shaken continuously at 900 rpm. After 2 hours, the hydrolysis reaction was stopped by adding 100. mu.l glycine buffer to each well, pH 10. Hydrolysis reaction products were analyzed by the PAHBAH assay.

PAHBAH assay: an aliquot of 150. mu.l of PAHBAH reducing sugar reagent (5% w/v para-hydroxybenzoic acid hydrazide (PAHBAH, Sigma # H9882 in 0.5N HCl) (Lever, Anal biochem, 47: 273-279, 1972)) was added to all wells of an empty microtiter plate. Add 10 μ l of hydrolysis supernatant to PABAH reaction plates. All plates were sealed, incubated at 69 ℃ and shaken continuously at 900 rpm. After 1 hour, the plate was placed on ice for 5 minutes and centrifuged at 720Xg for 5 minutes at room temperature. An 80. mu.l sample of the developed PAHBAH reaction mixture was transferred to a fresh (read) plate and the absorbance at 410nm was measured in a spectrophotometer. A cellobiose standard was included as a control. Dose response curves were generated for the wild-type CBH2 protein. To calculate the performance index (Pi or Pi), the ratio of (average) total sugars produced by the variants to (average) total sugars produced by the same dose of wild type was averaged.

Specific activity determination by hydrolysis of diluted acid Pretreated Corn Stover (PCS)

Pretreated Corn Stover (PCS): as described (Schell et al, J Appl biochem Biotechnol, 105: 69-86[2003 ]]) With 2% w/w H₂SO₄Corn stover was pretreated and then washed several times with deionized water to obtain a paste with a pH of 4.5. Then, sodium acetate buffer (pH5.0) was added to a final concentration of 50mM sodium acetate, and the mixture was then titrated with 1N NaOH to pH5.0, if necessary. The cellulose concentration in the reaction mixture was about 7%. To each well of a 96-well microtiter plate (Nunc Flat Bottom PS) was added 65. mu.l of this cellulose suspension. 10 microliters of 5mg/ml culture supernatant from the CBH 2-deleted strain was added to the PCS, and 5,10, 15 or 20 μ l of the supernatant from the culture expressing the wild-type CBH2 or CBH2 variant was added theretoMixed culture supernatants of hypocrea jecorina cells. Make up volumes of acetic acid buffer were added to make up for the difference in total volume. After sealing the microtiter plates, the plates were placed in an incubator at 50 ℃ and shaken continuously at 1300rpm for 5 minutes. The plate was then incubated at 50 ℃ while shaking at 220rpm in 80% humidity to prevent drying. After 7 days, the plates were placed on ice for 5 minutes and the hydrolysis reaction was stopped by adding 100. mu.l glycine buffer to each well, pH 10. Hydrolysis reaction products were analyzed by the PAHBAH assay.

PAHBAH assay: an aliquot of 150. mu.l of PAHBAH reducing sugar reagent (5% w/v para-hydroxybenzoic acid hydrazide (PAHBAH, Sigma # H9882 in 0.5N HCl) (Lever, Anal biochem, 47: 273-279, 1972)) was added to all wells of an empty microtiter plate. Add 10 μ l of hydrolysis supernatant to PABAH reaction plates. All plates were sealed, incubated at 69 ℃ and shaken continuously at 900 rpm. After 1 hour, the plate was placed on ice for 5 minutes and centrifuged at 720Xg for 5 minutes at room temperature. An 80. mu.l sample of the developed PAHBAH reaction mixture was transferred to a fresh (read) plate and the absorbance at 410nm was measured in a spectrophotometer. A cellobiose standard was included as a control. Dose response curves were generated for the wild-type CBH2 protein. To calculate the performance index (Pi or Pi), the ratio of (average) total sugars produced by the variants to (average) total sugars produced by the same dose of wild type was averaged.

Stability of CBH2 variants in the Presence of ethanol

The stability of wild-type CBH2 and CBH2 variants was tested in the presence of 4.5% ethanol (EtOH) at 49 ℃. Mixed culture supernatants (80. mu.L) of hypocrea jecorina cells expressing the CBH2 variant were added to 96-well plates (Greiner V-bottom PS) containing 10. mu.l of 40.5% EtOH/well. The plates were sealed, incubated in an incubator at 49 ℃ for 16 hours and shaken at 900 rpm. After incubation, the plates were placed on ice for 5 minutes. The remaining CBH2 activity was determined using a Phosphoric Acid Swollen Cellulose (PASC) hydrolysis assay as described above.

To calculate the remaining activity, the values of the products formed by the addition of 5,10, 15 and 20 μ l of EtOH incubated CBH2 to the remaining activity PASC assay were divided by the values of the products formed by the addition of 5,10, 15 and 20 μ l of EtOH free CBH2 to the PASC assay. Then, individual values of the above four ratios were averaged to produce an average residual activity. To determine the PI value of the variants, the value of the average residual activity of the variants was divided by the average of the residual activity values of the wild-type CBH2 control.

