WO2010006446A1

WO2010006446A1 - Modified family 6 glycosidases with altered substrate specificity

Info

Publication number: WO2010006446A1
Application number: PCT/CA2009/001020
Authority: WO
Inventors: John Tomashek; Annie Tremblay; Patrick St-Pierre; James Lavigne
Original assignee: Iogen Energy Corporation
Priority date: 2008-07-16
Filing date: 2009-07-16
Publication date: 2010-01-21
Also published as: EP2313500A4; MX2011000552A; BRPI0916767A2; US8263379B2; AU2009270398A1; US20100016570A1; EP2313500A1; CA2730662A1

Abstract

A modified Family 6 glycosidase enzyme comprising amino acid substitutions at one or more positions selected from the group 182, 367, 399, 400 and 427 is provided (the position determined form alignment of a parental Family 6 glycosidase with SEQ ID NO: 1). Genetic constructs and genetically modified microbes comprising nucleic sequences encoding the modified Family 6 glycosidase are also provided. Family 6 glycosidase of the invention display decreased hydrolysis activity of beta 1-4 linked polysaccharides and increased hydrolysis activity of beta 1-3, 1-4 linked polysaccharides compared with a parental Family 6 glycosidase. Such glycosidases find use in a variety of applications in industry, e.g., in hydrolysis of beta 1-3, 1-4 linked polysaccharides during the processing of cereal grains or the production of alcohol, animal feed or food products.

Description

MODIFIED FAMILY 6 GLYCOSIDASES WITH ALTERED SUBSTRATE SPECIFICITY

TECHNICAL FIELD

[0001] The present invention relates to modified glycosyl hydrolase (GH) enzymes. More specifically, the invention relates to modified enzymes of the GH Family 6 (GH6) with altered substrate specificity. The present invention also relates to genetic constructs comprising nucleotide sequences that encode and direct the expression and secretion of modified GH enzymes, methods for the production of modified GH enzymes from host strains, and the use of the modified GH enzymes.

BACKGROUND OF THE INVENTION

[0002] Glycosyl hydrolases (GHs) are a large group of enzymes that cleave glycosidic bonds between individual carbohydrate monomers in large polysaccharide molecules. For example, cellulases cleave the beta 1-4 bond between glucose monomers in the cellulose polymer; arabinofuranosidases cleave the alpha 1-2 and/or alpha 1-3 bonds between arabinose and xylose in arabinoxylan; amylases cleave the alpha 1-4 bonds between glucose molecules in starch, etc. As a result of the diversity of polysaccharide molecules, there are also many different GH enzymes. However, these enzymes all share one of two common mechanisms, called inverting and retaining, for introducing a water molecule at a glycosidic bond thus cleaving the polysaccharide. The majority of GH enzymes utilize the retaining mechanism.

[0003] The GH enzymes are grouped into more than 100 different families based on commonality in their primary and tertiary structures and their catalytic mechanism (CAZy website, URL: cazy.org: Coutinho and Henrissat, 1999). Some GH enzymes families are grouped into larger clans. Depending upon the particular family (all numbers are according to the CAZy website as of 13 March 2008), it may have only a few known examples (e.g., family GH82) or many (e.g. family GH34); more than half of the families have fewer than 200 members. Similarly, all the members of a particular family may represent essentially a single activity, which is to say activity against a single specific substrate (e.g., GHl 1, all of which are xylanases), whereas other families may have enzymes that cover a wide range of activities (e.g., GH5, comprising cellulases, xylanases, mannanases, chitosanases, galactanases, etc.). Individually, most enzymes have their highest activity for a single substrate, although there are examples of particular enzymes that have high activity against several substrates (e.g. Cel7B from Trichoderma reesei, which has both cellulase and xylanase activity).

[0004] The GH Family 6 belongs to no clan; it comprises over 100 members, all of which exhibit primarily cellulase activity using the inverting mechanism. Both endo- and exo- cellulases have been identified from a variety of bacterial and fungal sources. In addition, some GH6 members, including CelόA from Trichoderma reesei, have been shown to have hydrolytic activity against beta-glucan, which is a linear polymer of glucose with mixed linkages (Henriksson et al., 1995).

[0005] The beta-glucans form a large group of industrially important polysaccharides.

Because of their mixed linkages, the beta-glucans have higher solubility in aqueous solutions than more regular polymers such as cellulose. In the soluble form, the beta-glucans confer viscosity and/or gel-like properties to a solution. There are two major types: beta 1-3, 1-6 glucan, also known as laminaran because a major source of this glucan is Laminaria brown algae (kelp), and beta 1-3, 1-4 glucan, also known as lichenan because a major source of this glucan is lichen. However, beta 1-3, 1-4 glucan is also found as a major component of oat and barley endosperm. Hydrolysis of beta 1-3, 1-4 glucan from grains is desirable on the industrial scale to reduce viscosity in processes such as brewing, in the production of grain ethanol for fuel, and also to increase nutrient accessibility in animal feeds. In particular, Trichoderma reesei CelδA expressed in brewer's yeast is used to aid in the malting and brewing processes (Enari et al., 1987).

[0006] Some efforts to engineer GH enzymes in order to switch their activity from one substrate to another have been made, although experts in protein engineering generally concede that this is one of the more difficult protein engineering challenges (c.f. Tao and Cornish, 2002). The research group of W. M de Vos identified three key amino acid residues of a GHl beta-glucosidase that determined substrate specificity based on a structural comparison to a beta-galactosidase from the same family. By converting the residues of the beta-glucosidase to those found in the beta-galactosidase, they converted the beta - glucosidase into a beta-galactosidase. Similarly, key residues of a GHlO xylanase that discriminate between xylan and cellulose have been identified and mutagenized to change the enzyme from a xylanase to a cellulase (Andrews et al., 2000). [0007] The GH6 family of enzymes have been the target of mutational and protein engineering studies. The exocellulase CelόA from Trichoderma reesei, the exocellulase CelόA from Humicola insolens, and the cellulases CelόA (endo) and CelόB (exo) from Thermobifida fusca are representative enzymes that have been particularly well characterized. Specific sites of investigation include what are known as the loop regions. These are the principal determinants of whether an enzyme is an endocellulase (lacking loops) or an exocellulase (possessing loops). Mutations in the loops (Varrot et al., 2002) or deletion of the loops (Meinke et al., 1995) will alter the interaction between CelόA and cellulose. An extensive series of point mutations were studied in the two T. fusca enzymes, CelόA and CelόB (Zhang et al., 2000a; Zhang et al., 2000b). Changes in the relative activities towards different substrates - specifically filter paper, carboxymethyl cellulose, swollen cellulose and bacterial microcrystalline cellulose - were observed. Other studies have examined the role of aromatic amino acids in substrate binding (Koivula et al., 1996; Koivula et al., 1998; Zou et al., 1999) and the role of charged amino acids in activity and stability (Koivula et al., 2002; Wohlfahrt et al, 2003).

[0008] T. reesei CelόA (or TrCelόA) is one of the two major cellobiohydrolases secreted by this fungus and has been shown to be efficient in the enzymatic hydrolysis of crystalline cellulose, with low but measurable activity in the hydrolysis of beta 1,3-1,4 mixed linkage glucans such beta-glucan and lichenan. The tryptophan amino acid residue at position 367 (W367) of the Trichoderma reesei CelόA represents a highly conserved residue within a strongly conserved region of the enzyme (Figure 1). Generally, mutation of conserved residues results in enzyme inactivation, or a severe loss of activity.

SUMMARY OF THE INVENTION

[0009] The present invention relates to modified glycosyl hydrolase (GH) enzymes. More specifically, to modified enzymes of the GH Family 6 (GH6) with altered substrate specificity. The present invention also relates to genetic constructs comprising nucleotide sequences that encode and direct the expression and secretion of modified GH enzymes, methods for the production of modified GH enzymes from host strains, and the use of the modified GH enzymes.

[0010] It is an object of the invention to provide a modified glycosidase with an altered substrate specificity. [0011] The present invention provides modified glycosidase with an altered substrate preference from EC 3.2.1.91 (cellulase) to EC 3.2.1.73 (beta-glucanase).

[0012] The present invention relates to a modified Family 6 glycosidase comprising one or more amino acid substitutions selected from the group consisting of: N182X, W367X, E399X, C/S400X and A427X, the modified Familiy 6 glycosidase having an amino acid sequence in which the amino acids corresponding to those from position 83 to position 447 of TrCelόA (SEQ ID NO: 1) exhibit from about 47% to about 99.9% identity to amino acids 83-447 (TrCelόA numbering) of SEQ ID NO: 1. Furthermore, the one or more amino acid substitutions may be selected from the group consisting of N182S, N182R, N182G, N 182 A, W367A, W367C, W367G, W367N, W367R, W367S, W367T, W367V, E399H, E399S, E399T, C400V, C400M, C400T, C400S, A427V, A427L, and A427S.

[0013] The present invention also provides a modified Family 6 glycosidase comprising one or more amino acid substitutions selected from the group consisting of: N182X, W367X, E399X, C/S400X and A427X, the modified Family 6 glycosidase having an amino acid sequence in which the amino acids corresponding to those from position 83 to position 447 of TrCelόA (SEQ ID NO: 1) exhibit from about 70% to about 99.9% identity to amino acids 83-447 (TrCelόA numbering) of any one of SEQ ID NO: 1 through SEQ ID NO: 36. Furthermore, the one or more amino acid substitutions may be selected from the group consisting of N182S, N182R, N182G, N182A, W367A, W367C, W367G, W367N, W367R, W367S, W367T, W367V, E399H, E399S, E399T, C400V, C400M, C400T, C400S, A427V, A427L, and A427S.

[0014] The position of the one or more amino acid substitution defined above may be determined from sequence alignment of the amino acids corresponding to positions 83-447 of SEQ ID NO: 1 of a parental Family 6 glycosidase enzyme with amino acids 83-447 of the Trichoderma reesei CelόA amino acid sequence as defined in SEQ ID NO: I.

[0015] The modified Family 6 glyocosidase may be derived from a parental Family 6 glycosidase that is otherwise identical to the modified Family 6 glycosidase except for the substitution of the naturally occurring amino acid at one or more of positions 182, 367, 399, 400 and 427. For example, this invention includes a modified Family 6 glycosidase as defined above and further comprising a proline residue at position 413. [0016] The modified Family 6 glycosidase comprising these mutations may be from a filamentous fungus, such as Trichoderma reesei.

[0017] The present invention also relates to a modified Family 6 glycosidase as defined above and that has from about a 1.2-fold increase in activity in the hydrolysis of beta 1-3, 1- 4-linked or beta 1-3, 1-6-linked polysaccharides and may also exhibit at least a 1.2-fold decrease in activity in the hydrolysis of beta 1-4 -linked polysaccharides relative to a parental Family 6 glycosidase from which it is derived.

