# Patent

## Title
Carbohydrate binding modules with reduced binding to lignin

## Abstract

Provided is a modified Family 1 carbohydrate binding module (CBM) comprising amino acid substitutions at one or more of positions 10, 11, 12, 14, 17, 21, 24, 29, 31, 33, and 37, said position determined from alignment of a Family 1 CBM amino acid sequence with SEQ ID NO: 30, and exhibiting from about 50% to about 99.9% amino acid sequence identity to SEQ ID NO: 30. Also provided are modified glycosidase enzymes comprising the modified Family 1 CBM, genetic constructs and genetically modified microbes for expressing the modified Family 1 CBM or modified glycosidase enzyme. The modified Family 1 CBM confers reduced lignin binding and/or increased hydrolyzing activity in the presence of lignin to the modified glycosidase enzyme, which may be used in a process for hydrolyzing cellulose or hemicellulose in the presence of lignin.

## Background
This application is a continuation application of U.S. patent application Ser. No. 13/575,046, filed on Jul. 25, 2012, now U.S. Pat. No. 9,206,406, which is a national stage entry of International Application No. PCT/CA2011/000167, filed Feb. 11, 2011, which claims the benefit of U.S. Provisional Patent Application No. 61/303,400 filed Feb. 11, 2010. The contents of the aforementioned applications are incorporated herein by reference in their entireties.