Thermostability of CBH2 variants

The wild-type CBH2 and CBH2 variants were tested for thermostability at 53 ℃. Mixed culture supernatants (80. mu.L) of hypocrea jecorina cells expressing the CBH2 variant were added to 96-well plates (Greiner V-bottom PS). The plates were sealed, incubated in an incubator at 53 ℃ for 16 hours and shaken at 900 rpm. After incubation, the plates were placed on ice for 5 minutes. The remaining CBH2 activity was determined using a Phosphoric Acid Swollen Cellulose (PASC) hydrolysis assay as described above.

To calculate the remaining activity, the values of the products formed by adding 5,10, 15 and 20 μ l of heat-treated CBH2 to the remaining activity PASC assay were divided by the values of the products formed by adding 5,10, 15 and 20 μ l of non-heat-treated CBH2 to the PASC assay. Then, individual values of the above four ratios were averaged to produce an average residual activity. To determine the PI value of the variants, the value of the average residual activity of the variants was divided by the average of the residual activity values of the wild-type CBH2 control.

Example 2

Evaluation of lignin binding

Lignin is a complex biopolymer of the phenylpropanoid type (phenylpropane), which is the main non-carbohydrate constituent of wood, which binds to cellulose fibers to harden and strengthen the cell walls of plants. Lignin minimizes the accessibility of cellulose and hemicellulose to cellulose degrading enzymes because it crosslinks with other cell wall components. Thus, lignin is generally associated with reduced digestibility of all plant biomass. In particular, the binding of cellulase enzymes to lignin reduces the degradation of cellulose by cellulase enzymes. Lignin is hydrophobic and is apparently negatively charged. Thus, it is contemplated that the addition of negative charges to cellulase enzymes reduces its binding to lignin.

As described herein, the reaction was set up to measure the effect of chemical modification on the ability of a cellulase preparation of trichoderma species to bind to a component of a plant polymer (i.e. lignin). The samples were returned to pH5 by extensive digestion of acid pretreated bagasse with cellulase enzyme (100mg Laminex BG/g cellulose) and then recovery of lignin by hydrolysis of cellulase enzyme by non-specific serine proteases, as described by Berlin et al (Applied Biochemistry and Biotechnology, 121: 163-. Briefly, 50 μ L of 1.16% lignin (recovered from complete saccharification of bagasse) prepared in 50mM sodium acetate buffer (pH 5) was combined with 4 μ L of a desalted modified or unmodified Trichoderma cellulase preparation. The microcentrifuge tube containing the reaction mixture was incubated at room temperature for 1 hour, and then centrifuged at high speed to separate the soluble material from the insoluble material. Collect 10. mu.l of supernatant from each tube. The reaction tubes were re-mixed and incubated for an additional 2 hours before a second 10 μ l aliquot of supernatant was collected from each tube. Supernatant samples were analyzed by SDS-PAGE. In the modified Trichoderma species cellulase preparation, a decrease in band intensity is indicative of decreased lignin binding.

Example 3

Bagasse binding was evaluated.

Bagasse is the residual biomass after sugar cane is crushed to extract its juice. A bagasse solution (acid treated, 28% solids, 57% polysaccharide) containing 2% cellulose was prepared in 50mM sodium acetate (pH 5). Samples of unmodified or chemically modified cellulase preparations of Trichoderma species were diluted 10-fold in the same sodium acetate buffer. An aliquot of the diluted enzyme was mixed separately with bagasse solution or buffer and incubated for 1 hour at room temperature. The supernatant was collected and assayed for the activity of the cellulase component (i.e., β -glucosidase).

Beta-glucosidase activity was measured using a chloro-nitro-phenyl-beta-D-glucoside (CNPG) assay. The CNPG assay is a kinetic assay in which β -glucosidase converts CNPG to the colored product 2-chloro-4-nitrophenol (CNP). OD was measured at 405nm during 10 minutes at 37 ℃. Rates were obtained as Vmax using SpectraMax software and then converted to specific activity (μ M CNP/sec/mg protein). Briefly, 200. mu.l of 50mM sodium acetate buffer (pH5.0) was added to each well of a 96-well microtiter plate. The plate was covered and placed in an Eppendorf thermostatic mixer at 37 ℃ for 15 minutes to allow it to equilibrate to that temperature. After equilibration, 5. mu.l of enzyme sample serially diluted in 50mM sodium acetate buffer (pH5.0) was added to each well. 10mM CNPG stock was diluted 1: 5 with 50mM sodium acetate buffer (pH5.0) and then 20. mu.l of the diluted CNPG solution (2mM) was added to each well containing the enzyme sample. The microtiter plates were transferred to a spectrophotometer set at 37 ℃ (SpectraMAX model 340; molecular devices) and read for OD 0-15 minutes at 405nm, with intervals ≤ 9 seconds.