[0018] The present invention also relates to a modified Family 6 glycosidases selected from the group consisting of:

TrCel6A-N182S-S413P (SEQ ID NO: 83);

TrCel6A-N182R-D350E-S413P (SEQ ID NO: 84);

TrCel6A-N182G-S413P (SEQ ID NO: 85);

TrCel6A-N182A-S413P (SEQ ID NO: 86);

TrCel6A-W367A-S413P (SEQ ID NO: 37); TrCel6A-W367C-S413P (SEQ ID NO: 38);

TrCel6A-W367G-S413P (SEQ ID NO: 39);

TrCel6A-W367N-S413P (SEQ ID NO: 40);

TrCel6A-W367R-S413P (SEQ ID NO: 41);

TrCel6A-W367S-S413P (SEQ ID NO: 42); TrCel6A-W367T-S413P (SEQ ID NO: 43);

TrCel6A-W367V-S413P (SEQ ID NO: 44);

HiAvi2-W367G (SEQ ID NO: 45);

PcCel6A-W367G (SEQ ID NO: 46);

TrCel6A-S25G-T60S-E399H-S413P (SEQ ID NO: 87); TrCel6A-E399T-S413P (SEQ ID NO: 88);

TrCel6A-E399S-S413P (SEQ ID NO: 89);

TrCel6A-C400V-S413P (SEQ ID NO: 90);

TrCel6A-C400M-S413P (SEQ ID NO: 91);

TrCel6A-C400T-S413P (SEQ ID NO: 92); TrCel6A-C400S-S413P (SEQ ID NO: 93);

TrCel6A-A427V-S413P (SEQ ID NO: 94);

TrCel6A-A427L-S413P (SEQ ID NO: 95); and TrCel6A-A427S-S413P (SEQ ID NO: 96).

[0019] The present invention relates to genetic constructs comprising a nucleic acid sequence encoding a modified Family 6 glycosidase comprising one or more amino acid substitutions selected from the group consisting of: N 182X, W367X, E399X, C/S400X and A427X, the modified Family 6 glycosidase having an amino acid sequence that exhibits from 47% to 99.9% identity to amino acids 83-447 (TrCelόA numbering) of SEQ ID NO: 1 or an amino acid sequence that exhibits from 70% to 99.9% identity to amino acids 83-447 (TrCelόA numbering) of any one of SEQ ID NO: 1 through SEQ ID NO: 36. The nucleic acid sequence may be operably linked to other nucleic acid sequences regulating its expression and secretion from a host microbe. Preferably, the other nucleic sequences regulating the expression and secretion of the modified Family 6 glycosidase are derived from the host microbe used for expression of the modified Family 6 glycosidase. The host microbe may be a yeast, such as Saccharomyces cerevisiae, or a filamentous fungus, such as Tήchoderma reesei.

[0020] The invention also relates to a genetic construct as defined above, wherein the modified Family 6 glycosidase encoded by the genetic construct further comprises a substitution of the amino acid at position 413 with a proline or any other additional mutations at positions other than 182, 367, 399, 400 or 427.

[0021] The invention also relates to a genetically modified microbe comprising a genetic construct encoding the modified Family 6 glycosidase and capable of expression and secretion of a modified Family 6 glycosidase comprising one or more amino acid substitutions selected from the group consisting of: N182X, W367X, E399X, C/S400X and A427X, the modified Family 6 glycosidase having an amino acid sequence that exhibits 70% to 99.9% identity to amino acids 83-447 (TrCelόA numbering) of SEQ ID NO: 1 or an amino acid sequence that exhibits from 70% to 99.9% identity to amino acids 83-447 (TrCelόA numbering) of any one of SEQ ID NO: 1 through SEQ ID NO: 36. In one embodiment, the genetically modified microbe is capable of expression and secretion of a modified Family 6 glycosidase further comprising a substitution of the amino acid at position 413 with a proline or any other additional mutations at positions other than 182, 367, 399, 400 or 427. The genetically modified microbe may be a yeast or filamentous fungus. For example, the genetically modified microbe may be a species of Saccharomyces, Pichia, Hansenula, Tήchoderma, Hypocrea, Aspergillus, Fusarium, Humicola or Neurospora.

[0022] The present invention also relates to a process for hydrolysing a beta 1-3, 1 -4 -linked polysaccharide substrate with modified Family 6 glycosidase.

[0023] The invention also relates to a process of producing the modified Family 6 glycosidase as defined above, including transformation of a yeast or fungal host with a genetic construct comprising a DNA sequence encoding the modified Family 6 glycosidase, selection of recombinant yeast or fungal strains expressing the modified Family 6 glycosidase, culturing the selected recombinant strains in submerged liquid fermentations under conditions that induce the expression of the modified Family 6 glycosidase and recovering the modified Family 6 glycosidase by separation of the culture filtrate from the host microbe.

[0024] The inventors have made the surprising discovery that although substitution of Nl 82, W367, E399, C/S400 or A427 by another amino acid generally results in loss of activity against the beta 1 -4 linked substrate cellulose, several of these mutations significantly increase the activity of the enzyme towards beta 1-3, 1-4 glucans. Since these amino acids all participate in substrate binding within the active site, the inventors postulate, without wishing to be bound by theory, that the altered substrate specificity of such modified Family 6 glycosidases may be a consequence of an expansion of the enzyme active site to accommodate the branched beta 1-3, 1-4 linked substrates. This altered substrate specificity has potential value applied to industries where reduction of viscosity caused by beta 1-3, 1-4 glucan is desirable, as described above. The modified Family 6 glycosidase exhibits at least about 1.2-fold increase in activity on a beta-1-3, 1-4 linked polysaccharide and may also exhibit at least a 1.2-fold decrease in activity on a beta 1-4 linked polysaccharide such as cellulose. For example, the modified Family 6 glycosidase may exhibit from about a 1.2- to about a 4-fold increase in activity on a beta 1-3, 1-4 linked polysaccharide and may also exhibit from about a 1.2-fold to about a 10-fold decrease in activity on a beta 1-4 linked polysaccharide such as cellulose

[0025] The modified Family 6 glycosidases of the present display increased activity on beta 1-3, 1-4 -linked polysaccharides and decreased activity on beta 1-4 linked polysaccharides relative to the parental Family 6 glycosidase from which they are derived. [0026] Such glycosidases find use in a variety of applications in industry that require high activity on beta 1-3, 1-4 -linked or beta 1-3, 1-6-linked polysaccharide substrates. For example, modified Family 6 glycosidases, as described herein, may be used in industrial grain processing applications such as brewing, production of grain ethanol for fuel, and also to increase nutrient accessibility in animal feeds.

DESCRIPTION OF THE DRAWINGS

[0027] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0028] FIGURE 1 shows an amino acid sequence alignment among selected fungal glycosidases from Glycosyl Hydrolase Family 6 and a consensus Family 6 glycosidase sequence. A graphical representation of the frequency of occurrence of the amino acid at each position of the consensus Family 6 glycosidase among the 36 fungal Family 6 glycosidases is shown underneath the aligned sequences. The catalytic aspartic acid residues at the equivalent positions 175 and 221 in TrCelόA are indicated by arrows. The highly conserved amino acids at the equivalent of positions 182, 367, 399, 400 and 427 in TrCelόA are indicated with an asterisk. For cellulases with a cellulose-binding domain, only the catalytic core sequences are presented.

[0029] FIGURE 2 shows an identity matrix for the alignment of the amino acids corresponding to amino acids 83-447 of SEQ ID NO: 1 for each of 36 Family 6 glycosidase amino acid sequences to each other.

[0030] FIGURE 3 depicts plasmid vectors a) YΕp352/?GK9l-\ANhel-a_ss-TrCel6A-S413P, and b) YEpFLAG AXpnlO-cbh2 directing the expression and secretion of native and modified TrCelόA from recombinant Saccharomyces cerevisiae, c) YEp/PGK-αss-NKE-PcCelόA directing the expression and secretion of native and modified PcCelόA from recombinant Saccharomyces cerevisiae (The same organization if found for the PcCelό variants cloned in the same vectors), d) YEp/PGK-αss-NKE-HiAvi2 directing the expression and secretion of native and modified HiA vi2 from recombinant Saccharomyces cerevisiae (The same organization if found for the HiAvi2 variants cloned in the same vectors). [0031 ] FIGURE 4 shows the relative activity of modified TrCelόA glycosidases on cellulose, barley beta-glucan and lichenan to the activity of a parental TrCelόA glycosidase on each substrate.

[0032] FIGURE 5 shows the relative activity of parental and modified TrCelόA, PcCelόA and HiA vi2 glycosidases on (A) barley betaglucan: cellulose and (B) lichenan: cellulose.

[0033] FIGURE 6 shows the maps of Trie hoderma transformation vectors pCelόApst- S413P-pyr4-TV (A) and pCel6A413pst-hph-BB (B).

[0034] FIGURE 7 shows the verification of targeting of the TrCelόA genetic locus to native celόA locus by Southern hybridization. Genomic DNA was isolated from transformants P577A, B, C and parental strains BTR213, BTR213aux28 digested with EcoRl restriction enzyme, separated on a 1% agarose gel, transferred to a nitrocellulose membrane and hybridized using the TrCelόA coding nucleic acid sequence as a probe. pCel6ApXT-S413P- pyr4-TV transformation plasmid digested with EcoRl was used as a control (lane pCel6A_PXt-pyr4-TV).

[0035] FIGURE 8 shows the expression of the modified TrCelόA- W367G-S413P glycosidase by Trichoderma reesi transformants (P988A, P989A, B, C, P990A, P991B, P992A, P 1005 A, C, D) and the expression of wild-type TrCelόA by the host strain (P577C) and parental strain BTR213aux in microcultures. The abundance of TrCelόA- W367G-S413P or TrCelόA protein is indicated on each bar as a percent of total protein.

[0036] FIGURE 9 shows the crystal structure of TrCelόA (using coordinates from PDB file 1QK2) represented in ribbon form with the active-site ligand (cellotetraose) in black sticks and the amino acids at positions 182, 367, 399, 400 and 427 represented as black ball-and- sticks and are labeled. Residues 403 to 424 were removed for ease of visualization.

DESCRIPTION OF PREFERRED EMBODIMENT

[0037] The present invention relates to modified glycosidases. More specifically, the invention relates to modified Family 6 glycosidases with altered substrate specificity. The present invention also relates to genetic constructs comprising nucleotide sequences encoding for modified Family 6 glycosidases, methods for the production of the modified Family 6 glycosidase from host strains and the use of the modified Family 6 glycosidase in the hydrolysis of beta-glucan.

[0038] The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

Modified Glycosidases of Glycosyl Hydrolase Family 6

[0039] A glycosyl hydrolase enzyme is classified as a Family 6 glycosidase if exhibits similarity in its primary, secondary and tertiary protein structures to those of other Family 6 glycosidases. For example, all Family 6 glycosidases comprise two aspartic acid (D) residues which may serve as catalytic residues. These aspartic acid residues are found at positions 175 and 221 (see Figure 1 ; based on TrCelόA, Trichoderma reesei CelόA, amino acid numbering). Most of the Family 6 glycosidases identified thus far are mesophilic; however, this family also includes thermostable cellulases from Thermobifida fusca (TfCelόA and TfCelόB) and the alkalophilic cellulases from Humicola insolens (HiCelόA and HiCelόB). Family 6 glycosidases also share a similar three dimensional structure: an alpha/beta-barrel with a central beta-barrel containing seven parallel beta-strands connected by five alpha- helices. The three dimensional structures of several Family 6 glycosidases are known, such as TrCelόA (Rouvinen, J., et al. 1990), Thermobifida fusca endo-beta-l,4-glucanase CelόA (TfCelόA, Spezio, M., et al. 1993), Humicola insolens cellobiohydrolase CelόA (HiCelόA, Varrot, A., et al. 1999), Humicola insolens endo-beta-l,4-glucanase CelόB (HiCelόB, Davies, G.J., et al. 2000) and Mycobacterium tuberculosis H37Rv CelόA (MtCelόA, Varrot, A., et al. 2005).

