Patent Application
20140287471
Cellulose is an unbranched polymer of glucose linked by β(1→4)-glycosidic bonds. Cellulose chains can interact with each other via hydrogen bonding to form a crystalline solid of high mechanical strength and chemical stability. The cellulose chains are depolymerized into glucose and short oligosaccharides before organisms, such as the fermenting microbes used in ethanol production, can use them as metabolic fuel. Cellulase enzymes catalyze the hydrolysis of the cellulose (hydrolysis of β-1,4-D-glucan linkages) in the biomass into products such as glucose, cellobiose, and other cellooligosaccharides. Cellulase is a generic term denoting a multienzyme mixture comprising exo-acting cellobiohydrolases (CBHs), endoglucanases (EGs) and β-glucosidases (BGs) that can be produced by a number of plants and microorganisms. Enzymes in the cellulase of Trichoderma reesei include CBH I (more generally, Cel7A), CBH2 (Cel6A), EG1 (Cel7B), EG2 (Cel5), EG3 (Cel12), EG4 (Cel61A), EG5 (Cel45A), EG6 (Cel74A), Cip1, Cip2, β-glucosidases (including, e.g., Cel3A), acetyl xylan esterase, β-mannanase, and swollenin.
Cellulase enzymes work synergistically to hydrolyze cellulose to glucose. CBH I and CBH II act on opposing ends of cellulose chains (Barr et al., 1996, Biochemistry 35:586-92), while the endoglucanases act at internal locations in the cellulose. The primary product of these enzymes is cellobiose, which is further hydrolyzed to glucose by one or more β-glucosidases.
The cellobiohydrolases are subject to inhibition by their direct product, cellobiose, which results in a slowing down of saccharification reactions as product accumulates. There is a need for new and improved cellobiohyrolases with improved productivity that maintain their reaction rates during the course of a saccharification reaction, for use in the conversion of cellulose into fermentable sugars and for related fields of cellulosic material processing such as pulp and paper, textiles and animal feeds.
The present disclosure relates to variant CBH I polypeptides. Most naturally occurring CBH I polypeptides have arginines at positions corresponding to R268 and R411 of T. reesei CBH I (SEQ ID NO:2). The variant CBH I polypeptides of the present disclosure include a substitution at either or both positions resulting in a reduction or decrease in product (e.g., cellobiose) inhibition. Such variants are sometimes referred to herein as “product tolerant.” In some instances, the variants have an increased specific activity towards a CBH I substrate.
Accordingly, the present invention provides polypeptides (variant CBH I polypeptides) in which the CBH I catalytic domain has been engineered to incorporate an amino acid substitution that results in increased tolerance to cellobiose, increased specific activity, or both. The variant CBH I polypeptides of the disclosure minimally contain at least a CBH I catalytic domain, comprising (a) a substitution at the amino acid position corresponding to R268 of T. reesei CBH I (“R268 substitution”); (b) a substitution at the amino acid position corresponding to R411 of T. reesei CBH I (“R411 substitution”); or (c) both an R268 substitution and an R411 substitution. The amino acid positions of exemplary CBH I polypeptides into which R268 and/or R411 substitutions can be introduced are shown in Table 1, and the amino acid positions corresponding to R268 and/or R411 in these exemplary CBH I polypeptides are shown in Table 2.
The polypeptides of the disclosure show at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 500-fold or at least 700-fold greater tolerance to cellobiose, and in some cases up to 750-fold or up to 1,000-fold greater tolerance to cellobiose, a wild type CBH I which does not have a substitution at the amino acid corresponding to R268 or the amino acid position corresponding to R411. Product tolerance can suitably be determined by assaying the IC50, the half maximal inhibitory concentration, of cellobiose towards the polypeptide.
In certain aspects, the polypeptides of the disclosure are characterized by an IC50 of cellobiose is at least 0.1 mM, at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, at least 5 mM, at least 7 mM, at least 10 mM, at least 12 mM, at least 15 mM, at least 20 mM, at least 25 mM or at least 30 mM.
In certain embodiments, a polypeptide of the disclosure comprises an R268 substitution. The R268 substitution preferably results in an IC50 of cellobiose that is at least 2-fold, at least 5-fold, at least 7.5-fold or at least 10-fold the IC50 of cellobiose on the reference CBH I (e.g., a CBH I without an R268 or R411 substitution). In certain embodiments, the R411 substitution results in an IC50 of cellobiose of at least 0.1 mM, at least 0.25 mM, or at least 0.5 mM. Exemplary R268 substituents are (a) histidine or lysine; (b) isoleucine, leucine, valine, phenylalanine, tyrosine, asparagine, serine, threonine, cysteine, or glycine; (c) alanine, tryptophan, aspartate, glutamate, or proline; or (d) glutamine or methionine. R268 substitutions were generally found to increase the specific activity of CBH I, in some cases up to 4.4-fold (see Table 13).
In certain embodiments, a polypeptide of the disclosure comprises an R411 substitution. The R411 substitution preferably results in an IC50 of cellobiose that is at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold or at least 140-fold the IC50 of cellobiose on the reference CBH I (e.g., a CBH I without an R268 or R411 substitution). In certain embodiments, the R411 substitution results in an IC50 of cellobiose of at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM or at least 8 mM. Exemplary R411 substituents are (a) alanine, aspartate, serine, cysteine, or proline; (b) valine, glutamate, histidine, lysine, threonine, glycine, methionine, or, optionally, glutamine; (c) leucine, phenylalanine, tryptophan, tyrosine, or asparagine; or (d) isoleucine. R411 substitutions were generally found to not impact or slightly decrease the specific activity of CBH I.
It was surprisingly discovered that introducing both R268 and R411 substitutions resulted in synergistic effects on CBH I product tolerance (see Table 12), without meaningfully affecting, and in several cases increasing, specific activity of the enzyme (see Table 13). Accordingly, introducing both R268 and R411 substitutions into a CBH I molecule is particularly beneficial.
The CBH I polypeptides the disclosure with both R268 and R411 substitutions preferably show a 100-fold to 1,000-fold improvement in tolerance to cellobiose, and a specific activity of 0.7-fold to 3-fold the specific activity, of a wild type CBH I which does not have either R268 or R411 substitutions. In some embodiments of the foregoing ranges, the improvement in cellobiose tolerance is at least 200- or 300-fold, and the specific activity is at least 1-fold or at least 1.5-fold the specific activity of said wild type CBH I.
In certain aspects, a CBH I polypeptide of the disclosure is any variant having the amino acid substitutions enumerated in Table 14, which shows 399 possible R268 and/or R411 amino acid substitutions (with a dash “-” indicating a wild type “R” residue). Thus, the variant can be characterized by a single R268 or R411 substitution or a double R268/R411 substitution. Variants with single R268 substitutions can be selected from variant nos. 281-299 in Table 14, and variants with single R411 substitutions can be selected from variant nos. 15, 35, 55, 75, 95, 115, 135, 155, 175, 215, 235, 255, 275, 314, 334, 354, 374, and 396 in Table 14. Variants with a double R268/R411 substitution can be selected from variant nos. 1-14, 16-34, 36-54, 56-74, 76-94, 96-114, 116-134, 136-154, 156-174, 176-194, 196-214, 216-234, 236-254, 256-74, 276-280, 300-313, 315-333, 335-353, 355-373, 375-393, and 395-399. In specific embodiments, the variant does not have the same substitutions as one or more of variants 1, 9, 15, 161, 169, 175, 281 and/or 289 of Table 14.
In certain embodiments, R268 and/or R411 substituents can include lysines and/or alanines. Accordingly, the present disclosure provides a variant CBH I polypeptide comprising a CBH I catalytic domain with one of the following amino acid substitutions or pairs of R268 and/or R411 substitutions: (a) R268K and R411K; (b) R268K and R411A; (c) R268A and R411K; (d) R268A and R411A; (e) R268A; (f) R268K; (g) R411A; and (h) R411K. In some embodiments, however, the amino acid sequence of the variant CBH I polypeptide does not comprise or consist of SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, or SEQ ID NO:302.
The variant CBHI polypeptides of the disclosure typically include a CD comprising an amino acid sequence having at least 50% sequence identity to a CD of a reference CBH I exemplified in Table 1. The CD portions of the CBH I polypeptides exemplified in Table 1 are delineated in Table 3. The variant CBH I polypeptides can have a cellulose binding domain (“CBD”) sequence in addition to the catalytic domain (“CD”) sequence. The CBD can be N- or C-terminal to the CD, and the CBD and CD are optionally connected via a linker sequence.
The variant CBH I polypeptides can be mature polypeptides or they may further comprise a signal sequence.
Additional embodiments of the variant CBH I polypeptides are provided in Section 1.1.
The variant CBH I polypeptides of the disclosure typically exhibit reduced product inhibition by cellobiose. In certain embodiments, the IC50 of cellobiose towards a variant CBH I polypeptide of the disclosure is at least 1.2-fold, at least 1.5-fold, or at least 2-fold the IC50 of cellobiose towards a reference CBH I lacking the R268 substitution and/or R411 substitution present in the variant. Additional embodiments of the product inhibition characteristics of the variant CBH I polypeptides are provided in Section 1.1.
The variant CBH I polypeptides of the disclosure typically retain some cellobiohydrolase activity. In certain embodiments, a variant CBH I polypeptide retains at least 50% the CBH I activity of a reference CBH I lacking the R268 substitution and/or R411 substitution present in the variant. Additional embodiments of cellobiohydrolase activity of the variant CBH I polypeptides are provided in Section 1.1.
The present disclosure further provides compositions (including cellulase compositions, e.g., whole cellulase compositions, and fermentation broths) comprising variant CBH I polypeptides. Additional embodiments of compositions comprising variant CBH I polypeptides are provided in Section 1.3. The variant CBH I polypeptides and compositions comprising them can be used, inter alia, in processes for saccharifying biomass. Additional details of saccharification reactions, and additional applications of the variant CBH I polypeptides, are provided in Section 1.4.
The present disclosure further provides nucleic acids (e.g., vectors) comprising nucleotide sequences encoding variant CBH I polypeptides as described herein, and recombinant cells engineered to express the variant CBH I polypeptides. The recombinant cell can be a prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or filamentous fungal) cell. Further provided are methods of producing and optionally recovering the variant CBH I polypeptides. Additional embodiments of the recombinant expression system suitable for expression and production of the variant CBH I polypeptides are provided in Section 1.2.
TABLE 1: Amino acid sequences of exemplary “reference” CBH I polypeptides that can be modified at positions corresponding to R268 and/or R411 in T. reesei CBH I (SEQ ID NO:2). The database accession numbers are indicated in the second column. Unless indicated otherwise, the accession numbers refer to the Genbank database. “#” indicates that the CBH I has no signal peptide; “&” indicate that the sequence is from the PDB database and represents the catalytic domain only without signal sequence; * indicates a nonpublic database. These amino acid sequences are mostly wild type, with the exception of some sequences from the PDB database which contain mutations to facilitate protein crystallization.
TABLE 2: Amino acid positions in the exemplary reference CBH I polypeptides that correspond to R268 and R411 in T. reesei CBH I. Database descriptors are as for Table 1.
TABLE 3: Approximate amino acid positions of CBH I polypeptide domains. Abbreviations used: SS is signal sequence; CD is catalytic domain; and CBD is cellulose binding domain. Database descriptors are as for Table 1.
TABLE 4: Table 4 shows a segment within the catalytic domain of each exemplary reference CBH I polypeptide containing the active site loop (shown in bold, underlined text) and the catalytic residues (glutamates in most CBH I polypeptides) (shown in bold, double underlined text). Database descriptors are as for Table 1.
TABLE 5: MUL and bagasse assay results for variants of BD29555. ND means not determined. ±% Activity (+/− cellobiose)=[(Activity with cellobiose)/(Activity without cellobiose)]*100. ¥ % Activity (−/+BG)=[(Activity without BG)/(Activity with BG)]*100]
TABLE 6: MUL and bagasse assay results for variants of T. reesei CBH I. ND means not determined. +% Activity (+/− cellobiose)=[(Activity with cellobiose)/(Activity without cellobiose)]*100. ¥% Activity (−/+BG)=[(Activity without BG)/(Activity with BG)]*100.
TABLE 7: Informal sequence listing. SEQ ID NO:1-149 correspond to the exemplary reference CBH I polypeptides. SEQ ID NO:299 corresponds to mature T. reesei CBH I (amino acids 26-529 of SEQ ID NO:2) with an R268A substitution. SEQ ID NO:300 corresponds to mature T. reesei CBH I (amino acids 26-529 of SEQ ID NO:2) with an R411A substitution. SEQ ID NO:301 corresponds to full length BD29555 with both an R268K substitution and an R411K substitution. SEQ ID NO:302 corresponds to mature BD29555 with both an R268K substitution and an R411K substitution.