The amount of beta-glucosidase activity of the cellulase sample that remains unbound to the bagasse substrate is considerably higher than that of the chemically modified Trichoderma cellulase preparation. In particular, less than 50% of the unmodified β -glucosidase remained unbound (e.g., 50% bound) to the bagasse substrate, while nearly 80% of the modified bglu remained unbound (e.g., 20% bound) to the bagasse substrate as determined by the CNPG assay. Taken together, the data for modified cellulase binding indicates that decreasing the positive charge of the cellulase results in decreased binding to the more negatively charged plant polymer substrate. In this case, the plant polymer substrate is the lignin remaining in the acid-treated biomass. Acid treated biomass from corn stover (plant biopolymers with similar chemical composition) showed to accept an increased more negative charge during the saccharification process, as determined by measuring zeta potential (see tables 1-2).

Example 4

Saccharification of acid pretreated bagasse

Saccharification of cellulose present in pre-acid treated bagasse (APB) containing various amounts of other lignin was assessed using chemically modified and unmodified trichoderma species cellulase preparations and measured by HPLC to monitor release of sugars DP1 to DP 7. The results are shown in figure 1 as percent conversion of the polymer substrate. In microtiter plates, 200 μ L of APB (3.5% dextran) was prepared in 50mM sodium acetate buffer (pH 5), adjusted to various amounts of lignin. To the wells, 20 microliters of cellulase solution (unmodified or modified LAMINEX BG) was added. The plate was covered with an aluminum plate sealer and placed in an incubator at 50 ℃ and incubated for 24 hours or 48 hours with shaking. The reaction was stopped by adding 100. mu.l of 100mM glycine pH10 to each well. After thorough mixing, the contents of the microtiter plate wells were filtered through a Millipore 96-well filter plate (0.45 μm, PES). The filtrate was diluted into plates containing 100. mu.l of 10mM glycine pH10 and the amount of soluble sugars produced (DP1 to DP7) was measured by HPLC. The Agilent 1100 series HPLC was equipped with a deliming (de-ashing)/guard column (Biorad Cat No. 125-. The mobile phase used was water at a flow rate of 0.6 ml/min. Soluble sugar standards (DP1-DP7) obtained from Sigma were all diluted in Milli-Q water to 100mg/mL, which was used to convert the peak area for each sugar to the actual sugar concentration. Percent conversion was calculated by dividing the sugars measured from HPLC by 100% conversion of cellulose to glucose.

Cellulose bound to lignin will reduce its efficiency in degrading cellulose. This is shown as: in the presence of an increased amount of lignin, the conversion of cellulose present in the saccharification reaction is reduced. This trend continues as well in modified cellulase preparations. However, the cellulose conversion in the modified cellulase sample was increased by 10% compared to the unmodified cellulase sample. This result indicates that the increased negative charge of cellulase reduces the non-productive binding of cellulase to lignin.

Example 5

Chemically modified CBH2 enhances saccharification of APB

Purified trichoderma CBH1, CBH2 variant, EG1, EG2 and β -glucosidase were chemically modified as described in example 1. The CBH2 variant used in this experiment had multiple substitutions (P98L/M134V/T154A/I2112V/S316P/S413Y, numbers corresponding to wild-type mature CBH2 cellulase) as described in U.S. publication No. 2006/0205042. The amino acid sequence of the mature CBH2 variant is shown below:

QACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCLPGAASSSSSTRAASTTSRVSPTTSRSSSATPPPGSTTTRVPPVGSGTATYSGNPFVGVTLWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFVWLDTLDKTPLMEQTLADIRAANKNGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDTIRQIVVEYSDVRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSPRALRGLATNVANYNGWNITSPPPYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFITDQGRSGKQPTGQQQWGDWCNVIGTGFGIRPSANTGDSLLDSFVWVKPGGECDGTSDSSAPRFDYHCALPDALQPAPQAGAWFQAYFVQLLTNANPSFL(SEQ ID NO：14).

chemical modifications of CBH1, CBH2 variants, EG1, EG2 and β -glucosidase were verified by their altered migration on native gels (relative to the unmodified protein). The modified CBH1, CBH2 variants, EG1, EG2 and β -glucosidase have more negative charges. All protein concentrations were performed using NanoDrop^TMMeasured with a spectrophotometer (Thermo). The saccharification reaction was set up in microtiter plates, 150uL of APB (7% dextran prepared for PCS as described above) was prepared in 50mM sodium acetate buffer (pH 5) in each well of the microtiter plates, and 20. mu.g of total protein was addedEnzyme mix such that the final protein to cellulose ratio in each well is 20 mg/g. A mixture of 6 enzymes was generated by adding purified modified or unmodified Bglu, CBH2 variant, EG1 or EG2 to a background of trichoderma reesei (t.reesei), in which genes encoding cellobiohydrolase I (CBHI, Cel7a), cellobiohydrolase II (CBHII, Cel6a), endoglucanase I (EGI, Cel7b) and endoglucanase II (EGII, Cel5a) had been inactivated (see US 2007/0128690). To each mixture, 72.5% background of trichoderma reesei, 2.5% Bglu, 15% variant of CBH2, 5% EG1, 5% EG2 were added, the first four mixtures having an unmodified protein, the fifth mixture having all unmodified proteins, and the sixth mixture having all modified proteins. The plates were incubated at 50 ℃ for 72 hours. The reaction was stopped by adding 100. mu.l of 100mM glycine (pH 10) to each well. After thorough mixing, the contents of the microtiter plate wells were filtered through Millipore 96-well filter plates (0.45 μm, PES). The filtrate was diluted into a plate containing 100. mu.l of 10mM glycine, pH10, and the amount of soluble sugars produced (DP1 to DP7) was measured by HPLC. Agilent 1100 series HPLC were equipped with a deliming/guard column (Biorad Cat No. 125-. The mobile phase used was water at a flow rate of 0.6 ml/min. The soluble sugar standards (DP1-DP7) obtained from Sigma were all dissolved in Milli-Q water to 100mg/mL, which was used to convert the peak area for each sugar to the actual sugar concentration. Percent conversion was calculated by dividing the sugars measured from HPLC by 100% conversion of cellulose to glucose.