[0040] As shown in Figures 1 and 2, there is a high degree of conservation of primary amino acid sequence among Family 6 glycosidases. Multiple alignment across 36 currently known Family 6 glycosidase amino acid sequences of fungal origin shows that the most naturally occurring Family 6 glycosidases exhibit from about 47% to about 100% amino acid sequence identity to amino acids 83-447 comprising the catalytic domain of TrCelόA (Table 1) and from about 70% to 100% amino acid sequence identity to at least one other Family 6 glycosidase. Family 6 glycosidases of bacterial origin show a much lower degree of amino acid sequence identity to TrCelόA or to other Family 6 glycosidases of fungal origin. [0041] There are several positions where a particular amino acid is universally conserved at the same corresponding position across all Family 6 members. For example, W135, W269, W272 and W367 are highly conserved amino acids that interact with the glucose subunits in the cellulose substrate at the -2, +1, +2 and +4 subsites in the active site tunnel of TrCelόA. Nl 82, E399, and A427 are other highly conserved residues found in the -2 subsite in the active site tunnel of TrCelόA.

Table 1: % Amino Acid Sequence Identity of Fungal Family 6 Glycosidases to TrCelόA

[0042] By "TrCelόA numbering", it is meant the numbering corresponding to the position of amino acids based on the amino acid sequence of TrCelόA (Table 1; Figure 1; SEQ ID NO:1). As set forth below, and as is evident by Figure 1, Family 6 glycosidases exhibit a substantial degree of sequence similarity. Therefore, by aligning the amino acids to optimize the sequence similarity between glycosidase enzymes, and by using the amino acid numbering of TrCelόA as the basis for numbering, the positions of amino acids within other Family 6 glycosidases can be determined relative to TrCelόA.

[0043] Methods to align amino acid sequences are well known and available to those of skill in the art and include BLAST (Basic Local Alignment Search Tool, URL: blast.ncbi.nlm.nih.gove/Blast.chi; Altschul et al., 1990; using the published default settings) which is useful for aligning two sequences and CLUSTALW (URL: ebi.cak.ak/Tools/clustalw2/index.html) for alignment of two or more sequences.

[0044] By "modified Family 6 glycosidase" or "modified glycosidase", it is meant a Family 6 glycosidase which comprises one or more amino acid substitutions, introduced by genetic engineering techniques, selected from the group consisting of: Nl 82X(i.e. N at position 182 is substituted by X), W367X, E399X, C/S400X, and A427X, where X is any amino acid and the position is determined from sequence alignment of the modified Family 6 glycosidase with a Trichoderma reesei CelόA amino acid sequence as defined in SEQ ID NO: 1. For example, the modified Family 6 glycosidase comprises one or more amino acid substitutions selected from the group consisting of: N182S, N182R, N182G, N182A, W367A, W367C, W367G, W367N, W367R, W367S, W367T, W367V, E399H, E399S, E399T, C400V, C400M, C400T, C400S, A427V, A427L, and A427S.

[0045] It will be understood that modified Family 6 glycosidase may be derived from any Family 6 glycosidase. For example, the modified Family 6 glycosidase may be derived from a wild-type glycosidase or from a glycosidase that already contains other amino acid substitutions.

[0046] A "modified Family 6 glycosidase" may also be defined as an enzyme capable of hydrolyzing polysaccharides using an inverting mechanism and having one or more amino acid substitutions, introduced by genetic engineering techniques, selected from the group consisting of: Nl 82X, W367X, E399X, C/S400X, and A427X, which is characterized by having an amino acid sequence that is from about 47% to about 99.9% identical to the amino acids 83 to 447 of the TrCelόA amino acid sequence (SEQ ID NO: 1) or having an amino acid sequence that is from about 70% to about 99.9% identical to amino acids 83-447 (TrCelόA) of any of the Family 6 glycosidases of SEQ ID NO: 1, SEQ ID NO: 2, 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, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. For example, a modified Family 6 glycosidase may have an amino acid sequence that is about 47%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9% identical to the amino acids 83- 447 of SEQ ID NO: 1 or that is about 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%. 92%. 94%, 96%. 98% or 99.9% identical to at amino acids 83-447 (TrCelόA numbering) of any of the Family 6 glycosidases of SEQ ID NO: 1, SEQ ID NO: 2, 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, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,

SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. One of skill in the art recognizes that the amino acid sequence of a given Family 6 glycosidase may be modified by the addition, deletion or substitution of one or more amino acids and still be considered a modified Family 6 glycosidase. Non-limiting examples of Family 6 glycosidases that may be modified following the general approach and methodology as outlined herein are provided in Table 1.

[0047] Examples of Family 6 glycosidases useful for the present invention, which are not meant to be limiting, include Trichoderma reesei CelόA, Humicola insolens CelόA, Phanerochaete chrysospoήum CelόA, Celhdomonas fimi CelόB, Thermobifida fusca CelόB. Preferably, the modified Family 6 glycosidase of the present invention comprises a modified Trichoderma reesei CelόA glycosidase. [0048] As used herein in respect of modified Family 6 glycosidase amino acid sequences, "derived from" refers to the isolation of a target nucleic acid sequence element encoding the desired modified Family 6 glycosidase using genetic material or nucleic acid or amino acid sequence information specific to the corresponding parental Family 6 glycosidase. As is known by one of skill in the art, such material or sequence information can be used to generate a nucleic acid sequence encoding the desired modified Family 6 glycosidase using one or more molecular biology techniques including, but not limited to, cloning, sub-cloning, amplification by PCR, in vitro synthesis, and the like.

[0049] In one embodiment of the invention, the modified Family 6 glycosidase comprises an amino acid sequence that is from about 70% to 99.9% identical to amino acids 83-447 of

SEQ ID NO: 1 and exhibits from about a 1.2-fold, for example from about 1.2-fold to 4-fold, increase in activity in the hydrolysis of beta 1-3, 1-4-linked polysaccharides and may also exhibit at least a 1.2-fold, for example from about 1.2-fold to 10-fold, decrease in activity in the hydrolysis of beta 1-4-linked polysaccharides relative to a parental Family 6 glycosidase from which it is derived.

[0050] In another embodiment of the invention, the modified Family 6 glycosidase comprises an amino acid sequence that is from about 80% to about 99.9% identical to amino acids 83- 447 (TrCelόA numbering) of any one of SEQ ID NO: 1 through 36 and exhibits from about a 1.2-fold increase in activity in the hydrolysis of beta 1-3, 1-4-linked polysaccharides and may also exhibit at least a 1.2-fold decrease in activity in the hydrolysis of beta 1-4 -linked polysaccharides relative to a parental Family 6 glycosidase from which it is derived.

[0051] In other embodiments of the invention, the modified Family 6 glycosidase comprises an amino acid sequence that is from about 90% to about 99.9% identical to amino acids 83- 447 of SEQ ID NO: 1 or from about 95% to about 99.9% identical to amino acids 83-447 (TrCelόA numbering) of any one of SEQ ID NO: 1 through 36 and exhibits from about a 1.2- fold increase in activity in the hydrolysis of beta 1-3, 1-4-linked polysaccharides and may also exhibit at least a 1.2-fold decrease in activity in the hydrolysis of beta 1-4 -linked polysaccharides relative to a parental Family 6 glycosidase from which it is derived.

[0052] Techniques for altering amino acid sequences include, but are not limited to, site- directed mutagenesis, cassette mutagenesis, random mutagenesis, synthetic oligonucleotide construction, cloning and other genetic engineering techniques (Eijsink VG, et al. 2005). It will be understood that the modified Family 6 glycosidase may be derived from any Family 6 glycosidase — i.e., it may be derived from a naturally-occurring or "wild-type" Family 6 glycosidase or from a Family 6 glycosidase that already contains other amino acid substitutions.

[0053] By "wild type" or "native" Family 6 glycosidase, it is meant a Family 6 glycosidase having an amino acid sequence as encoded by the genome of the organism that naturally produces such Family 6 glycosidase without the introduction of any substitutions, deletions, insertions, or modifications. For example, by wild type TrCelόA, wild type HiCelόA and wild type PcCelόA it is meant the cellulases of SEQ ID NO: 1, SEQ ID NO: 23 and SEQ ID NO: 30 respectively, without any amino acid substitutions.

[0054] For the purposes of the present invention, a "parental Family 6 glycosidase" or "parental glycosidase" is a Family 6 glycosidase that does not contain the amino acid substitution(s) in the modified Family 6 glycosidases, namely at one or more position selected from the group consisting of 182, 367, 399, 400 and 427 (TrCelόA numbering) but that is otherwise identical to the modified Family 6 glycosidase. As such, the parental Family 6 glycosidase may be a Family 6 glycosidase that contains amino acid substitutions at other positions that have been introduced by genetic engineering or other techniques. However, a parental Family 6 glycosidase does not include those Family 6 enzymes in which one or more of the naturally occurring amino acid at positions 182, 367, 399, 400 and 427 are, respectively, tryptophan, asparagine, tryptophan, glutamic acid, cysteine or serine, and alanine.

[0055] Alternatively, after production of a modified Family 6 glycosidase comprising amino acid substitutions at one or more of positions 182, 367, 300, 400 and 427, the modified Family 6 glycosidase may be subsequently further modified to contain additional amino acid substitutions.

[0056] In order to assist one of skill in the art regarding those amino acid positions of a given Family 6 glycosidase at which amino acid substitutions (other than N182X, W367X, E399X, C/S400X and W427X) may be made and produce an active enzyme, an alignment of 36 Family 6 glycosidases derived from fungal sources is provided in Figure 1 along with a graph showing the frequency of occurrence of each amino acid of the consensus sequence at each position. Using the information provided in Figure 1, one of skill in the art would recognize regions of low sequence conservation among Family 6 glycosidases and could introduce additional amino acid substitutions in these regions.

Altering the Substrate Specificity of Family 6 glycosidases

[0057] The substrate specificity of the modified Family 6 glycosidase is determined by incubation of the enzyme in the presence of several different polysaccharides substrate and measuring the release of soluble sugars from those substrates. The release of soluble sugars can be measured by subsequent chemical or chemienzymatic assays known to one of skill in the art, including reaction with dinitrosalisylic acid (DNS). Hydrolysis of polysaccharides can also be monitored by chromatographic methods that separate and quantify soluble mono-, di- and oligo-saccharidses released by the enzyme activity. In addition, soluble colorimetric substrates may be incorporated into agar-medium on which a host microbe expressing and secreting a parental or modified Family 6 glycosidase is grown. In such an agar-plate assay, activity of the glycosidase is detected as a colored or colorless halo around the individual microbial colony expressing and secreting an active glycosidase. The practice of the present invention is not limited by the method used to assess the substrate specificity of the modified Family 6 glycosidase.

[0058] The effect of amino acid substitutions at positions 182, 367, 399, 400 and 427 was determined via a comparative study of the substrate specificity of modified and the parental TrCelόA glycosidases. As shown in Figures 4 and 5 and summarized for activity on barley beta 1-3, 1-4 glucan in Table 2

Table 2: Altered Substrate Specificity of Modified Family 6 Glycosidases

[0059] In a preferred embodiment, the modified Family 6 glycosidase exhibits at least a 1.2- fold, for example from about 1.2- fold to about 4-fold, increase in its hydrolysis activity of beta 1-3, 1-4 linked polysaccharides and may also exhibit at least a 1.2-fold, for example from about 1.2-fold to about 10-fold, decrease in its hydrolysis activity of beta 1-4 linked polysaccharides.

[0060] Without wishing to be bound by theory, the inventors hypothesize that the increased activity on beta 1-3, 1-4 glucans exhibited by the modified Family 6 glycosidases is due to the location of the substituted amino acids within or near the active site of the enzyme. Figure 9 shows that, for TrCelόA, amino acids W367, E399, C400 are involved in substrate binding while amino acids Nl 82 and A427 are located within the loop regions that enclose the active site tunnel. Therefore, mutations of these highly conserved amino acids may result in a more open or flexible geometry within the TrCelόA active site that allow for the accommodation of the branched beta 1-3, 1-4 glucans.