TABLE 8: Primary Screening Results (10 μL enzyme; cellobiose range: 0.0001-100 mM; n=1)
TABLE 9: Secondary Screening IC50s (CBH I levels normalized to 5 μg/μL; cellobiose range: 0.0001-100 mM)
TABLE 10: Secondary Screening IC50, (CBH I levels normalized to 5 μg/μL, cellobiose range: 0.00085-100 mM)
TABLE 11: Secondary Screening IC50s (304 harvested supernatant; cellobiose range: 0.00085-100 mM)
TABLE 12: Merged IC50 values (from Tables 8-11) showing increased tolerance by single mutations and synergistic increase by double mutation. ND=not determined; ¥=data with fewer than 3 replicates and/or curve fitting with R2<0.95; * Improvement of variant IC50 value over wild type=variant/WT (where WT IC50=0.046); ̂ expected=additive IC50 value based on single measurements; ** synergistic increase=measured/expected.
TABLE 13: Specific Activity (SA, μmol 4 MU/min/mg CBH I) values. *Δ SA: change in specific activity; ratio of variant: WT; ¥ data derived from variants with low protein quantification, with fewer than 3 replicates and/or curve fitting with R2<0.95; WT Specific Activity=0.76.
TABLE 14: Table of possible single and double R268 and/or R411 substitutions that can be introduced into a CBH I polypeptide.
The present disclosure relates to variant CBH I polypeptides. Most naturally occurring CBH I polypeptides have arginines at positions corresponding to R268 and R411 of T. reesei CBH I (SEQ ID NO:2). The variant CBH I polypeptides of the present disclosure include a substitution at either or both positions resulting in a reduction of product (e.g., cellobiose) inhibition, and/or an improved specific activity. The following subsections describe in greater detail the variant CBH I polypeptides and exemplary methods of their production, exemplary cellulase compositions comprising them, and some industrial applications of the polypeptides and cellulase compositions.
1.1. Variant CBH I Polypeptides
The present disclosure provides variant CBH I polypeptides comprising at least one amino acid substitution that results in reduced product inhibition. “Variant” means a polypeptide which differs in sequence from a reference polypeptide by substitution of one or more amino acids at one or a number of different sites in the amino acid sequence. Exemplary reference CBH I polypeptides are shown in Table 1.
The variant CBH I polypeptides of the disclosure have an amino acid substitution at the amino acid position corresponding to R268 of T. reesei CBH I (SEQ ID NO:2) (an “R268 substitution”), (b) a substitution at the amino acid position corresponding to R411 of T. reesei CBH I (“R411 substitution”); or (c) both an R268 substitution and an R411 substitution, as compared to a reference CBH I polypeptide. It is noted that the R268 and R411 numbering is made by reference to the full length T. reesei CBH I, which includes a signal sequence that is generally absent from the mature enzyme. The corresponding numbering in the mature T. reesei CBH I (see, e.g., SEQ ID NO:4) is R251 and R394, respectively.
Accordingly, the present disclosure provides variant CBH I polypeptides in which at least one of the amino acid positions corresponding to R268 and R411 of T. reesei CBH I, and optionally both the amino acid positions corresponding to 8268 and R411 of T. reesei CBH I, is not an arginine.
The amino acid positions in the reference polypeptides of Table 1 that correspond to R268 and R411 in T. reesei CBH I are shown in Table 2. Amino acid positions in other CBH 1 polypeptides that correspond to R268 and R411 can be identified through alignment of their sequences with T. reesei CBH I using a sequence comparison algorithm. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2:482-89; by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443-53; by the search for similarity method of Pearson & Lipman, 1988, Proc. Nat'l Acad. Sci. USA 85:2444-48, by 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 by visual inspection.
The R268 and/or R411 substitutions can be selected from Table 14, which includes all possible 399 possible single and double R268 and R411 substitutions. In certain embodiments, the variants (a) R268K and R411K; (b) R268K and R411A; (c) R268A and R411K; (d) R268A and R411A; (e) R268A; (f) R268K; (g) R411A; or (h) R411K. In other embodiments, the variants are any variants in Table 14 except one or more of the variants (a) R268K and R411K; (b) R268K and R411A; (c) R268A and R411K; (d) R268A and R411A; (e) R268A; (f) R268K; (g) R411A; and (h) R411K.
CBH I polypeptides belong to the glycosyl hydrolase family 7 (“GH7”). The glycosyl hydrolases of this family include endoglucanases and cellobiohydrolases (exoglucanases). The cellobiohydrolases act processively from the reducing ends of cellulose chains to generate cellobiose. Cellulases of bacterial and fungal origin characteristically have a small cellulose-binding domain (“CBD”) connected to either the N or the C terminus of the catalytic domain (“CD”) via a linker peptide (see Suumakki et al., 2000, Cellulose 7: 189-209). The CD contains the active site whereas the CBD interacts with cellulose by binding the enzyme to it (van Tilbeurgh et al., 1986, FEBS Lett. 204(2): 223-227; Tomme et al., 1988, Eur. J. Biochem. 170:575-581). The three-dimensional structure of the catalytic domain of T. reesei CBH I has been solved (Divne et al., 1994, Science 265:524-528). The CD consists of two β-sheets that pack face-to-face to form a β-sandwich. Most of the remaining amino acids in the CD are loops connecting the β-sheets. Some loops are elongated and bend around the active site, forming cellulose-binding tunnel of (˜50 Å). In contrast, endoglucanases have an open substrate binding cleft/groove rather than a tunnel. Typically, the catalytic residues are glutamic acids corresponding to E229 and E234 of T. reesei CBH I.
The loops characteristic of the active sites (“the active site loops”) of reference CBH I polypeptides, which are absent from GH7 family endoglucanases, as well as catalytic glutamate residues of the reference CBH I polypeptides, are shown in Table 4. The variant CBH I polypeptides of the disclosure preferably retain the catalytic glutamate residues or may include a glutamine instead at the position corresponding to E234, as for SEQ ID NO:4. In some embodiments, the variant CBH I polypeptides contain no substitutions or only conservative substitutions in the active site loops relative to the reference CBH I polypeptides from which the variants are derived.
Many CBH I polypeptides do not have a CBD, and most studies concerning the activity of cellulase domains on different substrates have been carried out with only the catalytic domains of CBH I polypeptides. Because CDs with cellobiohydrolase activity can be generated by limited proteolysis of mature CBH I by papain (see, e.g., Chen et al., 1993, Biochem. Mol. Biol. Int. 30(5):901-10), they are often referred to as “core” domains. Accordingly, a variant CBH I can include only the CD “core” of CBH I. Exemplary reference CDs comprise amino acid sequences corresponding to positions 26 to 455 of SEQ ID NO:1, positions 18 to 444 of SEQ ID NO:2, positions 26 to 455 of SEQ ID NO:3, positions 1 to 427 of SEQ ID NO:4, positions 24 to 457 of SEQ ID NO:5, positions 18 to 448 of SEQ ID NO:6, positions 27 to 460 of SEQ ID NO:7, positions 27 to 460 of SEQ ID NO:8, positions 20 to 449 of SEQ ID NO:9, positions 1 to 424 of SEQ ID NO:10, positions 18 to 447 of SEQ ID NO:11, positions 18 to 434 of SEQ ID NO:12, positions 18 to 445 of SEQ ID NO:13, positions 19 to 454 of SEQ ID NO:14, positions 19 to 443 of SEQ ID NO:15, positions 2 to 426 of SEQ ID NO:16, positions 23 to 446 of SEQ ID NO:17, positions 19 to 449 of SEQ ID NO:18, positions 23 to 446 of SEQ ID NO:19, positions 19 to 449 of SEQ ID NO:20, positions 2 to 416 of SEQ ID NO:21, positions 19 to 454 of SEQ ID NO:22, positions 19 to 447 of SEQ ID NO:23, positions 19 to 447 of SEQ ID NO:24, positions 20 to 443 of SEQ ID NO:25, positions 18 to 447 of SEQ ID NO:26, positions 19 to 442 of SEQ ID NO:27, positions 18 to 451 of SEQ ID NO:28, positions 23 to 446 of SEQ ID NO:29, positions 18 to 444 of SEQ ID NO:30, positions 18 to 451 of SEQ ID NO:31, positions 18 to 447 of SEQ ID NO:32, positions 19 to 449 of SEQ ID NO:33, positions 18 to 447 of SEQ ID NO:34, positions 26 to 459 of SEQ ID NO:35, positions 19 to 450 of SEQ ID NO:36, positions 19 to 453 of SEQ ID NO:37, positions 18 to 448 of SEQ ID NO:38, positions 19 to 443 of SEQ ID NO:39, positions 19 to 442 of SEQ ID NO:40, positions 18 to 444 of SEQ ID NO:41, positions 24 to 457 of SEQ ID NO:42, positions 18 to 449 of SEQ ID NO:43, positions 19 to 453 of SEQ ID NO:44, positions 26 to 456 of SEQ ID NO:45, positions 19 to 451 of SEQ ID NO:46, positions 18 to 443 of SEQ ID NO:47, positions 18 to 448 of SEQ ID NO:48, positions 19 to 451 of SEQ ID NO:49, positions 18 to 444 of SEQ ID NO:50, positions 2 to 419 of SEQ ID NO:51, positions 27 to 461 of SEQ ID NO:52, positions 21 to 445 of SEQ ID NO:53, positions 19 to 449 of SEQ ID NO:54, positions 19 to 448 of SEQ ID NO:55, positions 18 to 443 of SEQ ID NO:56, positions 20 to 443 of SEQ ID NO:57, positions 18 to 448 of SEQ ID NO:58, positions 18 to 447 of SEQ ID NO:59, positions 26 to 455 of SEQ ID NO:60, positions 19 to 449 of SEQ ID NO:61, positions 19 to 449 of SEQ ID NO:62, positions 26 to 460 of SEQ ID NO:63, positions 18 to 448 of SEQ ID NO:64, positions 19 to 451 of SEQ ID NO:65, positions 19 to 447 of SEQ ID NO:66, positions 1 to 424 of SEQ ID NO:67, positions 19 to 448 of SEQ ID NO:68, positions 19 to 443 of SEQ ID NO:69, positions 23 to 447 of SEQ ID NO:70, positions 17 to 448 of SEQ ID NO:71, positions 19 to 449 of SEQ ID NO:72, positions 18 to 444 of SEQ ID NO:73, positions 23 to 458 of SEQ ID NO:74, positions 20 to 452 of SEQ ID NO:75, positions 18 to 435 of SEQ ID NO:76, positions 18 to 446 of SEQ ID NO:77, positions 22 to 457 of SEQ ID NO:78, positions 18 to 448 of SEQ ID NO:79, positions 1 to 431 of SEQ ID NO:80, positions 19 to 453 of SEQ ID NO:81, positions 21 to 440 of SEQ ID NO:82, positions 19 to 442 of SEQ ID NO:83, positions 18 to 448 of SEQ ID NO:84, positions 17 to 446 of SEQ ID NO:85, positions 18 to 447 of SEQ ID NO:86, positions 18 to 443 of SEQ ID NO:87, positions 23 to 448 of SEQ ID NO:88, positions 18 to 451 of SEQ ID NO:89, positions 21 to 447 of SEQ ID NO:90, positions 18 to 444 of SEQ ID NO:91, positions 19 to 442 of SEQ ID NO:92, positions 20 to 436 of SEQ ID NO:93, positions 18 to 450 of SEQ ID NO:94, positions 22 to 453 of SEQ ID NO:95, positions 16 to 472 of SEQ ID NO:96, positions 21 to 445 of SEQ ID NO:97, positions 19 to 447 of SEQ ID NO:98, positions 19 to 450 of SEQ ID NO:99, positions 19 to 451 of SEQ ID NO:100, positions 18 to 448 of SEQ ID NO:101, positions 19 to 442 of SEQ ID NO:102, positions 20 to 457 of SEQ ID NO:103, positions 19 to 454 of SEQ ID NO:104, positions 18 to 440 of SEQ ID NO:105, positions 18 to 439 of SEQ ID NO:106, positions 27 to 460 of SEQ ID NO:107, positions 23 to 446 of SEQ ID NO:108, positions 17 to 446 of SEQ ID NO:109, positions 21 to 447 of SEQ ID NO:110, positions 19 to 447 of SEQ ID NO:111, positions 18 to 449 of SEQ ID NO:112, positions 22 to 457 of SEQ ID NO:113, positions 18 to 445 of SEQ ID NO:114, positions 18 to 448 of SEQ ID NO:115, positions 18 to 448 of SEQ ID NO:116, positions 23 to 435 of SEQ ID NO:117, positions 21 to 442 of SEQ ID NO:118, positions 23 to 435 of SEQ ID NO:119, positions 20 to 445 of SEQ ID NO:120, positions 21 to 443 of SEQ ID NO:121, positions 20 to 445 of SEQ ID NO:122, positions 23 to 443 of SEQ ID NO:123, positions 20 to 445 of SEQ ID NO:124, positions 21 to 435 of SEQ ID NO:125, positions 20 to 437 of SEQ ID NO:126, positions 21 to 442 of SEQ ID NO:127, positions 23 to 434 of SEQ ID NO:128, positions 20 to 444 of SEQ ID NO:129, positions 21 to 435 of SEQ ID NO:130, positions 20 to 445 of SEQ ID NO:131, positions 21 to 446 of SEQ ID NO:132, positions 21 to 435 of SEQ ID NO:133, positions 22 to 448 of SEQ ID NO:134, positions 23 to 433 of SEQ ID NO:135, positions 23 to 434 of SEQ ID NO:136, positions 23 to 435 of SEQ ID NO:137, positions 23 to 435 of SEQ ID NO:138, positions 20 to 445 of SEQ ID NO:139, positions 20 to 437 of SEQ ID NO:140, positions 21 to 435 of SEQ ID NO:141, positions 20 to 437 of SEQ ID NO:142, positions 21 to 435 of SEQ ID NO:143, positions 26 to 435 of SEQ ID NO:144, positions 23 to 435 of SEQ ID NO:145, positions 24 to 443 of SEQ ID NO:146, positions 20 to 445 of SEQ ID NO:147, positions 21 to 441 of SEQ ID NO:148, and positions 20 to 437 of SEQ ID NO:149.