FIELD OF THE INVENTION
The present invention relates to modified carbohydrate binding modules. More specifically, the invention relates to a modified Family 1 carbohydrate binding module exhibiting reduced binding to lignin. The present invention also relates to modified glycosidase enzymes comprising the modified Family 1 carbohydrate binding module, genetic constructs comprising nucleotide sequences encoding the modified Family 1 carbohydrate binding modules or the modified glycosidase enzyme, and the use of the modified glycosidase enzyme comprising the modified Family 1 carbohydrate binding module in the hydrolysis of cellulose or hemicellulose substrates in the presence of lignin.
BACKGROUND OF THE INVENTION
More than 50% of organic carbon on earth is found in the cell walls of plants. Plant cell walls consist mainly of the compounds: cellulose, hemicellulose, and lignin. Collectively these compounds are called “lignocellulose,” and they represent a potential source of sugars and other organic molecules for fermentation to ethanol or to other high-value products.
The conversion of lignocellulose (or lignocellulosic biomass) to ethanol has become a key feature of emerging energy policies due to the environmentally favorable and sustainable nature of cellulosic ethanol. There are several technologies being developed for cellulose conversion. Of interest here is a method by which lignocellulosic biomass is subjected to a pretreatment that increases its susceptibility to hydrolytic enzymes, followed by enzymatic hydrolysis of the pretreated lignocellulose to sugars and the fermentation of those sugars to ethanol or other high-value organic molecules (e.g. butanol). Common pretreatment methods include dilute acid steam explosion (U.S. Pat. No. 4,461,648), ammonia freeze explosion (AFEX; Holtzapple et al., 1991), and organosolv extraction (U.S. Pat. No. 4,409,032). Hydrolysis and fermentation systems may be either separate (sequential hydrolysis and fermentation; SHF) or coincident (simultaneous saccharification and fermentation; SSF). In all instances, the hemicellulose and cellulose are broken down to sugars that may be fermented, while the lignin becomes separated and may be used either as a solid fuel or as a source for other organic molecules.
The enzymatic hydrolysis of the pretreated lignocellulose is carried out by cellulase enzymes. The term cellulase (or cellulase enzymes) broadly refers to a class of glycoside hydrolase enzymes (or glycosidases) that catalyze the hydrolysis of the beta-1,4-glucosidic bonds joining individual glucose units in the cellulose polymer. The catalytic mechanism involves the synergistic actions of endoglucanases (Enzyme Commission number E.C. 3.2.1.4), cellobiohydrolases (E.C. 3.2.1.91) and beta-glucosidase (E.C. 3.2.1.21). Endoglucanases hydrolyze accessible glucosidic bonds in the middle of the cellulose chain, while cellobiohydrolases release cellobiose from these chain ends processively. Beta-glucosidases hydrolyze cellobiose to glucose and, in so doing, minimize product inhibition of the cellobiohydrolases. Collectively, the enzymes operate as a system that can hydrolyze a cellulose substrate.
Cellulase enzymes, as well as other glycoside hydrolases or glycosidases that hydrolyze poly- or oligo-saccharides, typically have a similar modular structure, consisting of one or more catalytic domain(s) and one or more carbohydrate-binding modules (CBM) joined together by flexible linker peptide(s). Many hemicellulases, e.g., xylanases (E.C. 3.2.1.8), mannanases (E.C. 3.2.1.78) and arabinofuranosidases (E.C. 3.2.1.55), are known to have a similar modular structure of a catalytic domain joined to a CBM via a flexible linker. Hemicellulases are enzymes that catalyze hydrolysis of the glycosidic linkages in the xylan backbone polysaccharide of hemicellulose or glycosidic linkages between xylose units in the xylan backbone and other sugars attached to the backbone.
The catalytic domain is a distinct structural domain that catalyzes the hydrolysis of the glycosidic linkages in the substrate. Many glycoside hydrolase catalytic domains have been isolated and characterized. The catalytic domain is typically, though not necessarily, the larger of the two domains. Glycoside hydrolases sharing a common three-dimensional structure and catalytic mechanism, though not necessarily substrate specificity, have been grouped into Families (Davies and Henrissat, 1995). To date, there are over 150 Glycoside Hydrolase (GH) families. Cellulase enzymes are found in many GH families including, but not limited to, Family 5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 61 and 74; xylanase enzymes are found in Family 5, 8, 10, 11 and 43; mannanase enzyme are found in Family 5, 26 and 113; arabinofuranosidase enzymes are found in Family 3, 43, 51, 54 and 62; and beta-glucosidase enzymes are found in Family 1 and 3.
Linker peptides are extended yet flexible structures that maintain the spatial orientation of the catalytic domain relative to the CBM (Shen et al., 1991; Receveur et al., 2002; Boisset et al., 1995). Naturally-occurring linker peptides in cellulase and hemicellulase enzymes, whether from bacterial or fungal sources, vary from 6-60 amino acids in length. These peptides are similar in their chemical properties and amino acid composition, if not their specific sequences, with the amino acids serine, threonine, and proline accounting for more than 50% of the amino acids in the linker peptide (reviewed in Gilkes et al. (1991). Linkers also contain several charged residues of a common type, either all negative (such as Glu or Asp) or all positive (such as Lys, Arg or His). The serine and threonine residues may be modified with O-linked glycans, which, in fungi, are predominantly mannose (Fagerstam et al., 1984). Results from small-angle x-ray or dynamic light scattering suggest that glycosylation of the linker peptide favours a more extended conformation, altering the relative positioning of the catalytic domain and CBM.
The carbohydrate binding module (CBM) is typically, though not always, smaller than the catalytic domain. The role of the CBM is to bring the enzyme into close and prolonged contact with the carbohydrate substrate and to increase the rate of substrate degradation. CBMs are found in a variety of enzymes involved in the degradation of carbohydrate substrates, including cellulases, hemicellulases, glucanases, amylases, glucoamylases, chitinases and the like. Thus, CBMs can recognize and bind to crystalline cellulose, non-crystalline cellulose, chitin, beta-1,3 glucans, mixed beta-1,3-1,4 glucans, xylan, mannan, galactan, and starch.
As is the case for catalytic domains, CBMs assume a variety of structures that govern their substrate binding affinities and can therefore also be classified into Families based on their structural and functional relationships. To date there are 59 known CBM Families (see URL cazy.org/fam/acc_CDM.html). Much research has been conducted over the past two decades to elucidate the function and structure of CBMs (as reviewed by Boraston et al., 2004; Hashimoto 2006 and Shoseyov et al., 2006).
The present application relates to Family 1 CBMs. These CBMs are found almost exclusively in fungal enzymes, including cellulase and hemicellulase enzymes produced by Trichoderma ssp., Aspergillus ssp., Hypocrea ssp., Humicola ssp., Neurospora ssp., Orpinomyces ssp., Gibberella ssp., Emericella ssp., Chaetomium ssp., Chrysosporium ssp., Fusarium ssp., Penicillium ssp., Magnaporthe ssp., Phanerochaete ssp., Trametes ssp., Lentinula edodes, Gleophyllum trabeiu, Ophiostoma piliferum, Corpinus cinereus, Geomyces pannorum, Cryptococcus laurentii, Aureobasidium pullulans, Amorphotheca resinae, Leucosporidium scotti, Cunninghamella elegans, Thermomyces lanuginosa, Sporotrichum thermophile, and Myceliophthora thermophilum. 
Family 1 CBMs were initially identified as cellulose binding domains (or CBDs) of fungal cellulases. Family 1 CBMs comprise approximately 40 amino acids and may be found at either the N- or C-terminus of the enzyme. Family 1 CBMs assume a small, wedge-shaped beta-sandwich structure with a flat binding surface containing three aromatic amino acids (usually tryptophan) spaced about 10 angstroms apart (Kraulis et al., 1989; Mattinen et al., 1997). These aromatic residues facilitate binding to the surfaces of crystalline substrates such as cellulose and chitin via van der Waal's contacts with the substrate surface (Mattinen et al., 1997; Reinikainen et al., 1992, Tormo et al., 1996).
The enzymatic hydrolysis of pretreated lignocellulosic feedstocks is an inefficient step in the production of cellulosic ethanol and its cost constitutes one of the major barriers to commercial viability. Improving the enzymatic activity of cellulases or increasing cellulase production efficiency has been widely regarded as an opportunity for significant cost savings.
The negative effects of lignin on cellulase enzyme systems are well documented. Removal of lignin from hardwood (aspen) was shown to increase sugar yield by enzymatic hydrolysis (Kong et al., 1992). Similarly, removal of lignin from softwood (Douglas fir) was shown to improve enzymatic hydrolysis of the cellulose, an effect attributed to improved accessibility of the enzymes to the cellulose (Mooney et al., 1998). Other groups have demonstrated that cellulases purified from Trichoderma reesei bind to isolated lignin (Chernoglazov et al., 1988) and have speculated on the role of the different binding domains in the enzyme-lignin interaction (Palonen et al., 2004). Binding to lignin and inactivation of Trichoderma reesei cellulases has been observed when lignin is added back to a pure cellulose system (Escoffier et al., 1991). Another study showed that lignin did not have any significant effect on cellulases (Meunier-Goddik and Penner, 1999). While other reports suggest that some hemicellulases may be resistant to, and even activated by, lignin and lignin breakdown products (Kaya et al., 2000). Nonetheless, it is generally recognized that lignin is a serious limitation to enzymatic hydrolysis of cellulose.
Cellulases purified from Trichoderma reesei have been shown to bind to isolated lignin (Chernoglazov et al., 1988). Further work has shown that all three domains, catalytic core, linker and CBM, will bind to lignin (Palonen et al., 2004). For example, Cel7B from Humicola sp., which exists naturally as just a catalytic domain without a CBM, is bound extensively by lignin (Berlin et al., 2005). Similarly Trichoderma Cel5A core, devoid of a CBM, does not bind enzymic lignin and binds alkali extracted lignin to a lesser extent than does the full-length protein (Palonen et al., 2004). CBMs are reportedly involved in lignin binding. For example, removal of the CBM from Trichoderma Cel7A essentially eliminates binding to alkali extracted lignin and to residual lignin prepared by enzyme hydrolysis (Palonen et al., 2004).
The absence of lignin resistant cellulases represents a large hurdle in the commercialization of cellulose conversion to soluble sugars including glucose for the production of ethanol and other products. The development of lignin resistant enzymes must preserve their cellulolytic activity. A variety of methods have been suggested to reduce the negative impact of lignin on the cellulase system. Non-specific binding proteins (e.g. bovine serum albumin; BSA) have been shown to block interactions between cellulases and lignin surfaces (Yang and Wyman, 2006; U.S. Publication No. 2004/0185542A1, U.S. Publication No. 2006/0088922A1; WO05024037 A2, A3; WO09429474 A1). Other chemical blocking agents and surfactants have been shown to have a similar effect (Tu et al., 2007; U.S. Pat. No. 7,354,743).
Modified glycosidase enzymes and methods for modification have been extensively described. In most instances, mutations are specifically directed to the catalytic domain of the enzyme. For example, variants of Trichoderma reesei Cel7A and Cel6A catalytic domains to improve thermostability have been reported (U.S. Pat. No. 7,375,197; WO 2005/028636; U. S. Publication No. 2007/0173431; Publication No. 2008/167214; WO 2006/074005; Publication No. 2006/0205042; U.S. Pat. No. 7,348,168; WO 2008/025164). In particular, substitution of the amino acid at the equivalent of position 413 in T. reesei Cel6A with a proline in Family 6 cellulases, e.g., a S407P mutation in the Phanerochaete chrysosporium Cel6A, confers increased thermostability (WO 2008/025164). Mutations at the equivalent of positions 103, 136, 186, 365 and 410 within the catalytic domain of T. reesei Cel6A and other Family 6 cellulases have been shown to lead to reduce inhibition by glucose (U.S. Publication No. 2009/0186381A1). Variants with resistance to proteases and to surfactants for detergent formulations have been created for textile applications (WO 99/01544; WO 94/07998; and U.S. Pat. No. 6,114,296).
Recently, modified cellulases exhibiting reduced interactions with, or reduced inactivation by, lignin have been reported. For example, WO2010/012102 reports that mutations at the equivalent of positions 129, 322, 363, 365 and 410 within the catalytic domain of T. reesei Cel6A (TrCel6A) and other Family 6 cellulases results in increased hydrolytic activity in the presence of lignin. Similarly, WO2009/149202 discloses cellulase variants with mutations that remove positive charges or introduce negative charges at the equivalents of positions 63, 77, 129, 147, 153, 161, 194, 197, 203, 237, 247, 254, 281, 285, 289, 294, 327, 339, 344, 356, 378 and 382 in the linker peptide and catalytic domain of Cel6A from Hypocrea jecorina. Such cellulase variants show reduced affinity to lignin, ethanol or heat treatment.
Only in a few instances has the linker peptide been identified as playing a critical role or as a target for modification. The linker peptide of the Humicola Family 45 endoglucanase was modified to reduce proteolysis (WO 94/07998; U.S. Pat. No. 6,114,296) and the linker peptide of the Trichoderma Cel7A was modified to promote thermostability (U.S. Pat. No. 7,375,197). U.S. Publication No. 2010/0221778A1 reports that mutations that reduce the isoelectric point and/or increase the Ser/Thr ratio of the linker peptide can also lead to increased hydrolytic activity in the presence of lignin.
There are relatively few reports of modifying CBMs. In one instance, Linder et al. (1995) showed that mutations of the tyrosine residues on the binding face of the Family 1 CBM from T. reesei Cel7A significant reduce its binding to cellulose while mutations at other highly conserved, but non-aromatic, amino acids on the binding surface result in less of a reduction of cellulose binding. In another instance, it was reported that substitution of the tyrosine residue at the “tip” of the wedge-shape structure, equivalent to Tyr33 in the TrCel6A-CBM to a histidine resulted in pH-dependent binding to cellulose (Linder et al., 1999). However, while it has been observed that Family 1 CBMs interact with lignin, there are no reports on the development of modified Family 1 CBMs with reduced binding to lignin.
SUMMARY OF THE INVENTION
The present invention relates to modified carbohydrate binding modules. More specifically, the invention relates to a modified Family 1 carbohydrate binding module exhibiting reduced binding to lignin. The present invention also relates to modified glycosidase enzymes comprising the modified Family 1 carbohydrate binding module, genetic constructs comprising nucleotide sequences encoding the modified Family 1 carbohydrate binding modules or the modified glycosidase enzyme, and the use of the modified glycosidase enzyme comprising the modified Family 1 carbohydrate binding module in the hydrolysis of cellulose or hemicellulose substrates in the presence of lignin.
The present invention provides a modified Family 1 carbohydrate binding module with reduced binding to lignin and the ability to confer not only reduced lignin binding but also increased substrate hydrolyzing activity, in the presence of lignin, to a modified glycosidase enzyme comprising the modified Family 1 carbohydrate binding module. Such modified glycosidase enzymes may also be more easily recovered and reused from any residual lignin present at the end of the hydrolysis reaction.
The present invention also relates to a modified Family 1 carbohydrate binding module comprising amino acid substitutions at one or more one positions selected from the group consisting of 10, 11, 12, 14, 17, 21, 24, 29, 31, 33, and 37. The one or more positions containing amino acid substitutions is determined from alignment of a Family 1 carbohydrate binding module amino acid sequence with a Trichoderma reesei Cel6A carbohydrate binding module (TrCel6A-CBM) amino acid sequence as defined in SEQ ID NO: 30. The modified Family 1 carbohydrate binding module of the present invention comprises an amino acid sequence that is from about 50% to about 99.9% identical to SEQ ID NO: 30 and has the ability to bind to crystalline cellulose. For example, the modified Family 1 carbohydrate binding module of the present invention comprises an amino acid sequence that is from about 60% to about 99.9% identical to SEQ ID NO: 30 or is from about 75% to about 99.9% identical to SEQ ID NO: 30.
In an alternate embodiment, the modified Family 1 carbohydrate binding module of the present invention comprises substitution of a basic or charge-neutral amino acid at one or more positions selected from the group consisting of 11, 12, 14, 17, 24, 29, and 31 to an acidic amino acid and exhibits from about 50% to about 99.9% identity to SEQ ID NO: 30 as well as the ability to bind to crystalline cellulose. For example, the amino acid at one or more of positions 11, 12, 14, 17, 24, 29, and 31 is substituted by an aspartic acid.
And furthermore, the modified Family 1 carbohydrate binding module of the present invention comprises amino acid substitutions at one or more positions selected from the group consisting of 10, 21, 33 and 37 and exhibits from about 50% to about 99.9% identity to SEQ ID NO: 30 as well as the ability to bind to crystalline cellulose. For example, the amino acid substitution at position 10 is to serine, the amino acid substitution at position 21 is to an aromatic amino acid such as tyrosine, the amino acid substitution at position 33 is to asparagine, and the amino acid substitution at position 37 is to an aromatic amino acid such as tyrosine.
The present invention also relates to a modified glycosidase enzyme comprising one or more catalytic domain and one or more carbohydrate binding module, at least one of the one or more carbohydrate binding module(s) being a modified Family 1 carbohydrate binding module as defined above, functionally joined by one or more linker peptides. The modified glycosidase enzyme exhibits an increase in hydrolyzing activity in the presence of lignin and/or a reduction in lignin binding relative to a parental glycosidase comprising a parental Family 1 carbohydrate binding module from which the modified carbohydrate binding module is derived, the same one or more one or more catalytic domain and the same one or more carbohydrate binding module joined by the same one or more linker peptide.
The one or more catalytic domain in the modified glycosidase of the present invention may be a cellulase catalytic domain, a hemicellulase catalytic domain, a beta-glucosidase catalytic domain and an accessory component catalytic domain. The one or more catalytic domain in the modified glycosidase of the present invention may be a wild-type catalytic domain or a modified catalytic domain comprising amino acid substitutions, insertions or deletions relative to a wild-type catalytic domain.
The one or more catalytic domain in the modified glycosidase of the present invention may be a cellulase catalytic domain of Glycoside Hydrolase Family 5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 61 and 74. For example, the cellulase catalytic domain may comprise amino acids 1-436 of Trichoderma reesei Cel7A (SEQ ID NO: 124), amino acids 83-447 of Trichoderma reesei Cel6A (SEQ ID NO: 1), amino acids 97-460 of Humicola insolens Avi2 (SEQ ID NO: 2), or amino acids 81-440 of Phanerochaete chrysosporium Cel6A (SEQ ID NO: 3). For example, the cellulase catalytic domain may comprise amino acids 83-447 of Trichoderma reesei Cel6A (TrCel6A as in SEQ ID NO: 1) with one or more amino acid substitutions selected from the group consisting of Y103H, Y103K, Y103R, Y103A, Y103V, Y103L, Y103P, K129E L136V, L1361, S186K, S186T, S186Y, Q204K, G2131D, A322D, Q363E, G365D, G365E, G365Q, G365S, R410A, R410F, R410L, R410Q, R410S and S413P.
The one or more catalytic domain in the modified glycosidase of the present invention may also be a hemicellulase catalytic domain from Glycoside Hydrolase Family 5, 8, 10, 11, 26, 43, 51, 54, 62 or 113, a beta-glucosidase catalytic domain from Glycoside Hydrolase Family 1 or 3, or an accessory component catalytic domain such as a swollenin, CIP or expansin catalytic domain. For example, a beta-glucosidase catalytic domain may be Trichoderma reesei Cel3A of SEQ ID No: 100 with one or more amino acid substitutions selected from the group consisting of V43X, V66X, S72X, V101X, T235X, N248X, F260X, N369X, A386X, and I543X.
The one or more carbohydrate binding module, other than the modified Family 1 carbohydrate binding module, in the modified glycosidase of the present invention may be a wild-type carbohydrate binding module or a modified carbohydrate binding module comprising amino acid substitutions, insertions or deletions relative to a wild-type catalytic domain. Similarly, the one or more linker peptide in the modified glycosidase of the present invention may be a wild-type linker peptide or a modified linker peptide comprising amino acid substitutions, insertions or deletions relative to a wild-type linker peptide. For example, the one or more linker peptide may be a modified linker peptide being about 6 to about 60 amino acids in length, with least about 50% of the amino acids being either proline, serine or threonine and comprising one or more amino acid substitutions, insertions, or deletions that result in a decrease in the calculated isoelectric point of the linker peptide and/or an increase in the ratio of threonine:serine in the linker peptide relative to a parental linker peptide from which the modified linker peptide is derived. Such a modified linker peptide confers to the modified glycosidase an increase in hydrolyzing activity in the presence of lignin and/or a reduction in lignin binding relative to a parental glycosidase comprising the parental linker peptide.
Any one or all of the modified Family 1 carbohydrate binding module, catalytic domain, other carbohydrate binding module or linker peptide may be derived from one or more fungal glycosidase enzymes produced by such organisms including, but not limited to, Trichoderma ssp., Aspergillus ssp., Hypocrea ssp., Humicola ssp., Neurospora ssp., Orpinomyces ssp., Gibberella ssp., Emericella ssp., Chaetomium ssp., Chrysosporium ssp., Fusarium ssp., Penicillium ssp., Magnaporthe ssp., Phanerochaete ssp., Trametes ssp., Lentinula elodes, Gleophyllum trabeiu, Ophiostoma piliferum, Corpinus cinereus, Geomyces pannorum, Cryptococcus laurentii, Aureobasidium pullulans, Amorphotheca resinae, Leucosporidium scotti, Cunninghamella elegans, Thermomyces lanuginosus, Myceliophthora thermophila and Sporotrichum thermophile. 
The present invention further relates to genetic constructs comprising nucleic acid sequences encoding the modified Family 1 carbohydrate binding module or modified glycosidase enzyme as described above and to genetically modified microbes comprising such genetic constructs for the expression and secretion of the Family 1 carbohydrate binding module or modified glycosidase enzyme. The genetically modified microbe may be a bacterium, yeast or filamentous fungus, such as a species of Streptomyces, Saccharomyces, Pichia, Hansenula, Hypocrea, Trichoderma, Aspergillus, Fusarium, Neurospora, Chrysoporium or Myceliophthora. 
The present invention also relates to a process for producing the modified Family 1 carbohydrate binding module or modified glycosidase enzyme as described above comprising the steps of growing the genetically modified microbe comprising a genetic construct encoding the modified Family 1 carbohydrate binding module or modified glycosidase enzyme under conditions that induce the expression and secretion of the modified Family 1 carbohydrate binding module or modified glycosidase enzyme and recovering the modified Family 1 carbohydrate binding module or modified glycosidase enzyme from the culture medium. Such process for producing the modified Family 1 carbohydrate binding module or modified glycosidase enzyme as described above may include a step of transforming a host cell with a genetic construct encoding the modified cellulase enzyme.
The present invention also relates to a process for hydrolyzing a cellulose or hemicellulose substrate to sugars comprising contacting the substrate with the modified glycosidase as described above. In one embodiment of such a process, the cellulose or hemicellulose substrate may be a pretreated lignocellulosic substrate. In another embodiment of such a process, the modified glycosidase enzyme exhibits improved recovery from the process relative to a parental glycosidase enzyme comprising the same one or more catalytic domain, one or more linker peptide and one or more carbohydrate binding module in which at least one of the one or more carbohydrate binding module is a parental Family 1 carbohydrate binding module from which the modified Family 1 carbohydrate binding module in the modified glycosidase is derived.
The process for hydrolyzing a cellulose or hemicellulose substrate to sugars may be conducted as a continuous, semi-continuous or fed-batch process. In addition, the process for hydrolyzing a cellulose or hemicellulose substrate to sugars may be followed by microbial fermentation of the sugars to alcohol or sugar alcohol.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 contains the SDS-PAGE and Western blot analysis of the purified cellulase components from Trichoderma reesei. Panel A shows Coomassie Blue stain of purified Cel7A, Cel6A, Cel7B and Cel5A after SDS-PAGE. A Trichoderma cellulase mixture was analyzed in parallel for comparison. Panel B shows component-specific Western blots (as indicated in the lower left or lower right corner of each blot) of these samples performed following SDS-PAGE separation and electro-transfer to a PVDF membrane.
FIG. 2 shows the effects of lignin on T. reesei Cel7A with and without a CBM (Cel7A and Cel7Acore). Panel A shows the loss of T. reesei Cel7A protein and Cel7A activity and Panel B shows the loss of Cel7Acore protein and Cel7Acore activity in the presence of lignin at 50° C. Cel7A and papain-treated Cel7A (Cel7Acore) were incubated with acid extracted lignin for up to 96 h. The concentrations Cel7A and Cel7Acore protein in the supernatant, free from lignin, were measured in samples taken at different times throughout the experiment. Residual Cel7A and Cel7Acore activities on pretreated wheat straw were measured in the lignin slurry over time.
FIG. 3 shows that adding the CBM from T. reesei Cel7A to T. reesei Cel3A increased lignin-binding and lignin-associated inactivation of Cel3A. Cel3A (Panel A) and Cel3A-CBM (Panel B) were incubated with acid extracted lignin for up to 96 h at 50° C. The concentrations of these proteins in their respective supernatants, free from lignin, were measured in samples taken at different times throughout the experiment. Their residual activities were also measured in their respective lignin slurries over time.
FIG. 4 shows the effects of lignin on T. reesei Cel6A with and without a CBM (Cel6A and Cel6Acore). Panel A shows compares the loss of Cel6A protein and Ce6A activity and Panel B shows loss of Cel6Acore protein and Cel6Acore activity in the presence of lignin at 30° C. Cel6A (Panel A) and Cel6Acore produced by papain treatment (Panel B) were incubated with acid extracted lignin for up to 96 h at 30° C. The concentrations of these proteins in their respective supernatants and their residual activities on pretreated wheat straw were measured over time.
FIG. 5 depicts plasmid vectors YEp352/PGK91-1-αss-NKE-HiAvi2 (Panel A) and YEp352/PGK91-1-αss-NKE-PcCel6A-S407P (Panel B) directing the expression and secretion of native and modified HiAvi2 and PcCel6A from recombinant Saccharomyces cerevisiae, respectively.
FIG. 6 depicts plasmid vector YEp352/PGK91-1ΔNheI-αss-TrCel6A-S413P directing the expression and secretion of parental and modified TrCel6A from recombinant Saccharomyces cerevisiae. 
FIG. 7 contains two scatter plots. Panel A is a scatter plot of enzyme activity in the presence of BSA-treated lignin (+BSA activity) versus enzyme activity in the presence of untreated lignin (−BSA activity) for the high-throughput assay described in Example 10b. The data relate to the screening of filtrates from micro plate cultures (Example 9) containing parental and modified HiAvi2 cellulases or filtrates from empty vector transformants. Panel B is a scatter plot of enzyme activity in the presence of BSA-treated lignin (+BSA) activity versus enzyme activity in the presence of untreated lignin (−BSA activity for the high-throughput assay described in Example 10c. The data relate to the screening of filtrates from microplate cultures (Example 9) containing parental (PcCel6A-S407P) and modified PcCel6A cellulases or filtrates from empty vector transformants.
FIG. 8 shows a scatter plot of enzyme activity in the presence of BSA-treated lignin (+BSA activity) versus enzyme activity in the presence of untreated lignin (−BSA activity) for the high-throughput assay described in Example 10a. The data relate to the screening of filtrates from microplate cultures (Example 9) containing parental (TrCel6A-S413P) and modified TrCel6A cellulases or filtrates from empty vector transformants.
FIG. 9 depicts vector pTrCel7A-pyr4-TV directing the expression and secretion of native and modified TrCel7A glycosidases from recombinant Trichoderma reesei. 
FIG. 10 depicts the distribution of amino acid substitutions within the CBM domains among two populations of modified glycosidases—i.e., lignin-resistant “hits” and non-selected, but active glycosidases—from the TrCel6A, HiAvi2 and PcCel6A-S407P error-prone PCR libraries. Amino acid changes were grouped as those that introduce positive charge (i.e., convert a neutral amino acid to a basic amino acid such as His, Lys or Arg) or those changes than introduce negative charge (i.e., convert a neutral amino acid to Glu or Asp).
FIG. 11 demonstrates that recovery of wild-type TrCel7A glycosidase from pre-treated lignocellulose increases upon removal or blocking of in situ lignin. A Trichoderma reesei cellulase mixture with enhance levels of TrCel3A beta-glucosidase was incubated with pretreated wheat straw (WS), hypochlorite-bleached pretreated wheat straw (bWS) and pretreated wheat straw that was pre-incubated with bovine serum albumin to block lignin (BSA-WS). Sample supernatants were collected throughout the hydrolysis time-course and assayed for glucose and TrCel7A concentrations. At a fractional conversion of 0.98, more TrCel7A was present in the supernatant from the reactions involving bWS and BSA-WS, compared to the WS control.
FIG. 12 shows the structure of the Family 1 CBM from TrCel7A (PCB entry laz6) and the calculated structures of CBMs from TrCel6A, HiAvi2 and PcCel6A, based from the structure of the Family 1 CBM from TrCel7A (PDB entry laz6). The beta-sheet structures are shown as ribbons. Amino acids equivalent to those observed to participate in cellulose binding in the CBM of TrCel7A are depicted by gray stick structures while those amino acids that interact with lignin are shown as black ball and stick structures. Residues that interact with lignin and cellulose are also shown as black ball and stick structures.
FIG. 13 shows a Clustal W alignment of 34 Family 1 CBMs as obtained from the ProSite URL expasy.ch/cgi-bin/aligner?psa=PS00562&color-1&maxinsert=10&linelen=0.
FIG. 14 shows the amino acid sequence identity between pairs of CBM sequences from FIG. 13.
FIG. 15 shows an alignment of the Family 1 CBMs from TrCel6A, HiAvi2, PcCel6A and TrCel7A. Amino acids that were found to be substituted in modified Family 1 CBMs with reduced lignin binding using the method of Examples 4 are shown in bold font.
FIG. 16 shows the relative residual activity of parental (WT) and modified PcCel6A glycosidases after a 24 h incubation with lignin as described in Example 4.
FIG. 17 shows the relative lignin dissociation constants (KL) of parental (WT) and modified TrCel6A glycosidases determined as described in Example 4.
FIG. 18 shows the relative lignin dissociation constants (KL) of parental (WT) and modified TrCel7A glycosidases determined as described in Example 4.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to modified carbohydrate binding modules. More specifically, the invention relates to a modified Family 1 carbohydrate binding module exhibiting reduced binding to lignin. The present invention also relates to modified glycosidase enzymes comprising the modified Family 1 carbohydrate binding module, genetic constructs comprising nucleotide sequences encoding the modified Family 1 carbohydrate binding modules or the modified glycosidase enzyme, and the use of the modified glycosidase enzyme comprising the modified Family 1 carbohydrate binding module in the hydrolysis cellulose or hemicellulose substrates in the presence of lignin.
The present invention provides a modified Family 1 carbohydrate binding module with reduced binding to lignin, and the ability to confer not only reduced lignin binding but also increased substrate hydrolyzing activity, in the presence of lignin, to a modified glycosidase enzyme comprising the modified Family 1 carbohydrate binding module.
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. The headings provided are not meant to be limiting of the various embodiments of the invention. Terms such as “comprises”, “comprising”, “comprise”, “includes”, “including” and “include” are not meant to be limiting. In addition, the use of the singular includes the plural, and “or” means “and/or” unless otherwise stated. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Family 1 Carbohydrate Binding Modules
Carbohydrate binding modules or CBMs are non-catalytic domains in glycoside hydrolases and other proteins that recognize and bind to polysaccharides. CBMs are often found in fungal and bacterial proteins that contain a glycoside hydrolase domain that degrade insoluble polysaccharides. However, CBMs have also been identified in proteins that do not contain a glycoside hydrolase domain but are involved in the degradation of insoluble polysaccharides such as cellulose. These include but are not limited to Cip1 (Foreman et al., 2003) and swollenin (Saloheimo et al., 2002). CBMs are divided into families based on amino acid sequence similarity; there are currently 59 families of CBMs (http://www.cazy.org/Carbohydrate-Binding-Modules.html). Amongst these CBMs, different members have been shown to recognize crystalline cellulose, non-crystalline cellulose, chitin, beta-glucans, xylan, mannan, galactan and starch. CBMs that bind to cellulose are sometimes referred to by the term “cellulose-binding domain” or “CBD”. Family 1 CBMs have a high binding affinity for crystalline cellulose while CBMs from other families have a high binding affinity for amorphous cellulose or single-chain polysaccharides (Boraston, et al., 1004).
As summarized by Shoseyov et al. (2006), the carbohydrate-binding activity of CBMs has been exploited for a number of uses. For example, isolated CBMs have been shown to play a role in the non-hydrolytic disruption of cellulose fibres as well as in the alteration of fibre properties. In addition, CBMs have been used in biotechnological applications as affinity tags for bio-specific affinity purification of recombinant fusion proteins or for targeting enzymes that normally do not contain a CBM to natural fibres (such as targeting oxidative enzymes to textiles surfaces). CBMs have also found utility as analytical tools for characterization of fibre surfaces or detection of polysaccharides in plant cell walls. CBM dimers have been developed as novel cellulose cross-linking proteins that have shown to be effective in enhancing mechanical properties or altering surface properties of paper. Finally, CBMs, when expressed in transgenic plants, have been shown to increase the rate of cellulose biosynthesis and/or growth.
In fungi, CBMs are homologous and members of CBM Family 1 (CBM1). Sequences of CBMs from T. reesei cellulases, hemicellulases and related proteins are shown in Table 1. Four cysteines are highly conserved and form two disulfide bridges. Three aromatic amino acids (tryptophan, tyrosine or phenylalanine) are also conserved, form a planar surface and interact directly with the glucose units of the cellulose polymer via van der Waals' interactions. Family 1 CBMs have a high binding affinity for crystalline cellulose.
A Family 1 CBM is defined herein as any protein sequence that is classified as such according to the CAZy system (see http://www.cazy.org/Carbohydrate-Binding-Modules.html for reference). A Family 1 CBM may exhibit from about 50% amino acid sequence identity with amino acids sequence of the CBM of Trichoderma reesei Cel6A (also known as cellobiohydolase II or CBH2) as defined in SEQ ID NO: 30. For example, the Family 1 CBM may show from about 50%, 60%, 70%, 80%, 90%, or 95% amino acid identity with the Trichoderma reesei TrCel6A CBM as provided in SEQ ID NO: 30. One of skill in the art recognizes that the amino acid sequence of a given CBM may be modified by the addition, deletion or substitution of one or more amino acids and still be considered a CBM.
When the CBM is located at the N-terminus of the secreted glycosidase, one of skill in the art recognizes that amino acids which compose a secretion signal peptide are discounted when numbering the amino acids the CBM. Herein, numbering of the amino acids in the Family 1 CBMs begins at the equivalent of the first glutamine (Q) in TrCel6A (SEQ ID NO: 1).