Genetic Constructs Encoding Modified Family 6 Glycosidase

[0061] The present invention also relates to genetic constructs comprising a nucleic acid sequence encoding the modified Family 6 glycosidase. The modified glycosidase-encoding nucleic acid sequence may be operably linked to regulatory nucleic acid sequences directing the expression and secretion of the modified Family 6 glycosidase from a host microbe. By "regulatory DNA sequences" it is meant a promoter and a DNA sequence encoding a secretion signal peptide. The regulatory DNA sequences are preferably functional in a fungal host. The regulatory DNA sequences may be derived from genes that are highly expressed and secreted in the host microbe under industrial fermentation conditions. In a preferred embodiment, the regulatory sequences are derived from one or more of the Tήchoderma reesei cellulase or hemicellulase genes.

[0062] The genetic construct may further comprise a selectable marker gene to enable isolation of a genetically modified microbe transformed with the construct as is commonly known to those of skill in the art. The selectable marker gene may confer resistance to an antibiotic or the ability to grow on medium lacking a specific nutrient to the host organism that otherwise could not grow under these conditions. The present invention is not limited by the choice of selectable marker gene, and one of skill in the art may readily determine an appropriate gene. In a preferred embodiment, the selectable marker gene confers resistance to hygromycin, phleomycin, kanamycin, geneticin, or G418, complements a deficiency of the host microbe in one of the trp, arg, leu, pyr4, pyr, ura3, ura5, his, or ade genes or confers the ability to grow on acetamide as a sole nitrogen source.

[0063] The genetic construct may further comprise other nucleic acid sequences, for example, transcriptional terminators, nucleic acid sequences encoding peptide tags, synthetic sequences to link the various nucleic acid sequences together, origins of replication, and the like. The practice of the present invention is not limited by the presence of any one or more of these other nucleic acid sequences.

Genetically Modified Microbes Producing Modified Family 6 Glycosidases

[0064] The modified Family 6 glycosidase may be expressed and secreted from a genetically modified microbe produced by transformation of a host microbe with a genetic construct encoding the modified Family 6 glycosidase. The host microbe may be a yeast or a filamentous fungus, particularly those microbes that are members of the phylum Ascomycota. Genera of yeasts useful as host microbes for the expression of modified TrCel3A beta- glucosidases of the present invention include Saccharomyces, Pichia, Hansenula, Kluyveromyces, Yarrowia, and Arxula. Genera of fungi useful as microbes for the expression of modified TrCel3A beta-glucosidases of the present invention include Trichoderma, Hypocrea, Aspergillus, Fusarium, Humicola, Neurospora, and Penicillium . Typically, the host microbe is one from which the gene(s) encoding any or all Family 6 glycosidase have been deleted. In a most preferred embodiment, the host microbe is an industrial strain of Trichoderma reesei. [0065] The genetic construct may be introduced into the host microbe by any number of methods known by one skilled in the art of microbial transformation, including but not limited to, treatment of cells with CaCl₂, electroporation, biolistic bombardment, PEG- mediated fusion of protoplasts (e.g. White et al., WO 2005/093072). After selecting the recombinant fungal strains expressing the modified Family 6 glycosidase, the selected recombinant strains may be cultured in submerged liquid fermentations under conditions that induce the expression of the modified Family 6 glycosidase. Preferably, the modified Family 6 glycosidase is produced in submerged liquid culture fermentation and separated from the cells at the end of the fermentation. The cells may be separated by filtration, centrifugation, or other processes familiar to those skilled in the art. The cell-free glycosidase-containing fraction may then be concentrated (for example, via ultrafiltration), preserved, and/or stabilized prior to use.

[0066] Therefore the present invention also provides a process for producing a modified Family 6 glycosidase. The method comprises growing a genetically modified microbe comprising a nucleotide sequences encoding a modified Family 6 glycosidase, in a culture medium under conditions that induce expression and secretion of the modified Family 6 glycosidase, and recovering the modified Family 6 glycosidase from the culture medium. The modified Family 6 glycosidase comprising one or more amino acid substitution at a position selected from the group consisting of N182X, W367X, E399X, C/S400X, and A427X, the position determined from alignment of a parental Family 6 glycosidase amino acid sequence with a Trichoderma reesei CelδA amino acid sequence as defined in SEQ ID NO: 1, wherein amino acids 83-447 (TrCelόA numbering) of the modified Family 6 glycosidase are from about 47% to about 99.9% identical to amino acids 83-447 of SEQ ID NO: 1, or from about 70-90% identical to amino acids 83-447 of any one of SEQ ID NO: 1 through 36.

Production of Modified TrCeBA Beta-glucosidases

[0067] A modified Family 6 glycosidase of the present invention may be produced in a fermentation process using a genetically modified microbe comprising a genetic construct encoding the modified Family 6 glycosidase, e.g., in submerged liquid culture fermentation.

[0068] Submerged liquid fermentations of microorganisms, including Trichoderma and related filamentous fungi, are typically conducted as a batch, fed-batch or continuous process. In a batch process, all the necessary materials, with the exception of oxygen for aerobic processes, are placed in a reactor at the start of the operation and the fermentation is allowed to proceed until completion, at which point the product is harvested. A batch process for producing the modified Family 6 glycosidase of the present invention may be carried out in a shake-flask or a bioreactor.

[0069] In a fed-batch process, the culture is fed continuously or sequentially with one or more media components without the removal of the culture fluid. In a continuous process, fresh medium is supplied and culture fluid is removed continuously at volumetrically equal rates to maintain the culture at a steady growth rate,

[0070] One of skill in the art is aware that fermentation medium comprises a carbon source, a nitrogen source and other nutrients, vitamins and minerals which can be added to the fermentation media to improve growth and enzyme production of the host cell. These other media components may be added prior to, simultaneously with or after inoculation of the culture with the host cell.

[0071 ] For the process for producing the modified Family 6 glycosidase of the present invention, the carbon source may comprise a carbohydrate that will induce the expression of the modified Family 6 glycosidase from a genetic construct in the genetically modified microbe. For example, if the genetically modified microbe is a strain of Trichoderma, the carbon source may comprise one or more of cellulose, cellobiose, sophorose, and related oligo- or poly-saccharides known to induce expression of cellulases and beta-glucosidase in Trichoderma.

[0072] In the case of batch fermentation, the carbon source may be added to the fermentation medium prior to or simultaneously with inoculation. In the cases of fed-batch or continuous operations, the carbon source may also be supplied continuously or intermittently during the fermentation process. For example, when the genetically modified microbe is a strain of Trichoderma, the carbon feed rate is between 0.2 and 2.5 g carbon/L of culture/h, or any amount therebetween.

[0073] The process for producing the modified Family 6 glycosidase of the present invention may be carried at a temperature from about 20°C to about 40°C, or any temperature therebetween, for example from about 25°C to about 37°C, or any temperature therebetween, or from 20, 22, 25, 26, 27, 28, 29, 30, 32, 35, 37, 40⁰C or any temperature therebetween. [0074] The process for producing the modified Family 6 glycosidase of the present invention may be carried out at a pH from about 3.0 to 6.5, or any pH therebetween, for example from about pH 3.5 to pH 5.5, or any pH therebetween, for example from about pH 3.0, 3.2, 3.4, 3.5, 3.7, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.5, 5.7, 5.8, 6.0, 6.2, 6.5 or any pH therebetween.

[0075] Following fermentation, the fermentation broth containing the modified Family 6 glycosidase may be used directly, or the modified Family 6 glycosidase may be separated from the fungal cells, for example by filtration or centrifugation. Low molecular solutes such as unconsumed components of the fermentation medium may be removed by ultra- filtration. The modified Family 6 glycosidase may be concentrated, for example, by evaporation, precipitation, sedimentation or filtration. Chemicals such as glycerol, sucrose, sorbitol and the like may be added to stabilize the cellulase enzyme. Other chemicals, such as sodium benzoate or potassium sorbate, may be added to the cellulase enzyme to prevent growth of microbial contamination.

The Use of Modified Family 6 Glycosidase

[0076] The modified Family 6 glycosidase of the present invention is used for the enzymatic hydrolysis of polysaccharides containing both beta 1-3 , 1-4 and/or beta 1-3, 1-6 glycosidic linkages. More preferably, the modified Family 6 glycosidase of the present invention is used for the enzymatic hydrolysis of beta 1-3, 1-4 glucans present in cereal grains. The modified Family 6 glycosidases of the present invention may be used in industrial processes such as brewing, production of grain ethanol for fuel, and also to increase nutrient accessibility in animal feeds.

[0077] By the term "enzymatic hydrolysis", it is meant a process by which glycosidase enzymes or mixtures, including those comprising the modified Family 6 glycosidase of the present invention, act on polysaccharides to convert all or a portion thereof to soluble sugars. Examples

[0078] The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.

Example 1: Strains and Vectors

[0079] Saccharomyces cerevisiae strain BY4742 (MATα his3Δl leu2Δ0 lys2Δ0 ura3Δ0 Δkre2) was obtained from ATCC (#4014317). The YEp352/PGK91-l vector was obtained from the National Institute of Health. The YEPFLAGΔJ-/7H10-S413P vector is described in U.S. Publication No. 2008/0076152Al . The YEpFLAG-I vector was obtained from Sigma as a part of the Amino-Terminal Yeast FLAG Expression Kit.

Example 2: Cloning of modified glycosidase genes and transformation of Saccharomyces cerevisiae

a. Cloning of the TrCel6A-S413P gene into the YEp352/PGK91-l vector and transformation of S. cerevisiae BY 4742

[0080] In order to facilitate cloning using Nhel and Kpnl restriction enzymes, the unique Nhel site at position 1936 of the YEp352/PGK91-l vector was blunted using the DNA Polymerase I large (Klenow) fragment to generate YEp352/PGK91-lΔMe/. The TrCelόA- S413P gene was amplified by PCR from YEρFLAGΔ/ζt?«10-S413P vector (U.S. Publication No. 2008/0076152Al) using primers 5'NheCel6A and 3'BglKpnCel6A. In parallel, the yeast α-factor leader sequence was amplified by PCR from the YEpFLAG-I vector (Sigma) using primers (5'BglAlphaSS and 3'NheAlphaSS) to introduce %/II at the 5' end and an Nhel site at 3' end of the amplicon. SEQ ID NOS: 47-50 were utilized as primer sequences.

5 'BglAlphaSS: 5 'ACC AAA AGA TCT ATG AGA TTT CCT TCA ATT (SEQ ID NO: 47)

3 'NheAlphaSS: 5 'TGA GCA GCT AGC CCT TTT ATC CAA AGA TAC (SEQ ID NO: 48) 5'NheCel6A: 5'AAA AGG GCT AGC TGC TCA AGC GTC TGG GGC (SEQ ID NO: 49)

3 BglKpnCel6A: 5'GAG CTC AGA TCT GGT ACC TTA CAG GAA CGA TGG GTT (SEQ ID NO:

50) [0081] The yeast alpha-factor leader sequence was isolated by Bglll/Nhel digestion and a three piece ligation performed with the TrCel6A-S413P gene (isolated by Nhel/Bglll digestion) and YEp352/PGK91-lΔMzd vector (isolated by BgIU digestion). The resulting vector YEp352/?GK9l-\ANhel-a_ss-TrCel6A-S4J3P (Figure 3) was transformed in yeast strain BY4742 using the procedure described by Gietz, R. D. and Woods, R. A. (2002).

b. Cloning of the Pccelόa, Pccel6A-W361G, HiAvH and HiAvi2-W374G genes into the YEp/PGK-a_ss-NKE and transformation in yeast

[0082] Generation ofYEep/PGK-alpha_ss-NKE: Vector YEp352/PGK91-l was digested with NAeI and EcoRl and the plasmid band was isolated from gel. A DΝA adapter was made by annealing of AT046 and AT047 5'-phosphorylated primers and was ligated with the digested vector. To eliminate possible concatemerization, the plasmid was then digested with Kpnl and self-ligated. The resulting vector is named YEp/PGK-alpha_sS-ΝKE and its sequence integrity was confirmed by sequencing.