The CBDs are particularly involved in the hydrolysis of crystalline cellulose. It has been shown that the ability of cellobiohydrolases to degrade crystalline cellulose decreases when the CBD is absent (Linder and Teed, 1997, Journal of Biotechnol. 57:15-28). The variant CBH I polypeptides of the disclosure can further include a CBD. Exemplary CBDs comprise amino acid sequences corresponding to positions 494 to 529 of SEQ ID NO:1, positions 480 to 514 of SEQ ID NO:2, positions 494 to 529 of SEQ ID NO:3, positions 491 to 526 of SEQ ID NO:5, positions 477 to 512 of SEQ ID NO:6, positions 497 to 532 of SEQ ID NO:7, positions 504 to 539 of SEQ ID NO:8, positions 486 to 521 of SEQ ID NO:13, positions 556 to 596 of SEQ ID NO:15, positions 490 to 525 of SEQ ID NO:18, positions 495 to 530 of SEQ ID NO:20, positions 471 to 506 of SEQ ID NO:23, positions 481 to 516 of SEQ ID NO:27, positions 480 to 514 of SEQ ID NO:30, positions 495 to 529 of SEQ ID NO:35, positions 493 to 528 of SEQ ID NO:36, positions 477 to 512 of SEQ ID NO:38, positions 547 to 586 of SEQ ID NO:39, positions 475 to 510 of SEQ ID NO:40, positions 479 to 513 of SEQ ID NO:41, positions 506 to 541 of SEQ ID NO:42, positions 481 to 516 of SEQ ID NO:43, positions 503 to 537 of SEQ ID NO:45, positions 488 to 523 of SEQ ID NO:46, positions 476 to 511 of SEQ ID NO:48, positions 488 to 523 of SEQ ID NO:49, positions 479 to 513 of SEQ ID NO:50, positions 500 to 535 of SEQ ID NO:52, positions 493 to 528 of SEQ ID NO:55, positions 479 to 514 of SEQ ID NO:58, positions 494 to 529 of SEQ ID NO:60, positions 490 to 525 of SEQ ID NO:61, positions 497 to 532 of SEQ ID NO:62, positions 475 to 510 of SEQ ID NO:64, positions 477 to 512 of SEQ ID NO:65, positions 486 to 521 of SEQ ID NO:66, positions 470 to 505 of SEQ ID NO:67, positions 491 to 526 of SEQ ID NO:68, positions 476 to 511 of SEQ ID NO:69, positions 480 to 514 of SEQ ID NO:73, positions 506 to 540 of SEQ ID NO:74, positions 471 to 504 of SEQ ID NO:76, positions 501 to 536 of SEQ ID NO:78, positions 473 to 508 of SEQ ID NO:79, positions 481 to 516 of SEQ ID NO:83, positions 488 to 523 of SEQ ID NO:86, positions 475 to 510 of SEQ ID NO:92, positions 468 to 504 of SEQ ID NO:93, positions 501 to 536 of SEQ ID NO:96, positions 482 to 517 of SEQ ID NO:98, positions 481 to 516 of SEQ ID NO:99, positions 488 to 523 of SEQ ID NO:100, positions 472 to 507 of SEQ ID NO:101, positions 481 to 516 of SEQ ID NO:102, positions 471 to 505 of SEQ ID NO:105, positions 481 to 516 of SEQ ID NO:106, positions 495 to 530 of SEQ ID NO:107, positions 488 to 523 of SEQ ID NO:111, positions 478 to 513 of SEQ ID NO:112, positions 501 to 536 of SEQ ID NO:113, positions 491 to 526 of SEQ ID NO:115, and positions 503 to 538 of SEQ ID NO:116.
The CD and CBD are often connected via a linker. Exemplary linker sequences correspond to positions 456 to 493 of SEQ ID NO:1, positions 445 to 479 of SEQ ID NO:2, positions 456 to 493 of SEQ ID NO:3, positions 458 to 490 of SEQ ID NO:5, positions 449 to 476 of SEQ ID NO:6, positions 461 to 496 of SEQ ID NO:7, positions 461 to 503 of SEQ ID NO:8, positions 446 to 485 of SEQ ID NO:13, positions 444 to 555 of SEQ ID NO:15, positions 450 to 489 of SEQ ID NO:18, positions 450 to 494 of SEQ ID NO:20, positions 448 to 470 of SEQ ID NO:23, positions 443 to 480 of SEQ ID NO:27, positions 445 to 479 of SEQ ID NO:30, positions 460 to 494 of SEQ ID NO:35, positions 451 to 492 of SEQ ID NO:36, positions 449 to 476 of SEQ ID NO:38, positions 444 to 546 of SEQ ID NO:39, positions 443 to 474 of SEQ ID NO:40, positions 445 to 478 of SEQ ID NO:41, positions 458 to 505 of SEQ ID NO:42, positions 450 to 480 of SEQ ID NO:43, positions 457 to 502 of SEQ ID NO:45, positions 452 to 487 of SEQ ID NO:46, positions 449 to 475 of SEQ ID NO:48, positions 452 to 487 of SEQ ID NO:49, positions 445 to 478 of SEQ ID NO:50, positions 462 to 499 of SEQ ID NO:52, positions 449 to 492 of SEQ ID NO:55, positions 449 to 478 of SEQ ID NO:58, positions 456 to 493 of SEQ ID NO:60, positions 450 to 489 of SEQ ID NO:61, positions 450 to 496 of SEQ ID NO:62, positions 449 to 474 of SEQ ID NO:64, positions 452 to 476 of SEQ ID NO:65, positions 448 to 485 of SEQ ID NO:66, positions 425 to 469 of SEQ ID NO:67, positions 449 to 490 of SEQ ID NO:68, positions 444 to 475 of SEQ ID NO:69, positions 445 to 479 of SEQ ID NO:73, positions 459 to 505 of SEQ ID NO:74, positions 436 to 470 of SEQ ID NO:76, positions 458 to 500 of SEQ ID NO:78, positions 449 to 472 of SEQ ID NO:79, positions 443 to 480 of SEQ ID NO:83, positions 448 to 487 of SEQ ID NO:86, positions 443 to 474 of SEQ ID NO:92, positions 437 to 467 of SEQ ID NO:93, positions 473 to 500 of SEQ ID NO:96, positions 448 to 481 of SEQ ID NO:98, positions 451 to 480 of SEQ ID NO:99, positions 452 to 487 of SEQ ID NO:100, positions 449 to 471 of SEQ ID NO:101, positions 443 to 480 of SEQ ID NO:102, positions 441 to 470 of SEQ ID NO:105, positions 440 to 480 of SEQ ID NO:106, positions 461 to 494 of SEQ ID NO:107, positions 448 to 487 of SEQ ID NO:111, positions 450 to 478 of SEQ ID NO:112, positions 458 to 500 of SEQ ID NO:113, positions 449 to 490 of SEQ ID NO:115, and positions 449 to 502 of SEQ ID NO:116.
Because CBH I polypeptides are modular, the CBDs, CDs and linkers of different CBH I polypeptides, such as the exemplary CBH I polypeptides of Table 1, can be used interchangeably. However, in a preferred embodiment, the CBDs, CDs and linkers of a variant CBH I of the disclosure originate from the same polypeptide.
The variant CBH I polypeptides of the disclosure preferably have at least a two-fold reduction of product inhibition, such that cellobiose has an IC50 towards the variant CBH I that is at least 2-fold the IC50 of the corresponding reference CBH I, e.g., CBH I lacking the R268 substitution and/or R411 substitution. More preferably the IC50 of cellobiose towards the variant CBH I is at least 3-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 500-fold or at least 700-fold greater tolerance to cellobiose, and in some cases up to 750-fold or up to 1,000-fold, the IC50 of the corresponding reference CBH I. In specific embodiments the IC50 of cellobiose towards the variant CBH I is ranges from 2-fold to 15-fold, from 2-fold to 10-fold, from 3-fold to 10-fold, from 5-fold to 12-fold, from 4-fold to 12-fold, from 5-fold to 10-fold, from 5-fold to 12-fold, from 2-fold to 8-fold, from 8-fold to 20-fold, from 20-fold to 100-fold, from 50-fold to 150-fold, from 150-fold to 500-fold, from 200-fold to 750-fold, from 50-fold to 700-fold, or from 100-fold to 1,000-fold the IC50 of the corresponding reference CBH I.
The IC50 can be determined in a phosphoric acid swollen cellulose (“PASC”) assay (Du et al., 2010, Applied Biochemistry and Biotechnology 161:313-317) or a methylumbelliferyl lactoside (“MUL”) assay (van Tilbeurgh and Claeyssens, 1985, FEBS Letts. 187(2):283-288), as exemplified in the Examples below.
The variant CBH I polypeptides of the disclosure preferably have a cellobiohydrolase activity that is at least 30% the cellobiohydrolase activity of the corresponding reference CBH I, e.g., CBH I lacking the R268 substitution and/or R411 substitution. More preferably, the cellobiohydrolase activity of the variant CBH I is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% the cellobiohydrolase activity of the corresponding reference CBH I, and in some cases 150%, 200%, 250%, 300%, 350%, 400% or 450% the cellobiohydrolase activity of the corresponding reference CBH I. In specific embodiments the cellobiohydrolase activity of the variant CBH I is ranges from 30% to 80%, from 40% to 70%, 30% to 60%, from 50% to 80%, from 60% to 80%, from 70% to 450%, from 80% to 350%, from 100% to 450%, from 150% to 450%, from 100% to 400%, from 150% to 400%, or from 90% to 450% of the cellobiohydrolase activity of the corresponding reference CBH I. Assays for cellobiohydrolase activity are described, for example, in Becker et al., 2011, Biochem J. 356:19-30 and Mitsuishi et al., 1990, FEBS Letts. 275:135-138, each of which is expressly incorporated by reference herein. The ability of CBH I to hydrolyze isolated soluble and insoluble substrates can also be measured using assays described in Srisodsuk et al., 1997, J. Biotech. 57:4957 and Nidetzky and Claeyssens, 1994, Biotech. Bioeng. 44:961-966. Substrates useful for assaying cellobiohydrolase activity include crystalline cellulose, filter paper, phosphoric acid swollen cellulose, cellooligosaccharides, methylumbelliferyl lactoside, methylumbelliferyl cellobioside, orthonitrophenyl lactoside, paranitrophenyl lactoside, orthonitrophenyl cellobioside, paranitrophenyl cellobioside. Cellobiohydrolase activity can be measured in an assay utilizing PASC as the substrate and a calcofluor white detection method (Du et al., 2010, Applied Biochemistry and Biotechnology 161:313-317). PASC can be prepared as described by Walseth, 1952, TAPPI 35:228-235 and Wood, 1971, Biochem. J. 121:353-362.
Other than said R268 and/or R411 substitution, the variant CBH I polypeptides of the disclosure preferably:
An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul et al., 1990, J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1992, Proc. Nat'l. Acad. Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparison of both strands.
