EP2576775A1 - Cellulases modifiées à thermostabilité améliorée - Google Patents

Cellulases modifiées à thermostabilité améliorée

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Publication number
EP2576775A1
EP2576775A1 EP11729340.7A EP11729340A EP2576775A1 EP 2576775 A1 EP2576775 A1 EP 2576775A1 EP 11729340 A EP11729340 A EP 11729340A EP 2576775 A1 EP2576775 A1 EP 2576775A1
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European Patent Office
Prior art keywords
cel8a
variant
sequence
bio
thermocellum
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EP11729340.7A
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German (de)
English (en)
Inventor
Edward A. Bayer
Michael Anbar
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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Publication of EP2576775A1 publication Critical patent/EP2576775A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • the present invention relates to variant family-8 cellulases comprising at least one amino acid substitution introduced into their catalytic domain and having enhanced thermostability compared to the wild-type enzymes.
  • Such cellulases are advantageous for the bioconversion process of cellulosic substrates.
  • Efficient enzymatic saccharification of cellulose to soluble sugars is of growing interest in the biofuel industry as a source of renewable energy.
  • Cellulose the major component of the plant cell wall, is composed of long ⁇ -1,4 linked D-glucose molecules and is the largest carbon source on earth.
  • the last two decades have seen tremendous progress in research on conversion of cellulosic biomass to biofuels. Nevertheless, many techno-economic challenges must be overcome before cellulosic fuel will be able to compete with corn ethanol and conventional sources of fossil fuel.
  • a major bottleneck in converting cellulose to fuels is the hydrolysis of plant cell wall biopolymers, especially the attack on highly recalcitrant cellulose fibers.
  • Cellulases are structurally and functionally diverse set of enzymes which hydrolyze the ⁇ -1,4 glycosidic bonds in the cellulose. Enzymatic hydrolysis of cellulose requires the synergistic action of at least three classes of enzymes: endoglucanases, also referred to as endocellulases, which catalyze the hydrolysis of internal bonds inside the cellulose chain and randomly produce new chain ends; exoglucanases, also referred to as exocellulases, which cleave the cellulose chain at the exposed ends, typically producing cellobiose; and ⁇ -glucosidases, which cleave short cellodextrins, notably cellobiose, into glucose. These three groups of enzymes act synergistically in order to efficiently degrade recalcitrant cellulosic substrates.
  • the cellulases are part of a larger group of enzymes collectively referred as glycoside hydrolases, which hydrolyze glycosidic bonds between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety.
  • glycoside hydrolases are divided into families numbered in ascending order based on sequence similarities of the catalytic domain. Cellulases are assigned to several glycoside hydrolases families. Information about the classification system is available on the Carbohydrate-Active Enzymes (CAZy) server (www.cazy.org) and CAZypedia database (www.cazypedia.org) (Cantarel et al., Nucleic Acids Res, 2009, 37(Database issue): p. D233-8).
  • CAZy Carbohydrate-Active Enzymes
  • cellulases are characterized by a multi-modular organization, where the catalytic module is associated with one or more ancillary, helper, modules which modulate the enzyme activity.
  • Each module or domain comprises a consecutive portion of the polypeptide chain and forms an independently folding, structurally and functionally distinct unit.
  • one of the main ancillary modules is the carbohydrate-binding module.
  • Cellulolytic microorganisms produce a wide range of enzymes that hydrolyze cellulose.
  • the thermophilic anaerobic bacterium Clostridium thermocellum
  • Clostridium thermocellum produces a large multi-enzyme complex of cellulases, hemicellulases and other carbohydrate-active enzymes termed the cellulosome, which can efficiently degrade and solubilize crystalline cellulosic substrates.
  • the cellulosome complex is characterized by a strong bi-modular protein-protein interaction between "cohesin” and "dockerin” modules that integrates the various enzymes into the complex.
  • the cohesin modules are part of "scaffoldin” subunits (non-enzymatic protein components), which incorporate the enzymes into the complex via their resident dockerins.
  • the primary scaffoldin subunit also includes a carbohydrate (e.g., cellulose)-binding module (CBM) through which the complex recognizes and binds to the cellulosic substrate.
  • CBM carbohydrate-binding module
  • Cellulase 8A (Cel8A). This family-8 glycoside hydrolase is the most prevalent endoglucanase secreted extracellularly as a component of the cellulosome complex.
  • the enzyme consists of a signal peptide segment (cleaved upon secretion), a catalytic module, which folds into an ( ⁇ / ⁇ ) 6 barrel formed by six inner and six outer a helices, and a type I dockerin at its C- terminus that anchors the enzyme to the extracellular cellulosome complex of the bacterium.
  • the enzyme was cloned and expressed previously in Escherichia coli (Schwarz et al., Appl Environ Microbiol 1986, 51 : 1293) and crystallized to elucidate its enzyme structure and catalytic mechanism (Alzari et al., Structure 1996, 4: 265).
  • Optimizing the biodegradation of lignocellulose substrates requires either the search for novel enzymes which are robust enough to withstand the industrial process or alternatively, enzymes that can be engineered to enhance desired qualities, such as high specific activity, low levels of end-product inhibition, tolerance to broad ranges of pH and inhibitors of byproducts of degradation.
  • desired qualities such as high specific activity, low levels of end-product inhibition, tolerance to broad ranges of pH and inhibitors of byproducts of degradation.
  • one of the major challenges today is to reduce the cost of biofuel production in order to reach future goals of substituting renewable sources of energy for fossil-based fuels.
  • Cellulases are relatively costly enzymes and a reduction in cost can greatly benefit their commercial use.
  • Thermostable cellulases may offer many benefits in the bioconversion process; including, for example, improvement in stability for longer periods, enhancement of specific activity, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess.
  • thermostability of protein biocatalysts There are three main approaches to enhancing the thermostability of protein biocatalysts: i) directed evolution; ii) rational design and iii) data-driven design (also referred to as consensus-guided mutagenesis), by construction of synthetic consensus genes. It has been demonstrated that large stability differences could be accomplished by inducing only one or very few amino acid substitutions (Bloom et al., Proc Natl Acad Sci USA 2009, 106 Suppl 1 : 9995). Other factors, such as increased internal hydrophobicity, increased hydrophobicity of the protein surface and electrostatic interactions as well as hydrogen bonding are responsible for a more rigid and stable protein (Machius et al., J Biol Chem 2003, 278: 11546).
  • thermostable structures Because the structure-function relationship is not known or fully understood for the majority of proteins, many mutational strategies that lead to high stability cannot easily be defined or rationalized.
  • Consensus-guided mutagenesis takes advantage of the large number of available protein sequences. This semi-rational approach is a well-established strategy to improve the thermostability and has been used successfully on both enzymatic and non-enzymatic proteins (see for example, Amin et al., Protein Eng Des Sel, 2004. 17(11): p. 787-93; Lehmann et al., Protein Eng, 2002 15(5): p. 403-1 1; and Polizzi et al., Biotechnol J, 2006. 1(5): p. 531 -6). The approach is based on the substitution of specific amino acids in a particular protein with the most prevalent amino acid present at these positions among homologous family members.
  • thermostability There is an unmet need for cellulolytic enzymes with improved thermostability. For example, it would be beneficial to have modified cellulases that show high thermostability while maintaining high specific activity towards the substrate.
  • the present invention provides modified family-8 cellulases that exhibit enhanced thermostability compared to the corresponding wild-type enzymes.
  • derivatives of the endocellulase Cel8A from Clostridium thermocellum are provided.
  • the present invention further provides polynucleotides encoding the modified cellulases, compositions comprising same and uses thereof.