AT046: 5' CTA GCT GAT CAC TGA GGT ACC G (SEQ ID NO: 54) AT047: 5' AAT TCG GTA CCT CAG TGA TCA G (SEQ ID NO: 55)

[0083] Generation ofPcCelόA and PcCelόA- W361 G vectors: The Pccelόa gene was amplified by PCR from YEpFLAGΔiφwlO-PcCelόA vector (U.S. Publication No. 2008/0076152Al) using primers 5ΥH098 and 3ΥH099. Pccelόa was cloned NheVKpnl in YEp/PGK-alpha_ss-NKE. PcCel6A-W361G was generated by two step PCR by mutating PcCelόA in YEp/PGK-alphas_S-NKE using primers 5 ' VH067 and 3 'PGK-term for fragment one and YalphaN21-2 and 3ΥH066 to generate fragment two. Fragments 1 and 2 were combined using primers YalphaN21-2 and 3 'PGK-term.

5ΥH098: 5' GGT ATC TTT GGA TAA AAG GGC TAG CTC GGA GTG GGG ACA G (SEQ ID NO:

56 ) 3ΥH099: 5' GGA GAT CGA ATT CGG TAC CTA CAG CGG CGG GTT GG (SEQ ID NO: 57)

5'VH067: 5' CAG TGG GGA GAC GGG TGC AAC ATC AAG (SEQ ID NO: 58)

3ΥH066: 5' GTC TCC CCA CTG TTG GCG GAT G (SEQ ID NO: 59)

YalphaN21-2 5 ' GCC AGC ATT GCT GCT AAA G (SEQ ID NO: 60) The resulting vectors, YEpFLAGΔAT/w 10-PcCel6A and YEpFLAGAKpn 10-PcCel6A - W361G were used to transform Saccharomyces cerevisiae strain BY4742 using the procedure described by Gietz, R. D. and Woods, R. A. (2002).

[0084] Generation of HiAvH and Hi AvH-W 374G vectors: The Hiavi2 gene was amplified by PCR from YEpFLAGΔXp«10-HiAvi2 vector (US Patent Provisional No. 60/841,507) using primers 5'NM083 and 3'NM084. HiAvH was cloned NhellKpnl in YEp/PGK-alpha_sS- NKE. HiAvi2-W374G was generated by two step PCR by mutating HiAvi2 in YEp/PGK- alpha_ss-NKE using primers 5ΥH065 and 3'PGK-term for fragment one and YalphaN21-2 and 3 'VH 064 to generate fragment two. Fragments 1 and 2 were combined using primers YalphaN21 -2 and 3 'PGK-term.

5'NM083: 5' AAG GAT GAC GAT GAC AAG GAA TTC CTC GAG GCT AGC TGT GCC CCG ACT

TGG GGC (SEQ ID NO: 61)

3'NM084: 5' AGC GGC CGC TTA CCG CGG GTC GAC GGG CCC GGT ACC TCA GAA CGG CGG ATT GGC (SEQ ID NO: 62)

5'VH065: 5' GAA TGG GGC CAC GGG TGC AAT GCC ATT GG (SEQ ID NO: 63)

3ΥH064: 5' GTG GCC CCA TTC CTT CTG GCC G (SEQ ID NO: 64)

The resulting vectors, YEpFLAGΔXpw 10-HiAvi2 and YEpFL AGΔφ/i 10-HiAvi2-W374G were used to transform Saccharomyces cerevisiae strain BY4742 using the procedure described by Gietz, R. D. and Woods, R. A. (2002).

Example 3: Making Error Prone-PCR Libraries

[0085] Random mutagenesis libraries were generated using two methods: a Mutazyme^® II DNA polymerase method and a Mn²⁺/biased dNTP mix method. For the Mutazyme^® II DNA polymerase method, a series of four independent PCR were performed using 10, 20, 30, 40 ng of YEp352/PGK9l-l ANhel-a_ss-TrCel6A-S413P vector and the Mutazyme⁸¹ II DNA polymerase with primers YalphaN21 and 3'PGK-term. The amplification was done for 25 cycles. The four PCR products were pooled and diluted to 10 ng/μL. A second PCR mutagenesis step was performed using 30 ng of pooled PCR product with Mutazyme* II DNA polymerase using the same primers for 30 amplification cycles. The YEp352/PGK91- lANhel-a_ss-TrCel6A-S413P vector was digested with Nhel and Kpnl and the empty vector fragment was isolated. This linear fragment and the final amplicon were transformed simultaneously and cloned by in vivo recombination into yeast strain BY4742 (Butler et ai, 2003).

[0086] For the Mn²⁺7biased dNTP mix method, a PCR was performed using 25 ng YE^352/?GK9\-\ ANhel-a_ss-TrCel6A-S413P vector, 0.2 mM dATP, 0.2 mM dCTP, 0.24 mM dGTP, 0.2 mM dTTP, and 0.64 mM Mn²⁺ with Taq DNA polymerase (Sigma) with primers YalphaN21 and 3'PGK-term for 30 amplification cycles. The final amplicon was cloned into YEp352/PGK9\-\ ANhel-a_ss-TrCel6A-S413P vector as described above.

YalphaΝ21 : 5'AGC ACA AAT AAC GGG TTA TTG (SEQ ID NO: 49) 3'PGK-term: 5'GCA ACA CCT GGC AAT TCC TTA CC (SEQ ID NO: 50)

Example 4: Screening of Error-Prone PCR Library of TrCelόA

a. Primary Screening of TrCelόA EP-PCR Library — Plate Assay

[0087] Saccharomyces cerevisiae transformants were grown on plates containing synthetic complete medium (SC: 2% agar w/v, 0.17% yeast nitrogen base w/v, 0.078% -Ura drop-out supplement w/v, 2% glucose w/v, 2% casamino acids w/v, 0.5% ammonium sulfate w/v, pH 5.5) and 0.12% Azo-barley-β-glucan (Megazyme) for 2 days at 30⁰C. Colonies showing bigger clearing halos, after an overnight incubation at 45⁰C, compared to the parent enzyme TrCel6A-S413P were selected and sequenced as described below in section c.

b. Primary Screening of TrCelόA EP-PCR Library - Liquid Assay

[0088] Clones from the EP-PCR (Example 3) or SSM (Example 5) libraries expressing variants of TrCel6A-S413P were selected for liquid media pre-cultures by toothpick inoculation of 150 μL synthetic complete media (SC: 0.17% yeast nitrogen base w/v, 0.078% -Ura drop-out supplement w/v, 2% glucose w/v, 2% casamino acids w/v, 0.5% ammonium sulfate w/v, pH 5.5) in 96-well microplates. Pre-cultures were grown overnight (16 - 18 h) at 30°C and 300 rpm to stationary phase. For expression culture inoculation, 25 μL of pre- culture was used to inoculate 1 mL of SC media in deep-well microplates containing one glass bead. The remaining pre-cultures were used to prepare culture stocks by the addition of glycerol to a final concentration of 15% and stored at -80⁰C. [0089] Expression cultures were grown for 3 days at 30⁰C with orbital shaking and humidity control Plates were centπfuged at 710 x g for 5 minutes to pellet cells and supernatant was aspirated for screening assays An aliquot (0 05 mL) of yeast supernatant was incubated with 0.5% beta-glucan in a 0.1 mL citrate buffered (50 mM; pH 5) reaction. Activity assays were performed for 3 hours in a PCR plate at 50⁰C Contained in each 96-well PCR plate were 6 parental TrCel6A-S413P controls used for compaπson. A glucose standard curve was placed in the first column of the PCR plate ranging from 3 to 0 05 mg/mL. Following incubation, 0 08 mL of DNS reagent was added to all wells and the plates were boiled for 10 mm. An aliquot (0.15 mL) was transferred to a microplate and the absorbance was measured at 560 nm.

DNS reagent contains

Component g/L

3,5-Dinitosalicylic acid (Acros) 20

Sodium hydroxide (Fisher) 20 Phenol (Sigma) 4

Sodium metabisulfate (Fisher) 1

[0090] The concentration of parental or modified TrCelόA glycosidases in yeast filtrates was determined by ELISA Filtrate and purified component standard were diluted 0.01-10 μg/mL (based on total protein) in phosphate-buffered saline, pH 7.2 (PBS) and incubated overnight at 4°C in microtitre plates (Costar EIA #9018). These plates were washed with PBS containing 0.1% Tween-20 (PBS/Tween) and then incubated in PBS containing 1% bovine serum albumin (PBS/BSA) for 1 h at room temperature. Blocked microtitre wells were washed with PBS/Tween Rabbit polyclonal antisera specific for TrCelόA was diluted (1 16,000) in PBS/BSA, added to separate microtitre plates and incubated for 2 h at room temperature Plates were washed and incubated with a goat anti-rabbit antibody coupled to horseradish peroxidase (Sigma #A6154), diluted 1/2000 in PBS/BSA, for 1 hr at room temperature. After washing, tetramethylbenzidine was added to each plate and incubated for 30 mm at room temperature. The absorbance at 360 nm was measured in each well and converted into protein concentration using the TrCelόA standard curve [0091] Enzyme activity was determined by converting As₆₀ values to reducing equivalents using the glucose standard curve. A specific activity was calculated for all modified and parental TrCelόA glycosidases by dividing the enzyme activity by the enzyme concentration determined by ELISA. The specific activity for each modified TrCelόA glycosidase was compared to the average of 6 parental TrCelόA glycosidase controls on a particular microplate and positives were selected at the 95% confidence level using a t-test. All positive variants were produced again in microculture and re-screened to reduce the number of false positives.

c. Sequencing of genes encoding modified glycosidases

[0092] Plasmid DNA comprising genes encoding modified TrCelόA 6 glycosidases with altered substrate specificity was isolated from yeast cultures grown from the glycerol stocks prepared in Example 4b. The modified TrCelόA glycosidase genes were subjected to DNA sequencing to identify mutations that confer altered substrate specificity.'

Example 5: Making Site-Saturation Mutagenesis (SSM) Libraries

[0093] Site-saturation mutagenesis of residue W367 was performed by megaprimer PCR (two-step PCR reaction) using the mutagenic primer 3' W367X (SEQ ID NO: 51), the YEp352/PGK91-lΔM?eI-alpha_ss-rrCe/<5Λ-&/73/^J vector as template, and the Platinum^® Taq DNA Polymerase High Fidelity (Invitrogen). The first-step PCR was done using the mutagenic primer 3' W367X and the complementary external primer (YalphaN21 or 3 'PGK- term, SEQ ID NOS: 52 and 53, respectively). The purified amplicon served as a megaprimer for the second-step PCR and the other complementary external primers were used to amplify the complete mutated gene. The YEp352/PGK91 - 1 ANhel-alpha_ss-TrCel6A-S413P vector was digested with Nhel and Kpnl and the empty vector fragment was isolated. This linear fragment and the final amplicon were transformed simultaneously and cloned by in vivo recombination into yeast strain BY4742 (Butler et al. 2003).