TABLE 1







Sequences of Family 1 CBMs from Trichoderma reesei proteins











% Identity with




T. reesei Cel6A


Enzyme
CBM Sequence (delete SEQ ID No′s)
CBM (aa 3-39)





CBH1
PTQSHYGQCGGIGYSGPTVCASGTTCQVLNPY
63.9


(TrCel7A)
YSQCL (SEQ ID NO: 27)



 


CBH2
QACSSVVVGQCGGQNWSGPTCCASGSTCVYSN
100.0 


(TrCel6A)
DYYSQCL (SEQ ID NO: 30)



 


EG1
CTQTHWGQCGGIGYSGCKTCTSGTTCQYSND
63.9


(TrCel7B)
YYSQCL (SEQ ID NO: 11)



 


EG2
AQQTVWGQCGGIGWSGPTNCAPGSACSTLNP
61.1


(TrCel5A)
YYAQCI (SEQ ID NO: 12)



 


EG4
PTQTLYGQCGGSGYSGPTRCAPPATCSTLNPY
52.8


(TrCel61A)
YAQCL (SEQ ID NO: 14)



 


EG5
GQQTLYGQCGGAGWTGPTTCQAPGTCKVQN
50.0


(TrCe145A)
QWYSQCL (SEQ ID NO: 15)



 


TrCel74A
GHYAQCGGIGWTGPTQCVAPYVCQKQNDYY
56.0



YQ (SEQ ID NO: 40)



 


Cip1
HYGQCGGIGYSGPTVCASGTTCQVLNPYYSQC
61.1



L (SEQ ID NO: 42)



 


Cip2
WGQCGGIGWSGPTTCVGGAYCVSYNPYY
64.0



(SEQ ID NO: 43)



 


Swollenin
ALFGQCGGIGWSGTTCCVAGAQCSFVNDWYS
58.3



QCL (SEQ ID NO: 44)



 


Man5A
LYGQCGGSGYTGPTCCAQGTCIYSNTWTSQCL
65.0



(SEQ ID No: 41)



 


Axe1
PTQTHWGQCGGQGWTGPTQCESGTTCQVISQ
70.0



WYSQCL (SEQ ID NO: 7)









As shown in FIG. 13, there is a high degree of conservation of primary amino acid sequence among Family 1 cellulose binding domains. Multiple alignment across 34 currently known Family 1 CBM amino acid sequences of fungal origin shows that most naturally occurring Family 1 CBMs exhibit from about 45% to about 100% amino acid sequence identity to amino acids 3-39 comprising the Family 1 CBM of TrCel6A and from about 40% to about 95% amino acid sequence identity to at least one other Family 1 CBM (FIG. 14).
Sequence identity can be readily determined by alignment of the amino acids of the two sequences, either using manual alignment, or any sequence alignment algorithm as known to one of skill in the art. The alignments and identity calculations present in FIGS. 13 and 14, respectively, were determined using ClustalW Multiple Alignment tool with default settings, found in the BioEdit software version 7.0.9.0 (Jun. 27, 2007). Other alignment algorithms known by one of skill in the art include, but not limited to, BLAST algorithm (BLAST and BLAST 2.0; Altschul et al., 1997 and 1990), the algorithm disclosed by Smith & Waterman (1981), by the homology alignment algorithm of Needleman & Wunsch (1970), \search for similarity method of Pearson & Lipman (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or manual alignment and visual inspection.
By “modified Family 1 carbohydrate binding module” or “modified Family 1 CBM”, it is meant a Family 1 CBM which exhibits binding to crystalline cellulose and comprises amino acid substitution at one or more positions selected from the group consisting of 10, 11, 12, 14, 17, 21, 24, 29, 31, 33, and 37, said position determined from alignment of a Family 1 carbohydrate binding module amino acid sequence with a Trichoderma reesei Cel6A carbohydrate binding module amino acid sequence as defined in SEQ ID NO: 30. As used herein, a modified Family 1 CBM does not include naturally-occurring CBMs.
As known to one of skill in the art, binding of a protein, such as a CBM, to its ligand or substrate, such as cellulose, can be quantified by establishing a binding isotherm. In such experiments, the fractional absorption of the protein added to a solution or suspension containing constant amount of substrate or ligand will increase with increasing amount of added protein until the substrate is saturated with protein. Methods to assess and quantify the binding of Family 1 CBMs to cellulose using binding isotherms are described in Linder et al. (1995 and 1999), Mattinen et al. (1997) and Reinikainen et al. (1992). Binding of a glycosidases comprising parental or modified Family 1 CBMs to a cellulose substrate may be assessed and quantified using the methods of Nidetzky et al. (1994) or using the methods provided in Examples 4 and 14.
For example, the modified Family 1 CBM may comprise substitution of a basic or charge-neutral amino acid at one or more positions selected from the group consisting of 11, 12, 14, 17, 24, 29, and 31 to an acidic amino acid. As defined herein, “basic amino acid” refers to any one of histidine, lysine or arginine, “acid amino acid” refers to any one of aspartic acid or glutamic acid and “charge-neutral amino acid” is any amino acid that is not a basic or acidic amino acid.
The modified Family 1 CBM may also comprise amino acid substitution at one or more positions selected from the group consisting of 10, 21, 33, and 37. For example, the amino acid substitution at position 10 is to serine, the amino acid substitution at position 21 is to an aromatic amino acid such as tyrosine, the amino acid substitution at position 33 is to asparagine, and the amino acid substitution at position 37 is to an aromatic amino acid such as tyrosine.
The modified Family 1 carbohydrate binding module amino acid sequence exhibits from about 50% to about 99.9% amino acid sequence identity to SEQ ID NO: 30, or any amount therebetween. For example, a modified Family 1 CBM may have an amino acid sequence that exhibits about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9% amino acid sequence identity to SEQ ID NO: 30. By “wild type” or “native” Family 1 CBM, it is meant a Family 1 CBM as it is found in nature, without any amino acid substitutions, insertions or deletions.
It will be understood that modified Family 1 CBM may be derived from a wild-type Family 1 CBM or from a Family 1 CBM that already contains other amino acid substitutions.
The modified Family 1 CBM of the present invention is encoded by a nucleic acid sequence that can be generated using genetic material or nucleic acid or amino acid sequence information specific to the desired modified Family 1 CBM or to a corresponding parental Family 1 CBM. As is known by one of skill in the art, such genetic material or sequence information can be used to generate a nucleic acid sequence encoding a desired modified Family 1 CBM using one or more molecular biology techniques for altering amino acid sequences including, but not limited to, site-directed mutagenesis, cassette mutagenesis, random mutagenesis, synthetic oligonucleotide construction, cloning, sub-cloning, amplification by PCR, in vitro synthesis and other genetic engineering techniques known to one of skill in the art. It will be understood that the modified Family 1 may be derived from any parental Family 1 CBM—i.e., it may be derived from a naturally-occurring or “wild-type” Family 1 CBM or from a Family 1 CBM that already contains other amino acid substitutions.
For example, the modified Family 1 CBM may exhibit reduced binding to lignin. In another embodiment, the modified Family 1 carbohydrate binding module may confer reduced binding to lignin, or increased substrate hydrolyzing activity in the presence of lignin, to a glycosidase enzyme comprising the modified Family 1 carbohydrate binding module, one or more catalytic domain and one or more carbohydrate binding module joined by one or more linker peptide.
For the purposes of the present invention, a “parental Family 1 CBM” or “parental Family 1 carbohydrate binding module” is a Family 1 CBM that does not contain the amino acid substitution(s) present in the modified Family 1 CBM. As such, the parental Family 1 CBM may be a Family 1 CBM that contains amino acid substitutions at other positions that have been introduced by genetic engineering or other techniques and that is capable of binding to cellulose. The parental Family 1 CBM could also be a wild-type Family 1 CBM. Alternatively, after production of a modified Family 1 CBM, the modified Family 1 CBM may be subsequently further modified to contain additional amino acid substitutions.
Modified Glycosidase Enzymes
A glycosidase enzyme, as used herein, comprises a one or more catalytic domain and one or more carbohydrate binding module (CBM) joined by one or more linker peptide positioned between the domains. The one or more catalytic domain, one or more CBM and one or more linker peptide may be homologous with respect to each other—i.e., belonging to the same glycosidase as isolated in nature or heterologous with respect to at least one other domain—i.e., being isolated from two or more different naturally occurring glycosidase from the same, or different, source organism(s). The amino acid sequences of the one or more catalytic domain, one or more CBM and one or more linker peptide may be “native” or “wild type”—i.e., as found in unmodified glycosidase enzymes produced in nature—or they may be “derived” from native or wild-type glycosidase enzymes by modification of their amino acid sequences. The term “glycosidase enzyme” may be used interchangeably with the term “glycoside hydrolase” or “glycoside hydrolase enzyme”.
A glycosidase enzyme may comprise additional functional domains, e.g., cohesins, dockerins, or fibronectin-like (Fn3) domains and still be considered a glycosidase enzyme.
Examples of glycosidase enzymes from which the one or more catalytic domain, one or more CBM and one or more linker peptide may be isolated or derived include glycosidase enzymes from various microorganism such as Trichoderma ssp., Aspergillus ssp., Hypocrea ssp., Humicola ssp., Neurospora ssp., Orpinomyces ssp., Gibberella ssp., Emericella ssp., Chaetomium ssp., Chrysosporium ssp., Fusarium ssp., Penicillium ssp., Magnaporthe ssp., Phanerochaete ssp., Trametes ssp., Lentinula edodes, Gleophyllum trabeiu, Ophiostoma piliferum, Corpinus cinereus, Geomyces pannorum, Cryptococcus laurentii, Aureobasidium pullulans, Amorphotheca resinae, Leucosporidium scotti, Cunninghamella elegans, Thermomyces lanuginosa, Sporotrichum thermophile, or Myceliophthora thermophila. The practice of the invention is not limited by the glycosidase(s) from which the one or more catalytic domain, one or more CBM and one or more linker peptide may be derived.
A “modified glycosidase enzyme” as used herein, is a glycosidase enzyme comprising the one or more catalytic domain, one or more CBM, and one or more linker peptide with at least one of the one or more CBM being the modified Family 1 CBM comprising amino acid substitution at one or more positions selected from the group consisting of 10, 11, 12, 14, 17, 21, 24, 29, 31, 33, and 37, and exhibiting binding to crystalline cellulose and being from about 50% to about 99.9% identical to SEQ ID NO: 30. The one or more catalytic domain may be a wild-type or modified catalytic domain and the one or more linker peptide may be a wild-type or modified linker peptide. As used herein, the term “modified glycosidase enzyme” does not include naturally-occurring glycosidase enzymes.
As used herein, a “parental glycosidase enzyme” is a glycosidase enzyme comprising the same one or more catalytic domain, one or more CBM and one or more linker peptide as the modified glycosidase enzyme except that at least one or more CBM is a parental Family 1 CBM from which the modified Family 1 CBM in the modified glycosidase is derived. Furthermore, the parental Family 1 CBM in the parental glycosidase is identical to the modified Family 1 CBM of the modified glycosidase enzyme except that it has does not contain amino acid substitution at one or more positions selected from the group consisting of 10, 11, 12, 14, 17, 21, 24, 29, 31, 33, and 37. One of skill in the art recognizes that the one or more catalytic domain, one or more CBM, the parental Family 1 CBM, and one or more linker peptide may contain amino acid substitutions, insertions or deletions relative to naturally-occurring catalytic domains, CBMs, or linker peptides provided that these amino acid substitutions are also present in the modified glycosidase enzyme.
In the modified glycosidase of the present invention, the one or more catalytic domain may be a cellulase catalytic domain, a hemicellulase catalytic domain, a beta-glucosidase catalytic domain or an accessory protein catalytic domain.
A “cellulase catalytic domain” is defined as any domain that is capable of cleaving the beta-1,4 glycosidic linkages in a cellulose polymer. A cellulase catalytic domain can be an endoglucanase (EC 3.2.1.4), which cleaves internal beta-1,4 glycosidic linkages in the cellulose polymer to decrease the degree of polymerization of the polymer and/or release oligosaccharides. A cellulase catalytic domain can also be an exoglucanase or cellobiohydrolase (EC 3.2.1.91), which releases small oligosaccharides, primarily cellobiose, from the ends of the cellulose polymer. A cellulose polymer can be natural cellulose, such as that produced by plants or algae or other organisms, and may be pure or be one of several constituents in plant biomass, which also comprises lignin and hemicellulose. The cellulose polymer may also be a cellulose derivative, such as carboxymethyl cellulose or hydroxyethyl cellulose. A cellulase catalytic domain may be a member of GH Family 5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 61 and 74. For example, the cellulase catalytic domain may comprise amino acids 1-436 of Trichoderma reesei Cel7A SEQ ID NO: 124), amino acids 83-447 of Trichoderma reesei Cel6A (SEQ ID NO: 1), amino acids 97-460 of Humicola insolens Avi2 (SEQ ID NO: 2), or amino acids 81-440 of Phanerochaete chrysosporium Cel6A (SEQ ID NO: 3). For example, the cellulase catalytic domain may comprise amino acids 83-447 of Trichoderma reesei Cel6A (SEQ ID NO: 1) with one or more amino acid substitutions selected from the group consisting of Y103H, Y103K, Y103R, Y103A, Y103V, Y103L, Y103P, K129E L136V, L1361, S186K, S186T, S186Y, Q204K, G231D, A322D, Q363E, G365D, G365E, G365Q, G365S, R410A, R410F, R410L, R410Q, and R410S.
A “hemicellulase catalytic domain” is defined as any domain that is capable of cleaving the beta-1,4 glycosidic linkages in a hemicellulose polymer. For example, a hemicellulase catalytic domain may be a xylanase (E. C. 3.2.1.8), a beta-mannanase (E.C. 3.2.1.78), or an arabinofuranosidase (E.C. 3.2.1.55). Alternatively, a hemicellulase catalytic domain may be a member of Glycoside Hydrolase Family 5, 8, 10, 11, 26, 43, 51, 54, 62 or 113.
A “beta-glucosidase” catalytic domain is defined as any domain that is capable of producing glucose from small beta-1,4 linked oligosaccharides, such as cellobiose. Beta-glucosidases (E.C. 3.2.1.21) may be a member of Glycoside Hydrolase Family 1 or 3. For example, a beta-glucosidase catalytic domain may be a Trichoderma reesei Cel3A (SEQ ID NO: 100) with one or more amino acid substitutions selected from the group consisting of V43X, V66X, S72X, V101X, T235X, N248X, F260X, N369X, A386X, and I543X, which confer improved stability and/or catalytic efficiency to the TrCel3A beta-glucosidase (U.S. Publication No. 2010/0093040A1 and U.S. Publication No. 2010/0304438A1).
Finally, an “accessory protein catalytic domain” includes proteins that interact with cellulose to facilitate its hydrolysis including, but not limited to, Cip1, Cip2, swollenins and expansins. Accessory protein catalytic domain also includes other proteins that assist in the hydrolysis of lignocellulose, such as acetyl xylan esterases (E.C. 3.1.1.72), ferulic acid esterases (E.C. 3.1.1.73), and cellobiose dehydrogenase (E.C. 1.1.99.18).
One of skill in the art recognizes that the amino acid sequence of a given catalytic domain may be modified by the addition, deletion or substitution of one or more amino acids and still be considered a cellulase catalytic domain.
CBMs and catalytic domains are often separated by linker peptides. The term “linker peptide” is intended to be understood as a stretch of amino acids located between two functional domains and comprising from about 6 to about 60 amino acids. Linker peptides can be identified from amino acid sequence information using models such as described by Bae et al. (2008) and Suyama et al. (2003). Gilkes et al., (1991) presents the sequences of linkers from a variety of cellulases and other bacterial and fungal proteins encompassed by this definition. Linker peptides are typically basic peptides, particularly enriched in serine, threonine and proline, relative to non-linker sequences. As presented in Table I of Gilkes et al (1991), proline, serine and threonine account for 50% or more of the amino acids in all linker peptide sequences from bacterial and fungal glycoside hydrolases (xylanases, endoglucanases, exoglucanases). For the purposes defined herein, a linker peptide maybe be defined as a stretch of about 6 to about 60 amino acids, at least 50% of which are proline, serine or threonine, that is naturally found between a catalytic domain and a CBM, two catalytic domains, two CBMs, or between another functional domain and either a catalytic domain or a CBM. Proline, serine and threonine may account for 50%, 60%, 70%, 80% 90% or 100% of the amino acids in the linker peptide ((# proline+threonine+serine)/# amino acids in linker×100%). One of skill in the art recognizes that the amino acid sequence of a given linker may be modified by the addition, deletion or substitution of one or more amino acids and still be considered a linker peptide.
The modified glycosidase may comprise additional CBMs, in addition to the modified Family 1 CBM as defined above. These additional CBMs may be derived from any of the 59 CBM Families defined using the CAZy system (see http://www.cazy.org/Carbohydrate-Binding-Modules.html for reference).
Finally, the modified glycosidase may comprises other domains including, but not limited to fibronectin-like (Fn3) domains, cohesions, dockerins or other carbohydrate-active domains such amylases, glucoamylases, chitinases and the like.
Measuring Lignin Binding
The extent to which parental or modified Family 1 CBMs, or parental and modified glycosidase enzymes, as defined above, bind to lignin can be determined by pre-incubating the CBM or glycosidase enzyme with purified lignin for a set period of time and then measuring the residual protein concentration and/or enzyme activity in solution, and/or in the lignin-protein slurry, using assay methods known to one of skill in the art. The relative residual activities of parental and modified glycosidases comprising a Family 6 cellulase catalytic domain and a parental or modified Family 1 CBM after a 24 h incubation with lignin are shown in FIG. 16.
If the purified lignin is insoluble, the protein-lignin complexes can be readily separated from the bulk solution containing unbound protein by centrifugation or filtration. The lignin may be purified from a lignocellulosic feedstock (described below) by acid-extraction, alkali extraction, extraction with organic solvents, or enzymatic digestion of the lignocellulose with hydrolytic enzymes. The determination of the relative binding of parental and modified Family 1 CBMs or glycosidases is not dependent on the method used to purify the lignin, the source of the lignin or the assay methods used to detect the protein in solution. Methods for measuring the relative binding of parental and modified Family 1 CBMs, and parental and modified glycosidase enzymes, are provided in Example 4.
The relative lignin binding of parental and modified Family 1 CBMs or parental and modified glycosidases may be determined by calculating the lignin dissociation constant (KL) for the modified Family 1 CBM or glycosidase and dividing by the lignin dissociation constant (KO calculated for the parental CBM or glycosidase as described in Example 4. The relative KL values for modified glycosidases comprising Family 6 or 7 cellulase catalytic domains are shown in FIGS. 17 and 18.
The decrease in the inactivation of the modified glycosidase enzymes by lignin can be determined by measuring the degradation of a substrate (such as azo-glucan or cellulose) in the presence and absence of lignin and then taking the ratio of activity in the presence of lignin to the activity in the absence of lignin. The lignin present in such a hydrolysis reaction can be part of the insoluble substrate, such as in pre-treated lignocellulose, or be isolated in a soluble or insoluble form. If the lignin is isolated or purified, the inactivation of the modified or parental glycosidase enzyme by lignin is determined by measuring the activity in equivalent hydrolysis reactions, wherein one of the reactions contains a sufficient amount of lignin to reduce the glycosidase activity. Alternatively, isolated lignin that has been treated to be less deactivating by coating with a non-specific protein such as bovine serum albumin (BSA), a surfactant or other chemical can be added to the control reaction in the same amounts as the untreated lignin. If the lignin is part of the insoluble substrate, the inactivation of the modified or parental glycosidase enzyme by lignin is determined by taking the ratio of glycosidase activity on a bleached substrate (from which the lignin has been removed, for example, by an oxidant such as chlorine dioxide) and the glycosidase activity on an unbleached, lignin-containing substrate. A modified glycosidase enzyme with decreased inactivation by lignin will show a higher activity ratio (untreated, isolated lignin:no lignin or treated lignin) than the parental glycosidase enzyme. Methods for measuring the relative activity of parental and modified glycosidases comprising, respectively, parental and modified Family 1 CBMs, in the presence of lignin enzymes, are provided in Example 10.
There are several assays for measuring substrate hydrolyzing activity of the modified and parental glycosidase enzymes known to one of skill in the art. For example, hydrolysis of cellulose or hemicellulose can be monitored by measuring the enzyme-dependent release of reducing sugars, which are quantified in subsequent chemical or chemoenzymatic 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 oligosaccharides 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 cellulase enzyme is grown. In such an agar plate assay, activity of the cellulase is detected as a colored or colorless halo around the individual microbial colony expressing and secreting an active cellulase. It will be appreciated, however, that the practice of the present invention is not limited by the method used to assess the activity of the modified glycosidase enzyme.
The effect of the presence or absence of a Family 1 CBM on protein stability and substrate hydrolyzing activity of cellulase catalytic domains, for example, Family 7 and Family 6 catalytic domains, or of a Family 3 beta-glucosidase catalytic domain may be determined after pre-incubation in a lignin slurry. The data FIGS. 2, 3 and 4 show that the presence of a Family 1 CBM dramatically increases the sequestration of protein from solution by the lignin in the hydrolysis reaction, but has little effect on the hydrolyzing activity of the catalytic domain to which it is attached. Furthermore, FIG. 11 shows that Cel7A with a wild-type Family 1 CBM is more recoverable from hydrolysis reactions from which the substrate was made to be lignin-free or in which the lignin was “blocked” by non-specific protein.
The cellulose-hydrolyzing activity of the parental and modified glycosidase enzymes, comprising parental or modified Family 1 CBMs, in the presence of untreated lignin (−BSA) and treated lignin (+BSA), was determined via a comparative study of the parental and modified glycosidase enzymes as described in Example 10. The results are shown in Table 2, below. All of the modified glycosidase enzymes comprising Family 1 CBMs show at least a 20% decrease in lignin binding (20% higher KO and/or 11% higher ratio of activity in the presence of untreated lignin:activity in the presence of BSA-treated lignin (10% increase in ±BSA activity ratio).