Most CBH I polypeptides are secreted and are therefore expressed with a signal sequence that is cleaved upon secretion of the polypeptide from the cell. Accordingly, in certain aspects, the variant CBH I polypeptides of the disclosure further include a signal sequence. Exemplary signal sequences comprise amino acid sequences corresponding to positions 1 to 25 of SEQ ID NO:1, positions 1 to 17 of SEQ ID NO:2, positions 1 to 25 of SEQ ID NO:3, positions 1 to 23 of SEQ ID NO:5, positions 1 to 17 of SEQ ID NO:6, positions 1 to 26 of SEQ ID NO:7, positions 1 to 27 of SEQ ID NO:8, positions 1 to 19 of SEQ ID NO:9, positions 1 to 17 of SEQ ID NO:11, positions 1 to 17 of SEQ ID NO:12, positions 1 to 17 of SEQ ID NO:13, positions 1 to 18 of SEQ ID NO:14, positions 1 to 18 of SEQ ID NO:15, positions 1 to 22 of SEQ ID NO:17, positions 1 to 18 of SEQ ID NO:18, positions 1 to 22 of SEQ ID NO:19, positions 1 to 18 of SEQ ID NO:20, positions 1 to 18 of SEQ ID NO:22, positions 1 to 18 of SEQ ID NO:23, positions 1 to 18 of SEQ ID NO:24, positions 1 to 19 of SEQ ID NO:25, positions 1 to 17 of SEQ ID NO:26, positions 1 to 18 of SEQ ID NO:27, positions 1 to 17 of SEQ ID NO:28, positions 1 to 22 of SEQ ID NO:29, positions 1 to 18 of SEQ ID NO:30, positions 1 to 17 of SEQ ID NO:31, positions 1 to 17 of SEQ ID NO:32, positions 1 to 18 of SEQ ID NO:33, positions 1 to 17 of SEQ ID NO:34, positions 1 to 25 of SEQ ID NO:35, positions 1 to 18 of SEQ ID NO:36, positions 1 to 18 of SEQ ID NO:37, positions 1 to 17 of SEQ ID NO:38, positions 1 to 18 of SEQ ID NO:39, positions 1 to 18 of SEQ ID NO:40, positions 1 to 17 of SEQ ID NO:41, positions 1 to 23 of SEQ ID NO:42, positions 1 to 17 of SEQ ID NO:43, positions 1 to 18 of SEQ ID NO:44, positions 1 to 25 of SEQ ID NO:45, positions 1 to 18 of SEQ ID NO:46, positions 1 to 17 of SEQ ID NO:47, positions 1 to 17 of SEQ ID NO:48, positions 1 to 18 of SEQ ID NO:49, positions 1 to 17 of SEQ ID NO:50, positions 1 to 26 of SEQ ID NO:52, positions 1 to 20 of SEQ ID NO:53, positions 1 to 18 of SEQ ID NO:54, positions 1 to 18 of SEQ ID NO:55, positions 1 to 17 of SEQ ID NO:56, positions 1 to 19 of SEQ ID NO:57, positions 1 to 17 of SEQ ID NO:58, positions 1 to 17 of SEQ ID NO:59, positions 1 to 25 of SEQ ID NO:60, positions 1 to 18 of SEQ ID NO:61, positions 1 to 18 of SEQ ID NO:62, positions 1 to 25 of SEQ ID NO:63, positions 1 to 17 of SEQ ID NO:64, positions 1 to 18 of SEQ ID NO:65, positions 1 to 18 of SEQ ID NO:66, positions 1 to 18 of SEQ ID NO:68, positions 1 to 18 of SEQ ID NO:69, positions 1 to 23 of SEQ ID NO:70, positions 1 to 17 of SEQ ID NO:71, positions 1 to 18 of SEQ ID NO:72, positions 1 to 17 of SEQ ID NO:73, positions 1 to 22 of SEQ ID NO:74, positions 1 to 19 of SEQ ID NO:75, positions 1 to 17 of SEQ ID NO:76, positions 1 to 17 of SEQ ID NO:77, positions 1 to 21 of SEQ ID NO:78, positions 1 to 18 of SEQ ID NO:79, positions 1 to 18 of SEQ ID NO:81, positions 1 to 20 of SEQ ID NO:82, positions 1 to 18 of SEQ ID NO:83, positions 1 to 17 of SEQ ID NO:84, positions 1 to 16 of SEQ ID NO:85, positions 1 to 17 of SEQ ID NO:86, positions 1 to 17 of SEQ ID NO:87, positions 1 to 22 of SEQ ID NO:88, positions 1 to 17 of SEQ ID NO:89, positions 1 to 20 of SEQ ID NO:90, positions 1 to 17 of SEQ ID NO:91, positions 1 to 18 of SEQ ID NO:92, positions 1 to 19 of SEQ ID NO:93, positions 1 to 17 of SEQ ID NO:94, positions 1 to 21 of SEQ ID NO:95, positions 1 to 15 of SEQ ID NO:96, positions 1 to 20 of SEQ ID NO:97, positions 1 to 18 of SEQ ID NO:98, positions 1 to 18 of SEQ ID NO:99, positions 1 to 18 of SEQ ID NO:100, positions 1 to 17 of SEQ ID NO:101, positions 1 to 18 of SEQ ID NO:102, positions 1 to 19 of SEQ ID NO:103, positions 1 to 18 of SEQ ID NO:104, positions 1 to 17 of SEQ ID NO:105, positions 1 to 17 of SEQ ID NO:106, positions 1 to 26 of SEQ ID NO:107, positions 1 to 22 of SEQ ID NO:108, positions 1 to 16 of SEQ ID NO:109, positions 1 to 20 of SEQ ID NO:110, positions 1 to 18 of SEQ ID NO:111, positions 1 to 17 of SEQ ID NO:112, positions 1 to 21 of SEQ ID NO:113, positions 1 to 17 of SEQ ID NO:114, positions 1 to 17 of SEQ ID NO:115, positions 1 to 18 of SEQ ID NO:116, positions 1 to 22 of SEQ ID NO:117, positions 1 to 20 of SEQ ID NO:118, positions 1 to 22 of SEQ ID NO:119, positions 1 to 19 of SEQ ID NO:120, positions 1 to 20 of SEQ ID NO:121, positions 1 to 19 of SEQ ID NO:122, positions 1 to 22 of SEQ ID NO:123, positions 1 to 19 of SEQ ID NO:124, positions 1 to 20 of SEQ ID NO:125, positions 1 to 19 of SEQ ID NO:126, positions 1 to 21 of SEQ ID NO:127, positions 1 to 22 of SEQ ID NO:128, positions 1 to 19 of SEQ ID NO:129, positions 1 to 20 of SEQ ID NO:130, positions 1 to 19 of SEQ ID NO:131, positions 1 to 20 of SEQ ID NO:132, positions 1 to 20 of SEQ ID NO:133, positions 1 to 21 of SEQ ID NO:134, positions 1 to 22 of SEQ ID NO:135, positions 1 to 22 of SEQ ID NO:136, positions 1 to 22 of SEQ ID NO:137, positions 1 to 22 of SEQ ID NO:138, positions 1 to 19 of SEQ ID NO:139, positions 1 to 19 of SEQ ID NO:140, positions 1 to 20 of SEQ ID NO:141, positions 1 to 19 of SEQ ID NO:142, positions 1 to 20 of SEQ ID NO:143, positions 1 to 25 of SEQ ID NO:144, positions 1 to 22 of SEQ ID NO:145, positions 1 to 23 of SEQ ID NO:146, positions 1 to 19 of SEQ ID NO:147, positions 1 to 20 of SEQ ID NO:148, and positions 1 to 19 of SEQ ID NO:149.
1.2. Recombinant Expression of Variant CBH I Polypeptides
1.2.1. Cell Culture Systems
The disclosure also provides recombinant cells engineered to express variant CBH I polypeptides. Suitably, the variant CBH I polypeptide is encoded by a nucleic acid operably linked to a promoter. The promoters can be homologous or heterologous, and constitutive or inducible.
Suitable host cells include cells of any microorganism (e.g., cells of a bacterium, a protist, an alga, a fungus (e.g., a yeast or filamentous fungus), or other microbe), and are preferably cells of a bacterium, a yeast, or a filamentous fungus.
Where recombinant expression in a filamentous fungal host is desired, the promoter can be a fungal promoter (including but not limited to a filamentous fungal promoter), a promoter operable in plant cells, a promoter operable in mammalian cells.
As described in U.S. provisional application No. 61/553,901, filed Oct. 31, 2011, the contents of which are hereby incorporated in their entireties, promoters that are constitutively active in mammalian cells (which can derived from a mammalian genome or the genome of a mammalian virus) are capable of eliciting high expression levels in filamentous fungi such as Trichoderma reesei. An exemplary promoter is the cytomegalovirus (“CMV”) promoter.
As described in U.S. provisional application No. 61/553,897, filed Oct. 31, 2011, the contents of which are hereby incorporated in their entireties, promoters that are constitutively active in plant cells (which can derived from a plant genome or the genome of a plant virus) are capable of eliciting high expression levels in filamentous fungi such as Trichoderma reesei. Exemplary promoters are the cauliflower mosaic virus (“CaMV”) 35S promoter or the Commelina yellow mottle virus (“CoYMV”) promoter.
Mammalian, mammalian viral, plant and plant viral promoters can drive particularly high expression when the associated 5′ UTR sequence (i.e., the sequence which begins at the transcription start site and ends one nucleotide (nt) before the start codon) normally associated with the mammalian or mammalian viral promoter is replaced by a fungal 5′ UTR sequence.
The source of the 5′ UTR can vary provided it is operable in the filamentous fungal cell. In various embodiments, the 5′ UTR can be derived from a yeast gene or a filamentous fungal gene. The 5′ UTR can be from the same species one other component in the expression cassette (e.g. the promoter or the CBH I coding sequence), or from a different species. The 5′ UTR can be from the same species as the filamentous fungal cell that the expression construct is intended to operate in. In an exemplary embodiment, the 5′ UTR comprises a sequence corresponding to a fragment of a 5′ UTR from a T. reesei glyceraldehyde-3-phosphate dehydrogenase (gpd). In a specific embodiment, the 5′ UTR is not naturally associated with the CMV promoter
Examples of other promoters that can be used include, but are not limited to, a cellulase promoter, a xylanase promoter, the 1818 promoter (previously identified as a highly expressed protein by EST mapping Trichoderma). For example, the promoter can suitably be a cellobiohydrolase, endoglucanase, or β-glucosidase promoter. A particularly suitable promoter can be, for example, a T. reesei cellobiohydrolase, endoglucanase, or β-glucosidase promoter. Non-limiting examples of promoters include a cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, pki1, gpd1, xyn1, or xyn2 promoter.
Suitable host cells of the bacterial genera include, but are not limited to, cells of Escherichia, Bacillus, Lactobacillus, Pseudomonas, and Streptomyces. Suitable cells of bacterial species include, but are not limited to; cells of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa, and Streptomyces lividans.
Suitable host cells of the genera of yeast include, but are not limited to, cells of Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, and Phaffia. Suitable cells of yeast species include, but are not limited to, cells of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Pichia pastoris, P. canadensis, Kluyveromyces marxianus, and Phaffia rhodozyma.
Suitable host cells of filamentous fungi include all filamentous forms of the subdivision Eumycotina. Suitable cells of filamentous fungal genera include, but are not limited to, cells of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysoporium, Coprinus, Coriolus, Corynascus, Chaetomium, Cryptococcus, Filobasidium, Fusarium, Gibberella, Humicola, Hypocrea, Magnaporthe, Mucor, Myceliophthora, Mucor, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Scytaldium, Schizophyllum, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma. More preferably, the recombinant cell is a Trichoderma sp. (e.g., Trichoderma reesei), Penicillium sp., Humicola sp. (e.g., Humicola insolens); Aspergillus sp. (e.g., Aspergillus niger), Chrysosporium sp., Fusarium sp., or Hypocrea sp. Suitable cells can also include cells of various anamorph and teleomorph forms of these filamentous fungal genera.
Suitable cells of filamentous fungal species include, but are not limited to, cells of Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminurn, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.
The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the nucleic acid sequence encoding the variant CBH I polypeptide. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. As noted, many references are available for the culture and production of many cells, including cells of bacterial and fungal origin. Cell culture media in general are set forth in Atlas and Parks (eds.), 1993, The Handbook of Microbiological Media, CRC Press, Boca Raton, Fla., which is incorporated herein by reference. For recombinant expression in filamentous fungal cells, the cells are cultured in a standard medium containing physiological salts and nutrients, such as described in Pourquie et al., 1988, Biochemistry and Genetics of Cellulose Degradation, eds. Aubert, et al., Academic Press, pp. 71-86; and Ilmen et al., 1997, Appl. Environ. Microbiol. 63:1298-1306. Culture conditions are also standard, e.g., cultures are incubated at 28° C. in shaker cultures or fermenters until desired levels of variant CBH I expression are achieved. Preferred culture conditions for a given filamentous fungus may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection (ATCC). After fungal growth has been established, the cells are exposed to conditions effective to cause or permit the expression of a variant CBH I.
In cases where a variant CBH I coding sequence is under the control of an inducible promoter, the inducing agent, e.g., a sugar, metal salt or antibiotics, is added to the medium at a concentration effective to induce variant CBH I expression.
In one embodiment, the recombinant cell is an Aspergillus niger, which is a useful strain for obtaining overexpressed polypeptide. For example A. niger var. awamori dgr246 is known to product elevated amounts of secreted cellulases (Goedegebuur et al., 2002, Curr. Genet. 41:89-98). Other strains of Aspergillus niger var awamori such as GCDAP3, GCDAP4 and GAPS-4 are known (Ward et al., 1993, Appl. Microbiol. Biotechnol. 39:738-743).
In another embodiment, the recombinant cell is a Trichoderma reesei, which is a useful strain for obtaining overexpressed polypeptide. For example, RL-P37, described by Sheir-Neiss et al., 1984, Appl. Microbiol. Biotechnol. 20:46-53, is known to secrete elevated amounts of cellulase enzymes. Functional equivalents of RL-P37 include Trichoderma reesei strain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). It is contemplated that these strains would also be useful in overexpressing variant CBH I polypeptides.