3 W367X: 5'CAG CAA CAG TGG GGA GAC NNS TGC AAT GTG ATC GGC ACC (SEQ ID NO: 51) YalphaN21: 5'AGC ACA AAT AAC GGG TTA TTG (SEQ ID NO: 52) 3 'PGK-term: 5 'GCA ACA CCT GGC AAT TCC TTA CC (SEQ ID NO: 53) [0094] The amino acids Nl 82, E399, C400 and A427 of TrCelόA were substituted separately for all amino acids (via SSM) by two-step PCR (Table 3) using the following primers:

YalphaN21-2 5'GCC AGC ATT GCT GCT AAA G (SEQ ID NO:60)

3'PGK-term 5'GCA ACA CCT GGC AAT TCC TTA CC (SEQ ID NO:53)

N182X-F 5'CC CTT GCC TCG NNS GGC GAA TAC TC (SEQ ID NO:66)

N182X-R 5'CGA GGC AAG GGC AGC GCA ATC G (SEQ ID NO:65)

E399X-F 5'G CCA GGC GGC NNS TGT GAC GGC ACC (SEQ ID NO:68)

E399X-R 5'GCC GCC TGG CTT GAC CCA GAC AAA CG (SEQ ID NO:67)

C400X-F 5 'CA GGC GGC GAG NNS GAC GGC ACC AG (SEQ ID NO:70)

C400X-R 5 'CTC GCC GCC TGG CTT GAC CCA GAC (SEQ ID NO:69)

A427X-F 5'CCG GCG CCT CAA NNS GGT GCT TGG TTC C (SEQ ID NO:72)

A427X-R 5'GAG GCG CCG GTT GCA AGG CAT CTG GG (SEQ ID NO:71

Table 3: Two-step PCR performed to generate site-saturated mutagenesis for all four positions.

PCR 1 and 2, Step 1 PCR Step 2

Position Primer 1 Primer 2 Size (bp) Primer 1 Primer 2 Size (bp)

N182X-1 YαN21 Wl N182X-R 588

YccN21 #2 3'PGK-Term 1473

N182X-2 N182X-F 3'PGK-Term 896

E399X-1 YαN21 #2 E399X-R 1239

YαN21 #2 3'PGK-Term 1473 E399X-2 E399X-F 3'PGK-Term 244

C400X-1 YαN21 #2 C400X-R 1242

YαN21 #2 3'PGK-Term 1473 C400X-2 C400X-F 3'PGK-Term 242

A427X-1 YαN21 #2 A427X-R 1321

YαN21 #2 3'PGK-Term 1473 A427X-2 A427X-F 3'PGK-Term 162

[0095] To perform a gap repair the vector Yep/PGK-alphas_S-6H-NKE was digested with Nhel and Kpnl and purified on gel. Saccharomyces cerevisiae strain kre2Δ (MATα his3 Δl Ieu2 Δ0 lys2Δ0 ura3Δ0 Δkre2) was used as the host. The digested YEp/PGK-alpha_ss-όH- NKE vector and the PCR Step 2 amplicons were transformed in the yeast strain kre2 Δ using the procedure described by Gietz, R. D. and Woods, R. A. (2002).

Example 6: Liquid assays of modified glycosidases to detect altered substrate preference [0096] TrCel6A-S413P variants from yeast supernatant were tested in liquid assays using three different substrates: barley-β-glucan (Medium Viscosity; Megazyme), lichenan and acid swollen cellulose (ASC, produced from Sigmacell50 using the methods described by Tansey, M. R. 1971).

[0097] The activity of each enzyme was determined by measuring the release of reducing sugars from the soluble barley-β-glucan or lichenan substrates. Specifically, in a 300 μL PCR plate, 50 μL of yeast supernatant (dilution series) was mixed with 50 μL of pre-heated 1% (w/v) barley-β-glucan or lichenan in 100 mM sodium citrate pH 5.0. Mixtures were incubated for up to 2 h at 50⁰C. Following the incubation, 80 μL of DNS reagent was added to each well and the plate was boiled for 10 minutes.

DNS reagent contains:

Component g/L

3,5-Dinitosalicylic acid (Acros) 10

Sodium hydroxide (Fisher) 10 Phenol (Sigma) 2

Sodium metabisulfate (Fisher) 0.5

[0098] Once the temperature decreased below 40⁰C, 150 μL of each reaction mixture was transferred to individual wells of a 96-well microplate and OD₅₆₀ was measured using a Fluostar Galaxy microplate reader. Blank value was measured by treating the supernatant from the strain carrying the empty vector the same way and was subtracted from each value. The data were fit with Equation A by the method of least squares using the Excel solver and by varying the a and b parameters for each enzyme.

Equation A: y = (a ^• E) / (b + E) where E represents enzyme concentration.

[0099] To determine the initial rate of each enzyme, the slope of Equation A was determined as the enzyme concentration approached zero. This was done by substituting E = 0 into the first derivative of Equation A. Initial rates for each variant were normalized to wild-type TrCelόA (Figure 4).

[00100] The activity of each enzyme on ASC was tested in a 0.25 mL cellulose hydrolysis assay. TrCelόA variants from yeast supernatant as described in Example 4 were diluted in 50 mM citrate buffer (pH 5.0), complemented with Trichoderma reesei Cel7B and Cel5A (10 mg protein / g cellulose) and A. niger beta-glucosidase (125 IU / g cellulose) and incubated with 0.067% ASC. Incubation was at 50⁰C for 19 hr. Microplates were centrifuged for 3 min at 2800 x g and an aliquot of supernatant was sampled for glucose. Enzyme activity was measured via the detection of glucose using a standard glucose oxidase / peroxidase coupled reaction assay (Trinder, 1969). The data were fit with Equation A by the method of least squares using the Excel solver and by varying the a and b parameters for each enzyme.

Equation A: y = (a ^• E) / (b + E) where E represents enzyme concentration.

[00101 ] To determine the initial rate of each enzyme, the slope of Equation A was determined as the enzyme concentration approached zero. This was done by substituting E = 0 into the first derivative of Equation A. Initial rates for each variant were normalized to wild-type TrCelόA (Figure 4).

[00102] Figures 4 and 5 show the relative activity of parental modified Family 6 glycosidases on cellulose and two beta-glucan substrates: barley beta-glucan, with a ratio of 3:1 ( beta 1-3 : beta 1-4) and lichenan, with a ratio of 2:1 (beta 1-3 : beta 1-4). All variants show at least a 1.2-fold increase in activity against one or both of the beta-glucan substrates. Some variants also exhibit more than a 1.2-fold decrease in activity against acid swollen cellulose.

Example 7: Expression of PcCelόA, HiA vi2 and their variant in flasks cultures

[00103] Saccharomyces cerevisiae transformants were grown on plates containing synthetic complete medium (SC: 2% agar w/v, 0.17% yeast nitrogen base w/v, 0.192% -Ura drop-out supplement w/v, 2% glucose w/v, 2% casamino acids w/v, 0.5% ammonium sulfate w/v, pH 5.5) for 3 days at 30⁰C.

[00104] A single colony of these streaks was used to inoculate 150 μL of synthetic complete medium in a 96-well microplate containing a small sterile glass bead. Pre-cultures were grown overnight (16 - 18 hr) at 30⁰C and 300 rpm to stationary phase. For expression culture inoculation, 25 μL of pre-culture was used to inoculate 50 mL of SC media. Expression cultures were grown for 3 days at 3O⁰C and 250 rpm with humidity control. Cultures were centrifuged at 3000 rpm for 5 min and the buffer of the supernatant was changed to 50 mM citrate buffer pH 5.0 using a Sartorius filtration device with a 5000 kDa cut-off membrane. All centrifugations for the buffer exchange were done at 4000 rpm at room temperature. The enzymes were washed twice with 20 mL of 50 mM citrate buffer pH 5.0, concentrated in a final volume of 3 mL (approx.15 fold concentration) of 50 mM citrate buffer pH 5.0, and stored at -20⁰C.

[00105] The activity of each parental and modified PcCelόA and HiAvi2 glycosidase was measured using barley beta-glucan, lichenan and acid-swollen cellulose as described in Example 6 except that HiA vi2 activity assays were performed at pH 6.5.

Example 8: Expression of modified TrCeiόA glycosidase in Trichoderma reesei

a. Trichoderma reesei strains

[00106] Apyr4 auxotrophic T. reesei strain (strain BTR213) was used as a host strain for expression of TrCel6A-W367G-S413 . BTR213 is a derivative of RutC30 (ATCC #56765; Montenecourt and Eveleigh, 1979) produced by random mutagenesis and first selected for ability to produce larger clearing zones on minimal media agar containing 1% acid swollen cellulose and 4 g L^"1 2-deoxyglucose and then selected for the ability to grow on lactose media containing 0.2 μg/ml carbendazim. Thepyr4 auxotroph of strain BTR213 was isolated by the ability to grow on 5-FOA (5-fluororotic acid) and inability to grow prototrophically in the absence of uridine.

b. Construction of transformation vectors

[00107] Two intermediate vectors, pCelό Apst-hph-TV and pCelό ApXt-hph-TV, containing either genomic celόa or cDNA celόa gene versions, respectively, were constructed.

[00108] For generation of pCelό Apst-hph-TV, the celόa promoter, secretion signal, coding sequence, and terminator were isolated from pZUK636 (U.S. Patent No. 6,015,703) as a 5.1 kb SphVBglll fragment and inserted into the same sites of pUC-NSNB, a derivative of the standard cloning vector pUCl 19 containing an adaptor comprising Nhel-Sphl-Notl-Bglll restriction sites, make pCel6A-Not. In order to increase the size of the 3' flanking fragment, a 1.7 kb fragment containing part of the celόa terminator (downstream of the Bglll site) and 3' flanking sequence, was amplified from BTR213 using primers KW008 and KW052 (Table 5) and cloned into pGEM T-easy (Promega). KW008 anneals to the internal BgIW site located 1 kb downstream of the stop codon while K.W052 introduces a Smal site 2.7 kb downstream of the stop codon. The CelόA 3' flanking fragment was amplified as a 1.7 kb fragment using BTR213 genomic DNA as a template, digested with BgWl and Smal restriction enzymes and cloned into the same sites of pCel6A-Not to make pCel6Apst-Not. pCel6Apst-Not was linearized with Sacll and blunt-ended with T4 polymerase. The hph selection marker cassette was isolated as a 3.1 kb XhoVEcoKV fragment from pHPTl 36, blunt-ended, and cloned into the blunted Sacll site to make pCel6Apst-hph-TV.

[00109] For generation of pCel6ApXt-hph-TV vector the CelόA promoter was amplified from pZUK636 using primers KW053 and KW054 (Table 4) and cloned into pGEM T-easy (Promega). KWO53 spans the Sphl site 2.5 kb upstream from the start codon while KW054 introduces a Ncol site at the start codon. The xyn2 secretion signal was amplified from BTR213 genomic DΝA using primers KW055 and K.W056 with introduced Ncol and Nhel sites, respectively, and cloned into pGEM T-easy. A celόa gene fragment encoding the mature TrCel6A-S413P parental glycosidase and the celόa terminator were isolated from previously constructed pc/xC2-S413P-TV (U.S. Publication No. 2008/0076152Al) as an NheVSphl fragment. A three factor ligation with the CelόA promoter (Sphl/Ncol), the xyn2 secretion signal coding sequence (Ncol/Nhel) and the pc/xC2-S413P-TV vector fragment (Sphl/Nhel) was used to make pCel6ApX-S413P. The 5 kb SphllBgtll fragment containing gene encoding TrCel6A-S413P was isolated from pCel6ApX-S413P and cloned into the same sites of pUC-NSNB to make pCel6ApX-S413P-Not. The size of the 3' flanking fragment was increased as described above (pCelόApst-hph-TV vector construction) generating pCe!6AptX-S413P vector. The pCel6AptX-S413P vector was linearized with Sacll (located in the CelόA terminator) and blunt-ended with T4 polymerase. The hph selection marker cassette was isolated as a 3.1 kb XhoVEcoRV fragment from pHPT136, blunt-ended, and cloned into the blunt-ended 5αcII site to make pCel6ApXt-hph-TV. The 2.2. kb pyr4 selection marker was isolated as a Kpnl fragment from pNcBgl (U.S. Patent No. 6,939,704), blunted and cloned into the blunted Sacll site to make pCel6ApXt-S413P-pyr4- TV (Figure 6A).