TABLE 2







Modified Glycosidases Comprising Modified Family 1 CBMs


and Exhibiting Enhanced Hydrolytic Activity in the Presence


of Lignin (relative to a parental glycosidase)









SEQ ID
Normalized −/+



NO.
BSA ratio














Mutations in PcCel6A-S407P




(positions as listed in SEQ ID NO: 5)


5
1.00
None


66
1.69 ± 0.01
G12D


67
1.76 ± 0.04
W5R, S52P


68
1.75 ± 0.25
G22D, S64T, Q335E


69
1.86 ± 0.42
C8S


70
1.49 ± 0.01
G22D, Q197L


71
1.89 ± 0.33
G10D


72
1.64 ± 0.23
P30S, A276V


73
1.65 ± 0.13
G22D


74
1.73 ± 0.26
V20L, K288E


75
1.77 ± 0.05
P30S, I323T


77
1.70 ± 0.29
W5C, S83L, L131M


78
2.29 ± 0.53
S2N, G12S, A123V


79
2.05 ± 0.53
I11T, T423I, P439S


80
1.70 ± 0.40
P30S


81
1.71 ± 0.23
C8S, V54I


82
2.15 ± 0.71
G15D, P80L, A184T, V282I


83
1.93 ± 0.22
N29T


85
1.89 ± 0.60
G12D, A296S


86
1.87 ± 0.20
V27D, H60Y, P80T


87
1.61 ± 0.22
A1D, L28P, N437K


102
2.36 ± 0.08
G12D


76
1.57 ± 0.30
L36S


84
2.00 ± 0.72
L36S, Q201H, A304G




Mutations in HiAvi2




(positions as listed in SEQ ID NO: 2)


2
1.00
None


55
1.26
C21Y, I255V, R342H, G423S


56
1.36
S25C, N31S, L278F, A303T


57
1.35
A1D, G84D, V175A, K259R, A275T


58
1.63
I13T, T61A


59
1.22
C3Y, T26A, V43D, S320T


60
1.20
C10S, E157G


61
1.84
N31D, P324T, N389Y


62
1.25
C37Y


63
1.35
W7R, A75T, M270T


64
1.24
G11C, I13F, S47L, N237D


65
1.37
P18S




Mutations in TrCel6A-S413P




(positions as listed in SEQ ID NO: 4)


4
1.00
None


45
1.29
V28D, A112T, Q357E


46
1.25
G8N, T87M, H414Y


47
1.74
G17D, G231S


48
1.17
A22T


49
1.55
Y33N


50
1.15
G8D, V217I


51
1.13
N31S, G320D


52
1.13
N31S


53
1.50
L38F, V57E, K157M


54
1.14
S25N









Genetic Constructs Encoding the Modified Family 1 Carbohydrate Binding Module or Modified Glycosidase Enzyme