Figure 2A shows that the 6 th enzyme cocktail with all proteins modified (modified EG2, EG1, CBH2 variant and β -glucosidase) has the highest cellulose conversion and the 5 th enzyme cocktail with all proteins unmodified has the lowest conversion. Comparing the first 4 enzyme cocktails, the 2 nd enzyme cocktail had unmodified CBH2, resulting in a2 nd lowest conversion. FIG. 2B shows the advantage of modified proteins over unmodified proteins in cellulose conversion.

Example 6

Preparation of Trichoderma reesei CBH2 Charge ladder variants

As determined in the development of the present invention, succinylation of lysine residues on the surface of CBH2 improved the performance on APB and pretreated corn stover. The charge of the modified CBH2 variant was about-17 compared to the unmodified CBH2 variant. Based on this, the charge ladder of CBH2 was designed to determine the optimal surface charge for cellulase performance applications.

SEQ ID NO: 1 describes a DNA sequence encoding the DNA sequence referred to hypocrea jecorina CBH 2:

atgattgtcggcattctcaccacgctggctacgctggccacactcgcagctagtgtgcctctagaggagcggcaagcttgctcaagcgt

ctggggccaatgtggtggccagaattggtcgggtccgacttgctgtgcttccggaagcacatgcgtctactccaacgactattactccc

agtgtcttcccggcgctgcaagctcaagctcgtccacgcgcgccgcgtcgacgacttctcgagtatcccccacaacatcccggtcga

gctccgcgacgcctccacctggttctactactaccagagtacctccagtcggatcgggaaccgctacgtattcaggcaacccttttgttg

gggtcactccttgggccaatgcatattacgcctctgaagttagcagcctcgctattcctagcttgactggagccatggccactgctgcag

cagctgtcgcaaaggttccctcttttatgtggctagatactcttgacaagacccctctcatggagcaaaccttggccgacatccgcaccg

ccaacaagaatggcggtaactatgccggacagtttgtggtgtatgacttgccggatcgcgattgcgctgcccttgcctcgaatggcgaa

tactctattgccgatggtggcgtcgccaaatataagaactatatcgacaccattcgtcaaattgtcgtggaatattccgatatccggaccct

cctggttattgagcctgactctcttgccaacctggtgaccaacctcggtactccaaagtgtgccaatgctcagtcagcctaccttgagtgc

atcaactacgccgtcacacagctgaaccttccaaatgttgcgatgtatttggacgctggccatgcaggatggcttggctggccggcaaa

ccaagacccggccgctcagctatttgcaaatgtttacaagaatgcatcgtctccgagagctcttcgcggattggcaaccaatgtcgcca

actacaacgggtggaacattaccagccccccatcgtacacgcaaggcaacgctgtctacaacgagaagctgtacatccacgctattg

gacctcttcttgccaatcacggctggtccaacgccttcttcatcactgatcaaggtcgatcgggaaagcagcctaccggacagcaaca

gtggggagactggtgcaatgtgatcggcaccggatttggtattcgcccatccgcaaacactggggactcgttgctggattcgtttgtctg

ggtcaagccaggcggcgagtgtgacggcaccagcgacagcagtgcgccacgatttgactcccactgtgcgctcccagatgccttgc

aaccggcgcctcaagctggtgcttggttccaagcctactttgtgcagcttctcacaaacgcaaacccatcgttcctgtaa*

SEQ ID NO: 2 describes the full-length protein sequence of hypocrea jecorina CBH 2:

MIVGILTTLATLATLAASVPLEERQACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCL

PGAASSSSSTRAASTTSRVSPTTSRSSSATPPPGSTTTRVPPVGSGTATYSGNPFVGVTPWA

NAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFMWLDTLDKTPLMEQTLADIRTANKNGGNY

AGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLA

NLVTNLGTPKCANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANV

YKNASSPRALRGLATNVANYNGWNITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFI

TDQGRSGKQPTGQQQWGDWCNVIGTGFGIRPSANTGDSLLDSFVWVKPGGECDGTSDSSAPR

FDSHCALPDALQPAPQAGAWFQAYFVQLLTNANPSFL*

SEQ ID NO: 3 describes the mature protein sequence of hypocrea jecorina CBH 2:

QACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCLPGAASSSSSTRAASTTSRVSPTTS

RSSSATPPPGSTTTRVPPVGSGTATYSGNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAA

AAVAKVPSFMWLDTLDKTPLMEQTLADIRTANKNGGNYAGQFVVYDLPDRDCAALASNGEYS

IADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINY

AVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSPRALRGLATNVANYNGWN

ITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFITDQGRSGKQPTGQQQWGDWCNVIG

TGFGIRPSANTGDSLLDSFVWVKPGGECDGTSDSSAPRFDSHCALPDALQPAPQAGAWFQAY

FVQLLTNANPSFL*

residues selected for mutagenesis include non-conserved, exposed lysine, arginine, asparagine, and glutamine residues, which are selected for substitution to introduce a negative charge. Succinylated lysines in modified CBH2 were identified by mass spectrometry and were selected for mutagenesis to glutamate, each substitution resulting in a-2 charge difference. Other residues for substitution were selected by analyzing the three-dimensional structure of CBH2 in combination with an amino acid alignment to the homologous CBH2 sequence (see, e.g., figure 3 of U.S. publication No. us 2006/0205042, which is incorporated herein by reference). Highly variable surface residues in the alignment of CBH2 amino acid sequences are candidates for mutagenesis. However, accumulation of closely adjacent substitutions is to be avoided. Arginine was replaced with glutamine (charge-1), and glutamine and asparagine were replaced with their respective carboxy variants (charge-1). In addition, aspartic acid and glutamic acid residues were selected for substitution to respective amine residues to complete the charge step (charge + 1). Table 6-1 shows a specific CBH2 substitution, all positions shown (except R63 and R77) being located in the CBH2 catalytic domain. The net positive charge can be created by removing negatively charged residues or by introducing positively charged residues. Similarly, a net negative charge can be created by removing positively charged residues or by introducing negatively charged residues.

For the preparation of the CBH2 charge ladder, 10 CBH2 charge variants (C-1 to C-10) were designed, covering the charge range +8 to-32, compared to the 4-step wild-type CBH2, as shown in table 6-2.

The amino acid sequence of the variant was reverse translated into DNA and the codons were optimized for expression in trichoderma reesei using GeneDesigner software (DNA 2.0). The codon optimized CBH2 variant gene was synthesized and DNA for CBH2 Surface Charge Variants (SCV) was amplified from the DNA2.0 construct by PCR using primers: GGHTK22 forward:

5'-CACCATGATCGTGGGAATTCTTACTACTC-3' (SEQ ID NO: 15); and GGTHK23 reverse 5'-CTACAAAAACGAAGGGTTCGCATT-3' (SEQ ID NO: 16). In one experiment, site-directed mutagenesis was used to introduce the K129E and K157E mutations (CBH2 charge variant C-3) into the genomic DNA of wild-type CBH 2. The CBH2 charge variant C-3 was cloned into pTrex3GM and expressed as follows.

The PCR product was purified and cloned into pENTR/TOPO for transformation of E.coli TOP10 cells. Plasmid DNA was isolated from a single colony and the correct sequence was verified. CBH2 SCV was cloned into pTrex3GM, pTTTpyr (pcbh1) and pTTTpyr (pstp1) as shown in tables 6-3.

	pTrex3gM	pTTTpyr(Pcbh1)	pTTTpyr(Pstp1)
				C-1(pTK354a)	1	11	21
C-2(pTK355a)	2	12	22
				C-3(pTK356a)	3	13	23
C-4(pTK357a)	4	14	24
				C-5(pTK358a)	5	15	25
C-8(pTK361a)	6	16	26
				C-6(pTK359a)	(7)	(17)	(27)
C-7(pTK360a)	8	18	28
				C-9(pTK362a)	9	19	29
C-10(pTK363b)	10	20	30