Table 4: Primers used for PCR amplification during construction of Trichoderma transformation vectors

Primer Hybridization site/ Sequence SEQ ID NO: direction

KW008 celόa terminator / Forward CGAGATCTTCGAGGGCGTAAC 73 KW052 celόa 3' flank/ Reverse GCTCACCCGGGAAGACCACATGGC 74

KW053 celόa 5' flank / Forward CCGTATAGTATCGCATGCAATTGC 75

KW054 celόa secretion signal / GCCGACAACCATGGTGCAATACACAG 76 Reverse AGGGTGA

KW055 xyn2 secretion signal / CATCACCATGGTCTCCTTCACCTCCCT 77 Forward CCTCGC

KW056 xyn2 secretion signal / CTTGAGCAGCTAGCCTGGCGCTTCTCC 78 Reverse ACAGCC

[00110] The final vector for T. reesei transformation was generated from two previously constructed CelόA targeting vectors - pCel6Apst-hph-TV and pCel6ApXt-hph-TV. Both vectors were digested with %/II and Sail restriction enzymes. The fragment from pCelόAXt- hph-TV vector containing CelόA coding sequence, terminator and hph cassette and the fragment from pCel6Apst-hph-TV vector containing celόa flanks and Amp^R gene were purified from agarose gel and ligated into pCel6A413pst-hph-BB vector (Figure 6B).

[00111] The W367G mutation into celόa gene was introduced by 3 step PCR ligation as described below. Two pairs of primers (Table 5) were used to amplify partial CelόA coding sequence and C-terminal CelόA coding sequence with partial celόa terminator. Both PCR products have short overlapping ends and were used in the 2^nd step, ten-cycle PCR reaction as templates and primers to anneal to each over and fill the missing strands at each end. Subsequently, two outside primers, CelόA-BEII-Fl and CelόA- Apa-R2, were added and entire fragment was amplified in standard 35 cycle PCR reaction. Amplified PCR product was digested with BsiEλl and Apal enzymes and ligated into corresponding sites of pCel6A413pst-hph-BB vector generating pCel6A413/367pst-hph-BB vector.

Table 5: Primers used for introduction of W367G mutation into Trichoderma transformation vector.

Primer Hybridization site/ Sequence SEQ ID NO: direction

CelόA-BEII- celόa 5 'end at BstΕll CCTGGTGACCAACCTCGGTAC 79

Fl site / forward

CelόA-367- celόa 3' end at 367 GTGGGGAGACGGGTGCAATGTG 80 R3 amino acid position / reverse

Cel6A-367- celόa 3' end at 367 CACATTGCACCCGTCTCCCCAC 81 F3 amino acid position / forward

Cel6A-Apa- celόa terminator at CCTCTGGGCCCCCAGATAAG 82 R2 Apal site / reverse c. Generation o/Trichoderma reesei strains expressing modified TrCelόA glycosidases by direct replacement of wild type celόa gene

[00112] To facilitate screening of T. reesei transformants which are targeted to celόa locus resulting in replacement of wild type celόa gene with modified CelόA protein encoding gene we generated host strain with tagged celόa locus.

[00113] The vector pCel6ApXt-S413P-pyr4-TV was transformed into BTR213aux28 T. reesei strain using PEG-mediated protoplast transformation method. About 5 x 10⁶ spores of BTR213aux28 were plated onto sterile cellophane placed on potato dextrose agar (PDA) (Difco) supplemented with 5 mM uridine and incubated for 20 h at 30⁰C. Cellophane discs with mycelia were transferred to 10 mL of a protoplast preparation solution containing 7.5 g/L Driselase and 4 g/L beta-glucanase (InterSpex Products Inc., Cat. # 0465-1 and 0439-2, respectively) in 50 mM potassium phosphate buffer, pH 6.5 containing 0.6 M ammonium sulfate (Buffer P). The mycelia were digested for 5 h at 28⁰C with gentle agitation at 60 rpm. Protoplasts were collected by centrifugation at 1000-1500 x g for 10 min at room temperature and washed with 5 mL of Buffer P. The pellet was resuspended in 1 mL of STC buffer (1.2 M sorbitol, 10 mM CaCl₂, 10 mM Tris-HCL, pH 7.5), separated from undigested mycelia by filtration through sterile No. 60 MIRACLOTH™ and collected into a sterile microcentrifuge tube. For transformation, 0.1 mL of protoplast suspension (approximately 5 x 10⁶ protoplasts) was combined with 10 μg of vector DNA, linearized with restriction enzyme BgIU, and 25 μl of PEG solution (25% PEG 4000, 50 mM CaCl₂, 10 mM Tris-HCl, pH 7.5). Protoplasts with DNA were incubated on ice for 30 min then 1 mL of PEG solution was added and the mixture incubated for 5 min at room temperature. Transformation mix was diluted with 2 mL of 1.2 M sorbitol in PEG solution and 4 aliquots of 0.75 mL of the mix were added into 25 mL of molten MMSS agar media (see below) cooled to about 47-5O⁰C and the protoplast suspensions were poured over MM agar (see below). Plates were incubated at 30⁰C until colony growth is visible. Transformants were transferred to individual plates containing MM agar and allowed to sporulate. Spores are collected and plated at high dilution on MM agar to isolate homokaryon transformants, which are then plated onto PDA and incubated at 3O⁰C for sporulation and subsequent genetic analysis. Minimal medium (MM*) agar contains:

Component Amount for 1 L of medium

KH^PO₄ 1O g

(NH₄)₂SO₄ 6 g

Na₃Citrate-2H?O 3 g

FeSO₄-7H₂O 5 mg

MnSO₄-H₂O 1 6 mg

ZnSO₄-7H₂O 1 4 mg

CaCl₂-2H₂O 2 mg

Agar 2O g

20% Glucose f s. 5O mL

1 M MgSO4-7H₂O f.s. 4 mL pH to 5.5

*MMSS agar contains the same components as MM agar plus 1.2 M sorbitol, 4 mM MgSO₄, 1 g/L YNB (Yeast Nitrogen Base w/o Amino Acids from DIFCO Cat. No.291940) and 0.12 g/L amino acids (-Ura DO Supplement from CLONTECH Cat. No.8601-1 ). [00114] Three stable T. reesei transformants were isolated and integration site of CelόA targeting cassette was characterized by Southern hybridization analysis. For genomic DNA extraction mitotically stable transformants, P577A, P577B and P577C, and the parental strains, BTR213 and BTR213aux28, were sporulated on PDA. Spores were inoculated in 100 mL of minimal media (MM) media and incubated at 30⁰C and 150 rpm for 5 days. Biomass was filtered using GF/A filter, transferred to aluminum foil and frozen immediately at -80⁰C for 24 hrs. Frozen biomass was grinded to a fine powder using liquid nitrogen and resuspended in 3 mL of extraction buffer (100 mM Tris pH 8.0, 50 mM EDTA pH 7.5, 1% SDS). Homogenate was transferred to a sterile 15 mL falcon tube and pelleted by cetrifugation at 4000 rpm for 5 min. Supernatant was transferred to a sterile 15 mL falcon tube, equal volume of saturated phenol (pH 6.6) was added and vortexed for 1 min. Aqueous phase containing DNA was separated by centrifugation for 5 min at 4000 rpm and transferred to fresh 15 mL falcon tube. Genomic DNA was further purified by adding an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mixing and separating aqueous phase by centrifugation for 5 min at 4000 rpm. This purification step was repeated until no interphase was visible. Phenol was removed by extracting with an equal volume of chloroform, mixing and separating aqueous phase by centrifugation. Genomic DNA was precipitated overnight at -2O⁰C using 0.1 X volume of 3M NaOAc pH 5.2 and 2.5X volume of 100% EtOH, then pelleted by centrifugation at 4000 rpm for 10-15 min. The pellet was washed once with 1 volume of 70% EtOH and once with 95% EtOH. After the pellet was air dried, the DNA was resuspended in 1 mL of TE buffer (Tris-HCl 10 mM; EDTA 1 mM; pH 8). To remove RNA, 5 μL of RNase A (10 mg/mL) was added and incubated at 37⁰C for 1 hour. RNase then was extracted with 1 volume of saturated phenol (pH 6.6) followed by 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) and 1 volume of chloroform. DNA was precipitated from separated aqueous phase with 0.1 volume of 3M NaOAc pH 5.2 and 2.5 volume of 100% EtOH, incubated at -20⁰C for 30 min, pelleted by centrifugation at 12000 rpm for 15 min and washed once with 1 volume of 70% EtOH and once with 95% EtOH. Finally, the DNA was resuspended in 0.2 mL of TE buffer and used for Southern hybridization as described below.

[00115] Southern blot using DIG labeling and detection system was performed as described in the Roche Applied Science manual. The restriction pattern at the wild type celόa locus expected after digestion of genomic DNA from BTR213 and BTR213aux28 was predicted using celόa sequence from JGI database URL: genome.jgi- psf.org/Trire2/Trire2/home.html) and expected to be detectable as 4.2 kb band specifically hybridizing with celόa probe (Fig. 7). In the event of ectopic vector integration resulting in presence of two copies of celόa gene in the genome, two specific bands would be observed. The targeting of CelόA vectors into celόa locus in transformants P577A, P577B, and P577C would result in a 6.4 kb fragment, as seen after EcoRl digestion of transformation vector (Figure 7) due to the presence of tye pyr4 selection cassette. As demonstrated in Figure 7, the Southern blot confirmed the integration of CelόA-marker cassettes into the native celόa locus and replacement of native CelδA coding sequence with coding sequence from the transformation vector.

d. Generation o/Trichoderma reesei transformants expressing Tr Cel6A-W367G-S413P

[00116] The vector pCel6A413/367pst-hph-BB was transformed into generated new T. reesei host strain, P577C, using PEG-mediated protoplast transformation as described above (Example 8c). The selection of transformants was performed using hygromycin resistance as a selectable marker. Aliquots (0.75 mL) of transformed protoplasts were added into 25 mL of PDA media cooled to about 47-50⁰C and the protoplast suspensions were poured into 200 mm Petri dishes. After the PDA media containing transformed protoplasts solidified, another 25 mL of PDA media supplemented with 8OLVmL of hygromycin B was added as a top agar. Plates were incubated at 3O⁰C until colony growth was visible. Transformants were transferred twice to individual plates containing PDA media supplemented with 40 LVmL of hygromycin B (PDAH) and allowed to sporulate. Spores were collected and plated at high dilution on PDAH to isolate homokaryon transformants, which were then plated onto PDA and incubated at 30⁰C for sporulation and subsequent analysis.

[001 17] Transformants possessing targeted vector integration into celόa locus were identified by their ability to grow in the presence of hygromycin and inability to grow on minimal media lacking uridine supplement. This indicated that the pyr4 selectable marker cassette present in P577C host strain was replaced with modified CelόA expression and hph selectable marker cassettes.

Example 9: Production of modified glycosidase from Trichoderma reesei

a. Production o/TrCel6A-W367G-S413P in T. reesei microcultures

[00118] To confirm expression of TrCel6A-W367G-S413P protein, all strains possessing targeted replacement of wild type celόa gene with TrCel6A-W367G-S413P coding gene were grown in microcultures for CelόA protein analysis.