Cells expressing the variant CBH I polypeptides of the disclosure can be grown under batch, fed-batch or continuous fermentations conditions. Classical batch fermentation is a closed system, wherein the compositions of the medium is set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a fed-batch fermentation in which the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.
1.2.2. Recombinant Expression in Plants
The disclosure provides transgenic plants and seeds that recombinantly express a variant CBH I polypeptide. The disclosure also provides plant products, e.g., oils, seeds, leaves, extracts and the like, comprising a variant CBH I polypeptide.
The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). The disclosure also provides methods of making and using these transgenic plants and seeds. The transgenic plant or plant cell expressing a variant CBH I can be constructed in accordance with any method known in the art. See, for example, U.S. Pat. No. 6,309,872. T. reesei CBH I has been successfully expressed in transgenic tobacco (Nicotiana tabaccum) and potato (Solanum tuberosum). See Hooker et al., 2000, in Glycosyl Hydrolases for Biomass Conversion, ACS Symposium Series, Vol. 769, Chapter 4, pp. 55-90.
In a particular aspect, the present disclosure provides for the expression of CBH I variants in transgenic plants or plant organs and methods for the production thereof. DNA expression constructs are provided for the transformation of plants with a nucleic acid encoding the variant CBH I polypeptide, preferably under the control of regulatory sequences which are capable of directing expression of the variant CBH I polypeptide. These regulatory sequences include sequences capable of directing transcription in plants, either constitutively, or in stage and/or tissue specific manners.
The expression of variant CBH I polypeptides in plants can be achieved by a variety of means. Specifically, for example, technologies are available for transforming a large number of plant species, including dicotyledonous species (e.g., tobacco, potato, tomato, Petunia, Brassica) and monocot species. Additionally, for example, strategies for the expression of foreign genes in plants are available. Additionally still, regulatory sequences from plant genes have been identified that are serviceable for the construction of chimeric genes that can be functionally expressed in plants and in plant cells (e.g., Klee, 1987, Ann. Rev. of Plant Phys. 38:467-486; Clark et al., 1990, Virology 179(2):640-7; Smith et al., 1990, Mol. Gen. Genet. 224(3):477-81.
The introduction of nucleic acids into plants can be achieved using several technologies including transformation with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Non-limiting examples of plant tissues that can be transformed include protoplasts, microspores or pollen, and explants such as leaves, stems, roots, hypocotyls, and cotyls. Furthermore, DNA encoding a variant CBH I can be introduced directly into protoplasts and plant cells or tissues by microinjection, electroporation, particle bombardment, and direct DNA uptake.
Variant CBH I polypeptides can be produced in plants by a variety of expression systems. For instance, the use of a constitutive promoter such as the 35S promoter of Cauliflower Mosaic Virus (Guilley et al., 1982, Cell 30:763-73) is serviceable for the accumulation of the expressed protein in virtually all organs of the transgenic plant. Alternatively, promoters that are tissue-specific and/or stage-specific can be used (Higgins, 1984, Annu. Rev. Plant Physiol. 35:191-221; Shotwell and Larkins, 1989, In: The Biochemistry of Plants Vol. 15 (Academic Press, San Diego: Stumpf and Conn, eds.), p. 297), permit expression of variant CBH I polypeptides in a target tissue and/or during a desired stage of development.
1.3. Compositions Of Variant CBH I Polypeptides
In general, a variant CBH I polypeptide produced in cell culture is secreted into the medium and may be purified or isolated, e.g., by removing unwanted components from the cell culture medium. However, in some cases, a variant CBH I polypeptide may be produced in a cellular form necessitating recovery from a cell lysate. In such cases the variant CBH I polypeptide is purified from the cells in which it was produced using techniques routinely employed by those skilled in the art. Examples include, but are not limited to, affinity chromatography (Van Tilbeurgh et al., 1984, FEBS Lett. 169(2):215-218), ion-exchange chromatographic methods (Goyal et al., 1991, Bioresource Technology, 36:37-50; Fliess et al., 1983, Eur. J. Appl. Microbiol. Biotechnol. 17:314-318; Bhikhabhai et al., 1984, J. Appl. Biochem. 6:336-345; Ellouz et al., 1987, Journal of Chromatography, 396:307-317), including ion-exchange using materials with high resolution power (Medve et al., 1998, J. Chromatography A, 808:153-165), hydrophobic interaction chromatography (Tomaz and Queiroz, 1999, J. Chromatography A, 865:123-128), and two-phase partitioning (Brumbauer et al., 1999, Bioseparation 7:287-295).
The variant CBH I polypeptides of the disclosure are suitably used in cellulase compositions. Cellulases are known in the art as enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulase enzymes have been traditionally divided into three major classes: endoglucanases (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91) (“CBH”) and beta-glucosidases (EC 3.2.1.21) (“BG”) (Knowles et al., 1987, TIBTECH 5:255-261; Schulein, 1988, Methods in Enzymology 160(25):234-243).
Certain fungi produce complete cellulase systems which include exo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-type cellulases and β-glucosidases or BG-type cellulases (Schulein, 1988, Methods in Enzymology 160(25):234-243). Such cellulase compositions are referred to herein as “whole” cellulases. However, sometimes these systems lack CBH-type cellulases and bacterial cellulases also typically include little or no CBH-type cellulases. In addition, it has been shown that the EG components and CBH components synergistically interact to more efficiently degrade cellulose. See, e.g., Wood, 1985, Biochemical Society Transactions 13(2):407-410.
The cellulase compositions of the disclosure typically include, in addition to a variant CBH I polypeptide, one or more cellobiohydrolases, endoglucanases and/or β-glucosidases. In their crudest form, cellulase compositions contain the microorganism culture that produced the enzyme components. “Cellulase compositions” also refers to a crude fermentation product of the microorganisms. A crude fermentation is preferably a fermentation broth that has been separated from the microorganism cells and/or cellular debris (e.g., by centrifugation and/or filtration). In some cases, the enzymes in the broth can be optionally diluted, concentrated, partially purified or purified and/or dried. The variant CBH I polypeptide can be co-expressed with one or more of the other components of the cellulase composition or it can be expressed separately, optionally purified and combined with a composition comprising one or more of the other cellulase components.
When employed in cellulase compositions, the variant CBH I is generally present in an amount sufficient to allow release of soluble sugars from the biomass. The amount of variant CBH I enzymes added depends upon the type of biomass to be saccharified which can be readily determined by the skilled artisan. In certain embodiments, the weight percent of variant CBH I polypeptide is suitably at least 1, at least 5, at least 10, or at least 20 weight percent of the total polypeptides in a cellulase composition. Exemplary cellulase compositions include a variant CBH I of the disclosure in an amount ranging from about 1 to about 20 weight percent, from about 1 to about 25 weight percent, from about 5 to about 20 weight percent, from about 5 to about 25 weight percent, from about 5 to about 30 weight percent, from about 5 to about 35 weight percent, from about 5 to about 40 weight percent, from about 5 to about 45 weight percent, from about 5 to about 50 weight percent, from about 10 to about 20 weight percent, from about 10 to about 25 weight percent, from about 10 to about 30 weight percent, from about 10 to about 35 weight percent, from about 10 to about 40 weight percent, from about 10 to about 45 weight percent, from about 10 to about 50 weight percent, from about 15 to about 20 weight percent, from about 15 to about 25 weight percent, from about 15 to about 30 weight percent, from about 15 to about 35 weight percent, from about 15 to about 30 weight percent, from about 15 to about 45 weight percent, or from about 15 to about 50 weight percent of the total polypeptides in the composition.
1.4. Utility of Variant CBH I Polypeptides
It can be appreciated that the variant CBH I polypeptides of the disclosure and compositions comprising the variant CBH I polypeptides find utility in a wide variety applications, for example detergent compositions that exhibit enhanced cleaning ability, function as a softening agent and/or improve the feel of cotton fabrics (e.g., “stone washing” or “biopolishing”), or in cellulase compositions for degrading wood pulp into sugars (e.g., for bio-ethanol production). Other applications include the treatment of mechanical pulp (Pere et al., 1996, Tappi Pulping Conference, pp. 693-696 (Nashville, Tenn., Oct. 27-31, 1996)), for use as a feed additive (see, e.g., WO 91/04673) and in grain wet milling.
1.4.1. Saccharification Reactions
Ethanol can be produced via saccharification and fermentation processes from cellulosic biomass such as trees, herbaceous plants, municipal solid waste and agricultural and forestry residues. However, the ratio of individual cellulase enzymes within a naturally occurring cellulase mixture produced by a microbe may not be the most efficient for rapid conversion of cellulose in biomass to glucose. It is known that endoglucanases act to produce new cellulose chain ends which themselves are substrates for the action of cellobiohydrolases and thereby improve the efficiency of hydrolysis of the entire cellulase system. The use of optimized cellobiohydrolase activity may greatly enhance the production of ethanol.
Cellulase compositions comprising one or more of the variant CBH I polypeptides of the disclosure can be used in saccharification reaction to produce simple sugars for fermentation. Accordingly, the present disclosure provides methods for saccharification comprising contacting biomass with a cellulase composition comprising a variant CBH I polypeptide of the disclosure and, optionally, subjecting the resulting sugars to fermentation by a microorganism.
The term “biomass,” as used herein, refers to any composition comprising cellulose (optionally also hemicellulose and/or lignin). As used herein, biomass includes, without limitation, seeds, grains, tubers, plant waste or byproducts of food processing or industrial processing (e.g., stalks), corn (including, e.g., cobs, stover, and the like), grasses (including, e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g., Panicum species, such as Panicum virgatum), wood (including, e.g., wood chips, processing waste), paper, pulp, and recycled paper (including, e.g., newspaper, printer paper, and the like). Other biomass materials include, without limitation, potatoes, soybean (e.g., rapeseed), barley, rye, oats, wheat, beets, and sugar cane bagasse.
The saccharified biomass (e.g., lignocellulosic material processed by enzymes of the disclosure) can be made into a number of bio-based products, via processes such as, e.g., microbial fermentation and/or chemical synthesis. As used herein, “microbial fermentation” refers to a process of growing and harvesting fermenting microorganisms under suitable conditions. The fermenting microorganism can be any microorganism suitable for use in a desired fermentation process for the production of bio-based products. Suitable fermenting microorganisms include, without limitation, filamentous fungi, yeast, and bacteria. The saccharified biomass can, for example, be made into a fuel (e.g., a biofuel such as a bioethanol, biobutanol, biomethanol, a biopropanol, a biodiesel, a jet fuel, or the like) via fermentation and/or chemical synthesis. The saccharified biomass can, for example, also be made into a commodity chemical (e.g., ascorbic acid, isoprene, 1,3-propanediol), lipids, amino acids, polypeptides, and enzymes, via fermentation and/or chemical synthesis.
Thus, in certain aspects, the variant CBH I polypeptides of the disclosure find utility in the generation of ethanol from biomass in either separate or simultaneous saccharification and fermentation processes. Separate saccharification and fermentation is a process whereby cellulose present in biomass is saccharified into simple sugars (e.g., glucose) and the simple sugars subsequently fermented by microorganisms (e.g., yeast) into ethanol. Simultaneous saccharification and fermentation is a process whereby cellulose present in biomass is saccharified into simple sugars (e.g., glucose) and, at the same time and in the same reactor, microorganisms (e.g., yeast) ferment the simple sugars into ethanol.
Prior to saccharification, biomass is preferably subject to one or more pretreatment step(s) in order to render cellulose material more accessible or susceptible to enzymes and thus more amenable to hydrolysis by the variant CBH I polypeptides of the disclosure.
In an exemplary embodiment, the pretreatment entails subjecting biomass material to a catalyst comprising a dilute solution of a strong acid and a metal salt in a reactor. The biomass material can, e.g., be a raw material or a dried material. This pretreatment can lower the activation energy, or the temperature, of cellulose hydrolysis, ultimately allowing higher yields of fermentable sugars. See, e.g., U.S. Pat. Nos. 6,660,506; 6,423,145.
Another exemplary pretreatment method entails hydrolyzing biomass by subjecting the biomass material to a first hydrolysis step in an aqueous medium at a temperature and a pressure chosen to effectuate primarily depolymerization of hemicellulose without achieving significant depolymerization of cellulose into glucose. This step yields a slurry in which the liquid aqueous phase contains dissolved monosaccharides resulting from depolymerization of hemicellulose, and a solid phase containing cellulose and lignin. The slurry is then subject to a second hydrolysis step under conditions that allow a major portion of the cellulose to be depolymerized, yielding a liquid aqueous phase containing dissolved/soluble depolymerization products of cellulose. See, e.g., U.S. Pat. No. 5,536,325.
A further exemplary method involves processing a biomass material by one or more stages of dilute acid hydrolysis using about 0.4% to about 2% of a strong acid; followed by treating the unreacted solid lignocellulosic component of the acid hydrolyzed material with alkaline delignification. See, e.g., U.S. Pat. No. 6,409,841. Another exemplary pretreatment method comprises prehydrolyzing biomass (e.g., lignocellulosic materials) in a prehydrolysis reactor; adding an acidic liquid to the solid lignocellulosic material to make a mixture; heating the mixture to reaction temperature; maintaining reaction temperature for a period of time sufficient to fractionate the lignocellulosic material into a solubilized portion containing at least about 20% of the lignin from the lignocellulosic material, and a solid fraction containing cellulose; separating the solubilized portion from the solid fraction, and removing the solubilized portion while at or near reaction temperature; and recovering the solubilized portion. The cellulose in the solid fraction is rendered more amenable to enzymatic digestion. See, e.g., U.S. Pat. No. 5,705,369. Further pretreatment methods can involve the use of hydrogen peroxide H2O2. See Gould, 1984, Biotech, and Bioengr. 26:46-52.
Pretreatment can also comprise contacting a biomass material with stoichiometric amounts of sodium hydroxide and ammonium hydroxide at a very low concentration. See Teixeira et al., 1999, Appl. Biochem. and Biotech. 77-79:19-34. Pretreatment can also comprise contacting a lignocellulose with a chemical (e.g., a base, such as sodium carbonate or potassium hydroxide) at a pH of about 9 to about 14 at moderate temperature, pressure, and pH. See PCT Publication WO2004/081185.
Ammonia pretreatment can also be used. Such a pretreatment method comprises subjecting a biomass material to low ammonia concentration under conditions of high solids. See, e.g., U.S. Patent Publication No. 20070031918 and PCT publication WO 06/110901.
1.4.2. Detergent Compositions Comprising Variant CBH I Proteins
The present disclosure also provides detergent compositions comprising a variant CBH I polypeptide of the disclosure. The detergent compositions may employ besides the variant CBH I polypeptide one or more of a surfactant, including anionic, non-ionic and ampholytic surfactants; a hydrolase; a bleaching agents; a bluing agent; a caking inhibitors; a solubilizer; and a cationic surfactant. All of these components are known in the detergent art.
The variant CBH I polypeptide is preferably provided as part of cellulase composition. The cellulase composition can be employed from about 0.00005 weight percent to about 5 weight percent or from about 0.0002 weight percent to about 2 weight percent of the total detergent composition. The cellulase composition can be in the form of a liquid diluent, granule, emulsion, gel, paste, and the like. Such forms are known to the skilled artisan. When a solid detergent composition is employed, the cellulase composition is preferably formulated as granules. CL 2. Example 1
2.1. Materials and Methods
2.1.1. Preparation of CBH I Polypeptides for Biochemical Characterization
Protein expression was carried out in an Aspergillus niger host strain that had been transformed using PEG-mediated transformation with expression constructs for CBH I that included the hygromycin resistance gene as a selectable marker, in which the full length CBH I sequences (signal sequence, catalytic domain, linker and cellulose binding domain) were under the control of the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter. Transformants were selected on the regeneration medium based on resistance to hygromycin. The selected transformants were cultured in Aspergillus salts medium, pH 6.2 supplemented with the antibiotics penicillin, streptomycin, and hygromycin, and 80 g/L glycerol, 20 g/L soytone, 10 mM uridine, 20 g/L MES) in baffled shake flasks at 30° C., 170 rpm. After five days of incubation, the total secreted protein supernatant was recovered, and then subjected to hollow fiber filtration to concentrate and exchange the sample into acetate buffer (50 mM NaAc, pH 5). CBH I protein represented over 90% of the total protein in these samples. Protein purity was analyzed by SDS-PAGE. Protein concentration was determined by gel densitometry and/or HPLC analysis. All CBH I protein concentrations were normalized before assay and concentrated to 1-2.5 mg/ml.
2.1.2. CBH I Activity Assays
Methylumbelliferyl Lactoside (4-MUL) Assay:
This assay measures the activity of CBH I on the fluorogenic substrate 4-MUL (also known as MUL). Assays were run in a costar 96-well black bottom plate, where reactions were initiated by the addition of 4-MUL to enzyme in buffer (2 mM 4-MUL in 200 mM MES pH 6). Enzymatic rates were monitored by fluorescent readouts over five minutes on a SPECTRAMAX™ plate reader (ex/em 365/450 nm). Data in the linear range was used to calculate initial rates (Vo).
Phosphoric Acid Swollen Cellulose (PASC) Assay:
This assay measures the activity of CBH I using PASC as the substrate. During the assay, the concentration of PASC is monitored by a fluorescent signal derived from calcofluor binding to PASC (ex/em 365/440 nm). The assay is initiated by mixing enzyme (15 μl) and reaction buffer (85 μl of 0.2% PASC, 200 mM MES, pH 6), and then incubating at 35° C. while shaking at 225 RPM. After 2 hours, one reaction volume of calcofluor stop solution (100 μg/ml in 500 mM glycine pH 10) is added and fluorescence read-outs obtained (ex/em 365/440 nm).
Saccharification Assay (Bagasse Assay):
This assay measures the activity of CBH I on bagasse, a lignocellulosic substrate. Reactions were run in 10 ml vials with 5% dilute acid pretreated bagasse (250 mg solids per 5 ml reaction). Each reaction contained 4 mg CBH I enzyme/g solids, 200 mM MES pH 6, kanamycin, and chloramphenicol. Reactions were incubated at 35° C. in hybridization incubators (Robbins Scientific), rotating at 20 RPM. Time points were taken by transferring a sample of homogenous slurry (150 μl) into a 96-well deep well plate and quenching the reaction with stop buffer (450 μl of 500 mM sodium carbonate, pH 10). Time point measurements were taken every 24 hours for 72 hours.
Cellobiose Tolerance Assays (or Cellobiose Inhibition Assays):
Tolerance to cellobiose (or inhibition caused by cellobiose) was tested in two ways in the CBH I assays. A direct-dose tolerance method can be applied to all of the CBH I assays (i.e., 4-MUL, PASC, and/or bagasse assays), and entails the exogenous addition of a known amount of cellobiose into assay mixtures. A different indirect method entails the addition of an excess amount of β-glucosidase (BG) to PASC and bagasse assays (typically, 1 mg β-glucosidase/g solids loaded). BG will enzymatically hydrolyze the cellobiose generated during these assays; therefore, CBH I activity in the presence of BG can be taken as a measure of activity in the absence of cellobiose. Furthermore, when activity in the presence and absence of BG are similar, this indicates tolerance to cellobiose. Notably, in cases where BG activity is undesired, but may be present in crude CBH I enzyme preparations, the BG inhibitor gluconolactone can be added into CBH I assays to prevent cellobiose breakdown.
2.2. Library Screening Assays
The wild type CBH I polypeptide BD29555 was mutagenized to identify variants with improved product tolerance. A small (60-member) library of BD29555 variants was designed to identify variant CBH I polypeptides with reduced product inhibition. This product-release-site library was designed based on residues directly interacting with the cellobiose product in an attempt to identify variants with weakened interactions with cellobiose from which the product would be released more readily than the wild type enzyme. The 60-member evolution library contained wild-type residues and mutations at positions R273, W405, and R422 of BD29555 (SEQ ID NO:1), and included the following substitutions: R273 (WT), R273Q, R273K, R273A, W405 (WT), W405Q, W405H, R422 (WT), R422Q, R422K, R422L, and R422E (4 variants at position 273×3 variants at position 405×5 variants at position 422 equals 60 variants in total). All members of the library were screened using the 4-MUL assay in the presence and absence of 250 mg/L cellobiose and using gluconolactone to inhibit any BG activity. The R273A, R273Q, and R273K/R422K variants showed enhanced product tolerance. The R273K/R422K variant showed greatest activity, expression, and cellobiose tolerance at 250 mg/L (730 mM). Due to low expression, other variants were not tested further.
2.3. Characterization of Product Tolerant Variants of BD29555
The R273K/R422K substitutions were characterized in both a wild type BD29555 background and also in combination with the substitutions Y274Q, D281K, Y410H, P411G, which were identified in a screen of an expanded product release site evolution library.
The wild type, the R273K/R422K variant and the R273K/Y274Q/D281K/Y410H/P411G/R422K variants were tested for activity on 4-MUL in the presence and absence of 250 mg/L cellobiose, and the R273K/R422K variant was also tested in the bagasse assay in the presence and absence of BG. The results are summarized in Table 5.
The results from these activity assays were converted into the percentage of activity remaining with and without cellobiose present, where values close to 100% indicated cellobiose tolerance. The percent of activity remaining in the MUL assay in the presence cellobiose versus in the absence of cellobiose shows that the R273K/R422K variant was the most tolerant, followed by the R273K/Y274Q/D281K/Y410H/P411G/R422K variant, and then wild-type, at 95%, 78%, and 25% activity, respectively.
Cellobiose dose response curves of the wild-type and R273K/R422K variant of BD29555 were obtained during the 4-MUL assay. Enzyme rates (Vo) were measured in the presence of different concentrations of cellobiose (200 mM MES pH 6, 25° C.). Rates were measured in quadruplicate. The results are shown in
The bagasse assay results shown in Table 5, which lists the percentage of activity remaining in the absence vs. presence of BG, also demonstrate that the percentage activity of the wild type BD29555 is lower than the percentage activity of the R273K/R422K variant, indicating that the R273K/R422K variant is less sensitive to the presence of cellobiose than the wild type.
The wild type and R273K/R422K variant were also characterized in the PASC assay. Results are shown in
2.4. Characterization of Product Tolerant Variants of T. reesei CBH I
Cellobiose product tolerant substitutions were introduced into T. reesei CBH I (SEQ ID NO:2). A panel of variants with single and double alanine and lysine substitutions at R268 and R411 were expressed and analyzed. The variants were tested for activity on 4-MUL in the presence and absence of 250 mg/L cellobiose and also in the bagasse assay in the absence and presence of BG. The results from these assays were converted into the percentage activity remaining in the presence and absence of cellobiose and BG, respectively. Values are summarized in Table 6.
The 4-MUL assay results shown in Table 6 demonstrate that the activity of the wild type T. reesei CBH I was reduced to 23% in the presence of cellobiose, whereas the double mutants at R268 and R411 retained more than 90% of their activity under the same conditions.
The bagasse assay results shown in Table 6 demonstrate that the activity of the wild type T. reesei CBH I is more significantly impacted by the presence of BG than is the activity of the single or double substitution variants, indicating that the variants are less sensitive to the accumulation of cellobiose than the wild type.
3.1. Materials and Methods
3.1.1. Preparation of CBH I Polypeptides for Biochemical Characterization:
Protein Expression:
Protein expression was carried out in a strain of Trichoderma reesei in which the native CBH I gene had been knocked out. The strain was transformed with a library of CBH I variant expression constructs that included the hygromycin resistance gene as a selectable marker. Expression constructs contained full-length CBH I wild-type or variant sequences (signal sequence, catalytic domain, linker and carbohydrate binding domain) under the control of a constitutive promoter. Transformants were selected on potato dextrose agar containing hygromycin (50 μg/mL). The selected isolates were subsequently cultured on 96-well plates containing potato dextrose agar without hygromycin. After sporulation, the transformants were stocked in 20% glycerol at −80° C. For screening, transformants were grown in 96-deep-well format for 6 days at 26° C., shaking at 850 rpm in a Multitron II shaker (3 mm throw), in 0.4 mL of liquid medium (2.5 g/L sodium citrate; 5 g/L KH2PO4; 2 g/L NH4NO3; 0.2 g/L MgSO4.7H2O; 0.1 g/L CaCl2; 9.1 g/L soytone; 80 g/L glycerol; 10 g/L MES buffer pH 6; 5 mg/L citric acid; 5 mg/L ZnSO4.7H2O; 1 mg/L Fe(NH4)2(SO4)2; 0.25 mg/L CuSO4.5H2O; 0.05 mg/L MnSO4; 0.05 mg/L H3BO3; 0.05 mg/L Na2MoO4.2H2O; 5 μg/L biotin). Total secreted protein supernatants were harvested by filtration. The knock-out strain alone produced no CBH I protein. Protein concentration was determined by gel densitometry and/or RP-HPLC analysis.
Protein Quantification by Reverse-Phase (RP) High Performance Liquid chromatography (HPLC):
CBH I protein concentrations in supernatants were quantified using RP-HPLC. The system used was an Agilent 1100 series model, equipped with quaternary pump (connected to reservoirs A and B, where reservoir A contained water with 0.1% trifluoroacetic acid and reservoir B contained acetonitrile with 0.1% trifluoroacetic acid), a diode array detector (monitored at 225 nm and 280 nm), and a fluorescence detector (monitored at ex/em 280/340 nm). An Agilent Zorbax 300SB-C3 (5 μM, 4.6×150 mm) was used to separate samples using a 20 minute method (30-50% B over 10 minutes; 100% B for 5 minutes; 30% B for 5 min; at 60° C. at a flow rate of 1 mL/min). CBH I was identified by a retention time at 7.8-8.2 minutes and quantitated by area. Concentrations were determined by reference to a standard curve generated with a commercial CBH I (E-CBH I from Megazymes).