CBH2 Surface Charge Variants (SCV) in Trichoderma reesei. Biolistic transfer of Trichoderma reesei with pTrex3gM expression vector containing the open reading frame of cbh2 charge variant C3 (with K129E and K157E mutations)The following protocol was used. In the Trichoderma reesei used, genes encoding cellobiohydrolase I (CBH I, Cel7a), cellobiohydrolase II (CBH II, Cel6a), endoglucanase I (EG I, Cel7b) and endoglucanase II (EG II, Cel5a) had been inactivated (see, US 2007/0128690). Biolistic using Bio-Rad (Hercules, Calif.) according to the manufacturer's instructions (see WO05/001036 and US 2006/0003408)PDS-1000/he Particle Delivery System enables transformation of Trichoderma reesei strains using biolistic transformation methods. Transformants were transferred to new acetamide selection plates. The stable transformants were inoculated into filter microtiter plates (Millipore) containing 200 ul/well glycine minimal medium (6.0g/L glycine; 4.7g/L (NH)₄)₂SO₄；5.0g/L KH₂PO₄；1.0g/L MgSO₄·7H₂O; 33.0g/L PIPPS; pH 5.5), 2% glucose/Sophorose mixture as carbon source, 10ml/L of 100g/L CaCl were added after sterilization₂2.5ml/L Trichoderma reesei trace elements (400X): 175g/L of anhydrous citric acid; 200g/L FeSO₄·7H₂O；16g/L ZnSO₄·7H₂O；3.2g/LCuSO₄·5H₂O；1.4g/L MnSO₄·H₂O；0.8g/L H₃BO₃. Transformants were grown in liquid medium for 5 days in an oxygen-enriched chamber in an incubator at 28 ℃. Supernatant samples from the filter microtiter plates were obtained using a vacuum manifold. Samples were run on 4-12% NuPAGE gels (Invitrogen) according to the manufacturer's instructions. The gel was stained with Simply Blue dye (Invitrogen). The expression of other surface charge variants of CBH2 can be achieved using this method.

Example 7

Generation of CBH2 charge variants in Trichoderma reesei

The pTTTpyr-CBH2 plasmid containing The hypocrea jecorina CBH2 protein coding sequence (SEQ ID NO: 1) was sent to BASEClear (Leiden, The Netherlands), GeneArtAG (Regensburg, Germany) and Sloning Biotechnology GmbH (Puchheim, Germany) to generate a Site Evaluation library (Site Evaluation Libraries, SEL). FIG. 5 shows a plasmid map of pTTTpyr-cbh 2. The facilitator was asked to generate a library of positions of the various sites of the mature protein of hypocrea jecorina CBH2(SEQ ID NO: 3). SEQ ID NO: the amino acid sequence of the full-length protein of CBH2 is shown in fig. 2.

SEQ ID NO: 1 describes a DNA sequence encoding the DNA sequence referred to hypocrea jecorina CBH 2:

atgattgtcggcattctcaccacgctggctacgctggccacactcgcagctagtgtgcctctagaggagcggcaagcttgctcaagcgt

ctggggccaatgtggtggccagaattggtcgggtccgacttgctgtgcttccggaagcacatgcgtctactccaacgactattactccc

agtgtcttcccggcgctgcaagctcaagctcgtccacgcgcgccgcgtcgacgacttctcgagtatcccccacaacatcccggtcga

gctccgcgacgcctccacctggttctactactaccagagtacctccagtcggatcgggaaccgctacgtattcaggcaacccttttgttg

gggtcactccttgggccaatgcatattacgcctctgaagttagcagcctcgctattcctagcttgactggagccatggccactgctgcag

cagctgtcgcaaaggttccctcttttatgtggctagatactcttgacaagacccctctcatggagcaaaccttggccgacatccgcaccg

ccaacaagaatggcggtaactatgccggacagtttgtggtgtatgacttgccggatcgcgattgcgctgcccttgcctcgaatggcgaa

tactctattgccgatggtggcgtcgccaaatataagaactatatcgacaccattcgtcaaattgtcgtggaatattccgatatccggaccct

cctggttattgagcctgactctcttgccaacctggtgaccaacctcggtactccaaagtgtgccaatgctcagtcagcctaccttgagtgc

atcaactacgccgtcacacagctgaaccttccaaatgttgcgatgtatttggacgctggccatgcaggatggcttggctggccggcaaa

ccaagacccggccgctcagctatttgcaaatgtttacaagaatgcatcgtctccgagagctcttcgcggattggcaaccaatgtcgcca

actacaacgggtggaacattaccagccccccatcgtacacgcaaggcaacgctgtctacaacgagaagctgtacatccacgctattg

gacctcttcttgccaatcacggctggtccaacgccttcttcatcactgatcaaggtcgatcgggaaagcagcctaccggacagcaaca

gtggggagactggtgcaatgtgatcggcaccggatttggtattcgcccatccgcaaacactggggactcgttgctggattcgtttgtctg

ggtcaagccaggcggcgagtgtgacggcaccagcgacagcagtgcgccacgatttgactcccactgtgcgctcccagatgccttgc

aaccggcgcctcaagctggtgcttggttccaagcctactttgtgcagcttctcacaaacgcaaacccatcgttcctgtaa*

SEQ ID NO: 2 describes the full-length protein sequence of hypocrea jecorina CBH 2:

MIVGILTTLATLATLAASVPLEERQACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCL

PGAASSSSSTRAASTTSRVSPTTSRSSSATPPPGSTTTRVPPVGSGTATYSGNPFVGVTPWA

NAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFMWLDTLDKTPLMEQTLADIRTANKNGGNY

AGQFVVYDLPDRDCAALASNGEYSIADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLA

NLVTNLGTPKCANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANV

YKNASSPRALRGLATNVANYNGWNITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFI

TDQGRSGKQPTGQQQWGDWCNVIGTGFGIRPSANTGDSLLDSFVWVKPGGECDGTSDSSAPR

FDSHCALPDALQPAPQAGAWFQAYFVQLLTNANPSFL*

SEQ ID NO: 3 describes the mature protein sequence of hypocrea jecorina CBH 2:

QACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCLPGAASSSSSTRAASTTSRVSPTTS

RSSSATPPPGSTTTRVPPVGSGTATYSGNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAA

AAVAKVPSFMWLDTLDKTPLMEQTLADIRTANKNGGNYAGQFVVYDLPDRDCAALASNGEYS

IADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINY

AVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSPRALRGLATNVANYNGWN

ITSPPSYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFITDQGRSGKQPTGQQQWGDWCNVIG

TGFGIRPSANTGDSLLDSFVWVKPGGECDGTSDSSAPRFDSHCALPDALQPAPQAGAWFQAY

FVQLLTNANPSFL*

a purified pTTTpyr-CBH2 plasmid (p) containing an open reading frame encoding the CBH2 variant sequence was obtained from the above facilitator_cbh1，Amp^R，Acetamide^R). Protoplasts of hypocrea jecorina strains (Δ eg1, Δ eg2, Δ cbh1, Δ cbh2) were transformed with the pTTTpyr construct and grown on selective agar containing acetamide for 7 days at 28 ℃. Briefly, biolistic transformation of hypocrea jecorina was performed using the following protocol and strains in which the genes encoding cellobiohydrolase I (CBH I, Cel7a), cellobiohydrolase II (CBH II, Cel6a), endoglucanase I (EG I, Cel7b) and endoglucanase II (EG II, Cel5a) had been inactivated. Biolistic using Bio-Rad (Hercules, Calif.) according to the manufacturer's instructions (see WO05/001036 and US 2006/0003408)The PDS-1000/he Particle Delivery System enables transformation of hypocrea jecorina using a biolistic transformation method. Spores were harvested, replanted on acetamide agar, and incubated at 28 ℃ for 7 days. Spores were harvested in 15% glycerol and stored at-20 ℃ for further use. For CBH2 variant protein production, in PVDF filter plates, willA volume of 10. mu.l of spore suspension was added to 200ul of glycine minimal medium supplemented with a 2% glucose/sophorose mixture: 6.0g/L glycine; 4.7g/L (NH)₄)₂SO₄；5.0g/L KH₂PO₄；1.0g/LMgSO₄·7H₂O; 33.0g/L PIPPS; pH 5.5; after sterilization, a 2% glucose/sophorose mixture was added as a carbon source, 10ml/L of 100g/L CaCl₂2.5ml/L Trichoderma reesei trace elements (400X): 175g/L of anhydrous citric acid; 200g/L FeSO₄·7H₂O；16g/LZnSO₄·7H₂O；3.2g/L CuSO₄·5H₂O；1.4g/L MnSO₄·H₂O；0.8g/L H₃BO₃. Each CBH2 variant was grown in quadruplicate. After sealing the plates with oxygen permeable membranes, the plates were incubated at 28 ℃ for 6 days while shaking at 220 rpm. The supernatant was harvested by transferring the medium to a microtiter plate at low pressure, and the remaining glucose was tested using the hexokinase assay as described in example 1.

Example 8

Expression, Activity and Performance of CBH2 variants

Hypocrea jecorina CBH2 charge variants were tested for various target properties. In particular, as described in example 1, the cellulase variants were tested for protein expression using an HPLC assay (HPLC), specific activity using a PASC hydrolysis assay (act. PASC) and a PCS hydrolysis assay (act. PCS), and stability and thermostability (heat ratio) in the presence of ethanol (EtOH ratio). The performance data for the charge variants of CBH2 are shown in Table 8-1. The Performance Index (PI) is the ratio of the performance of the variant cellulase to the parent or reference cellulase. The following various terms are used to describe mutations: the ascending mutation has a PI > 1; neutral mutations have PI greater than or equal to 0.5; non-deleterious mutations have a PI > 0.05; deleterious mutations have a PI of 0.05; combinable mutations are mutations in which the variant has a performance index value of ≧ 0.5 in at least one property. Combinable mutations are mutations that can be combined to give a protein with an appropriate performance index for one or more properties of interest. The positions at which mutations occur are classified as follows: non-limiting positions have ≧ 20% neutral mutations for at least one property; the restriction positions had < 20% neutral mutations for activity and stability. The complete restriction site has no neutral mutations for activity and stability.

These data can be used to engineer any CBH 2. Even though the CBH2 to be engineered has an amino acid at a specific position that is different from hypocrea jecorina CBH2, by identifying the best substitution options, these data can be used to find substitutions that can alter the properties of interest, including substituting the hypocrea jecorina CBH2 wild-type amino acid.

Tables 8-11 show the performance index values (Pi or Pi) for the variants of hypocrea jecorina CBH 2. The performance index of less than or equal to 0.05 was fixed at 0.05 and is shown in bold italics in the table.