The present invention also relates to genetic constructs comprising a polynucleotide sequence encoding the modified Family 1 carbohydrate binding module or modified glycosidase enzyme operably linked to regulatory polynucleotide sequences directing the expression and secretion of the modified Family 1 carbohydrate binding module or modified glycosidase enzyme from a host microbe. By “regulatory polynucleotide sequences” it is meant a promoter and a polynucleotide sequence encoding a secretion signal peptide. The regulatory polynucleotide sequences may be derived from genes that are highly expressed and secreted in the host microbe under industrial fermentation conditions. For example, the regulatory sequences are derived from any one or more of the Trichoderma reesei cellulase or hemicellulase genes.
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. For example, the selectable marker gene may confer resistance to hygromycin, phleomycin, kanamycin, geneticin, or G418, complement 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.
The genetic construct may further comprise other polynucleotide sequences, for example, transcriptional terminators, polynucleotide encoding peptide tags, synthetic sequences to link the various polynucleotide 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 polynucleotide sequences.
Genetically Modified Microbes Producing the Modified Family 1 Carbohydrate Binding Module or Modified Glycosidase Enzyme
The modified Family 1 carbohydrate binding module or modified glycosidase enzyme 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 1 carbohydrate binding module or modified glycosidase enzyme. The host microbe may be a bacterium, such as Escherichia coli or Streptomyces lividans, a yeast such Saccharomyces, Pichia, or Hansenula, or a filamentous fungus such as Trichoderma, Hypocrea, Aspergillus, Fusarium, Humicola, Chrysosporium, Myceliophthora, Sporotrichum, Thielavia, or Neurospora. In a most preferred embodiment, the host microbe is an industrial strain of Trichoderma reesei. 
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 cellulase enzyme, the selected recombinant strains may be cultured in submerged liquid fermentations under conditions that induce the expression of the modified cellulase enzyme.
Production of the Modified Family 1 Carbohydrate Binding Module or the Modified Glycosidase Enzyme
A modified Family 1 carbohydrate binding module or modified glycosidase enzyme of the present invention may be produced in a fermentation process using a genetically modified microbe comprising a genetic construct encoding the modified Family 1 carbohydrate binding module or modified glycosidase enzyme, e.g., in submerged liquid culture fermentation.
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 1 carbohydrate binding module or modified glycosidase enzyme of the present invention may be carried out in a shake-flask or a bioreactor.
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.
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.
For the process for producing the modified Family 1 carbohydrate binding module or modified glycosidase enzyme of the present invention, the carbon source may comprise a carbohydrate that will induce the expression of the modified Family 1 carbohydrate binding module or modified glycosidase enzyme 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. 
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.
The process for producing the modified Family 1 carbohydrate binding module or modified glycosidase enzyme of the present invention may be conducted at a temperature from about 20° C. to about 50° 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, 40, 45 or 50° C. or any temperature therebetween.
The process for producing the modified Family 1 carbohydrate binding module or modified glycosidase enzyme 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.
Following fermentation, the fermentation broth containing the modified Family 1 carbohydrate binding module or modified glycosidase enzyme may be used directly, or the modified Family 1 carbohydrate binding module or modified glycosidase enzyme may be separated from the fungal cells, for example by filtration or centrifugation. Low molecular weight solutes such as unconsumed components of the fermentation medium may be removed by ultra-filtration. The modified Family 1 carbohydrate binding module or modified glycosidase enzyme 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.
Hydrolysis of Cellulose or Hemicellulose Using the Modified Glycosidase Enzymes
The modified glycosidase enzymes of the present invention are used for the enzymatic hydrolysis of cellulose or hemicellulose in a hydrolysis reaction further comprising lignin. For example, the modified glycosidase enzyme of the present invention is used for the enzymatic hydrolysis of a pretreated lignocellulosic substrate, such as in industrial processes producing fermentable sugars, sugar alcohols or fuel alcohols from lignocellulose, or in the enzymatic hydrolysis of pulp. The modified glycosidase enzymes of the present invention may be part of an enzyme mixture comprising other cellulase enzymes, hemicellulases, glucosidases, and non-hydrolytic proteins known to alter cellulose structure, such as swollenins and expansins.
By the term “enzymatic hydrolysis”, it is meant a process by which glycosidase enzymes or mixtures, including those comprising the modified glycosidase enzyme of the present invention, act on polysaccharides to convert all or a portion thereof to soluble sugars.
The modified glycosidase enzyme of the invention is used for the enzymatic hydrolysis of a “pretreated lignocellulosic substrate.” A pretreated lignocellulosic substrate is a material of plant origin that, prior to pretreatment, contains 20-90% cellulose (dry wt), more preferably about 30-90% cellulose, even more preferably 40-90% cellulose, for example 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90% or any % therebetween, and at least 10% lignin (dry wt), more typically at least 12% (dry wt) and that has been subjected to physical and/or chemical processes to make the fiber more accessible and/or receptive to the actions of cellulolytic enzymes.
After pretreatment, the lignocellulosic feedstock may contain higher levels of cellulose. For example, if acid pretreatment is employed, the hemicellulose component is hydrolyzed, which increases the relative level of cellulose. In this case, the pretreated feedstock may contain greater than about 20% cellulose and greater than about 12% lignin. In one embodiment, the pretreated lignocellulosic feedstock contains greater than about 20% cellulose and greater than about 10% lignin.
Lignocellulosic feedstocks that may be used in the invention include, but are not limited to, agricultural residues such as corn stover, wheat straw, barley straw, rice straw, oat straw, canola stover, and soybean stover; fiber process residues such as corn fiber, sugar beet pulp, pulp mill fines and rejects or sugar cane bagasse; forestry residues such as aspen wood, other hardwoods, softwood, and sawdust; grasses such as switch grass, miscanthus, cord grass, and reed canary grass; or post-consumer waste paper products.
The lignocellulosic feedstock may be first subjected to size reduction by methods including, but not limited to, milling, grinding, agitation, shedding, compression/expansion, or other types of mechanical action. Size reduction by mechanical action can be performed by any type of equipment adapted for the purpose, for example, but not limited to, a hammer mill.
Non-limiting examples of pretreatment processes include chemical treatment of a lignocellulosic feedstock with sulfuric or sulfurous acid, or other acids; ammonia, lime, ammonium hydroxide, or other alkali; ethanol, butanol, or other organic solvents; or pressurized water (See U.S. Pat. Nos. 4,461,648; 5,916,780; 6,090,595; 6,043,392; 4,600,590).
The pretreatment may be carried out to hydrolyze the hemicellulose, or a portion thereof, that is present in the lignocellulosic feedstock to monomeric sugars, for example xylose, arabinose, mannose, galactose, or a combination thereof. Preferably, the pretreatment is carried out so that nearly complete hydrolysis of the hemicellulose and a small amount of conversion of cellulose to glucose occurs. During the pretreatment, typically an acid concentration in the aqueous slurry from about 0.02% (w/w) to about 2% (w/w), or any amount therebetween, is used for the treatment of the lignocellulosic feedstock. The acid may be, but is not limited to, hydrochloric acid, nitric acid, or sulfuric acid. For example, the acid used during pretreatment is sulfuric acid.
One method of performing acid pretreatment of the feedstock is steam explosion using the process conditions set out in U.S. Pat. No. 4,461,648. Another method of pretreating the feedstock slurry involves continuous pretreatment, meaning that the lignocellulosic feedstock is pumped though a reactor continuously. Continuous acid pretreatment is familiar to those skilled in the art; see, for example, U.S. Pat. No. 5,536,325; WO 2006/128304; and U.S. Pat. No. 4,237,226. Additional techniques known in the art may be used as required such as the process disclosed in U.S. Pat. No. 4,556,430.
As noted above, the pretreatment may be conducted with alkali. In contrast to acid pretreatment, pretreatment with alkali does not hydrolyze the hemicellulose component of the feedstock, but rather the alkali reacts with acidic groups present on the hemicellulose to open up the surface of the substrate. The addition of alkali may also alter the crystal structure of the cellulose so that it is more amenable to hydrolysis. Examples of alkali that may be used in the pretreatment include ammonia, ammonium hydroxide, potassium hydroxide, and sodium hydroxide. The pretreatment is preferably not conducted with alkali that is insoluble in water, such as lime and magnesium hydroxide.
An example of a suitable alkali pretreatment is Ammonia Freeze Explosion, Ammonia Fiber Explosion or Ammonia Fiber Expansion (“AFEX” process). According to this process, the lignocellulosic feedstock is contacted with ammonia or ammonium hydroxide in a pressure vessel for a sufficient time to enable the ammonia or ammonium hydroxide to alter the crystal structure of the cellulose fibers. The pressure is then rapidly reduced, which allows the ammonia to flash or boil and explode the cellulose fiber structure. (See U.S. Pat. Nos. 5,171,592; 5,037,663; 4,600,590; 6,106,888; 4,356,196; 5,939,544; 6,176,176; 5,037,663 and 5,171,592). The flashed ammonia may then be recovered according to known processes.
The pretreated lignocellulosic feedstock may be processed after pretreatment but prior to the enzymatic hydrolysis by any of several steps, such as dilution with water, washing with water, buffering, filtration, or centrifugation, or a combination of these processes, prior to enzymatic hydrolysis, as is familiar to those skilled in the art.
The pretreated lignocellulosic feedstock is next subjected to enzymatic hydrolysis. By the term “enzymatic hydrolysis”, it is meant a process by which cellulase enzymes act on cellulose to convert all or a portion thereof to soluble sugars. Soluble sugars are meant to include water-soluble hexose monomers and oligomers of up to six monomer units that are derived from the cellulose portion of the pretreated lignocellulosic feedstock. Examples of soluble sugars include, but are not limited to, glucose, cellobiose, cellodextrins, or mixtures thereof. The soluble sugars may be predominantly cellobiose and glucose. The soluble sugars may predominantly be glucose.
The enzymatic hydrolysis using the cellulase mixture may be batch hydrolysis, continuous hydrolysis, or a combination thereof. The hydrolysis may be agitated, unmixed, or a combination thereof.
The enzymatic hydrolysis is preferably carried out at a temperature of about 30° C. to about 75° C., or any temperature therebetween, for example a temperature of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75° C., or any temperature therebetween, and a pH of about 3.5 to about 7.5, or any pH therebetween, for example a temperature of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or pH therebetween. The initial concentration of cellulose in the hydrolysis reactor, prior to the start of hydrolysis, is preferably about 0.5% (w/w) to about 15% (w/w), or any amount therebetween, for example 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 15% or any amount therebetween. The combined dosage of all primary cellulase enzymes may be about 0.001 to about 100 mg protein per gram cellulose, or any amount therebetween, for example 0.001, 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 mg protein per gram cellulose or any amount therebetween. The hydrolysis may be carried out for a time period of about 0.5 hours to about 200 hours, or any time therebetween, for example, the hydrolysis may be carried out for a period of 2 hours to 100 hours, or any time therebetween, or it may be carried out for 0.5, 1, 2, 5, 7, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200 or any time therebetween. It should be appreciated that the reaction conditions are not meant to limit the invention in any manner and may be adjusted as desired by those of skill in the art.
The enzymatic hydrolysis is typically carried out in a hydrolysis reactor. The enzyme mixture is added to the pretreated lignocellulosic feedstock (also referred to as the “substrate”) prior to, during, or after the addition of the substrate to the hydrolysis reactor.
Preferably, the modified glycosidase enzyme is produced in one or more submerged liquid culture fermentations and may be separated from the cells at the end of the fermentation by filtration, centrifugation, or other processes familiar to those skilled in the art. The cell-free cellulase-containing fraction may then be concentrated (for example, via ultrafiltration), preserved, and/or stabilized prior to use. Alternatively, the modified glycosidase enzyme(s) are not separated from the cells, but are added to the enzymatic hydrolysis with the cells.
EXAMPLES
Example 1: Preparation of Trichoderma reesei Cel7A, Cel7A Catalytic Domain, Cel6A and Cel6A Catalytic Domains
A strain of Trichoderma reesei was grown in submerged liquid fermentation under conditions that induce cellulase production as known to those skilled in the art. The crude mixture of Trichoderma proteins was secreted by the cells into the fermentation broth. The fungal cells were removed from the fermentation broth by filtration across a glass microfiber filter containing a Harborlite filter bed. Cel7A and Cel6A were separated from the crude filtrate by anion exchange chromatography using a DEAE-Sepharose column as described by Bhikhabhai et al. (1984). Cel7A and Cel6A were then further purified by p-aminophenyl-1-thio-β-D-cellobioside affinity chromatography as reported by Piyachomkwan et al. (1997, 1998). These components were concentrated and buffer exchanged into 50 mM sodium citrate, pH 5.0 using a stirred ultrafiltration cell (Amicon) and a 10 kDa NMWL polyethersulfone membrane.
To demonstrate that each component preparation was devoid of contaminating primary cellulases, purified Cel7A and Cel6A were analyzed by Western blotting using component-specific polyclonal antisera from rabbit (FIG. 1, panel B). Proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane at 100 V for 1 h using a Mini Trans-Blot® Cell from BioRad. Western blotting was performed using the method of Birkett et al. (1985). The component-specific polyclonal antisera were generated using synthetic peptides, the sequences of which were based on the primary amino acid sequence of Cel7A, Cel6A, Cel7B and Cel5A from Trichoderma reesei, as known to those skilled in the art.
These examples demonstrated that the purification methods used yielded substantially pure Cel7A, Cel6A, Cel7B and Cel5A. This also demonstrated the specificity of these antisera for each of these primary cellulase components.
The catalytic domains of T. reesei TrCel7A and Cel6A were prepared by incubating the purified full-length proteins with the protease papain. Papain cleaves cellulase enzymes within the linker peptide, separating the CBM from the catalytic (core) domain. This method is known to one of skill in the art and has been used to study the contribution of the CBM and catalytic domain in, for example, substrate binding and catalysis (Nidetsky et al., 2004; Herner et al., 1999). Papain treatment of a cellulase enzyme decreases its molecular mass. Therefore, the papain treatments of Cel7A and Cel6A were monitored by SDS-PAGE in order to ensure complete digestion of the full-length protein. The products of papain-treatment of Cel7A and Cel6A, referred to as Cel7Acore and Cel6Acore, respectively, were purified, concentrated and buffer exchanged as described above.
Protein concentrations were determined chemically using the method of Bradford et al. (1976).
Example 2: Preparation of Cel3A and Cel3A-CBM
Strains of Trichoderma reesei that over-express Cel3A (SEQ ID NO: 100) or Cel3A-CBM (SEQ ID NO: 101), as described in U.S. Publication No. 2009/0209009A1 were grown in submerged liquid fermentations under conditions that induce cellulase production as known to those skilled in the art. The crude mixtures of Trichoderma proteins were secreted by the cells into the fermentation broth. The fungal cells were removed from the fermentation broth by filtration across a glass microfiber filter containing a Harborlite filter bed. Cel3A and Cel3A-CBM were separated from their respective culture filtrates by anion exchange and cation exchange chromatography.
A column of DEAE-Sepharose was equilibrated in 5 mM sodium phosphate, pH 7.2. Trichoderma culture filtrate containing Cel3A or Cel3A-CBM was adjusted to pH 7.2 and applied to the column at 10 mL/min. The column was washed with 4 column volumes of the equilibration buffer and then bound protein was eluted with 4 column volumes of 5 mM sodium phosphate, pH 7.2 containing 0.5 M NaCl. Column fractions were assayed for activity on cellobiose. The flow-though peak contained greater than 95% of the total activity on cellobiose in the sample initially loaded on the DEAE column. These fractions were pooled and separated by cation exchange chromatography. A column of SP-Sepharose was equilibrated in 5 mM sodium acetate, pH 5.5. The flow-though pool from anion exchange chromatography was adjusted to pH 5.5 and diluted to a conductivity ≦0.6 mS. After loading, Cel3A or Cel3A-CBM was eluted using a linear gradient of 5-50 mM sodium acetate at pH 5.5. Purified Cel3A and Cel3A-CBM were concentrated and buffer exchanged into 50 mM sodium citrate, pH 5.0 using a stirred ultrafiltration cell (Amicon) and a 10 kDa NMWL polyethersulfone membrane. Protein concentrations were determined chemically using the method of Bradford et al. (1976).
Example 3: Preparation of Lignin
Wheat straw was pretreated using the methods described in U.S. Pat. No. 4,461,648. Following pretreatment, sodium benzoate was added at a concentration of 0.5% as a preservative. The pretreated material was then washed with six volumes of lukewarm ( ̃35° C.) tap water using a Buchner funnel and filter paper.
A sample of pretreated wheat straw (167 g wet wt; 30% solids; 60% cellulose) was added to 625 mL of 82% H2SO4 with stirring in a 1 L flask, then stoppered and incubated at 50° C. with shaking for 4 hours. The remaining solids were filtered to dampness using a Buchner funnel and a glass fiber filter, resuspended in 1 L of water and adjusted to pH 4.5 with NaOH. The solids were filtered and washed with  ̃8 L water. The solids, which were determined to contain less than 1% (dry wt) cellulose, are referred to herein as “lignin”.
Bovine serum albumin (BSA) treatment of lignin was performed by incubating equal amounts (w/w) of lignin and BSA, at a concentration of 30 g/L in 50 mM citrate buffer (pH 5) containing 0.1% sodium benzoate, for 5 days at 50° C. with shaking. The solids were filtered and washed with approximately 8 L of water.
Example 4: Characterizing the Inactivation of Purified Cellulase Components in the Presence of Lignin
Purified catalytic domains or intact glycosidase enzymes (comprising a catalytic domain and CBM joined by a linker peptide) prepared as in Examples 1, 2, and 12 (0.06 mg) were incubated with untreated lignin (29 mg) in stoppered, glass flasks in a total volume of 1.2 mL of 50 mM citrate buffer, pH 5.0. Incubations were done at 30 or 50° C. with orbital shaking. Under the conditions tested, the proteins were essentially stable in solution in the absence of lignin for up to 96 h. 0.2 mL samples were collected from each flask at times ranging from 0 up to a maximum of 96 h. Each sample was centrifuged to separate the lignin and stored at 4° C.
Upon completion of the time course, the protein concentration in the supernatant of each time course sample was measured using the method of Bradford. Samples were then mixed briefly to resuspend the pellet and 0.05 mL of slurry containing both soluble and insoluble material added to a microtitre plate containing 3 glass beads/well. To microtitre wells containing Cel7A, Cel7Acore, Cel6A and Cel6Acore and lignin, 0.02 mL of a dilute preparation of Trichoderma cellulase devoid of Cel7A and Cel6A cellobiohydrolases (1 μg total protein) was added to each well in the microtitre plate to complement the cellobiohydrolase activity. Purified Trichoderma Cel3A (1.4 μg) was also added to the microtitre plate wells to complement cellulose hydrolysis activity. Finally, 0.2 mL slurry of delignified cellulose (0.25% cellulose) was added to each well. For microtitre plate wells containing TrCel3A or TrCel3A-CBM and lignin, 0.02 mL of a dilute preparation of Trichoderma cellulase (1 μs total protein) was added to each well in the microtitre plate to complement Cel3A activity. Finally, 0.2 mL slurry of delignified cellulose (0.25% cellulose) was added to each well. The assay plates were incubated at 50° C. for 2 h with orbital shaking. The plate was then centrifuged at 710×g for 2 min and the glucose concentrations measured as described by Trinder et al. (1969).
Glucose concentrations were converted to enzyme activity, expressed as mg glucose produced/h/mg of protein. Activities measured throughout the time course were divided by the activity measured at t=0 h (prior to the addition of lignin) in order to calculate a relative residual activity for each enzyme throughout the time course. For the purposes of analyzing the results, measurements of relative residual activity were considered representative of the relative residual active enzyme concentration in the lignin slurry. Similarly, the protein concentrations measured throughout the time course were divided by the protein concentration at t=0 h for each reaction in order to calculate a relative residual protein concentration.
For the purpose of characterizing lignin binding and inactivation of cellulase components from Trichoderma reesei with and without a CBM, the relative residual protein and/or relative residual activity versus time data were modeled using Equation 1. In this equation, E represents the free enzyme, L represents lignin, EL represents a reversible enzyme-lignin complex and EL* represents an irreversible enzyme-lignin complex. KL represents [E][L]/[EL] at steady state while kL is a rate constant describing the rate of conversion of the reversible to the irreversible enzyme-lignin complex. The relative residual protein in the supernatant at each time was fit to the E parameter in Equation 1 while the relative residual activity in the slurry was fit to a sum of the E+EL parameters.
Modeling was done using a 4th order Runge-Kutta spreadsheet in Microsoft Excel. The data for each experiment involving one component were fit by varying KL and kL. Error minimization was done by the method of least squares as known to those of skill in the art.




  
    
      
        
          E
          +
          L
        
        ⁢
        
          ⇄
          
            
              
            
            ⁢
            
              K
              
                L
                ⁢
                
                  
                
              
            
          
        
        ⁢
        
          EL
          ⁢
          
            →
            
              
                
              
              ⁢
              
                k
                L
              
              ⁢
              
                
              
            
          
          ⁢
          
            EL
            *
          
        
      
    
    
      
        Equation
        ⁢
        
          
        
        ⁢
        1
      
    
  




The lignin inactivation profiles of Cel7A and Cel7Acore at 50° C. are shown in FIG. 2. Approximately 55% of Cel7A was lost from the supernatant within 0.5 h (panel A, solid circles). In this time period only about 10% of the total Cel7A activity was lost from the lignin slurry (Panel A, open squares). Throughout the rest of the time course, the Cel7A concentration in the supernatant remained essentially constant while Cel7A activity in the slurry decreased slowly. This indicates that Cel7A is rapidly bound by lignin in a manner that preserves its activity in this experiment, since incubating these samples with crystalline cellulose resulted in much higher relative residual Cel7A activity than Cel7A protein. No such rapid loss of Cel7Acore protein was observed (Panel B, solid circles), suggesting that T. reesei Cel7A rapidly associates with lignin via its CBM.
The lignin inactivation profiles of T. reesei Cel3A, which does not have a CBM, and Cel3A-CBM, T. reesei Cel3A linked to the Family 1 CBM from T. reesei Cel7A at its C-terminus, are shown in FIG. 3. The loss of Cel3A protein from the supernatant (Panel A, solid circles) and activity from the slurry (Panel A, open squares) occur at similar rates. Cel3A-CBM protein (FIG. 3, Panel B, solid circles) and activity (Panel B, closed squares) decreased much more rapidly, compared to Cel3A. Approximately 70% of Cel3A-CBM activity was lost from the supernatant within 0.5 h, while about 95% of Cel3A-CBM activity was lost from the slurry. These results demonstrate that Cel3A-CBM binds lignin much more rapidly than does Cel3A and further implicates the Family 1 CBM, from Cel7A in this case, in lignin binding.
Similar results were obtained for T. reesei Cel6A and Cel6A catalytic domain (Cel6Acore) in lignin inactivation experiments at 30° C. The Cel6A concentration in the supernatant decreased by about 60% within 0.5 h (FIG. 4, Panel A, solid circles) while Cel6A activity (open squares) decreased by about 14%. Any further changes in the concentration of Cel6A in the supernatant were negligible during the remainder of the experiment while Cel6A activity decreased slowly. As was observed for Cel7Acore, Cel6Acore protein concentrations (Panel B, solid circles) decreased slowly throughout the time course in parallel with Cel6Acore activity (open squares) in the slurry, suggesting T. reesei Cel6A rapidly associates with lignin via its CBM.
Further, the presence of the CBM significantly increased the binding affinity of the T. reesei Cel7A, Cel6A and Cel3A enzymes, as evidenced by a much lower value of KL for the glycosidase enzymes comprising CBMs as compared to those that do not (Cel7Acore and Cel6Acore) (Table 3).







TABLE 3







Effects of the CBM on Binding of Isolated


Cellulase Components to Lignin










Enzyme
Relative KL













Cel7A
1.0



Cel7Acore
62.5



Cel3A
1.0



Cel3A-CBM
0.2



Cel6A
1.0



Cel6Acore
40.5









Example 5: Construction of a Vector Expressing TrCel7A (SEQ ID NO: 124)
A vector was constructed to express and secrete parental and modified TrCel7A glycosidases and target the native cel7a locus in the genome of a host T. reesei strain. The vector was constructed using pUC19 vector (Fermentas, #SD0061) as a backbone. To facilitate targeting, sequences adjacent to the 5′ and 3′ ends of the native Trcel7a gene amplified from T. reesei genomic DNA were inserted into the transformation vectors so as to flank the expression and selection cassettes. The entire N. crassa pyr4 (orotidine-5′-monophosphate decarboxylase) gene (GenBank #AL669988.1, position 65346-66992) was used as a selection cassette. The expression cassette contains the following sequences from the native T. reesei cel7a gene: promoter (PCel7A), secretion signal (Cel7A ss) and mature protein coding sequences (Cel7A). These sequences are operatively linked to each other and to the transcriptional terminator of the native T. reesei cel6a gene (TCel6a). All Trichoderma sequences present in the final transformation vector are available from the complete Trichoderma reesei genome sequence (version 2) via the DOE Joint Genomics Institute, as described in Table 4. A map of the complete pTRCel7A-pyr4-TV vector is shown in FIG. 9.







TABLE 4







Origins of Trichoderma sequences


present in transformations vectors.











Fragment name
JGI scaffold
position














Cel7a 3′ flank
29
334132-336251



Pcel7a
29
330605-332455



Cel7A
29
332456-334131



Tcel6a
3
14184-14547





Sequence information can be found at URL: genome.jgi-psf.org/Trire2/Trere2.home.html






Example 6: Construction of Vectors Expressing HiAvi2 (SEQ ID NO: 2) and PcCel6A-S407P (SEQ ID NO: 5)
Construction of Vector YEp352/PGK91-1-αss-NKE
Saccharomyces cerevisiae strain YDR483W BY4742 [14317] (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 Δkre2) was obtained from ATCC (cat#4014317). Humicola insolens and Phanerochaete chrysosporium strains were obtained from ATCC® (#22082TM and #201542TM respectively). Escherichia coli strain DH5α (F− φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hdR17(rk−, mk+)phoA supE44 thi-1 gyrA96 relA1λ−) was obtained from Invitrogen (cat#18265-017).
A DNA adapter containing NheI, KpnI, and EcoRI restriction sites was prepared by annealing primers AT046 and AT047 together. The adapter was inserted into a YEp based-plasmid (YEp352/PGK91-1αss) containing the pgk1 promoter, alpha mating factor secretion signal, and pgk1 terminator sequences to make plasmid YEp352/PGK91-1/αss NKE. Specifically, the linker was inserted as a NheI/EcoRI fragment into the NheI and EcoRI sites located downstream of the alpha mating factor secretion signal and upstream of the pgk1 terminator. Primer sequences are shown below:









AT046



(SEQ ID NO: 88)



5′ CTA GCT GAT CAC TGA GGT ACC G 



 



AT047



(SEQ ID NO: 89)



5′ AAT TCG GTA CCT CAG TGA TCA G 






Construction of the YEp352/PGK91-1-αss-NKE-HiAvi2 Vector

Lyophilized H. insolens was resuspended in 300 μL sterile H2O and 50 μL was spread onto Emerson YPSS pH 7 agar plate (0.4% Yeast extract, 0.1% K2HPO4, 0.05% MgSO4.7H2O, 1.5% Glucose, 1.5% Agar). The agar plate was incubated for 6 days at 45° C., then spores were inoculated in Novo media (as per Barbesgaard U.S. Pat. No. 4,435,307): Incubation for 48 hours at 37° C. in 100 mL growth phase media (2.4% CSL, 2.4% Glucose, 0.5% Soy oil, pH adjusted to 5.5, 0.5% CaCO3), then 6 mL of pre-culture was transferred into 100 mL production phase media (0.25% NH4NO3, 0.56% KH2PO4, 0.44% K2HPO4, 0.075% MgSO4.7H2O, 2% Sigmacell, pH adjusted to 7, 0.25% CaCO3) and culture was incubated for up to 4 days prior to biomass harvest. Then, 50 mg of biomass was used to isolate total RNA with the Absolutely RNA Miniprep Kit (Stratagene) according to the manufacturer's procedure. Total cDNA was generated from the total RNA using the SuperScript® II Reverse Transcriptase (Invitrogen) according to the manufacturer's procedure. A polynucleotide encoding for HiAvi2 was amplified from the cDNA using the following primers (which introduced a NheI site upstream the gene and KpnI and EcoRI sites downstream the HiAvi2 coding region):







5′HiAvi2-cDNA


(SEQ ID NO: 90)


5′ CTA TTG CTA GCT GTG CCC CGA CTT GGG GCC AGT


GC 


 


3′HiAvi2-cDNA


(SEQ ID NO: 91)


5′ CTA TTG AAT TCG GTA CCT CAG AAC GGC GGA TTG


GCA TTA CGA AG 






The PCR amplicon was cloned into the pGEM®-T Easy vector by TA-cloning according to the manufacturer's recommendations. This vector was digested with NheI and KpnI and the released HiAvi2 gene was ligated to the NheI and KpnI digested YEp352/PGK91-1/αss-NKE vector. The ligation mix was transformed into DH5α chemically-competent E. coli cells, plasmid isolated, and sequenced to confirm sequence and cloning site integrity. Resulting vector is called YEp352/PGK91-1-αss-NKE-HiAvi2 (FIG. 5A). Introduction of the NheI site upstream the gene changed the first two amino acids of the mature protein for an alanine and a serine respectively. Thus the parental HiAvi2 glycosidase defined in SEQ ID NO: 2 is HiAvi2-Q1A-N2S.
Construction of Vector YEp352/PGK91-1-αss-NKE-PcCel6A-S407P
Lyophilized P. chrysosporium was resuspended in 300 μL sterile H2O and 50 μL were spread onto PDA plates. Plates were incubated at 24° C. for 4 days. Spores for P. chrysosporium were inoculated on a cellophane circle on top of a PDA plate and biomass was harvested after 4-6 days at 24° C. Then, 50 mg of biomass was used to isolate total RNA with the Absolutely RNA® Miniprep Kit (Stratagene) according to the manufacturer's procedure. Total cDNA was generated from the total RNA using the SuperScript® II Reverse Transcriptase (Invitrogen) according to the manufacturer procedure. A polynucleotide encoding for PcCel6A was amplified from the cDNA using the following primers (which introduced a NheI site upstream the gene and KpnI and EcoRI sites downstream the PcCel6A coding region):







5′PcCel6A-cDNA


(SEQ ID NO: 92)


5′ CTA TTG CTA GCT CGG AGT GGG GAC AGT GCG GTG


GC 


 


3′PcCel6A-cDNA


(SEQ ID NO: 93)


5′ CTA TTG AAT TCG GTA CCC TAC AGC GGC GGG TTG


GCA GCA GAA AC 






The PCR amplicon was cloned into the pGEM®-T Easy vector by TA-cloning according to the manufacturer's recommendations which yield to plasmid pGEM-PcCel6A. The coding sequence for PcCel6A was then amplified from that source to introduce mutation S407P. To do so, mutagenic primer NM088 and reverse primers VH099 were used to generate megaprimer PCR. The resulting PCR product was isolated and used as a reverse primer in conjunction with the forward primer VH098 to generate the final mutated construct. Primers sequences are listed below:







VH098


(SEQ ID NO: 94)


5′ GGT ATC TTT GGA TAA AAG GGC TAG CTC GGA GTG GGG


ACA G 


 


VH099


(SEQ ID NO: 95)


5′ GGA GAT CGA ATT CGG TAC CTA CAG CGG CGG GTT GG 


 


NM088


(SEQ ID NO: 96)


5′ CCC CGC TAC GAC CCT ACT TGT TCT CTG 






The PcCel6A-S407P amplicon was digested with NheI and KpnI then ligated to the YEp352/PGK91-1/αss-NKE vector digested with NheI and KpnI. The ligation mix was transformed into chemically-competent E. coli DH5α cells, plasmid isolated, and sequenced to confirm sequence and cloning sites integrity. Resulting vector is called YEp352/PGK91-1-αss-NKE-PcCel6A-S407P (FIG. 5B). Introduction of the NheI site upstream the PcCel6A coding region changed the first two amino acids of the mature protein for an alanine and a serine respectively. Thus the parental glycosidase PcCel6A-S407P defined in SEQ ID NO: 5 is PcCel6A-Q1A-A2S-S407P.
Example 7: Construction of a Vector Expressing TrCel6A-S413P (SEQ ID NO: 4)
In order to facilitate cloning using NheI and KpnI restriction enzymes, the unique NheI site at position 1936 of the YEp352/PGK91-1 vector was blunted using the DNA Polymerase I large (Klenow) fragment to generate YEp352/PGK91-1 ΔNheI. The TrCel6A-S413P gene was amplified by PCR from the vector YEpFLAG ΔKpn10-S413P (U.S. Pat. No. 7,785,854) using primers 5′NheCel6A and 3′BglKpnCel6A. In parallel, the yeast alpha-factor leader sequence was amplified by PCR from the YEpFLAG-1 vector (Sigma) using primers (5′BglAlphaSS and 3′NheAlphaSS) to introduce a BglII at the 5′ end and an NheI site at the 3′ end of the amplicon.
The yeast alpha-factor leader sequence was isolated by BglII/NheI digestion and a three-piece ligation performed with the TrCel6A-S413P gene (isolated by NheI/BglII digestion) and the YEp352/PGK91-1 ΔNheI vector (isolated by BglII digestion). The resulting vector YEp352/PGK91-1 ΔNheI-αss-TrCel6A-S413P (FIG. 6) was transformed into yeast strain BY4742 using the procedure described by Gietz, R. D. and Woods, R. A. (2002). Primer sequences are listed below:







5′BglAlphaSS:


(SEQ ID NO: 103)


5′ACC AAA AGA TCT ATG AGA TTT CCT TCA ATT 


 


3′NheAlphaSS:


(SEQ ID NO: 104)


5′TGA GCA GCT AGC CCT TTT ATC CAA AGA TAC 


 


5′NheCel6A:


(SEQ ID NO: 105)


5′AAA AGG GCT AGC TGC TCA AGC GTC TGG GGC 


 


3′BglKpnCel6A:


(SEQ ID NO: 106)


5′GAG CTC AGA TCT GGT ACC TTA CAG GAA CGA TGG GTT






Example 8: Generation of Error Prone-PCR Libraries
Random mutagenesis libraries were generated using the Mutazyme® II DNA polymerase contained in the GeneMorph® II Random Mutagenesis Kit (Stratagene®). To make a HiAvi2 library, a PCR was performed for 20 amplification cycles using 58 ng of YEp352/PGK91-1/αssNKE-HiAvi2 as template with primers YalphaN21 and 3′PGK-term. To make the PcCel6A-S407P library, a PCR was performed for 30 amplification cycles using 57 ng of YEp352/PGK91-1/αss NKE-PcCel6A-S407P as template with primers YalphaN21 and 3′PGK-term. The YEp352/PGK91-1/αss NKE vector was digested with NheI and KpnI and then purified. This vector fragment and each final amplicon were transformed simultaneously and cloned by in vivo recombination into yeast strain YDR483W BY4742 [14317] (Butler et al., 2003).









YalphaN21 



(SEQ ID NO: 97)



5′ AGC ACA AAT AAC GGG TTA TTG 



 



3′PGK-term



(SEQ ID NO: 98)



5′ GCA ACA CCT GGC AAT TCC TTA CC 






Example 9: Expression and Isolation of Parental and Modified TrCel6A, HiAvi2, and PcCel6A Cellulases from Microplate Cultures
This example describes the selection and expression of TrCel6A, HiAvi2 and PcCel6A and modified TrCel6A, HiAvi2 and PcCel6A cellulases from Saccharomyces cerevisiae for use in high-throughput screening assays.
Saccharomyces cerevisiae transformants were grown for 4 days at 30° C. 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). Replica plates were prepared by transferring colonies to synthetic complete medium plates containing 0.12% Azo-barley-beta-glucan (Megazyme) and incubated at 30° C. overnight.
Colonies showing visible clearing halos after 6 hours of incubation at 50° C. were selected for liquid media pre-cultures by toothpick inoculation of 0.15 mL 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) in 96-well microplates containing one glass bead. Pre-cultures were grown overnight (16-18 h) at 30° C. with orbital shaking 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. Expression cultures were grown for 3 days at 30° C. with orbital shaking and humidity control. Plates were centrifuged at 710×g for 5 minutes to pellet cells and the supernatant was aspirated for screening assays. To the remaining pre-culture, stocks were prepared by the addition of glycerol to a final concentration of 20% and stored at −80° C.
Example 10: High-Throughput Screening for Modified Glycosidase Enzymes Comprising Modified Family 1 CBMs
a. Screening of TrCel6A-S413P Libraries
This example describes the screening of modified TrCel6A glycosidases in order to identify those with resistance to inactivation by lignin in comparison to the parental TrCel6A-S413P glycosidase that had been cloned into Saccharomyces cerevisiae. 
An aliquot (0.15 mL) of yeast supernatant was pre-incubated with lignin (1.6% w/v) in a 0.25 mL citrate buffered (50 mM; pH 5) reaction. An equivalent aliquot of supernatant from each modified glycosidase was also pre-incubated with BSA pre-treated lignin (1.6% w/v). Pre-incubation was performed for 5.5 hour at 50° C. with orbital shaking (NB Innova 44) in a 96-well microplate containing 1 glass bead per well. Each 96-well microplate contained six parental TrCel6A-S413P controls for comparison. Following pre-incubation, microplates were centrifuged for 5 min at 2800×g and the supernatant was aspirated for residual activity assays.
Supernatant (0.05 mL) was incubated with 0.5% beta-glucan in a 100 μL citrate buffered (50 mM; pH 5) reaction. Residual activity assays were performed in a PCR plate at 50° C. for 16 hours for samples pre-incubated with lignin and 3 hours for samples pre-incubated with BSA-treated lignin. 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 min. An aliquot (0.15 mL) was transferred to a microplate and the absorbance was measured at 560 nm. Residual enzyme activity was determined by converting A560 values to reducing equivalents using the glucose standard curve and dividing by the appropriate incubation time (16 h or 3 h) to obtain mg/mL/h. An activity ratio was calculated for all modified TrCel6A glycosidases and the parental TrCel6A-S413P glycosidase controls by dividing the residual enzyme activity in the presence of untreated lignin by the residual enzyme activity in the presence of BSA-treated lignin. The activity ratio for each modified TrCel6A glycosidase was compared to the average of six parental TrCel6A-S413P glycosidase controls on a particular microplate and positives (those having increased ratios) were selected at the 95% confidence level using a t-test. All positive modified TrCel6A glycosidases were produced again in microculture and re-screened to reduce the number of false positives. A sample of the data from one screening plate is shown in FIG. 8.