  • the present invention discloses for the first time that by replacing one or more amino acids at the catalytic domain of family-8 cellulases, a significant increase in the thermostability of the enzyme could be achieved.
  • the present invention discloses several specific mutations in the catalytic domain of family-8 cellulases that confer enhanced thermostability.
  • thermostability can be enhanced while maintaining high specific activity towards the substrate.
  • the present invention is based in part on the unexpected increase in the thermostability of C. thermocellum Cel8A that was obtained using a combination of directed evolution strategy and consensus-guided mutagenesis. As exemplified herein below, the activity of the mutant is maintained even after exposure to 80°C or more.
  • the present invention provides a bio-engineered polypeptide variant of a family-8 cellulase comprising at least one amino acid substitution introduced into the catalytic domain of the enzyme and having an enhanced thermostability compared to the unaltered sequence.
  • bio-engineered indicates that the variant is made artificially and does not occur in nature. It is to be explicitly understood that naturally- occurring sequences are excluded from the scope of the present invention. Accordingly, naturally-occurring enzymes comprising the amino acid substitutions disclosed herein are excluded from the scope of the present invention.
  • the variant comprises a non-native glycine (G) at the position corresponding to position 329 of C. thermocellum Cel8A.
  • non-native when referring to an amino acid present at a certain position, means “does not naturally occur in nature”.
  • the positions of the amino acid substitutions of the present invention are determined from sequence alignment of the unaltered family-8 sequence to be modified with the amino acid sequence of the wild-type C. thermocellum Cel8A.
  • the sequence of the naturally occurring, wild-type Cel8A from C. thermocellum (Accession No. AAA83521) is set forth in SEQ ID NO.l .
  • the DNA encoding the wild-type Cel8A (Accession No. K03088) is set forth in SEQ ID NO.2.
  • the variant further comprises an additional substitution selected from the group consisting of a non-native arginine (R) at the position corresponding to position 276 of C. thermocellum Cel8A, and non-native threonine (T) at the position corresponding to position 375 of C. thermocellum Cel8A.
  • R non-native arginine
  • T non-native threonine
  • the variant comprises a non-native glycine (G) at the position corresponding to position 329 of C. thermocellum Cel8A, a non-native arginine (R) at the position corresponding to position 276 of C. thermocellum Cel8A, and a non- native threonine (T) at the position corresponding to position 375 of C. thermocellum Cel8A.
  • G non-native glycine
  • R non-native arginine
  • T non- native threonine
  • the variant comprises a non-native glycine (G) at the position corresponding to position 329 of C. thermocellum Cel8A, a non-native arginine (R) at the position corresponding to position 276 of C. thermocellum Cel8A, a non-native threonine (T) at the position corresponding to position 375 of C. thermocellum Cel8A., and a non-native proline (P) at the position corresponding to position 283 of C. thermocellum Cel8A.
  • G non-native glycine
  • R non-native arginine
  • T non-native threonine
  • P non-native proline
  • the variant comprises a non-native proline (P) at the position corresponding to position 283 of C. thermocellum Cel8A.
  • a bio-engineered polypeptide variant of the endoglucanase Cel8A from C. thermocellum is provided.
  • the variant Cel8A comprises a serine (S) to glycine (G) substitution at position 329 of the polypeptide chain.
  • the protein sequence of the variant is as set forth in SEQ ID NO: 5.
  • this single mutation serves to increase the T m by 7.0°C and the half-life of activity by 8 fold at 85°C.
  • the variant Cel8A further comprises an additional substitution selected from the group consisting of lysine (K) to arginine (R) substitution at position 276 of the polypeptide chain and serine (S) to threonine (T) substitution at position 375 of the polypeptide chain.
  • an additional substitution selected from the group consisting of lysine (K) to arginine (R) substitution at position 276 of the polypeptide chain and serine (S) to threonine (T) substitution at position 375 of the polypeptide chain.
  • the protein sequence of the variant is selected from the group consisting of the sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 43. Each possibility represents a separate embodiment of the invention.
  • an isolated polypeptide variant of Cel8A from C. thermocellum comprises a serine (S) to glycine (G) substitution at position 329 of the polypeptide chain, a lysine (K) to arginine (R) substitution at position 276 of the polypeptide chain and a serine (S) to threonine (T) substitution at position 375 of the polypeptide chain.
  • the protein sequence of the variant is as set forth in SEQ ID NO. 13.
  • an bio-engineered polypeptide variant of Cel8A from C. thermocellum comprises a serine (S) to glycine (G) substitution at position 329 of the polypeptide chain, a lysine (K) to arginine (R) substitution at position 276 of the polypeptide chain, a serine (S) to threonine (T) substitution at position 375 of the polypeptide chain and a glycine (G) to proline (P) substitution at position 283 of the polypeptide chain.
  • the protein sequence of the variant is as set forth in SEQ ID NO: 17.
  • a bio-engineered polypeptide variant of Cel8A from C. thermocellum the variant comprises a glycine (G) to proline (P) substitution at position 283 of the polypeptide chain.
  • the protein sequence of the variant is as set forth in SEQ ID NO: 21.
  • this single mutation displays a higher thermal stability than the wild-type enzyme.
  • bio-engineered polypeptide variants of the present invention exhibit increased thermostability compared to the wild-type enzymes from which they are derived.
  • Thermostability of a protein may be defined by its melting temperature (T m ), namely, the temperature at which 50% of the protein is unfolded.
  • T m melting temperature
  • An increased T m corresponds with better thermostability.
  • the variant cellulases of the present invention have a T m which is at least 4°C, at least 5 °C, at least 7 °C, at least 9 °C higher than the T m of the unaltered sequence from which they are derived. Each possibility represents a separate embodiment of the invention.
  • the present invention provides an isolated polynucleotide encoding a bio-engineered polypeptide variant family-8 cellulase of the present invention.
  • the polynucleotide encodes a variant of the endoglucanase Cel8A from C. thermocellum.
  • the polynucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 1 1, SEQ ID NO. 15, SEQ ID NO: 19, SEQ ID NO: 23 and SEQ ID NO: 44.
  • SEQ ID NO. 7 SEQ ID NO. 1 1
  • SEQ ID NO. 15 SEQ ID NO: 19
  • SEQ ID NO: 23 SEQ ID NO: 44.
  • the present invention provides an isolated construct comprising a polynucleotide of the present invention.
  • the present invention provides a genetically- modified cell capable of expressing and producing the variant cellulases of the present invention.
  • a genetically-modified cell comprising a polynucleotide encoding the variant cellulases of the present invention.
  • a host cell comprising a construct comprising a polynucleotide of the present invention.
  • the cell is selected from a prokaryotic and eukaryotic cell. Each possibility represents a separate embodiment of the invention.
  • the cell is a prokaryotic cell.
  • the present invention provides an artificial cellulosome complex comprising a bio-engineered polypeptide variant of a family-8 cellulase of the present invention.
  • the present invention provides a composition comprising a bio-engineered polypeptide variant of a family-8 cellulase of the present invention, for use in the bioconversion process of cellulosic substrates into degradation products.
  • the present invention provides a method for converting cellulosic material into degradation products, the method comprising exposing said cellulosic material to cells capable of expressing and producing a variant family-8 cellulases of the present invention, for example, the cells described above.
  • the present invention provides a method for converting cellulosic material into degradation products, the method comprising exposing said cellulosic material to any of the variant polypeptides described above.
  • FIG. 1 Residual activity of Cel8A and H5G2 mutants after heat treatment (82°C, 15 min). Each mutant was generated by site-directed mutagenesis of a single codon in either the wild-type Cel8A or the thermostable mutant H5G2 (mutl l-mutl4).
  • FIG. 1 Improvement of residual activity of mutants generated by recombination of selected Cel8A thermostable variants in the first generation library.
  • Wild-type Cel8A (WT) and the S329G (SG) mutant were used as controls.
  • DM1, DM2 and TM were generated as described in the text.
  • FIG 3. Thermal inactivation of CelSA and mutants at 85°C. The residual endoglucanase activity of wild-type (closed circles), SG (open circles), DM1 (closed triangles) and TM (open triangles) was assayed at different time points.
  • Figure 4. Schematic ribbon diagram of the overall three dimensional structure of Cel8A (PDB code 1CEM). The residues that were mutated in the thermal resistant variants are marked in white. The residues involved in catalysis are marked in black.
  • Figure 6 Distribution of consensus mutations in the shuffled library. Several isolates contained between 1-2 additional missense mutations that were not included in the analysis.
  • FIG. 8 Kinetics of thermal inactivation of Cel8A variants. The residual activities were measured at different time points. Wild-type Cel8A served as a control. The activity of unheated enzymes was taken as 100%. Each point represents the mean of duplicate determinations.
  • FIG. 9 Specific activities of wild-type Cel8A and thermostable mutants on CMC and PASC. Enzymes were incubated with 0.5% (wt/vol) solutions at 65°C for 1 h.
  • FIG. 10 Schematic ribbon diagram of the overall three dimensional structure of Cel8A (PDB code 1CEM). The localization of the residues that were replaced in the QM mutant in the Cel8A structure is shown.
  • the present invention provides variant forms of cellulases having a catalytic domain belonging to glycoside hydrolases family-8.
  • derivatives of the endocellulase Cel8A from C. thermocellum are provided.
  • the variant cellulases of the present invention comprise one or more amino acid substitutions introduced into their catalytic domain, and exhibit enhanced thermostability relative to the wild-type enzyme. According to some aspects, thermostability is enhanced while maintaining high specific activity towards the substrate.
  • family-8 cellulase refers to a cellulase having a catalytic domain classified as family-8 glycoside hydrolase, as defined in the Carbohydrate-Active Enzymes (CAZy) server (www.cazy.org) and/or CAZypedia (www.cazypedia.org).
  • CAZy Carbohydrate-Active Enzymes
  • catalytic domain and “catalytic module” are used interchangeably, and as used herein refer to their accepted interpretation for modular enzymes, for which the catalytic domain can be readily identified within the enzyme polypeptide sequence. Such modular enzymes are under the scope of the present invention.
  • C. thermocellum Cel8A As used herein, the terms “Cel8A”, “endoglucanase Cel8A”, “C. thermocellum Cel8A” are used interchangeably and refer to the endoglucanase Cel8A of C. thermocellum Accession No. AAA83521.
  • derivative As used herein, the terms “derivative”, “mutant”, “variant” are used interchangeably and refer to a polypeptide which differs from an unaltered, wild-type amino acid sequence due to one or more amino acid substitutions introduced into the sequence, and/or due to the inclusion of sequences not included in the wild-type protein.
  • wild type and “unaltered sequence” are used interchangeably and refer to the naturally occurring DNA/protein.
  • gene has its meaning as understood in the art.
  • a gene is taken to include gene regulatory sequences (e.g. promoters, enhancers, etc.) and/or intron sequences, in addition to coding sequences (open reading frames).
  • isolated means 1) separated from at least some of the components with which it is usually associated in nature; 2) prepared or purified by a process that involves the hand of man; and/or 3) not occurring in nature.
  • polypeptide peptide
  • protein protein
  • DNA construct refers to an artificially assembled or isolated nucleic acid molecule which comprises the gene of interest.
  • vector refers to any recombinant polynucleotide construct that may be used for the purpose of transformation, i.e. the introduction of heterologous DNA into a host cell.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another exemplary type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • a "primer” defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
  • transformation refers to the introduction of foreign DNA into cells.
  • thermo stability As used herein, the terms “thermal stability”, “thermostability”, “thermal resistance”, are used interchangeably and refer to the ability of an enzyme to retain activity after exposure to elevated temperatures.
  • the term "enhanced thermostability" when referring to a variant, indicates an enhanced ability of the variant to retain activity after exposure to elevated temperatures compared to the corresponding wild-type enzyme.
  • the present invention discloses several amino acid substitutions in the catalytic domain of family-8 cellulases that confer enhanced thermostability.
  • the position of each of the disclosed mutations is determined herein according to its position in Cel8A, i.e., the amino acid numbering of Cel8A is used as the basis for determining the position of the amino acid substitutions of the present invention.
  • the amino acid sequence of a wild-type family-8 cellulase to be modified is aligned with the amino acid sequence of the wild-type Cel8A in order to determine the position of a particular amino acid substitution of the present invention.
  • the present invention is based in part on the development of highly thermostable derivatives of Cel8A using two approaches, in-vitro directed evolution and limited mutagenesis in residues that occur in consensus sequences.
  • thermostability screen was used in order to isolate thermostable mutants that maintain high activity levels.
  • One of the mutants isolated in the screen for thermostability conferred a remarkable residual activity after heat treatment.
  • This mutant contained four amino acid changes, three of which were expendable for the enzyme properties as demonstrated by site-directed mutagenesis analysis of each of the mutations individually and in combination (Example 2 hereinbelow).
  • the fourth amino acid substitution a substitution of serine to glycine at position 329 of Cel8A, resulted in a significant increase in thermostability.
  • the present invention discloses a single replacement in the catalytic domain of family-8 cellulases, such as Cel8A, that is sufficient to provide a significant increase in thermostability.
  • thermostability upon substitution of a hydrophilic residue with a nonpolar side chain on solvent-exposed protein surfaces is not without precedence (Kotzia et al., Febs J 2009, 276, 1750). Yet, saturation mutagenesis of the S329 site of Cel8A followed by thermostability screening demonstrated that no other amino acid except glycine could confer thermostability at that position.
  • Glycine is the simplest of all amino acids and the lack of a ⁇ -carbon enables rotation around both ⁇ and ⁇ with much less restriction.
  • the present invention further discloses a second amino acid substitution, a non- native arginine (R) at the position corresponding to position 276 of Cel8A (K276R in the case of Cel8A), derived from several thermostable mutants identified in a second random mutagenesis screen (Example 3 hereinbelow).
  • a second amino acid substitution a non- native arginine (R) at the position corresponding to position 276 of Cel8A (K276R in the case of Cel8A), derived from several thermostable mutants identified in a second random mutagenesis screen (Example 3 hereinbelow).
  • the K276R confrered a more modest but significant increase in residual activity.
  • the K276 site is located in close tertiary proximity to S329, and borders the active-site cleft.
  • lysine to arginine mutations were shown to confer intrinsic stability to a number of unrelated enzymes.
  • thermostable structures since the structure-function relationship is not known or fully understood for the majority of proteins, and since there is no unique paradigm of thermostable structures, it is not guaranteed that a certain strategy used to increase thermostability of one protein would yield similar results in another protein. The increase in thermostability observed for the mutants of the present invention is therefore unexpected.
  • the present invention further discloses a third amino acid substitution, a non- native threonine at position 375 (S375T in the case of Cel8A), which was also identified in the second random mutagenesis screen (Example 3 hereinbelow).
  • S375T substitution showed an additive effect on residual activity.
  • This conservative substitution which is located in the midst of an a helix of Cel8A, may reflect improved internal packing of the protein.
  • sequence comparison of family 8 cellulases suggests that threonine is the most prevalent amino acid at position 375. Therefore the S375T substitution could be considered a type of 'back-to-consensus' mutation.