Example 9

Effect of Charge Change on CBH2 variant Activity

In this example, the effect of charge alteration on CBH2 activity in a Pretreated Corn Stover (PCS) assay for cellulase activity was evaluated. Briefly, the PCS winner in CBH2SEL was determined to be the nature of the net charge change. In Table 9-1, the ratio of observed to expected winners (o/e) was determined in the PCS assay. The value of bold italics is significantly different from the 10 randomly distributed means plus or minus the number of standard deviations (sd) listed in each column.

As shown in table 9-1 and fig. 4, decreased charge (e.g., -1, -2) resulted in a significantly higher CBH2 winner frequency in the PCS assay, while increased charge (e.g., +1) resulted in a significantly lower CBH2 winner frequency in the PCS assay. In summary, the activity of CBH2 on PCS was associated with charge reduction.

Claims (19)

1. An isolated cellulase variant derived from a parent hypocrea jecorina cellobiohydrolase II, wherein the variant is a mature form having cellulase activity and comprises a substitution of K327E or K327D, wherein position is determined by homology to SEQ ID NO: 3, and wherein the substitution results in the cellulase variant having a net charge that is more negative than the parent hypocrea jecorina cellobiohydrolase II.

2. The isolated cellulase variant of claim 1, wherein the amino acid sequence of said parent hypocrea jecorina cellobiohydrolase II is set forth in SEQ ID NO: 3, respectively.

3. The isolated cellulase variant of claim 1 or 2, wherein said variant comprises additional substitutions at one or more additional positions selected from the group consisting of: 63. 77, 129, 147, 153, 157, 161, 194, 197, 203, 237, 239, 247, 254, 281, 285, 288, 289, 294, 339, 344, 356, 378 and 382, and wherein the substitution results in the cellulase variant having a more negative net charge compared to the parent hypocrea jecorina cellobiohydrolase II.

4. The isolated cellulase variant of claim 3, wherein the additional substitution at one or more additional positions comprises removal of one or more positive charges.

5. The isolated cellulase variant of claim 4, wherein removing one or more positive charges comprises replacing lysine or arginine with a neutral amino acid.

6. The isolated cellulase variant of claim 3, wherein the additional substitution at one or more additional positions comprises the addition of one or more negative charges.

7. The isolated cellulase variant of claim 6, wherein adding one or more negative charges comprises replacing a neutral amino acid with a negatively charged amino acid.

8. The isolated cellulase variant of claim 3, wherein the other substitution at one or more other positions comprises removal of one or more positive charges and addition of one or more negative charges.

9. The isolated cellulase variant of claim 8, wherein removing one or more positive charges and adding one or more negative charges comprises replacing lysine or arginine with a negatively charged amino acid.

10. The isolated cellulase variant of claim 3, wherein the further substitution at one or more further positions is selected from the group consisting of K129E, K157E, K194E, K288E, K356E, R63Q, R77Q, R153Q, R203Q, R294Q, R378Q, N161D, N197D, N237D, N247D, N254D, N285D, N289D, N339D, N344D, N382D, Q147E, Q204E, Q239E and Q281E.

11. The isolated cellulase variant of any one of claims 1-10, wherein the variant comprises additional substitutions at one or more additional positions selected from the group consisting of 146, 151, 189, 208, 211, 244, 277 and 405.

12. The isolated cellulase variant of claim 11, wherein the additional substitution at one or more additional positions comprises a substitution of a neutral amino acid for aspartic acid or glutamic acid.

13. The isolated cellulase variant of claim 11, wherein the additional substitutions at one or more additional positions comprise one or more of D151N, D189N, D211N, D277N, D405N, E146Q, E208Q, and E244Q.

14. The isolated cellulase variant of claim 3, wherein the other substitutions of the variant are at 1,2, 3, 4, 5,6, 7, 8, 9, or 10 other positions.

15. The isolated cellulase variant of any one of claims 1-14, wherein the more negative net charge is-1 or-2 compared to the reference cellobiohydrolase II.

16. A method of converting biomass to sugars comprising contacting the biomass with the cellulase variant of any one of claims 1-15.

17. A method of producing a fuel, comprising:

contacting a biomass composition with an enzyme composition comprising a cellulase variant of any one of claims 1-15 to produce a sugar solution; and

the sugar solution is cultured with a fermenting microorganism under conditions sufficient to produce a fuel.

18. The cellulase variant of any one of claims 1-4, wherein the variant further comprises a chemical modification of a lysine residue to remove the positive charge of the lysine residue.

19. The cellulase variant of claim 18, wherein the chemical modification comprises treatment with a compound selected from the group consisting of succinic anhydride, acetoxysuccinic anhydride, maleic anhydride, tartaric anhydride, phthalic anhydride, trimetallitic anhydride, cis-aconitic anhydride, t-nitrophthalic anhydride, acetic anhydride, butyric anhydride, isobutyric anhydride, hexanoic anhydride, valeric anhydride, isovaleric anhydride, and pivalic anhydride.