DNS reagent contains:










Component
g/L













3,5-Dinitrosalicylic acid (Acros)
20



Sodium hydroxide (Fisher)
20



Phenol (Sigma)
4



Sodium metabisulfate (Fisher)
1









b. Screening of HiAvi2 Gene Libraries
This example describes the screening of modified HiAvi2 glycosidases in order to identify those with resistance to inactivation by lignin in comparison to the parental HiAvi2 that had been cloned into Saccharomyces cerevisiae. 
An aliquot (0.15 mL) of yeast supernatant was pre-incubated with lignin (0.4% w/v) in a 0.25 mL citrate buffered (50 mM; pH 5) reaction. An equivalent aliquot of supernatant from each modified cellulase was also pre-incubated with BSA pre-treated lignin (0.4% w/v). Pre-incubation was performed for 1 hour at 50° C. with orbital shaking (NB Innova 44) in a 96-well microplate containing 1 glass bead per well. Each 96-well microplate comprised six parental HiAvi2 controls for comparison. Following pre-incubation, microplates were centrifuged for 5 min at 2800×g and the supernatant was aspirated for residual activity assays.
Supernatant (0.05 mL) was incubated with 0.5% beta-glucan in a 100 μL citrate buffered (50 mM; pH 7) reaction. Residual activity assays were performed in a PCR plate at 65° C. for 16 hours for samples pre-incubated with lignin and 3 hours for samples pre-incubated with BSA-treated lignin. 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 min. An aliquot (0.15 mL) was transferred to a microplate and the absorbance was measured at 560 nm. Residual enzyme activity was determined by converting A550 values to reducing equivalents using the glucose standard curve and dividing by the appropriate incubation time (16 h or 3 h) to obtain mg/mL/h. An activity ratio was calculated for all modified HiAvi2 glycosidases and the parental HiAvi2 glycosidase controls by dividing the residual enzyme activity in the presence of untreated lignin by the residual enzyme activity in the presence of BSA-treated lignin. The activity ratio for each modified HiAvi2 glycosidase was compared to the average of six parental HiAvi2 glycosidase controls on a particular microplate and positives (those having increased ratios) were selected at the 95% confidence level using a t-test. All positive modified HiAvi2 glycosidases were produced again in microculture and re-screened to reduce the number of false positives. A sample of the data from one screening plate is shown in FIG. 7A.
c. Screening of PcCel6A-S407P Gene Libraries
This example describes the screening of modified PcCel6A glycosidase in order to identify those with resistance to inactivation by lignin in comparison to the parental PcCel6A-S407P glycosidase that had been cloned into Saccharomyces cerevisiae. 
An aliquot (0.15 mL) of yeast supernatant was pre-incubated with lignin (0.4% w/v) in a 0.25 mL citrate buffered (50 mM; pH 5) reaction. An equivalent aliquot of supernatant from each modified glycosidase was also pre-incubated with BSA pre-treated lignin (0.4% w/v). Pre-incubation was performed for 2 hour at 50° C. with orbital shaking (NB Innova 44) in a 96-well microplate containing 1 glass bead. Each 96-well microplate comprised six parental PcCel6A-S407P controls for comparison. Following pre-incubation, microplates were centrifuged for 5 min at 2800×g and the supernatant was aspirated for residual activity assays.
Supernatant (0.05 mL) was incubated with 0.5% beta-glucan in a 100 μL citrate buffered (50 mM; pH 5) reaction. Residual activity assays were performed in a PCR plate at 50° C. for 16 hours for samples pre-incubated with lignin and 3 hours for samples pre-incubated with BSA-treated lignin. 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 min. An aliquot (0.15 mL) was transferred to a microplate and the absorbance was measured at 560 nm. Residual enzyme activity was determined by converting A560 values to reducing equivalents using the glucose standard curve and dividing by the appropriate incubation time (16 h or 3 h) to obtain mg/mL/h. An activity ratio was calculated for all modified PcCel6A glycosidases and the parental PcCel6A-S407P controls by dividing the residual enzyme activity in the presence of untreated lignin by the residual enzyme activity in the presence of BSA-treated lignin. The activity ratio for each modified PcCel6A glycosidase was compared to the average of six parental PcCel6A-S407P controls on a particular microplate and positives (those having increased ratios) were selected at the 95% confidence level using a t-test. All positive modified PcCel6A glycosidases were produced again in microculture and re-screened to reduce the number of false positives. A sample of the data from one screening plate is shown in FIG. 7B.
Example 11: Statistical Analysis of EP-PCR Libraries
Amino acid charge changes within the CBM, was compared between glycosidase variants having lignin resistance to those of a random population of active glycosidase variants. Several variants showing activity on beta-glucan following pre-incubation with BSA-treated lignin were randomly picked and sequenced from all three libraries (TrCel6A-S413P, HiAvi2 and PcCel6A-S407P). From this population of random active variants, five charge change mutations were found: 4 neutral amino acids were changed to positive amino acids and 1 neutral amino acid was changed to a negative amino acid. For the population of lignin resistant variants, regardless of parental glycosidase, there were 16 charge change mutations: 2 neutral amino acids were changed to positive amino acids and 14 neutral amino acids were changed to negative amino acids (FIG. 10). A significant difference (P=0.0035) between neutral-to-positive versus neutral-to-negative charge change was observed between the two populations using the following equation:



  
    z
    =
    
      
      
        
          
            
              x
              1
            
            
              n
              1
            
          
          -
          
            
              x
              2
            
            
              n
              2
            
          
        
        
          
            
              
                p
                ^
              
              ⁡
              
                (
                
                  1
                  -
                  
                    p
                    ^
                  
                
                )
              
            
            ⁢
            
              (
              
                
                  1
                  
                    n
                    1
                  
                
                +
                
                  1
                  
                    n
                    2
                  
                
              
              )
            
          
        
      
      
    
  




  
    
      p
      ^
    
    =
    
      
        
          x
          1
        
        +
        
          x
          2
        
      
      
        
          n
          1
        
        +
        
          n
          2
        
      
    
  



These results support that the introduction of acidic amino acids on the surface of the CBM results in a modified Family 1 CBM with reduced binding to lignin.

Example 12: Construction of Modified TrCel6A Glycosidases
Using Yep352/PGK91-1-αss-Cel6A-S413P as a template, additional mutations were introduced into the Family 1 CBM of TrCel6A-S413P (SEQ ID NO: 4) using a two-step PCR method involving megaprimer synthesis followed by megaprimer PCR (Table 5). The internal primers were modified to introduce the desired amino acid substitutions into the TrCel6A-S413P construct. The external plasmid primers (YalphaN21 and 3′PGK-term) were used to amplify the final product. Megaprimers and final products were purified using the Wizard® SV Gel and PCR Clean-Up System.







TABLE 5







Generation of the modified TrCel6A enzymes by PCRα















Primer
Primer



PCR
Step
Template
1
2
Amplicon





1
1
Yep352/PGK91-1-αss-
YalphaN21
DKX02
PCR 1 Step 1




Cel6A(S413P)






1
Yep352/PGK91-1-αss-
DKX01
3′PGK-term
PCR 1 Step 1




Cel6A(S413P)






2
Both PCR 1 Step 1
YalphaN21
3′PGK-term
trcel6A-S413P-G17D




megaprimers





2
1
Yep352/PGK91-1-αss-
YalphaN21
DK270
PCR 2 Step 1




Cel6A(S413P)






1
Yep352/PGK91-1-αss-
DK269
3′PGK-term
PCR 2 Step 1




Cel6A(S413P)






2
Both PCR 2 Step 1
YalphaN21
3′PGK-term
trcel6A-S413P-Y29D




megaprimers





3
1
Yep352/PGK91-1-αss-
YalphaN21
DK274
PCR 3 Step 1




Cel6A(S413P)






1
Yep352/PGK91-1-αss-
DK273
3′PGK-term
PCR 3 Step 1




Cel6A(S413P)






2
Both PCR 3 Step 1
YalphaN21
3′PGK-term
Trcel6A-S413P-N31T




megaprimers









The final PCR products were digested with NheI+KpnI and ligated into vector Yep352/PGK91-1-αss-Cel6A-S413P linearized with NheI+KpnI. The ligation mix was transformed into chemically-competent DH5α E. coli cells, plasmid extracted, and sequenced.









5′YalphaN21



(SEQ ID NO: 97)



5′-AGCACAAATAACGGGTTATTG-3′ 



 



3′PGK-term



(SEQ ID NO: 98)



5′-GCAACACCTGGCAATTCCTTACC-3′ 



 



5′DKX01



(SEQ ID NO: 106)



5′-GAATTGGTCGGATCCGACTTGCTGTGCTTC-3′ 



 



3′DKX02



(SEQ ID NO: 107)



5′-AGCAAGTCGGATCCGACCAATTCTGGCC-3′ 



 



5′DK269



(SEQ ID NO: 108)



5′-GCACATGCGTCGACTCCAACGAC-3′ 



 



3′DK270



(SEQ ID NO: 109)



5′-GTCGTTGGAGTCGACGCATGTGC-3′ 



 



5′DK273



(SEQ ID NO: 110)



5′-CGTCTACTCCACCGACTATTACT-3′ 



 



3′DK274



(SEQ ID NO: 111)



5′-AGTAATAGTCGGTGGAGTAGACG-3′ 






Example 13: Construction of Modified TrCel7A Glycosidases
Using pTrCel7A-pyr4-TV as a template, additional mutations were introduced into T. reesei Cel7A (SEQ ID NO: 124) using a two-step PCR method involving megaprimer synthesis followed by megaprimer PCR (Table 6). The internal primers were modified to introduce the desired amino acid substitutions into the TrCel7A construct. The external plasmid primers (FT016 and AC413) were used to amplify the final product. Megaprimers and final products were purified using the Wizard® SV Gel and PCR Clean-Up System.







TABLE 6







Generation of the modified TrCel7A enzymes by PCR















Primer
Primer



PCR
Step
Template
1
2
Amplicon





1
1
pTrCel7A-pyr4-TV
FT016
DK298
PCR 1 Step 1



1
pTrCel7A-pyr4-TV
DK297
AC413
PCR 1 Step 1



2
Both PCR 1 Step 1
FT016
AC413
trcel7A-C469S




megaprimers





2
1
pTrCel7A-pyr4-TV
FT016
DK300
PCR 2 Step 1



1
pTrCel7A-pyr4-TV
DK299
AC413
PCR 2 Step 1



2
Both PCR 2 Step 1
FT016
AC413
trcel7A- G470C




megaprimers





3
1
pTrCel7A-pyr4-TV
FT016
DK302
PCR 3 Step 1



1
pTrCel7A-pyr4-TV
DK301
AC413
PCR 3 Step 1



2
Both PCR 3 Step 1
FT016
AC413
trcel7A-G471D




megaprimers





4
1
pTrCel7A-pyr4-TV
FT016
DK316
PCR 4 Step 1



1
pTrCel7A-pyr4-TV
DK315
AC413
PCR 4 Step 1



2
Both PCR 4 Step 1
FT016
AC413
trcel7A-C480Y




megaprimers





5
1
pTrCel7A-pyr4-TV
FT016
DK346
PCR 5 Step 1



1
pTrCel7A-pyr4-TV
DK345
AC413
PCR 5 Step 1



2
Both PCR 5 Step 1
FT016
AC413
trcel7A-C496Y




megaprimers









The final PCR products were digested with MluI+KpnI and ligated into vector pTrCel7A-pyr4-TV linearized with MluI+KpnI. The ligation mix was transformed into chemically-competent DH5α E. coli cells, plasmid extracted, and sequenced.









5′FT016



(SEQ ID NO: 112)



5′-GCCTGCACTCTCCAATCG-3′ 



 



3′AC413



(SEQ ID NO: 113)



5′-GTTGCTCATTTGCGGTCTAC-3′ 



 



5′DK297



(SEQ ID NO: 114)



5′-TACGGCCAGTCTGGCGGTATTGGCTACAG-3′ 



 



3′DK298



(SEQ ID NO: 115)



5′-AATACCGCCAGACTGGCCGTAGTGAGAC-3′ 



 



5′DK299



(SEQ ID NO: 116)



5′-GGCCAGTGCTGCGGTATTGGC-3′ 



 



3′DK300



(SEQ ID NO: 117)



5′-CAATACCGCAGCACTGGCCGT-3′ 



 



5′DK301



(SEQ ID NO: 118)



5′-AGTGCGGCGACATTGGCTACAGCGGCC-3′ 



 



3′DK302



(SEQ ID NO: 119)



5′-GTAGCCAATGTCGCCGCACTGGCCGT-3′ 



 



5′DK315



(SEQ ID NO: 120)



5′-CACGGTCTATGCCAGCGGCACAACTT-3′ 



 



3′DK316



(SEQ ID NO: 121)



5′-GCCGCTGGCATAGACCGTGGGGCCG-3′ 



 



5′DK345



(SEQ ID NO: 122)



5′-TACTACTCTCAGTACCTGTAAGGTACC-3′ 



 



3′DK346



(SEQ ID NO: 123)