  • the present invention further discloses a quadruple-mutant, which contains the optimal combination of the four substitutions disclosed herein.
  • the quadruple-mutant exhibits an increase in thermal stability, compared to the parental wild-type enzyme. Remarkably, no loss of catalytic activity was observed compared to the wild-type endoglucanase.
  • the consensus method may not always indicate the most stabilizing amino acid at a specific position, and a library of single mutations or combination of mutations will often be necessary.
  • Introducing the G283P mutation to increase the thermostability of the triple mutant (TM) generated by a random library resulted in a further enhanced thermostable protein.
  • the present invention provides isolated variants of family-8 cellulases comprising at least one non-native amino acid substitution artificially introduced into the catalytic domain of the enzyme, wherein the polypeptide variant has an enhanced thermostability compared to the corresponding wild-type sequence.
  • the variant comprises a non-native glycine (G) at the position corresponding to position 329 of C. thermocellum Cel8A, said position being determined from sequence alignment of the unaltered sequence with the amino acid sequence of C. thermocellum Cel8A set forth in SEQ ID NO: 1.
  • G non-native glycine
  • Methods for sequence alignment are known in the art and include, for example, the use of web-based servers such as ClustalW, www.ebi.ac.uk/Tools/msa/clustalw2.
  • the variant comprises a non-native glycine (G) at the position corresponding to position 329 of C. thermocellum Cel8A and further comprising an additional substitution selected from the group consisting of a non-native arginine (R) at the position corresponding to position 276 of C. thermocellum Cel8A, and non-native threonine (T) at the position corresponding to position 375 of C. thermocellum Cel8A.
  • R non-native arginine
  • T non-native threonine
  • the variant comprises a non-native glycine (G) at the position corresponding to position 329 of C.
  • thermocellum Cel8A a non-native arginine (R) at the position corresponding to position 276 of C. thermocellum Cel8A, and a non- native threonine (T) at the position corresponding to position 375 of C. thermocellum Cel8A.
  • R non-native arginine
  • T non- native threonine
  • the variant comprises a non-native glycine (G) at the position corresponding to position 329 of C. thermocellum Cel8A, a non-native arginine (R) at the position corresponding to position 276 of C. thermocellum Cel8A, a non-native threonine (T) at the position corresponding to position 375 of C. thermocellum Cel8A., and a non-native proline (P) at the position corresponding to position 283 of C. thermocellum Cel8A.
  • G non-native glycine
  • R non-native arginine
  • T non-native threonine
  • P non-native proline
  • the at least one amino acid substitution is a non-native proline (P) at the position corresponding to position 283 of C. thermocellum Cel8A.
  • a variant polypeptide of a family-8 cellulase comprises either a non-native glycine (G) at the position corresponding to position 329 of C. thermocellum Cel8A, a non-native arginine (R) at the position corresponding to position 276 of C. thermocellum Cel8A, a non-native threonine (T) at the position corresponding to position 375 of C. thermocellum Cel8A., a non-native proline (P) at the position corresponding to position 283 of C. thermocellum Cel8A or any combination thereof.
  • G non-native glycine
  • R non-native arginine
  • T non-native threonine
  • P non-native proline
  • variants of the endoglucanase Cel8A from C. thermocellum are provided.
  • the Cel8A variant comprises at least one mutation selected from the group consisting of S329G, K276R, S375T and G283P. Each possibility represents a separate embodiment of the invention.
  • the variants of the present invention comprise a Cel8A single mutant, double mutant, triple mutant and/or quadruple mutant.
  • the variant Cel8A comprises a serine (S) to glycine (G) substitution at position 329 of the polypeptide chain.
  • S serine
  • G glycine
  • An exemplary Cel8A variant comprising said substitution is provided in SEQ ID NO: 5.
  • a corresponding DNA sequence encoding the variant is provided in SEQ ID NO: 7.
  • the variant Cel8A comprises a glycine (G) to proline (P) at position 283 of the polypeptide chain.
  • G glycine
  • P proline
  • An exemplary Cel8A variant comprising said substitution is provided in SEQ ID NO: 21.
  • corresponding DNA sequence encoding the variant is provided in SEQ ID NO: 23.
  • the variant Cel8A comprises a serine (S) to glycine (G) substitution at position 329 of the polypeptide chain and a lysine (K) to arginine (R) substitution at position 276 of the polypeptide chain.
  • S serine
  • G glycine
  • K lysine
  • R arginine
  • SEQ ID NO: 9 A corresponding DNA sequence encoding the variant is provided in SEQ ID NO: 1 1.
  • the variant Cel8A comprises a serine (S) to glycine (G) substitution at position 329 of the polypeptide chain and a serine (S) to threonine (T) substitution at position 375 of the polypeptide chain.
  • An exemplary Cel8A variant comprising said substitutions is provided in SEQ ID NO: 43.
  • a corresponding DNA sequence encoding the variant is provided in SEQ ID NO: 44.
  • the variant Cel8A comprises a serine (S) to glycine (G) substitution at position 329 of the polypeptide chain, a lysine (K) to arginine (R) substitution at position 276 of the polypeptide chain and a serine (S) to threonine (T) substitution at position 375 of the polypeptide chain.
  • An exemplary Cel8A variant comprising said substitutions is provided in SEQ ID NO: 13.
  • a corresponding DNA sequence encoding the variant is provided in SEQ ID NO: 15.
  • the variant Cel8A comprises a serine (S) to glycine (G) substitution at position 329 of the polypeptide chain, a lysine (K) to arginine (R) substitution at position 276 of the polypeptide chain, a serine (S) to threonine (T) substitution at position 375 of the polypeptide chain, and a glycine (G) to proline (P) substitution at position 283 of the polypeptide chain.
  • An exemplary Cel8A variant comprising said substitutions is provided in SEQ ID NO: 17.
  • a corresponding DNA sequence encoding the variant is provided in SEQ ID NO: 19.
  • Protein thermostability may be defined, for example, by its melting temperature (T m ), the half-life (t ⁇ ) at a defined temperature, and the temperature at which 50% of the initial enzyme activity is lost after incubation at a defined time (T 50 ).
  • the variant cellulases of the present invention has a T m which is at least about 4°C, at least about 5 °C, at least about 7 °C, at least about 9 °C higher than the T m of the unaltered sequence from which they are derived.
  • the T m may be determined, for example, using circular dichroism, as exemplified below.
  • the variant polypeptides disclosed herein may be produced by either recombinant or chemical synthetic methods.
  • the variant family-8 cellulases are a product of recombinant expression.
  • the variant polypeptides of the present invention may be synthesized by expressing a polynucleotide molecule encoding the variant polypeptide in a host cell, for example, a microorganism cell transformed with the nucleic acid molecule.
  • a polynucleotide may be produced, for example, by mutation of a first polynucleotide encoding a wild type polypeptide, so as to provide a second polynucleotide which encodes a variant polypeptide having replacements of one or more residues which are normally present in the wild type cellulase.
  • DNA sequences encoding wild type family-8 cellulases may be isolated from any strain or subtype of a microorganism producing them, using various methods well known in the art (see for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y., (2001)).
  • a DNA encoding the wild-type polypeptide may be amplified from genomic DNA of the appropriate microorganism by polymerase chain reaction (PCR) using specific primers, constructed on the basis of the nucleotide sequence of the known wild type sequence.
  • PCR polymerase chain reaction
  • Suitable techniques are well known in the art, described for example in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159 and 4,965,188
  • the genomic DNA may be extracted from the bacterial cell prior to the amplification using various methods known in the art, see for example, Marek P. M et al., "Cloning and expression in Escherichia coli of Clostridium thermocellum DNA encoding p-glucosidase activity", Enzyme and Microbial Technology Volume 9, Issue 8, August 1987, Pages 474-478.
  • the isolated polynucleotide encoding the wild type family-8 cellulase may be cloned into a vector, such as the pET28a plasmid.
  • the desired mutation(s) may be introduced by modification at one or more base pairs, using methods known in the art, such as for example, site-specific mutagenesis (see for example, Kunkel Proc. Natl. Acad. Sci. USA 1985, 82:488-492; Weiner et al, Gene 1994, 151 : 1 19-123; Ishii et al., Methods Enzymol. 1998, 293:53-71); cassette mutagenesis (see for example, Kegler-Ebo et al., Nucleic Acids Res.
  • introduction of two and/or three mutations can be performed using commercially available kits, such as the QuickChange® site-directed mutagenesis kit (Stratagene).
  • kits such as the QuickChange® site-directed mutagenesis kit (Stratagene).
  • a polynucleotide encoding a variant of family- 8 cellulase may be prepared synthetically, for example using the phosphoroamidite method (see, Beaucage et al, Curr Protoc Nucleic Acid Chem. 2001 May; Chapter 3:Unit 3.3; Caruthers et al, Methods Enzymol.1987, 154:287-313).
  • the polynucleotide thus produced which encodes a variant of a family-8 cellulase, may then be subjected to further manipulations, including one or more of purification, annealing, ligation, amplification, digestion by restriction endonucleases and cloning into appropriate vectors.
  • the polynucleotide may be ligated either initially into a cloning vector, or directly into an expression vector that is appropriate for its expression in a particular host cell type.
  • Polypeptides of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences, such as a thrombin cleavage site.
  • the polynucleotide encoding the polypeptide of the invention may be incorporated into a wide variety of expression vectors, which may be transformed into in a wide variety of host cells.
  • the host cell may be prokaryotic or eukaryotic.
  • Introduction of a polynucleotide into the host cell can be effected by well known methods, such as chemical transformation (e.g. calcium chloride treatment), electroporation, conjugation, transduction, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, scrape loading, ballistic introduction and infection.
  • bacterial cells such as cells of E. coli and Bacillus subtilis.
  • the polypeptides may be expressed in any vector suitable for expression.
  • the appropriate vector is determined according the selected host cell.
  • Vectors for expressing proteins in E. coli include, but are not limited to, pET, pK233, pT7 and lambda pSKF.
  • Other expression vector systems are based on beta-galactosidase (pEX); maltose binding protein (pMAL); and glutathione S-transferase (pGST).
  • the proteins may be desgined to include a tag.
  • a non-limiting example of a fusion construct is His-Tag (six consecutive histidine residues), which can be isolated and purified by conventional methods.
  • Selection of a host cell transformed with the desired vector may be accomplished using standard selection protocols involving growth in a selection medium which is toxic to non-transformed cells.
  • E. coli may be grown in a medium containing an antibiotic selection agent; cells transformed with the expression vector which further provides an antibiotic resistance gene, will grow in the selection medium.
  • the desried polypeptide Upon transformation of a suitable host cell, and propagation under conditions appropriate for protein expression, the desried polypeptide ma y be identified in cell extracts of the transformed cells.
  • Transformed hosts expressing a variant family-8 cellulase may be identified by analyzing the proteins expressed by the host using SDS- PAGE and comparing the gel to an SDS-PAGE gel obtained from the host which was transformed with the same vector but not containing a nucleic acid sequence encoding a family-8 cellulase or a variant of a family-8 cellulase.
  • Variant family-8 cellulases can also be identified by other known methods such as immunoblot analysis using anti- family-8 cellulase antibodies, dot blotting of total cell extracts, limited proteolysis, mass spectrometry analysis, and combinations thereof.
  • Variant family-8 cellulases which have been identified in cell extracts may be isolated and purified by conventional methods, including ammonium sulfate or ethanol precipitation, acid extraction, salt fractionation, ion exchange chromatography, hydrophobic interaction chromatography, gel permeation chromatography, affinity chromatography, and combinations thereof.
  • polypeptides of the invention can be produced as fusion proteins, attached to an affinity purification tag, such as a His-tag, in order to facilitate their rapid purification.
  • an affinity purification tag such as a His-tag
  • the isolated variant of the family-8 cellulase can be analyzed for its various properties, for example specific activity and thermal stability, using methods known in the art, some of them are described hereinbelow.
  • the polypeptides of the invention can be produced and/or used without their start codon (methionine or valine) and/or without their leader (signal) peptide to favor production and purification of recombinant polypeptides. It is known that cloning genes without sequences encoding leader peptides will restrict the polypeptides to the cytoplasm of the host cell and will facilitate their recovery (see for example, Glick, B. R. and Pasternak, J. J. (1998) In “Molecular biotechnology: Principles and applications of recombinant DNA", 2nd edition, ASM Press, Washington D.C., p. 109-143). Synthetic production
  • variant polypeptides of the present invention may also be produced by synthetic means using well known techniques, for example, solid phase synthesis (see for example, Merrifield, R. B., J. Am. Chem. Soc, 85:2149-2154, 1963; Stewart, J. M. and Young, J.D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, 111., pp. 1 1-12).
  • Synthetic peptides may be produced using commercially available laboratory peptide design and synthesis kits (see for example, Geysen et al, Proc. Natl. Acad. Sci., USA 1984, 81 :3998).
  • FMOC peptide synthesis systems are available.
  • Assembly of a polypeptide or fragment can be carried out on a solid support using for example, an Applied Biosystems, Inc. Model 431 A automated peptide synthesizer.
  • the polypeptides may be made by either direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.
  • the present invention provides a nucleic acid molecule encoding a bio-engineered variant of a family-8 cellulase, the variant comprising at least one amino acid substitution introduced into the catalytic domain of the enzyme and having an enhanced thermostability compared to the unaltered sequence.
  • the polynucleotide encodes a variant of the endoglucanase Cel8A from C. thermocellum.
  • the polynucleotide sequence encodes a variant Cel8A polypeptide comprising at least one amino acid substitution selected from the group consisting of S329G, K276R, S375T and G283P. Each possibility represents a separate embodiment of the invention.
  • the present invention provides polynucleotide sequences encoding the disclosed variants of family-8 cellulases.
  • the polynucleotides encode the disclosed Cel8A variants.
  • the codon used in the polynucleotide for encoding a particular amino acid which is to substitute an amino acid originally present in the sequence encoding the wild-type enzyme should be selected in accordance with the known and favored codon usage of the host cell which was selected for expressing the polynucleotide.
  • a skilled person will be aware of the relationship between nucleic acid sequence and polypeptide sequence, in particular, the genetic code and the degeneracy of this code, and will be able to construct nucleic acids encoding the polypeptides of the present invention without difficulty. For example, a skilled person will be aware that for each amino acid substitution in a polypeptide sequence, there may be one or more codons which encode the substitute amino acid.
  • one or more nucleic acid sequences may be generated corresponding to a certain variant polypeptide sequence.
  • the variant polypeptide comprises more than one substitution, for example S329G/K276R in a Cel8A variant
  • the corresponding nucleic acids may comprise pairwise combinations of the codons which encode respectively the two amino acid changes.
  • the polynucleotides of the present invention may include non-coding sequences, including for example, non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns and polyadenylation signals. Further included are polynucleotides that comprise coding sequences for additional amino acids heterologous to the variant polypeptide, in particular a marker sequence, such as a poly-His tag, that facilitates purification of the polypeptide in the form of a fusion protein.
  • non-coding 5' and 3' sequences such as transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns and polyadenylation signals.
  • polynucleotides that comprise coding sequences for additional amino acids heterologous to the variant polypeptide in particular a marker sequence, such as a poly-His tag, that facilitates purification of the polypeptide in
  • the present invention provides a construct comprising a polynucleotide of the present invention.
  • the present invention provides a genetically- modified cell capable of expressing and producing the variant cellulases of the present invention.
  • the cell comprises the construct described above.
  • the variant cellulase is designed to include a signal sequence in order to enable its secretion from the host cell.
  • a genetically-modified cell is provided, said cell is capable of producing and secreting the variant polypeptide of the present invention.
  • the cell is a prokaryotic cell.
  • suitable prokaryotic hosts include bacterial cells, such as cells of Escherictahia coli and Bacillus subtilis.
  • the cell is a eukaryotic cell.
  • the cell is a fungal cell, such as yeast.
  • suitable yeast cells include Saccharomyces cerevisiae and Pichia pastoris.
  • the cell is a plant cell.
  • variant polypeptides of the present invention compositions comprising same and cells producing same may be utilized for the bioconversion of cellulosic material into degradation products
  • cellulosic substrate encompasses any substrate derived from plant biomass and comprising cellulose, including but not limited to, lignocellulosic feedstocks for the production of ethanol or other high value products, animal feeds, forestry waste products, such as pulp and wood chips, and textiles.
  • Resulting sugars may be used for the production of alcohols such as ethanol, propanol, butanol and/or methanol, production of fuels, e.g., biofuels such as synthetic liquids or gases, such as syngas, and the production of other fermentation products, e.g. succinic acid, lactic acid, or acetic acid.
  • alcohols such as ethanol, propanol, butanol and/or methanol
  • fuels e.g., biofuels such as synthetic liquids or gases, such as syngas
  • other fermentation products e.g. succinic acid, lactic acid, or acetic acid.
  • variant polypeptides of the present invention may also be incorporated into artificial cellulosome complexes, also referred to as designer cellulosomes.
  • the designer cellulosome concept is based on the very high affinity and specific interaction between cohesin and dockerin modules from the same microorganism species.
  • Designer cellulosomes are typically constructed from recombinant chimeric scaffoldins containing divergent cohesins from different microorganism species to which matching dockerin-containing enzyme hybrids are prepared. In effect, in designer cellulosomes, enzymes are complexed together on a scaffoldin subunit via the very strong and specific cohesin-dockerin interaction.
  • the present invention provides an artificial cellulosome complex comprising a bio-engineered polypeptide variant of a family-8 cellulase of the present invention.
  • the present invention provides a composition comprising an isolated polypeptide variant of a family-8 cellulase of the present invention, for use in the bioconversion process of cellulosic substrates into degradation products.
  • the present invention provides a method for converting cellulosic material into degradation products, the method comprising exposing said cellulosic material to cells capable of expressing a variant family-8 cellulases of the present invention, for example, the host cells described above.
  • the present invention provides a method for converting cellulosic material into degradation products, the method comprising exposing said cellulosic material to any of the variant polypeptides described above.
  • polypeptides of the present invention may be added to bioconversion and other industrial processes for example, continuously, in batches or by fed-batch methods. Alternatively or additionally, the enzymes of the present invention may be recycled.
  • Cel8A from Clostridium thermocellum ATCC 27405 was cloned without the signal peptide into pET28a (Novagen, Madison, WI) with a C-terminal His tag.
  • the amino acid sequence and DNA sequence of the cloned Cel8A are set forth in SEQ ID NOs: 3 and 4, respectively.
  • E. coli DH5a was used for propagation of plasmids.
  • E. coli BL21 (DE3) was used for high level expression of the recombinant endoglucanase, and was cultivated at 37°C in Luria-Bertani (LB) medium containing 50 ⁇ g/ml kanamycin.
  • Random mutagenesis and construction of libraries A library of cel8A mutants was generated by error-prone PCR using a GeneMorph II Random Mutagenesis Kit (Stratagene, La Jolla, CA). pET28cel8A plasmid was used as a template and T7 promoter primer and T7 terminator primer were used for amplification. Reaction mixtures contained 8 ng of pETcel8A. Thermal cycling parameters were 95°C for 3 min and 28 cycles of 95°C for 40 s, 56°C for 40 s and 72°C for 1.2 min.
  • the resulting PCR product was treated with Dpnl (New England Biolabs, UK) to destroy the template plasmid, purified from agarose gel, and then used as a template for a nested PCR using ReadyMix PCR reaction mix (Thermo Scientific, Waltham, MA ) and primers 5'- AAGAAGGAGATATACCATGG-3' (SEQ ID NO: 25) and 5'- GTGGTGGTGGTGCTCG AG-3 ' (SEQ ID NO: 26) (boldface letters indicate Ncol and Xhol restriction sites, respectively).
  • the amplified product was purified and ligated into the expression vector pET28a through the restriction sites and transformed into E. coli DH5a cells, yielding ⁇ 10 5 transformants. Plasmid DNA was then extracted to obtain the library for subsequent transformations and screening.
  • thermostable endoglucanase variants Transformed E. coli cells derived from the library of Cel8A variants were spread onto LB plates containing 50 ⁇ g/ml kanamycin and incubated overnight at 37°C. The plates were overlaid with soft agar containing 0.3% CMC, 0.7% agar and 0.2 mM IPTG as an inducer in 25 mM sodium acetate (pH 6.0). The plates were incubated for 30 min at 37°C to induce enzyme expression and 2 h at 60°C to facilitate enzyme activity. The plates were then stained for 10 min with 0.25% fresh Congo red solution and destained with 1 M NaCl.
  • DMS 3,5-dinitrosalicylic acid
  • identification of active endoglucanase enzymes was performed using a high-throughput screening procedure employing double layered carboxymethyl cellulose (CMC)-containing plates.
  • CMC carboxymethyl cellulose
  • the cultures were streaked onto agar plates, and a few colonies from each were grown and reanalyzed for thermostability. Plasmids were then extracted from the positive clones and retransformed into fresh E. coli cells, in order to confirm the phenotype.
  • the cel8A ORF of the extracted plasmids from the positive clones were sequenced to determine the mutation(s) responsible for the observed thermostability (Table 1).
  • a gene library encoding all possible amino acids at position S329 of Cel8A was constructed by replacing the target codon with NNS (where N is A, G, C, or T and S is G or C).
  • the enzymes were purified to homogeneity by virtue of the attached His tag, and the S329G mutant was assayed for increased thermostability (the amino acid sequence and DNA sequence of the His-tagged S329G mutant are set forth in SEQ ID NOs: 6 and 8, respectively.
  • saturation mutagenesis was performed at the S329 site. To insure a 0.99 probability of all possible outcomes, the library size for one mutated site was calculated by a binomial probability approximation to be 140 colonies.
  • thermostability The four mutants that demonstrated a significant increase in thermostability were individually amplified, mixed, and digested with DNase I (Sigma). The resulting 50-200 bp fragments were assembled by PCR as described previously (Abecassis et al., Nucleic Acids Res 2000, 28, E88). The resulting library was cloned into the pET28 vector using the Ncol and Xhol restriction sites. Results
  • the four clones that showed increased thermostability were shuffled using in-vitro recombination to produce an assembly of different combinations of mutations.
  • the assembled PCR fragments were ligated into pET28a and transformed into E. coli cells. Approximately 1000 colonies were isolated. At this stage, initial CMC plate assay was not required, as over 95% of the colonies were positive for activity at 60°C.
  • the clones were subjected to heat treatment of 15 min at an increased temperature (87°C). Under these conditions, the S329G mutant retained 30% of its activity while the wild-type enzyme underwent near-complete inactivation.
  • Figure 2 shows the six variants that were selected for their increased thermostability compared to the S329G mutant.
  • thermostability Mutants DM1 and TM, which showed the highest thermostability, were purified to homogeneity and their thermostability properties were determined and compared with that of the SG single mutant and the wild-type enzyme.
  • E. coli BL21 (DE3) transformants were grown at 37°C in LB supplemented with 50 ⁇ g/ml kanamycin, until an OD 6 oo of -0.8 was reached. Overexpression was induced by adding 0.5 mM IPTG; the cultures were grown for another 3 h and harvested (4000 x g, 15 min, 4°C). The pellet was frozen overnight at -20°C.
  • the cells were resuspended in Tris-buffered saline (TBS, 137 mM NaCl, 2.7 mM KC1, 25 mM Tris-HCl, pH 7.4) supplemented with 5 mM imidazole (Merck KGaA, Darmstadt, Germany) and protease- inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride (PMSF), 0.4 mM benzamidine and 0.06 mM benzamide from Sigma-Aldrich, St. Louis, MO) and disrupted by sonication. The sonicate was heated for 30 min at 60°C then centrifuged (20,000 x g, 30 min, 4°C).
  • the soluble fraction was mixed with Ni-NTA (nitrilotriacetic acid), supplemented with 5-10 mM imidazole, for 1 h in an Econo-pack column at 4°C (batch purification system). The column was then washed by gravity flow with 50 mM imidazole. Elution was preformed first using 100 mM imidazole, followed by 250 mM imidazole. Fractions (2 ml) were collected and analyzed by SDS-PAGE. The fractions containing the purified proteins were pooled and extensively dialyzed against 50 mM sodium acetate buffer at pH 6.0.
  • Protein concentrations were determined by spectrophotometeric absorbance (280 nm) using the calculated molar absorption coefficient of the protein. Samples were stored at 4°C, supplemented with 0.02% sodium azide, or at -20°C with 50% glycerol.
  • Circular dichroism (CD) measurements Melting curves were recorded on a Chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, UK) in a 1 -mm path-length cuvette. The proteins were used at ⁇ 5 ⁇ concentration in 50 mM sodium acetate buffer at pH 6.0., the samples were heated at a rate of 1 °C/ min from 55 to 95°C, and the CD ellipticity signal at 222 nm, which showed the maximal change with the temperature, was monitored.
  • Endoglucanase activity was measured by incubating the purified enzymes with 0.5% (wt/vol) of CMC or phosphoric acid-swollen cellulose (PASC) at 65°C for 1 h with occasional shaking. Activities were determined by assaying the release of reducing sugars by the DNS method. Enzyme activity was expressed as units (U). One unit of endoglucanase activity corresponds to the release of 1 ⁇ of glucose equivalent per hour.
  • the catalytic efficiency was also determined on CMC and the specific activity was determined on both CMC and phosphoric acid-swollen cellulose (PASC). The results are shown in Table 2.
  • the kinetic parameters of the purified mutants and parent enzyme showed that the enhanced thermostability gained by the S329G mutation reduces its catalytic efficiency by 22%. This reduction is also reflected in the initial activity performances on both CMC and PASC.
  • DM1 showed a reduction of 42% in its catalytic efficiency and a greater reduction in the initial activity performances.
  • the triple mutant (TM) that contains an addition of a S375T mutation is able to almost restore the catalytic efficiency and initial activity of the mutant, compared to the wild-type enzyme.
  • a m app is the approximate mid-point temperature of melting determined by CD spectroscopy at 222 nra.
  • bPurified enzyme was incubated with 0.5% (wt/vol) solutions at 65°C for 1 h with occasional shaking. Activities were determined by assaying the release of reducing sugars.
  • thermocellum Cel8A the most influential mutation that conferred the most significant thermostability upon C. thermocellum Cel8A was the serine-to-glycine substitution at position 329.
  • Figure 4 shows the secondary structural elements that form the ( ⁇ / ) 6 barrel structure of the Cel8A catalytic module, as determined by Alzari et al.
  • S329 is located on a loop between helices 9 and 10 of the barrel on the surface of the protein. It forms a hydrogen bond with D319 and can also form hydrogen bonds with the water molecules of the solvent. Replacement of serine by glycine results in loss of hydrogen bonds and may contribute to an increase in structural flexibility.
  • the lysine at position 276 is located on a loop between helices 7 and 8 on the surface of the protein and is in close proximity to the active site cleft. It makes contact with D274 and can also form hydrogen bonds with the water molecules of the solvent.
  • the replacement of lysine by arginine is rather conservative. Both are positively charged and both have large hydrophobic aliphatic side chains (Berezovsky et al., PLoS Comput Biol 2005, 1, e47). However, the substitution of lysine by arginine could improve thermostability by replacing water-mediated hydrogen bonds made by the lysine side chain with direct hydrogen bonds of the guanidinium group that protrudes further in space.
  • the third mutation which showed an additive effect on residual activity was the substitution of serine to threonine at position 375 located in helix 12 of Cel8A. Threonine maintains the side chain hydroxyl group but introduces an extra methyl group. This conservative substitution may contribute to better internal packing and by enhancing the hydrophobic interaction in the interior of the protein molecule. This substitution has the least additive influence on the observed thermostability, but served to increase enzyme activity.
  • Plasmid pET28aCel8A containing the cel8A gene from Clostridium thermocellum ATCC 27405 was used to construct the library.
  • the cel8A gene was amplified and digested with DNase I (Sigma).
  • the resulting 50-200 bp fragments were assembled by PCR in the presence of an equimolar mixture of 8 oligonucleotides encoding the consensus mutations (total of 10 pmol) as described previously (Herman et al., Protein Eng Des Sel, 2007, 20(5): p. 219-26).
  • the primers are listed in Table 3 hereinbelow.
  • the resulting library was cloned into the pET28 vector using the Ncol and Xhol restriction sites.
  • thermostable endoglucanase variants Transformed E. coli cells derived from the library of Cel8A variants were spread onto LB plates containing 50 ⁇ g/ml kanamycin and incubated overnight at 37°C. The clones were picked and grown overnight at 37°C in 96-well plates containing 0.5 ml LB, 50 ⁇ g/ml kanamycin and 0.1 mM isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG). Proteins were extracted using PopCulture Reagent (Novagen) according to the product manual. A sample of the extracted solution was diluted in 50 mM sodium acetate (pH 6.0) and incubated at various temperatures and time periods.
  • Residual activity was determined with 1% CMC, 10 mM CaCl 2 and 50 mM sodium acetate (pH 6.0) at 65°C.
  • the amount of reducing sugars released by the enzyme was determined colorimetrically using 3,5-dinitrosalicylic acid (DNS) reagent.
  • the amino acid sequence of Cel8A from C. thermocellum was used to identify 18 homologous sequences in GenBank. The sequences were selected based on amino acid identity values of 30 to 60%. The following sequences were used for the consensus alignment: gi_220928180_ref_YP_002505089.1 (family-8 glycoside hydrolase Clostridium cellulolyticum H10);
  • gi_256756512_ref_ZP_05497268.1 family-8 glycoside hydrolase Clostridium papyrosolvens DSM 2782
  • gi_110639233_ref_YP_679442.1 beta-glycosidase-like protein Cytophaga hutchinsonii ATCC 33406;
  • gi_146298783_ref_YP_001193374.1 (licheninase Flavobacterium johnsoniae UWIOI);
  • gi_1 10640093_ref_YP_680303.1 (b-glycosidase C. hutchinsonii ATCC 33406); gi_289640415_ref_ZP_06472621.1 (family-8 glycoside hydrolase Ethanoligenens harbinense YUAN-3);
  • gi_159896826_ref_YP_001543073.1 glycoside hydrolase family protein Herpeto siphon aurantiacus ATCC 23779);
  • gi_159896827_ref_YP_001543074.1 cellulose-binding family II protein H. aurantiacus ATCC 23779;
  • gi_182414080_ref_YP_001819146.1 (glycoside hydrolase family protein Opitutus terrae PB90-1);
  • gi_l 52997715_ref_YP_001342550.1 (licheninase Marinomonas sp. MWYL1)
  • gi_261404389_ref_YP_003240630.1 (licheninase (Paenibacillus sp. Y412MC10)
  • gi_l 5552945_dbj_BAB64835.1 (chitosanase-glucanase Paenibacillus fukuinensis);
  • gi_229128022_ref_ZP_04257004.1 (endoglucanase Bacillus cereus BDRD-Cer4)
  • gi_228994572_ref_ZP_04154406.1 (endoglucanase Bacillus pseudomycoides DSM 12442);
  • the sequences were aligned using the ClustalW algorithm and either consensus positions or most abundant positions were determined. Overall, the Cel8A gene differed in 8 positions from the consensus sequence ( Figure 5). It should be noted that many of the proteins used for the alignment were from mesophilic bacteria, e.g., Clostridium cellulolyticum and Flavobacterium johnsoniae, with an optimal temperature well below that of Cel8A for C. thermocellum. Eight oligonucleotide primers were designed, each containing a single codon replacement of the Cel8A gene with the matching consensus residue. In-vitro recombination was then used to produce an assembly of the different combinations of mutations.
  • the resultant library was cloned into the pET28a expression vector and expressed in E. coli.
  • the diversity of the genes in the resulting unselected library is presented in Figure 6. All of the planned consensus mutations were observed in the library but in half of the genes one to two unplanned point mutations appeared that were introduced during the DNA fragment assembly.
  • thermostable mutants In various methods which use random mutagenesis in order to generate thermostable mutants, large numbers of clones have to be screened before identifying the desired mutants. Using the consensus approach it is possible to screen significantly less clones and still acquire thermostable variants. Here, less than 600 clones were screened before the identification of 1 1 thermostable mutants that showed considerably higher residual activity after heat treatment compared to the wild-type Cel8A enzyme. The Cel8A gene from each of the positive clones was sequenced in order to determine the mutation(s) responsible for the increased thermostability.
  • E. coli BL21 (DE3) transformants were grown at 37°C in LB supplemented with 50 ⁇ g/ml kanamycin, until an OD 600 of -0.8 was reached. Overexpression was induced by adding 0.5 mM IPTG; the cultures were grown for another 3 h and harvested (4000 x g, 15 min, 4°C). The pellet was frozen overnight at -20°C.
  • the cells were resuspended in Tris-buffered saline (TBS, 137 mM NaCl, 2.7 mM KC1, 25 mM Tris-HCl, pH 7.4) supplemented with 5 mM imidazole (Merck KGaA, Darmstadt, Germany) and protease- inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride (PMSF), 0.4 mM benzamidine and 0.06 mM benzamide from Sigma- Aldrich, St. Louis, MO) and disrupted by sonication. The sonicate was heated for 30 min at 60°C then centrifuged (20,000 x g, 30 min, 4°C).
  • the soluble fraction was mixed with Ni-NTA (nitrilotriacetic acid), supplemented with 5-10 mM imidazole, for 1 h in an Econo-pack column at 4°C (batch purification system). The column was then washed by gravity flow with 50 mM imidazole. Elution was preformed first using 100 mM imidazole, followed by 250 mM imidazole. Fractions (2 ml) were collected and analyzed by SDS-PAGE. The fractions containing the purified proteins were pooled and dialyzed extensively against 50 mM sodium acetate buffer at pH 6.0.
  • Protein concentrations were determined by spectrophotometeric absorbance (280 nm) using the calculated molar absorption coefficient of the protein. Samples were stored at 4°C, supplemented with 0.02% sodium azide, or at -20°C with 50% glycerol. Results
  • thermostable variants by combining multiple consensus mutations in different combinations.
  • the frequency of the individual consensus mutations in these variants were determined.
  • TM engineered triple mutant
  • the enzymes were purified to homogeneity and their properties were determined and compared with that of wild-type enzyme.
  • the 3.3°C increase in T m of the QM variant relative to the TM variant (Table 5) demonstrates that the thermostabihzing effect of G283P is additive.
  • thermostable quadruple-mutant QM
  • Enzymatic assays Endoglucanase activity was measured by incubating the purified enzymes with 0.5% (wt/vol) of CMC or phosphoric acid-swollen cellulose (PASC) at 65°C for 1 h with occasional shaking. Activities were determined by assaying the release of reducing sugars by the DNS method. Enzyme activity was expressed as units (U). One unit of endoglucanase activity corresponds to the release of 1 ⁇ ⁇ ⁇ of glucose equivalent per hour.
  • the specific activities of the mutants were determined on both CMC and phosphoric acid-swollen cellulose (PASC). As shown in Figure 9, both the G283P and the QM variants demonstrate similar specific activities on CMC compared to the TM and wild-type enzymes.
  • the G283P mutation demonstrated an increase in activity on PASC either as a single mutation or in combination with the TM variant (QM).
  • the stability of the mutants was also determined at pH values of 3.0 to 9.0 and showed similar residual activities as the wild-type Cel8A enzyme.
  • Circular dichroism (CD) measurements Melting curves were recorded on a Chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, UK) in a 1-mm path-length cuvette. The proteins were used at about 5 ⁇ concentration in 50 mM sodium acetate buffer at pH 6.0. The samples were heated at a rate of 1°C/ min from 55 to 95 °C, and the CD ellipticity signal at 222 nm, which showed the maximal change with the temperature, was monitored.
  • thermostable variants namely, G283P, the TM and the QM
  • the results showed that there were no significant changes between the wild-type enzyme and the mutants with increased thermostability.
  • Figure 10 shows the three- dimensional structure of Cel8A as determined by Alzari et al and the mutations that were introduced in the present work. Without being bound by any particular theory or mechanism of action, the explanation for the increased thermostability appears to lie in the reduction of the conformational freedom of the protein backbone in its unfolded state.
  • prolines at key positions namely the first turn of the a-helix, the second site of the ⁇ -turn and in flexible loops, can stabilize proteins. Indeed, in many thermostable proteins there is an increase in the number of prolines at the N-terminus of a-helices (see for example, Watanabe et al., J Mol Biol, 1997, 269(1): p. 142-53). Because proline residue has a pyrrolidine ring, the backbone conformation of proline is constrained.
  • the ⁇ and ⁇ values of the proline residue is restricted, and, in addition, the ⁇ and ⁇ values of the preceding residue are limited (Schimmel et al., J Mol Biol, 1968, 34(1): p. 105-20).
  • Tk-R ase HII from hyperthermophile Thermococcus kodakaraensis was thermostabilized by the introduction of prolines at the N-terminus of a-helices.
  • Barley a-glucosidase was thermostabilized by replacing its N-cap Thr340 residue with proline (Muslin et al., Protein Eng, 2002, 15(1): p. 29-33).

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Abstract

Cette invention concerne des cellulases modifiées appartenant à la famille 8 présentant une thermostabilité meilleure que la thermostabilité de l'enzyme correspondante du type sauvage ; l'invention concerne également des polynucléotides codant ces cellulases modifiées, des compositions comprenant lesdites cellulases modifiées et les utilisations de ces compositions. Les variants de ces cellulases sont avantageux pour le processus de bioconversion de substrats cellulosiques.
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