3.1.2. Biochemical Characterization:
Methylumbelliferyl Lactoside (4-MUL) Assay:
CBH I activity on was measured using the 4-MUL assay using gluconolactone to inhibit any BG activity. The fluorogenic 4-MUL substrate (SIGMA) was prepared at 100 mM concentration in DMSO. Assays were run in black 96-well-flat-bottomed plates (Costar) and 4-MU fluorescence was read on a BioTek H4 plate reader (ex/em 365/450 nm). Assay plates were filled with buffer (final concentrations of 100 mM MES, pH 6, 25 mM gluconolactone, with or without cellobiose; cellobiose concentrations are listed with appropriate data sets), to which enzyme mixture was added (10-30 μl, 5 μg/mL final) and then assays were initiated by addition of 4-MUL (0.5 mM final concentration in 100 μl total volume). Enzyme mixtures were either CBH I variants from harvested supernatants or standards. Standards included: a negative control, consisting of harvested supernatant from the CBH I knock-out strain; a positive control, consisting of wild-type CBH I from harvested supernatants; and, a commercial CBH I standard (E-CBHI from Megazymes). Activity standards were run by serial dilution of commercial CBH I from 40 to 0.02 μg/mL and 4-MU (SIGMA, prepared at 20 mM in DMSO) (in dilution increments of 2-fold; all dilutions were made using harvested supernatant from the knock-out control). Kinetic rates were monitored over the first 15 mins following 4-MUL addition; initial rates were calculated based on data in the linear range. After 1 hr, a final endpoint read was taken, both before and after reaction quenching (100 μL of 200 mM Sodium Carbonate, pH 10.0). Activity was calculated for kinetic and endpoint reads; background resulting from the CBH I knock-out supernatant remained negligible. 4MU standard curves and HPLC quantification values were used to calculate specific activity.
Saccharification Assay:
CBH I activity on a native lignocellulosic substrate was measured using the saccharification assay. Reactions were run in 96-well plates with the following composition in each well: 22 μL of variant/enzyme sample, 0.7% solids (dilute acid pretreated bagasse at 0.4% cellulose), β-glucosidase (50 ug/mL), and buffer (50 mM Sodium Citrate pH 5.5.), in a final volume of 227 μL. Time points were taken by transferring the reaction solution (15 into another 384-well plate and quenching the reaction with stop buffer (45 μl of 200 mM sodium carbonate, pH 10). Stop plates were sealed and stored at 4° C. for 14 hours before running a secondary BG digest: 15 ul of the stopped reaction into 35 ul of BG mix (50 ug/ml BG, 250 mM Sodium Citrate pH 5.5) and incubated at 37° C. for 14 hr. After the incubation, glucose was quantified by a glucose oxidase detection assay (GO assay), and percent cellulose conversion was calculated (based on 100% conversion at 25 mM) using a standard curve of known glucose concentrations (0.01-3.0 mM).
Cellobiose Tolerance/Inhibition Assays:
Tolerance/inhibition values represent activity ratios and/or percent activity remaining/percent activity decreased in the presence versus the absence of cellobiose. Tolerant variants show less inhibition in the presence of cellobiose as compared to wild type, where an activity ratio of 1 (with vs. without a given concentration of cellobiose) is equivalent to 0% inhibition by cellobiose, or 100% tolerance. The effect of cellobiose on CBH I variant performance was monitored by dose-response in the 4MUL assay. Dose-response curves were generated by assaying variant activity in the presence of 6-8 different cellobiose concentrations ranging up to 100 mM cellobiose. CBH I samples were diluted to 5 μg/mL final concentration or were used directly in the case of protein quantification levels below 5 μg/mL. Half maximal inhibitory concentration (IC50) values were determined by plotting 4MUL activity versus cellobiose concentration and fitting with a four parameter dose-response fitting algorithm, with zero activity (or 100% inhibition) constrained to background activity (as established by CBH I knockout values) and with automatic outlier elimination (on GraphPad Prism 5).
Remazolbrilliant Blue R Stained Carboxymethyl-Cellulose (Azo-CMC) Assay:
Endoglycosidase activity was measured using the Azo-CMC assay. The colorimetric substrate Azo-CMC was obtained from Megazymes. The substrate was used as provided in solution (4M partially depolymerized and dyed CM-cellulose containing approximately one Remazolbrilliant Blue R dye molecule per 20 sugar residues). Assays were run in clear 96-well-flat-bottomed plates (Costar) and released Remazolbrilliant Blue R was monitored at 590 nm on a BioTek H4 reader. Assay plates were charged with equal volumes (40 uL) of supernatant/standard and Azo-CM-cellulose, incubated 14 h at 35° C., and stopped (200 μL; 80% EtOH, 0.3 M NaOAc, 0.03 M ZnOAc, pH 5.0). After stopping, the reaction plates were centrifuged (4000 rpm, 5 mins), and the clarified supernatant was transferred to a second clear flat bottom plate for absorbance reading. Activity was calibrated using an endoglycosidase standard (20 μg/mL); in all cases, harvested supernatants had activity values below the standard.
3.1.3. Library Design, Screening, and Characterization:
Library Design:
Example 1 describes CBH I variants that retain activity in the presence of cellobiose levels which are inhibitory to the wild-type enzyme. These cellobiose-tolerant variants were garnered when two arginines found at positions 268 and 411 in the enzyme's product release site were mutagenized to any combination of lysine and alanine. To further characterize single amino acid mutations that contribute to CBH I variants with cellobiose tolerance, a 40-member library was designed to individually mutate position 268 and 411 to each of the 20 naturally occurring amino acids. Additionally, the contribution of double amino acid mutations to CBH I variants with cellobiose tolerance was scanned with a 40-member library introducing each of the 20 amino acids to positions 268 and 411, while the other position was held constant at alanine. The final 80-member library contained: 20 variants with site 268 mutagenized to all possible amino acids (R268aa); 20 variants with site 268 mutagenized to all possible amino acids, and site 411 mutated to alanine (R268aa/R411A); 20 variants with site 411 mutagenized to all possible amino acids (R411 aa); 20 variants s with site 411 mutagenized to all possible amino acids, and site 268 mutated to alanine (R268A/R411aa).
Transformation and Primary Screening for Active Isolates (Scheme 1 (FIG. 6)):
The variant library was successfully transformed with the exception of R268A/R411N and R268A/R411Y variants. For the 78 transformed variants, 8 isolates of each were picked, stocked, and grown. Supernatants were harvested for the primary screening by 4-MUL assay (see
Active Variants:
The harvested protein samples from active isolates were evaluated for CBH I activity, by 4-MUL assay, and CBH I concentration, by HPLC. EG activity was assessed by Azo-CMC assay to verify no background interference. Protein samples were then directly tested in a primary screen for cellobiose tolerance in the 4-MUL assay and for activity on native substrate in the saccharification assay, as shown in
Screening by 4-MUL:
Harvested supernatants from active variant isolates were evaluated for cellobiose tolerance at 1 mM cellobiose in the 4-MUL activity assay. Table 8 lists the tolerance of variants at 1 mM. All non-WT variants demonstrated enhanced tolerance compared with the wild-type enzyme, which is significantly inhibited (% tolerance=6%, or 94% inhibited). Notably, the library contained a wild-type sequence member; this isolate showed consistent behavior with 3% tolerance at 1 mM. Additional cellobiose concentrations at 0.25, 5, 10, 50, and 100 mM were tested leading to full dose-response curves for which half maximal inhibitory concentration (IC50) values were generated (Table 8). The IC50 values support that the variant library has decreased product inhibition, or increased tolerance to cellobiose, when compared to the wild-type enzyme (WT IC50=0.03 mM; see first entry, Table 8).
Primary Screening by Saccharification:
In one example, picked mutants were tested using the saccharification assay, which measures the extent to which CBH I converts polymeric cellulose into cellobiose. Saccharification was carried out for 48 hours and the percent of cellulose converted was calculated for each variant.
Secondary Screening: IC50 Values:
In one example, the cellobiose tolerance of the library was explored in more detail by generating dose-response curves and determining half maximal inhibitory concentration (IC50) values, the point at which the enzyme is 50% inhibited. In two instances, IC50 values were generated using samples with CBH I variant protein levels normalized to 5 μg/mL and using cellobiose concentrations in the range of 0.0001-100 mM (Table 9) or in the range of 0.00085-100 mM (Table 10). In another instance, IC50 curves were generated using 30 μl of variant supernatant characterized by CBH I levels lower than 5 μg/mL and using cellobiose concentrations in the range of 0.00085-100 mM (Table 11).
3.2. Results
Table 5 and
The specific activity (SA) of the variant library was evaluated in a secondary 4-MUL assay. Table 13 lists the specific activity for the variant library and
All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.
While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).
Trichoderma
reesei
Penicillium
occitanis
Trichoderma
reesei
Aspergillus
nidulans FGSC A4
Gibberella zeae
Aspergillus
fumigatus Af293
Aspergillus
clavatus NRRL 1
Claviceps
purpurea
Phanerochaete
chrysosporium
Neosartorya
fischeri NRRL 181
Leptosphaeria
maculans
Neurospora crassa
Coprinopsis
cinerea okayama
Botryotinia
fuckeliana B05-10
Phaeosphaeria
nodorum SN15
Humicola grisea
Humicola grisea
Chaetomium
thermophilum
Chaetomium
thermophilum
Phaeosphaeria
nodorum SN15
Coprinopsis
cinerea Okayama
Agaricus bisporus
Phaeosphaeria
nodorum SN15
Phanerochaete
chrysosporium
Aspergillus
fumigatus Af293
Phanerochaete
chrysosporium
Thermoascus
aurantiacus
Humicola grisea
Trichoderma
viride (also known
Thermoascus
aurantiacus
Magnaporthe
grisea (oryzae) 70-
Talaromyces
emersonii
Aspergillus oryzae
Penicillium
chrysogenum
Podospora
anserina
Pleurotus sp
Gibberella zeae
Sclerotinia
sclerotiorum 1980
Phanerochaete
chrysosporium
Trichoderma
reesei
Aspergillus terreus
Botryotinia
fuckeliana B05-10
Volvariella
volvacea
Penicillium
janthinellum
Pleurotus sp
Aspergillus
nidulans FGSC A4
Fusarium poae
Pleurotus sp
Trichoderma
viride
Alternaria
alternata
Neosartorya
fischeri NRRL 181
Neurospora crassa
Cryphonectria
parasitica
Corticium rolfsii
Magnaporthe
grisea 70-15
Magnaporthe
grisea 70-15
Fusarium
oxysporum
Aspergillus
clavatus NRRL 1
Penicillium
funiculosum
Humicola grisea
Chaetomium
thermophilum
Aspergillus oryzae
Gibberella
pulicaris
Volvariella
volvacea
Irpex lacteus
Chaetomium
thermophilum var
thermophilum
Irpex lacteus
Phanerochaete
chrysosporium
Melanocarpus
albomyces
Podospora
anserina
Cochliobolus
carbonum
Trichoderma
viride
Aspergillus
aculeatus
Dictyostelium
discoideum
Sclerotinia
sclerotiorum 1980
Chaetomium
globosum CBS
Aspergillus niger
Nectria
haematococca
Talaromyces
emersonii
Polyporus
arcularius
Leptosphaeria
maculans
Phanerochaete
chrysosporium
Penicillium
decumbens
Sclerotinia
sclerotiorum 1980
Acremonium
thermophilum
Aspergillus
nidulans (also
Emericella
nidulans)
Podospora
anserine (S mat+)
Thermoascus
aurantiacus var
levisporus
Pseudotrichonympha
grassii
Aspergillus terreus
Phanerochaete
chrysosporium
Phanerochaete
chrysosporium
Thermoascus
aurantiacus
Acremonium
thermophilum
Volvariella
volvacea
Chaetomium
globosum CBS
Irpex lacteus
Lentinula edodes
Pleurotus sp
Fusarium
venenatum
Phanerochaete
chrysosporium
Fusicoccum sp
Coprinopsis
cinerea okayama
Trichoderma
harzianum
Hypocrea lixii)
Neurospora crassa
Neosartorya
fischeri NRRL 181
Chaetomium
thermophilum
Botryotinia
fuckeliana B05-10
Pseudotrichonympha
grassii
Irpex lacteus
Gibberella
avenacea
Aspergillus niger
Aspergillus niger
Chrysosporium
lucknowense
Thielavia
australiensis
speratus
koshunensis
speratus
punctulatus
sjoestedti
punctutatus
speratus
punctuiatus
darwiniensis
koshunensis
koshunensis
speratus
punctulatus
darwiniensis
punctulatus
sjoestedti
darwiniensis
speratus
speratus
speratus
speratus
speratus
punctulatus
koshunensis
darwiniensis
koshunensis
darwiniensis
speratus
speratus
sjoestedti
punctuiatus
darwiniensis
koshunensis
Trichoderma reesei
Penicillium occitanis
Trichoderma reesei
Aspergillus nidulans FGSC A4
Gibberella zeae PH-1
Aspergillus fumigatus Af293
Aspergillus clavatus NRRL 1
Claviceps purpurea
Phanerochaete chrysosporium
Neosartorya fischeri NRRL 181
Leptosphaeria maculans
Neurospora crassa N150
Coprinopsis cinerea okayama
Botryotinia fuckeliana B05-10
Phaeosphaeria nodorum SN15
Humicola grisea
Humicola grisea
Chaetomium thermophilum
Chaetomium thermophilum
Phaeosphaeria nodorum SN15
Coprinopsis cinerea okayama
Agaricus bisporus
Phaeosphaeria nodorum SN15
Phanerochaete chrysosporium
Aspergillus fumigatus Af293
Phanerochaete chrysosporium
Thermoascus aurantiacus
Humicola grisea var thermoidea
Trichoderma viride (also known
Thermoascus aurantiacus
Magnaporthe grisea (oryzae) 70-
Talaromyces emersonii
Aspergillus oryzae
Penicillium chrysogenum
Podospora anserina
Pleurotus sp Florida
Gibberella zeae
Sclerotinia sclerotiorum 1980
Phanerochaete chrysosporium
Trichoderma reesei
Aspergillus terreus NIH2624
Botryotinia fuckeliana B05-10
Volvariella volvacea
Penicillium janthinellum
Pleurotus sp Florida
Aspergillus nidulans FGSC A4
Fusarium poae
Pleurotus sp Florida
Trichoderma viride
Alternaria alternata
Neosartorya fischeri NRRL 181
Neurospora crassa OR74A
Cryphonectria parasitica
Corticium rolfsii
Magnaporthe grisea 70-15
Magnaporthe grisea 70-15
Fusarium oxysporum
Aspergillus clavatus NRRL 1
Penicillium funiculosum
Humicola grisea var thermoidea
Chaetomium thermophilum
Aspergillus oryzae RIB40
Gibberella pulicaris
Volvariella volvacea
Irpex lacteus
Chaetomium thermophilum var
thermophilum
Irpex lacteus
Phanerochaete chrysosporium
Melanocarpus albomyces
Podospora anserina
Cochliobolus carbonum
Trichoderma viride
Aspergillus aculeatus
Dictyostelium discoideum
Sclerotinia sclerotiorum 1980
Chaetomium globosum CBS 148-
Aspergillus niger
Nectria haematococca mpVI
Talaromyces emersonii
Polyporus arcularius
Leptosphaeria maculans
Phanerochaete chrysosporium
Penicillium decumbens
Sclerotinia sclerotiorum 1980
Acremonium thermophilum
Aspergillus nidulans
Podospora anserina
Thermoascus aurantiacus var
levisporus
Pseudotrichonympha grassii
Aspergillus terreus NIH2624
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Thermoascus aurantiacus
Acremonium thermophilum
Volvariella volvacea
Chaetomium globosum CBS 148-
Irpex lacteus
Lentinula edodes
Pleurotus sp Florida
Fusarium venenatum
Phanerochaete chrysosporium
Fusicoccum sp BCC4124
Coprinopsis cinerea okayama
Trichoderma harzianum
Neurospora crassa
Neosartorya fischeri NRRL 181
Chaetomium thermophilum
Botryotinia fuckeliana B05-10
Pseudotrichonympha grassii
Irpex lacteus
Gibberella avenacea
Aspergillus niger
Aspergillus niger
Chrysosporium lucknowense
Thielavia australiensis
Reticulitermes speratus
Neotermes koshunensis
Reticulitermes speratus
Cryptocercus punciulatus
Hodotermopsis sjoestedti
Cryptocercus punctulatus
Reticulitermes speratus
Cryptocercus punctulatus
Mastotermes darwiniensis
Neotermes koshunensis
Neotermes koshunensis
Reticulitermes speratus
Cryptocercus punctulatus
Mastotermes darwiniensis
Cryptocercus punctulatus
Hodotermopsis sjoestedti
Mastotermes darwiniensis
Reticulitermes speratus
Reticulitermes speratus
Reticulitermes speratus
Reticulitermes speratus
Reticulitermes speratus
Cryptocercus punctulatus
Neotermes koshunensis
Mastotermes darwiniensis
Neotermes koshunensis
Mastotermes darwiniensis
Reticulitermes speratus
Reticulitermes speratus
Hodotermopsis sjoestedti
Cryptocercus punctulatus
Mastotermes darwiniensis
Neotermes koshunensis
Trichoderma reesei
Penicillium occitanis
Trichoderma reesei
Aspergillus nidulans
Gibberella zeae PH-1
Aspergillus
fumigatus Af293
Aspergillus clavatus
Claviceps purpurea
Phanerochaete
chrysosporium
Neosartorya fischeri
Leptosphaeria
maculans
Neurospora crassa
Coprinopsis cinerea
okayama
Botryotinia
fuckeliana B05-10
Phaeosphaeria
nodorum SN15
Humicola grisea
Humicola grisea
Chaetomium
thermophilum
Chaetomium
thermophilum
Phaeosphaeria
nodorum SN15
Coprinopsis cinerea
okayama
Agaricus bisporus
Phaeosphaeria
nodorum SN15
Phanerochaete
chrysosporium
Aspergillus
fumigatus Af293
Phanerochaete
chrysosporium
Thermoascus
aurantiacus
Humicola grisea var
thermoidea
Trichoderma viride
Hypochrea rufa)
Thermoascus
aurantiacus
Magnaporthe grisea
Talaromyces
emersonii
Aspergillus oryzae
Penicillium
chrysogenum
Podospora anserina
Pleurotus sp Florida
Gibberella zeae
Sclerotinia
sclerotiorum 1980
Phanerochaete
chrysosporium
Trichoderma reesei
Aspergillus terreus
Botryotinia
fuckeliana B05-10
Volvariella volvacea
Penicillium
janthinellum
Pleurotus sp Florida
Aspergillus nidulans
Fusarium poae
Pleurotus sp Florida
Trichoderma viride
Alternaria alternata
Neosartorya fischeri
Neurospora crassa
Cryphonectria
parasitica
Corticium rolfsii
Magnaporthe grisea
Magnaporthe grisea
Fusarium
oxysporum
Aspergillus clavatus
Penicillium
funiculosum
Humicola grisea var
thermoidea
Chaetomium
thermophilum
Aspergillus oryzae
Gibberella pulicaris
Volvariella volvacea
Irpex lacteus
Chaetomium
thermophilum var
thermophilum
Irpex lacteus
Phanerochaete
chrysosporium
Melanocarpus
albomyces
Podospora anserina
Cochliobolus
carbonum
Trichoderma viride
Aspergillus
aculeatus
Dictyostelium
discoideum
Sclerotinia
sclerotiorum 1980
Chaetomium
globosum CBS 148-
Aspergillus niger
Nectria
haematococca mpVI
Talaromyces
emersonii
Polyporus
arcularius
Leptosphaeria
maculans
Phanerochaete
chrysosporium
Penicillium
decumbens
Sclerotinia
sclerotiorum 1980
Acremonium
thermophilum
Aspergillus nidulans
Podospora anserina
Thermoascus
aurantiacus var
levisporus
Pseudotrichonympha
grassii
Aspergillus terreus
Phanerochaete
chrysosporium
Phanerochaete
chrysosporium
Thermoascus
aurantiacus
Acremonium
thermophilum
Volvariella volvacea
Chaetomium
globosum CBS 148-
Irpex lacteus
Lentinula edodes
Pleurotus sp Florida
Fusarium venenatum
Phanerochaete
chrysosporium
Fusicoccum sp
Coprinopsis cinerea
okayama
Trichoderma
harzianum
Neurospora crassa
Neosartorya fischeri
Chaetomium
thermophilum
Botryotinia
fuckeliana B05-10
Pseudotrichonympha
grassii
Irpex lacteus
Gibberella avenacea
Aspergillus niger
Aspergillus niger
Chrysosporium
lucknowense
Thielavia
australiensis
Reticulitermes
speratus
koshunensis
Reticulitermes
speratus
Cryptocercus
punctulatus
Hodotermopsis
sjoestedii
Cryptocercus
punctulatus
Reticulitermes
speratus
Cryptocercus
punctulatus
Mastotermes
darwiniensis
koshunensis
koshunensis
Reticulitermes
speratus
Cryptocercus
punctulatus
Mastotermes
darwiniensis
Cryptocercus
punctulatus
Hodotermopsis
sjoestedii
Mastotermes
darwiniensis
Reticulitermes
speratus
Reticulitermes
speratus
Reticulitermes
speratus
Reticulitermes
speratus
Reticulitermes
speratus
Cryptocercus
punctulatus
koshunensis
Mastotermes
darwiniensis
koshunensis
Mastotermes
darwiniensis
Reticulitermes
speratus
Reticulitermes
speratus
Hodotermopsis
sjoestedti
Cryptocercus
punctulatus
Mastotermes
darwiniensis
koshunensis
Trichoderma
reesei
Penicillium
occitanis
Trichoderma
reesei
Aspergillus
nidulans FGSC
Gibberella zeae
Aspergillus
fumigatus Af293
Aspergillus
clavatus NRRL 1
Claviceps
purpurca
Phanerochaete
chrysosporium
Neosartorya
fischeri NRRL
Leptosphaeria
maculans
Neurospora
crassa N150
Coprinopsis
cinerea okayama
Botryotinia
fuckeliana B05-
Phaeosphaeria
nodorum SN15
Humicola grisea
Humicola grisea
Chaetomium
thermophilum
Chaetomium
thermophilum
Phaeosphaeria
nodorum SN15
Coprinopsis
cinerea okayama
Agaricus
bisporus
Phaeosphaeria
nodorum SN15
Phanerochaete
chrysosporium
Aspergillus
fumigatus Af293
Phanerochaete
chrysosporium
Thermoascus
aurantiacus
Humicola grisea
Trichoderma
viride (also
Hypochrea rufa)
Thermoascus
aurantiacus
Magnaporthe
grisea (oryzae)
Talaromyces
emersonii
Aspergillus
oryzae
Penicillium
chrysogenum
Podospora
anserina
Pleurotus sp
Gibberella zeae
Sclerotinia
sclerotiorum
Phanerochaete
chrysosporium
Trichoderma
reesei
Aspergillus
terreus NIH2624
Botryotinia
fuckeliana B05-
Volvariella
volvacea
Penicillium
janthinellum
Pleurotus sp
Aspergillus
nidulans FGSC
Fusarium poae
Pleurotus sp
Trichoderma
viride
Alternaria
alternata
Neosartorya
fischeri NRRL
Neurospora
crassa OR74A
Cryphonectria
parasitica
Corticium rolfsii
Magnaporthe
grisea 70-15
Magnaporthe
grisea 70-15
Fusarium
oxysporum
Aspergillus
clavatus NRRL 1
Penicillium
funiculosum
Humicola grisea
Chaetomium
thermophilum
Aspergillus
oryzae RIB40
Gibberella
pulicaris
Volvariella
volvacea
Irpex lacteus
Chaetomium
thermophilum var
thermophilum
Irpex lacteus
Phanerochaete
chrysosporium
Melanocarpus
albomyces
Podospora
anserina
Cochliobolus
carbonum
Trichoderma
viride
Aspergillus
aculeatus
Dictyostelium
discoideum
Sclerotinia
sclerotiorum
Chaetomium
globosum CBS
Aspergillus niger
Nectria
haematococca
Talaromyces
emersonii
Polyporus
arcularius
Leptosphaeria
maculans
Phanerochaete
chrysosporium
Penicillium
decumbens
Sclerotinia
sclerotiorum
Acremonium
thermophilum
Aspergillus
nidulans (also
Emericella
nidulans)
Podospora
anserine (S
Thermoascus
aurantiacus var
levisporus
Pseudotrichonympha
grassii
Aspergillus
terreus NIH2624
Phanerochaete
chrysosporium
Phanerochaete
chrysosporium
Thermoascus
aurantiacus
Acremonium
thermophilum
Volvariella
volvacea
Chaetomium
globosum CBS
Irpex lacteus
Lentinula edodes
Pleurotus sp
Fusarium
venenatum
Phanerochaete
chrysosporium
Fusicoccum sp
Coprinopsis
cinerea okayama
Trichoderma
harzianum
Hypocrea lixii)
Neurospora
crassa (OR74A)
Neosartorya
fischeri NRRL
Chaetomium
thermophilum
Botryotinia
fuckeliana B05-
Pseudotrichonympha
grassii
Irpex lacteus
Gibberella
avenacea
Aspergillus niger
Aspergillus niger
Chrysosporium
luckowense
Thielavia
australiensis
speratus
koshunensis
speratus
punctulatus
sjoestedti
punctulatus
speratus
punctulatus
darwiniensis
koshunensis
koshunensis
speratus
punctulatus
darwiniensis
punctulatus
sjoestedti
darwiniensis
speratus
speratus
speratus
speratus
speratus
punctulatus
koshunensis
darwiniensis
koshunensis
darwiniensis
speratus
speratus
sjoestedti
punctulatus
darwiniensis
koshunensis
¥poor fit; R2 < 0.95
indicates data missing or illegible when filed
indicates data missing or illegible when filed
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/59005 | 10/5/2012 | WO | 00 | 4/2/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61622971 | Apr 2012 | US | |
| 61544256 | Oct 2011 | US |