5′-GGTACCTTACAGGTACTGAGAGTAGTA-3′ 






Example 14: Measuring Cellulase Recovery from Hydrolysis Residue
Cellulose hydrolysis experiments were done using steam exploded pretreated wheat straw, prepared as described in U.S. Pat. No. 4,461,648, and a cellulase mixture from a strain of Trichoderma reesei that over-expressed TrCel3A as described in U.S. Pat. No. 6,015,703. Samples of hydrolysis slurry were taken throughout the hydrolysis time-course and centrifuged to separate the solids from the supernatant. The glucose concentration in the supernatant was measured using a glucose oxidase-horseradish peroxidase coupled enzyme assay (Trinder et al., 1969). The concentration of Cel7A in the supernatant was measured by ELISA as described in U.S. Pat. No. 7,785,854. Glucose concentrations were converted to units of Fractional Cellulose Conversion and Cel7A protein or activity converted to units of fraction of initial Cel7A (Fractional Cel7A Recovery).
Immediately following the addition of enzyme to these substrates, only about 10% of the total Cel7A remained in the supernatant (FIG. 11). The concentration of Cel7A in the supernatant increased slowly as the fractional conversion increased from about 0 to about 0.60. As the conversion of BSA-WS increased above 0.60, the concentration of Cel7A in the supernatant increased gradually until 76% of the total Cel7A was recovered in the supernatant once cellulose conversion reached about 99%. The fractional concentration of Cel7A recovered in the supernatant from the hydrolysis of bWS increased markedly beginning at about 91% cellulose conversion, resulting in a total recovery of about 89% of the total Cel7A once cellulose conversion reached about 99%. By comparison the recovery of Cel7A from the hydrolysis of pretreated wheat straw was about 48% at the same level of cellulose conversion (99%). These experiments demonstrated that removal or blocking of in situ lignin markedly increases the recovery of cellulase, such as TrCel7A, from a hydrolysis reaction containing pre-treated lignocellulose.
Example 15: Expression and Purification of Modified Cel6A Glycosidases from Large Scale Cultures of S. cerevisiae 
500 mL of sterile YPD medium (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose) was inoculated with 10 mL of an overnight culture of transformed S. cerevisiae grown from cells freshly picked from an agar plate. The 500 mL cultures were then incubated for 96 hours at 30° C. with orbital shaking.
After incubation, the broth from each culture was centrifuged for 10 minutes at 16,700×g and the pellet (containing yeast cells) discarded. The pH of the supernatant was adjusted to 5.0 and then allowed to cool to 4° C. for an hour. Subsequent to cooling, 625 g (NH4)2SO4 was added to bring the yeast supernatant to 93% saturation. Precipitation was allowed to occur over a period of 2 hours at 4° C. with constant stirring. After centrifugation for 15 minutes at 16,700×g, the supernatant was discarded.
The pellet was resuspended with pipetting in 20 mL of 50 mM citrate, pH 5.0. Once the pellet was resuspended, 80 mL of 0.1 M sodium acetate, 200 mM glucose and 1 mM gluconic acid lactone, pH 5.0 was added. Samples were then incubated at 4° C. for 30 min with gentle stirring. Each sample was then centrifuged at 710×g for 3 minutes to pellet any insoluble material. The supernatant was removed carefully with a pipette to prevent disruption of the pellet and retained. The Cel6A cellulase in each sample was purified by APTC affinity chromatography as described by (Piyachomkwan et al., 1997). Purified Cel6A cellulases were buffer exchanged into 50 mM citrate, pH 5.0 and concentrated using a Centricon (Millipore) centrifugal concentrator with a 5 kDa NMWL polyethersulfone membrane. Protein concentrations were measured by the method of Bradford. Samples of the purified parental and modified Cel6A glycosidases were separated by SDS-PAGE and visualized by Coomassie Blue staining in order to confirm that each preparation was substantially pure and free of cored enzyme.
Example 16: Characterizing the Inactivation of Modified Cel6A Glycosidases Expressed from S. cerevisiae in the Presence of Lignin
The testing of purified parental and modified TrCel6A and PcCel6A glycosidases was done in a manner similar to that described in Example 4. The protein and lignin masses used in each of these experiments were 0.08 mg and 28 mg, respectively. The total reaction volume in these experiments was 2 mL and samples were taken over the course of 24 h.
TrCel6A lignin inactivation profiles were modeled in a manner similar to that described in Example 4. The KL associated with each of the modified TrCel6A glycosidases was divided by the KL, associated with the parental TrCel6A-S413P glycosidase in order to calculate a relative KL. The relative KL values for modified TrCel6A glycosidases are presented in FIG. 17. The modified TrCel6A-S413P glycosidase variants containing modified Family 1 CBMs with a G17D, a N29D or a N31T substitution all show reduced binding to lignin (as evidenced by a 1.3- to 1.7-fold higher KL than the parental TrCel6A-S413P glycosidase).
For the purpose of analyzing the modified PcCel6A glycosidases, a model free approach was used to identify modified glycosidases that were less inactivated in the presence of lignin, relative to the parental PcCel6A-S407P glycosidase. The residual PcCel6A activity was measured only before the addition of lignin (t=0 h) and in the lignin slurry 24 h after the addition of enzyme (t=24 h). The PcCel6A activity measured in the lignin slurry after 24 h of incubation was divided by the enzyme activity measured at t=0 h in order to calculate a fractional residual activity for each enzyme. The fractional residual activity for each modified PcCel6A glycosidase was then divided by the fractional residual activity for the parental PcCel6A-S407P in order to calculate a relative residual activity at 24 h. The relative residual activities of the parental and four modified PcCel6A glycosidases are shown in FIG. 16. These assays were done with four independent replicate experiments for each parental or modified PcCel6A glycosidase. The error bars represent the standard errors of these experiments for each modified PcCel6A glycosidase. The relative residual activity of the modified glycosidases comprising mutations at the equivalents of positions 12, 14 and 24 of SEQ ID NO: 30 (PcCel6A-S407P-G10D, PcCel6A-S407P-G12D and PcCel6A-S407P-G22D) were markedly higher (1.9- to 2.9-fold higher) than that of the parental glycosidase PcCel6A-S407P glycosidase, indicating that the mutations in the Family 1 CBM of the modified glycosidases conferred greater resistance to lignin binding and/or lignin inactivation.
Example 17: Expression of Modified TrCel7A Glycosidases
a. Host Trichoderma reesei Strain Construction
A uridine auxotroph Trichoderma reesei strain P297J (P297Jaux4) was used for expression of modified TrCel6A and TrCel7A cellulases. This strain contains disruption of the cel7a, cel7b and cel6a genes and is deficient in production of TrCel7A, TrCel7B and TrCel6A cellulases as described in WO2010/0096931A1.
b. PEG Transformation of Trichoderma reesei Protoplasts
5×106 spores of P297Jaux4 were plated onto sterile cellophane on Potato Dextrose agar supplemented with 5 mM uridine and were incubated for 20 hours at 30° C. to facilitate spore germination and mycelial growth. Cellophane discs with mycelia were transferred to 10 mL of a protoplasting solution containing 7.5 g/L Driselase and 4 g/L beta-glucanase (InterSpex Products Inc., Cat. Nos. 0465-1 and 0439-2, respectively) in 50 mM potassium phosphate buffer, pH 6.5 containing 0.6 M ammonium sulfate (Buffer P). The mycelial mat was digested for 5 hours with shaking at 60 rpm. Protoplasts were separated from undigested mycelia by filtration though sterile No. 30 MIRACLOTHTM and collected into a sterile 50 mL round-bottom centrifuge tube and recovered by centrifugation at 1000-1500×g for 10 min at room temperature. Protoplasts were washed with 5 mL of Buffer P and centrifuged again at 1000-1500×g for 10 min at room temperature. Protoplasts were resuspended in 1 mL of STC buffer (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCL, pH 7.5). For transformation, 0.1 mL of resuspended protoplasts were combined with 10 μg of vector pTrCel7A-pyr4-TV DNA (or a similar vector encoding the modified TrCel7A glycosidases constructed as described in Example 13) and 25 μL of PEG solution (25% PEG 3350, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5). After incubation in an ice water bath for 30 min, 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 STC buffer and the entire mix was added to 50 mL of molten MMSS agar media (see below) cooled to about 47° C., split in half, and poured over MMSS agar. Plates were incubated at 30° C. until colony growth was visible. Transformants were transferred to individual plates containing MM agar and allowed to sporulate. Spores were collected and plated at high dilution on MM agar to isolate homokaryon transformants, which were then plated onto PDA to allow for growth and sufficient sporulation to inoculate the screening cultures described below.
Minimal Medium (MM) Agar Contains:













Component*
Per L



















KH2PO4
10
g



(NH4)2SO4
6
g



Na3Citrate•2H2O
3
g



FeSO4•7H2O
5
mg



MnSO4•H2O
1.6
mg



ZnSO4•7H2O
1.4
mg



CaCl2•2H2O
2
mg



Agar
20
g



20% Glucose
50
mL



1M MgSO4-7H2O.
4
mL









pH to 5.5





*MMSS agar contains the same components as MM agar plus 1.2M sorbitol, 6.6 g/L YNB (Yeast Nitrogen Base w/o Amino Acids from DIFCO Cat. No. 291940) and 1.92 g/L amino acids (-Ura DO Supplement from Sigma Cat. No. Y1501-20G).






c. Production of Modified Glycosidases in Trichoderma reesei Microcultures
Sets of five random independent transformants expressing each modified TrCel7A glycosidase were selected for pre-screening in 24-well microcultures. Individual colonies of Trichoderma were transferred to PDA plates for the propagation of each culture. Sporulation was necessary for the uniform inoculation micro-cultures which were used in testing the ability of the culture to produce cellulase. The culture media was composed of the following:













Component
g/L


















(NH4)2SO4
12.7



KH2PO4
8.00



MgSO4•7H2O
4.00



CaCl2•2H2O
1.02



CSL
5.00



CaCO3
20.00



Carbon source**
30-35



Trace elements*
2 mL/L





*Trace elements solution contains 5 g/L FeSO4*7H20; 1.6 g/L MnSO4*H20; 1.4 g/L1 ZnSO4*7H20.


**glucose, Solka floc, lactose, cellobiose, sophorose, corn syrup, or Avicel. The carbon source can be sterilized separately as an aqueous solution at pH 2 to 7 and added to the remaining media initially or though the course of the fermentation.






Individual transformants were grown in the above media in 1 mL cultures in 24-well micro-plates. The initial pH was 5.5 and the media sterilized by steam autoclave for 30 minutes at 121° C. prior to inoculation. For both native and transformed cells, spores were isolated from the PDA plates, suspended in water and 104-106 spores per mL are used to inoculate each culture. The cultures were shaken at 250 rpm at a temperature of 30° C. for a period of 6 days. The biomass was separated from the filtrate containing the secreted protein by centrifugation at 12,000 rpm. The protein concentration was determined using the Bio-Rad Protein Assay (Cat. No. 500-0001).
The relative abundance (in weight % of total secreted protein) of TrCel7A in the microculture filtrates was determined by ELISA. Culture supernatants and purified component standards were diluted to 0.01-10 μg/mL 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 TrCel7A was diluted 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 h at room temperature. After washing, tetramethylbenzidine was added to each plate and incubated for 30 min at room temperature. The absorbance at 360 nm was measured in each well and converted into protein concentration using a TrCel7A standard curve.
Example 18: Characterization of Modified TrCel7A Glycosidases
One transformant expressing each modified TrCel7A glycosidase and exhibiting the highest TrCel7A expression levels in microculture filtrates (as described in Example 17) were grown in 50 mL of microculture media in shake flasks for 6 days at 30° C. with shaking at 250 rpm. Supernatants were collected and the lignin inactivation of the modified TrCel7A glycosidases was assessed as described in Example 4. The relative lignin dissociation constants (relative KL) of the modified TrCel7A glycosidases comprising mutations at the equivalents of positions 10, 11, 12, 14, 21 and 37 of SEQ ID NO: 30 (TrCel7A-C469S, TrCel7A-G470C, TrCel7A-G471D, TrCel7A-C480Y and TrCel7A-C496Y) were markedly higher (1.8- to 3.2-fold higher) than that of the parental glycosidase TrCel7A glycosidase, indicating that the mutations in the Family 1 CBM of the modified glycosidases conferred greater resistance to lignin binding and/or lignin inactivation.
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## Claims

1. A modified Family 1 carbohydrate binding module (CBM) comprising one or more amino acid substitutions selected from the group consisting of:
(a) substitution at position 10 to serine;
(b) substitution of at position 14 to aspartic acid;
(c) substitution at position 21 to tyrosine;
(d) substitution at position 29 to aspartic acid;
(e) substitution at position 31 to aspartic acid,
(f) substitution at position 33 to asparagine; and
(g) substitution at position 37 to tyrosine,

the position determined from alignment of the Family 1 CBM amino acid sequence with a Trichoderma reesei Cel6A carbohydrate binding module amino acid sequence as defined by amino acids 1 to 38 of SEQ ID NO: 1,
wherein the modified Family 1 carbohydrate binding module binds to crystalline cellulose and exhibits reduced binding to lignin relative to the unmodified Family 1 CBM, and
wherein the modified Family 1 carbohydrate binding module comprises an amino acid sequence that is 75% to 99.9% identical to amino acids 1 to 38 of SEQ ID NO:1.

2. The modified Family 1 carbohydrate binding module (CBM) of claim 1, wherein the amino acid sequence is 80% to 99.9% identical to amino acids 1 to 38 of SEQ ID NO:1.

3. The modified Family 1 carbohydrate binding module (CBM) of claim 1, wherein the amino acid sequence is 85% to 99.9% identical to amino acids 1 to 38 of SEQ ID NO:1.

4. The modified Family 1 carbohydrate binding module (CBM) of claim 1, wherein the amino acid sequence is 90% to 99.9% identical to amino acids 1 to 38 of SEQ ID NO:1.

5. The modified Family 1 carbohydrate binding module (CBM) of claim 1, wherein the amino acid sequence is 95% to 99.9% identical to amino acids 1 to 38 of SEQ ID NO:1.

6. The modified Family 1 carbohydrate binding module of claim 1, wherein
the amino acid substitution at position 10 is from cysteine to serine,
the amino acid substitution at position 14 is from asparagine to aspartic acid,
the amino acid substitution at position 21 is from cysteine to tyrosine,
the amino acid substitution at position 29 is from tyrosine to aspartic acid,
the amino acid substitution at position 31 is from asparagine to aspartic acid,
the amino acid substitution at position 33 is from tyrosine to asparagine, and
the amino acid substitution at position 37 is from cysteine to tyrosine.

7. The modified Family 1 carbohydrate binding module of claim 1, wherein the modified Family 1 carbohydrate binding module confers reduced binding to lignin of a glycosidase enzyme comprising the modified Family 1 carbohydrate binding module and one or more catalytic domain joined to the modified Family 1 carbohydrate binding module by one or more linker peptide.

8. A modified glycosidase enzyme comprising one or more catalytic domain and one or more carbohydrate binding module,
wherein the one or more catalytic domain and one or more carbohydrate binding modules are functionally joined by one or more linker peptide, and
wherein at least one of the one or more carbohydrate binding module is the modified Family 1 carbohydrate binding module of claim 1, the modified glycosidase enzyme exhibiting an increase in hydrolyzing activity in the presence of lignin and/or a reduction in lignin binding relative to a parental glycosidase comprising a parental Family 1 carbohydrate binding module from which the modified carbohydrate binding module is derived, the same one or more catalytic domain and the same one or more carbohydrate binding module joined by the same one or more linker peptide.

9. The modified glycosidase enzyme of claim 8, wherein the one or more catalytic domain is selected from the group consisting of a cellulase catalytic domain, a hemicellulase catalytic domain, a beta-glucosidase catalytic domain, and an accessory component catalytic domain.

10. The modified glycosidase enzyme of claim 9,
wherein the cellulase catalytic domain is a member of Glycoside Hydrolase Family 5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 61 or 74,
wherein the hemicellulase catalytic domain is a member of Glycoside Hydrolase Family 5, 8, 10, 11, 26, 43, 51, 54, 62 or 113,
wherein the beta-glucosidase catalytic domain is a member of Glycoside Hydrolase Family 1 or 3; and
wherein the accessory component catalytic domain is a swollenin, CIP or expansin catalytic domain.

11. The modified glycosidase enzyme of claim 10, comprising a Glycoside Hydrolase Family 6 or 7 cellulase catalytic domain.

12. The modified glycosidase enzyme of claim 11, wherein the cellulase catalytic domain comprises amino acids 83-447 of SEQ ID NO: 1 (Trichoderma reesei Cel6A), amino acids 1-436 of SEQ ID NO: 124 (Trichoderma reesei Cel7A), amino acids 97-460 of SEQ ID NO: 2 (Humicola insolens Avi2), or amino acids 81-440 of SEQ ID NO: 3 (Phanerochaete chrysosporium Cel6A).

13. The modified glycosidase enzyme of claim 11, wherein the cellulase catalytic domain is amino acids 83-447 of SEQ ID NO: 1 (Trichoderma reesei Cel6A) comprising one or more amino acid substitutions selected from the group consisting of Y103H, Y103K, Y103R, Y103A, Y103V, Y103L, Y103P, K129E L136V, L1361, S186K, S186T, S186Y, Q204K, G231D, A322D, Q363E, G365D, G365E, G365Q, G365S, R410A, R410F, R410L, R410Q, and R410S.

14. The modified glycosidase enzyme of claim 9, comprising a beta-glucosidase catalytic domain, wherein the beta-glucosidase catalytic domain is Trichoderma reesei Cel3A of SEQ ID NO: 100 comprising one or more amino acid substitutions selected from the group consisting of V43X, V66X, S72X, V101X, T235X, N248X, F260X, N369X, A386X, and I543X.

15. The modified glycosidase enzyme of claim 9, wherein the one or more catalytic domain is from a fungal glycosidase enzyme.

16. The modified glycosidase enzyme of claim 15, wherein the fungal glycosidase enzyme is from Trichoderma ssp., Aspergillus ssp., Hypocrea ssp., Humicola ssp., Neurospora ssp., Orpinomyces ssp., Gibberella ssp., Emericella ssp., Chaetomium ssp., Chrysosporium ssp., Fusarium ssp., Penicillium ssp., Magnaporthe ssp., or Phanerochaete ssp., Trametes ssp., Lentinulaedodes, Gleophyllumtrabeiu, Ophiostomapiliferum, Corpinuscinereus, Geomycespannorum, Cryptococcus laurentii, Aureobasidiumpullulans, Amorphothecaresinae, Leucosporidiumscotti, Cunninghamellaelegans, Thermomyceslanuginosa, Myceliophthora thermophilum or Sporotrichum thermophile.

17. The modified glycosidase enzyme of claim 16, wherein the fungal glycosidase is from Trichoderma reesei.

18. The modified glycosidase enzyme of claim 8, wherein the one or more linker peptide is a modified linker peptide from 6 to 60 amino acids in length and of which at least 50% of the amino acids are proline, serine or threonine,
wherein the modified linker peptide comprises one or more amino acid substitutions, insertions, or deletions that result in:
(a) a decrease in the calculated isoelectric point of the linker peptide;
(b) an increase in the ratio of threonine:serine in the linker peptide; or
(c) both (a) and (b), and
wherein relative to a parental linker peptide from which the modified linker peptide is derived, the modified linker peptide confers to the modified glycosidase an increase in hydrolyzing activity in the presence of lignin and/or a reduction in lignin binding relative to a parental glycosidase comprising the parental linker positioned between the same cellulase catalytic domain and carbohydrate binding module.

19. A process for hydrolyzing a cellulose or hemicellulose substrate to sugars comprising contacting the substrate with the modified glycosidase enzyme of claim 8 in the presence of lignin.

20. The process of claim 19, wherein the cellulose or hemicellulose substrate is a pretreated lignocellulosic substrate.

21. The process of claim 19, wherein the modified glycosidase enzyme exhibits improved recovery from the process relative to a parental glycosidase enzyme comprising the same one or more catalytic domain, one or more linker peptide and one or more carbohydrate binding module in which at least one of the one or more carbohydrate binding module is a parental Family 1 carbohydrate binding module from which the modified Family 1 carbohydrate binding module in the modified glycosidase is derived.

22. The process of claim 19, wherein the process is conducted as a continuous, semi-continuous or fed-batch process.

23. The process of claim 19, further comprising microbial fermentation of the sugars to alcohol or sugar alcohol.