[00119] T. reesei transformants and the parental strain BTR213aux28 were cultured on PDA plates supplemented with 5mM of uridine for 6-7 days at 3O⁰C. The spore suspensions were prepared by washing spores from the agar plate with sterile water. The composition of microculture media containing glucose with cellulase inducing carbohydrates as a carbon source is indicated below.

Trichoderma microculture media

Component Concentration g/L

Glucose with cellulase inducing 35 carbohydrates⁸

Ammonium sulphate 12.7

KH₂PO₄ 8.0

MgSO₄-7H2O 4.0

CaCl₂-2H₂O 1.0

FeSO₄-7H2O 0.1

MnSO₄-7H2O 0.032

ZnSO₄7H₂O 0.028

CaCO₃ 20

Corn Steep Liquor (powder) 5 pH4.24 aA cellulase- inducing cocktail comprising, as a function of total carbohydrate, 56% gentiobiose, 14% sophorose, 6% cellobiose, 10% trehalose, 6% maltotriose, 4% glucose and 14% other carbohydrates [00120] About 5000 T. reesei spores were inoculated in each well of 24-well culture dish (COSTAR) containing 1 mL of media. Plates were incubated for 5-7 days at 3O⁰C with shaking at 250 rpm.

[00121] The relative concentration of the TrCel6A-W367G-S413P produced by transformants was determined by ELISA (Example 4). The relative concentration of TrCel6A-W367G-S413P protein was calculated by dividing TrCel6A-W367G-S413P concentration by the total amount of protein produced, as determined using a Bradford protein assay. The expression levels of CelόA are presented in Figure 8.

b. Analysis of T. reesei transformants in 14L pilot fermentations

[00122] Two T. reesei transformants with the highest CelόA expression levels, strains P989B and P989B, were selected for 14L fed-batch pilot fermentation and enzyme analysis. Tήchoderma spores were inoculated onto standard 85 mm Petri plates containing potato dextrose agar (PDA). These plates were incubated at 28⁰C for 3-5 days to achieve a confluent growth of fresh green spores. To prepare the inoculum for fermentation, spores from a single PDA plate were transferred to 2 L, baffled Erlenmeyer flasks containing 750 mL of liquid Berkley media (pH 5.5) supplemented with 10 mM of uridine. Flasks were incubated at 28⁰C for 3 days using an orbital agitator (Model G-52 New Brunswick Scientific Co.) running at 100 rpm.

Berkley Media for Flasks

Component Concentration, g/L

(NH₄J₂SO₄ 1.4

KH₂PO₄ 2.0

MgSO₄-7H₂O 0.31

CaClr2H,O 0.53

Dry Corn Steep Liquor 5.1

Glucose 10

Trace elements* X mLIL *Trace elements solution contains 5 g/L FeSO₄ TH₂O; 1.6 g/L MnSO₄ H₂O; 1.4 g/L ZnSO₄-7H₂0.

[00123] The contents of an inoculum flask were transferred to a 14L pilot scale fermentation vessel (Model MFl 14 New Brunswick Scientific Co.) set up with 10 L of Initial Media for Feb-Batch fermentation (pH 5.5) supplemented with 10 mM of uridine. The vessel was run in batch mode until glucose in the media was depleted. At this point, the carbon source containing cellulase inducing carbohydrates (56% gentiobiose, 14% sophorose, 6% cellobiose, 10% trehalose, 6% maltotriose, 4% glucose and 14% other carbohydrates) was added, on a continuous basis, from a stock that was 35.5% w/v of solids dissolved in water. Peristaltic pumps were used to deliver the carbon source at a feed rate of 0.4 grams of carbon per liter culture per hour. Operational parameters during both the batch and fed-batch portions of the run were: mixing by impeller agitation at 500 rpm, air sparging at 8 standard liters per minute, and a temperature of 28°C. Culture pH was maintained at 4.0-4.5 during batch growth and pH 3.5 during cellulase production using an automated controller connected to an online pH probe and a pump enabling the addition of a 10% ammonium hydroxide solution. Periodically, 100 mL samples of broth were drawn for biomass and protein analysis. After 96 hours of fermentation time IL of fermentation media was collected and filtered for further protein analysis.

Initial Media for Fed-Batch Fermentations

Component Concentration, g/L

(NH₄)₂SO₄ 2.20

KH₇PO₄ 1.39

MgSO₄ TH₂O 0.70

CaClv2H₂O 0.185

Dry Corn Steep Liquor 6.00

Glucose 13.00

Trace elements* 0.38 mL/L

*Trace elements solution contains 5 g/L FeSO₄7H₂O; 1.6 g/L MnSO₄ H₂O; 1.4 g/L ZnSO₄ TH₂O.

Example 10: Hydrolysis of beta-glucan by T. reesei enzyme mixtures comprising parental and modified TrCelόA glycosidases.

[00124] Testing was performed on a Legacy Barley varietal from northern Saskatchewan. Solids 89.6%, -60% starch, 10- 14% NSP (non-starch polysaccharides).

[00125] Grain samples were ground to pass a 20 mesh screen using a Wiley Mill. Total carbohydrates were determined through acid hydrolysis and ion chromatography on a DX- 500 system with PAl column and amperometric detection. Total carbohydrates minus total starch was used to determine quantity of non-starch polysaccharides in the substrate in order to determine starting enzyme dose. Solids determination was used to correct for sample dry weights in all experiments.

[00126] Viscosity reduction by parental and modified Family 6 glycosidases was deteremined using a Perten SuperRVA4 can and paddle assembly, fixed retention time of 15 min, a 30 mL sample size at 35% solids, 50 mM citrate buffer, pH 4.5, and a temperature of 52°C. An initial sec mix at 900 rpm was followed by data collection at 4 sec intervals at 160 rpm. Data were collected in centepoise units (cP)

[00127] Samples were treated with dilute enzyme solutions of 1 mL based on a weight of protein per metric tonne of substrate. Viscosity reduction was calculated as a change from control over the last 1 minute of data collection. The results are presented in Tables 6 and 7.

[00128] A much greater reduction in viscosity of the barley beta-glucan substrate is achieved by the modified Family 6 glycosidase TrCel6A-W367G-S413P effects than by the wild type Family 6 glycosidase TrCelόA both when the Family 6 glycosidase is acting alone (Table 6) or in combination with other cellulases and hemicellulases (Table 7).

Table 6: Reduction of Barley beta-glucan viscosity by wild-type and modified Family 6 glycosidases

Table 7: Reduction of Barley beta-glucan viscosity by cellulase-hemicellulase mixtures comprising wild-type and modified Family 6 glycosidases

¹ Ultimase XTP is a commercial Trichoderma reesei whole cellulase with an enriched thermostable xylanase II component. REFERENCES

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS.

1. A modified Family 6 glycosidase comprising one or more amino acid substitution selected from the group consisting of N182X, W367X, E399X, C/S400X, and A427X, the position determined from alignment of a parental Family 6 glycosidase amino acid sequence with a Trichoderma reesei CelόA amino acid sequence as defined in SEQ ID NO: 1, wherein amino acids 83-447 (TrCelόA numbering) of the modified Family 6 glycosidase are from about 47% to about 99.9% identical to amino acids 83-447 of SEQ ID NO: 1.

2. A modified Family 6 glycosidase comprising one or more amino acid substitution selected from the group consisting of N182X, W367X, E399X, C/S400X, and A427X, the position determined from alignment of a parental Family 6 glycosidase amino acid sequence with a Trichoderma reesei CelόA amino acid sequence as defined in SEQ ID

NO: 1, wherein amino acids 83-447 (TrCelόA numbering) of the modified Family 6 glycosidase are from about 70% to about 99.9% identical to amino acids 83-447 (TrCelόA numbering) of any one of SEQ ID NO: 1 through SEQ ID NO: 36.

3. The modified Family 6 glycosidase of claim 1, wherein amino acids 83-447 (TrCelόA numbering) of the modified Family 6 glycosidase are from about 70% to about 99.9% identical to amino acids 83-447 of SEQ ID NO: 1 and wherein the modified Family 6 glycosidase exhibits at least a 1.2-fold increase in hydrolysis activity of beta 1-3, 1-4 - linked polysaccharides and at least a three- fold decrease in hydrolysis activity of beta 1-4 linked polysaccharides, compared with a parental Family 6 glycosidase from which the modified Family 6 glycosidase is derived.

4. The modified Family 6 glycosidase of claim 2, wherein amino acids 83-447 (TrCelόA numbering) of the modified Family 6 glycosidase are from about 80% to about 99.9% identical to amino acids 83-447 (TrCleόA numbering) of any one of SEQ ID NO: 1 through SEQ ID NO: 36 and wherein the modified Family 6 glycosidase exhibits at least a 1.2-fold increase in hydrolysis activity of beta 1-3, 1-4 -linked polysaccharides and at least a three-fold decrease in hydrolysis activity of beta 1-4 linked polysaccharides, compared with the hydrolysis activity of a parental Family 6 glycosidase from which the modified Family 6 glycosidase is derived.

5. The modified Family 6 glycosidase of any one of claim 3 or 4, wherein the modified Family 6 glycosidase exhibits at least a 1.2-fold decrease in hydrolysis activity of beta 1-

4-linked polysaccharides compared with the hydrolysis activity of a parental Family 6 glycosidase from which the modified Family 6 glycosidase is derived.

6. The modified Family 6 glycosidase of claim 3, wherein amino acids 83-447 (TrCelόA numbering) are from about 90% to about 99.9% identical to amino acids 83-447 of SEQ

ID NO: 1.

7. The modified Family 6 glycosidase of claim 4, wherein amino acids 83-447 (TrCelόA numbering) are from about 95% to about 99.9% identical to amino acids 83-447 (TrCelόA numbering) of any one of SEQ ID NO: 1 through SEQ ID NO: 36.

8. The modified Family 6 glycosidase of any one of claims 1 to 7, wherein the amino acid substitutions are selected from the group consisting of N182S, N182R, N182G, N182A, W367A, W367C, W367G, W367N, W367R, W367S, W367T, W367V, E399H, E399S, E399T, C400V, C400M, C400T, C400S, A427V, A427L, and A427S.

9. An isolated genetic construct comprising a nucleic acid sequence encoding the modified Family 6 glycosidase of any one of claims 1 through 8.

10. An isolated genetically modified microbe comprising the genetic construct of claim 9.

11. The isolated genetically modified microbe of claim 10, wherein the microbe is a species of yeast or filamentous fungus.

12. The isolated genetically modified microbe of claim 11, wherein the microbe is Saccharomyces cerevisiae or Trichoderma reesei.

13. A process for producing a modified Family 6 glycosidase comprising the steps of growing the genetically modified microbe of claim 10 in a culture medium under conditions that induce the expression and secretion of the modified Family 6 glycosidase and recovering the modified Family 6 glycosidase from the culture medium.

14. A process for hydrolyzing a beta-1, 3-1,4-linked polysaccharide substrate comprising contacting the substrate with the modified Family 6 glycosidase of any one of claims 1 to 8.

15. The process of claim 14, wherein the beta-1, 3-1,4-linked polysaccharide substrate is a constituent of a cereal grain.

16. The process of claim 15, wherein the process is part of an industrial process to produce alcohol, animal feed or food products.

17. A process for producing a modified Family 6 glycosidase, comprising the steps of (i) transforming fungal host cells with a genetic construct as defined in claim 9 to produce recombinant fungal strains; (ii) selecting the recombinant fungal strains expressing the modified Family 6 glycosidase; and (iii) culturing selected recombinant strains in submerged liquid fermentations under conditions that induce the expression of the modified Family 6 glycosidase.

18. A modified Family 6 glycosidase selected from the group consisting of: