# Patent

## Title
Bio-engineered multi-enzyme complexes comprising xylanases and uses thereof

## Abstract

The present invention relates to bio-engineered multi-enzyme complexes having synergistic enzyme activity comprising xylanases and optionally further comprising additional carbohydrate active enzymes. Such complexes are advantageous for degrading recalcitrant cellulosic biomass.

## Background
RELATED APPLICATION DATA
This application is the U.S. National Stage of International Application No. PCT/IL2011/000158, filed Feb. 15, 2011, which claims the benefit of U.S. Provisional Application No. 61/304,500, filed Feb. 15, 2010, the contents of each of which are herein expressly incorporated by reference for all purposes.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 166,172 byte ASCII (text) file named “Seq_List” created on Aug. 15, 2012.


FIELD OF THE INVENTION
The present invention relates to bio-engineered multi-enzyme complexes comprising at least two xylanases, and optionally other types of carbohydrate active enzymes, designed to attach to a scaffold molecule by the introduction of suitable docking modules. Such complexes are advantageous for degrading recalcitrant cellulosic biomass.
BACKGROUND OF THE INVENTION
When a plurality of enzymes attack their substrates at different sites and create new sites for each other, whereby the resulting activity is higher than the sum of the individual activities, they are said to act synergistically. Enhancing enzyme activity in order to improve industrial processes is one of the most important biotechnological and industrial challenges of recent years. In this context, one group of enzymes that has received much attention is the carbohydrate active enzymes, which is a large group of enzymes that catalyze the breakdown, biosynthesis or modification of carbohydrates and glycoconjugates. Members of this group play an important role in the degradation of cellulosic biomass to soluble sugars that can be converted by microorganisms into liquid fuels, and are therefore of great interest in the fields of bio-energy and bio-fuel production.
The broad group of carbohydrate active enzymes is divided into enzyme classes and further into enzyme families according to a standard classification system (Cantarel et al. 2009 Nucleic Acids Res 37:D233-238). According to this classification system, four enzyme classes are defined, namely glycoside hydrolases, glycosyl transferases, polysaccharide lyases and carbohydrate esterases. Each class includes various enzymatic activities and substrate specificities and is further divided into families numbered in ascending order based on sequence similarities. The different families within each class may display a very broad diversity. For example, one family may contain members from bacteria, fungi, plants and animals, with several different activities and substrate specificities. In addition, a certain activity (for example, a xylanase) may be found in several different families. An informative and updated classification of carbohydrate active enzymes is available on the Carbohydrate-Active Enzymes (CAZy) server.
Typically, carbohydrate active enzymes 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.
The complex structure of cellulosic materials (which may include, for example, cellulose, hemicellulose, lignin) requires cooperation of many types of carbohydrate active enzymes for degradation. Enhancing synergy between carbohydrate active enzymes could lead to improved degradation, and thus has a great impact in the field of energy production from cellulosic biomass.
Synergism has been demonstrated previously between different types of carbohydrate active enzyme, in particular between glycoside hydrolases such as cellulases and xylanases. For example, synergism has been demonstrated between cellulases from different microbial systems (Irwin et al. 1993 Biotechnol. Bioeng. 42:1002-1013), between cellulosomal and non-cellulosomal enzymes (Murashima et al. 2003 J. Bacteriol. 185(5):1518-24, Koukiekolo et al. 2005 Appl Environ Microbiol. 71(7):3504-11), between different types of enzymes from different families and between enzymes that have different substrate-degradation mechanisms (i.e. exoglucanase and endoglucanase).
As another example, synergism has been demonstrated among glycoside hydrolases from the bacterium Termobifida fusca, for example, between xylan-degrading enzymes (Bachmann et al. 1991 Appl. Environ. Microbiol. 57:2121-2130, Tuncer et al. 2003 J Appl Microbiol. 94(6):1030-5) and between cellulose-degrading enzymes (Wilson et al. 2004 Chem. Rec. 4:72-82). Attempts in enhancing T. fusca enzyme synergism have been undertaken while integrating its enzymes into designer cellulosomes (Caspi et al. 2006 Biocat. Biotransform. 24:3-12).
The cellulosome system is a multi-enzyme complex characterized by a strong bi-modular protein-protein interaction between “cohesin” and “dockerin” modules that integrates the various enzymes into the complex. “Scaffoldin” subunits (non-enzymatic protein components) contain the cohesin modules that incorporate the enzymes into the complex via their resident dockerins. The primary scaffoldin subunit also includes a carbohydrate (cellulose)-binding module (CBM) through which the complex recognizes and binds to the cellulosic substrate. Previous research has suggested that the multi-enzyme cellulosome complex from the bacterium Clostridium thermocellum is far more efficient than free cellulase systems that were tested in degrading polysaccharides.
The designer cellulosome concept is based on the very high affinity and specific interaction between cohesin and dockerin modules from the same microorganism species. Since the various scaffoldin-borne cohesins of a given species essentially show the same specificity of binding for the enzyme-borne dockerins, designer cellulosomes are constructed from recombinant chimeric scaffoldins containing divergent cohesins from different 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. In such designer cellulosome complexes, enzyme proximity, combined with substrate binding via a carbohydrate-binding module contained in the scaffoldin, resulted in enhanced enzymatic activities in several cases, for example, as described in Fierobe et al. 2005 J. Biol. Chem. 280:16325-16334.
Apart from the designer cellulosome approach, another attempt to increase enzyme synergism has been reported in the form of multifunctional enzyme conjugates. An increase in degradation of natural substrates was observed upon fusing two or three complementary xylan-degrading activities (xylanase, arabinofuranosidase and xylosidase) into the same polypeptide chain. This approach may be cost-reducing, however, strategies involving multifunctional enzyme are limited to small numbers of enzymes and restricted to sub-optimal equimolar ratios of enzymes.
International Patent Application Publication No. WO 1997/014789 discloses an enzymatic array, which composition comprises one or more enzymes non-covalently bound to a peptide backbone, wherein at least one of the enzymes is heterologous to the peptide backbone and the peptide backbone is capable of having bound thereto a plurality of enzymes. The array is useful, for example, in recovery systems, targeted multi-enzyme delivery systems, soluble substrate modification, quantification type assays, and other applications in the food industry, feed, textiles, bioconversion, pulp and paper production, plant protection and pest control, wood preservatives, topical lotions and biomass conversions.
International Patent Application Publication No. WO 2010/057064 discloses designer cellulosomes for efficient hydrolysis of cellulosic material and more particularly for the generation of ethanol.
International Patent Application Publication No. WO 2010/096562 discloses the engineering and expression of heterologous cellulosomes in microorganisms in order to facilitate the conversion of biomass to useful products. In some embodiments, the invention relates to the expression of scaffoldin proteins which form the nucleus of a cellulosome. Cellulases or other biomass-degrading enzymes can be non-covalently linked to the scaffoldin protein by virtue of a dockerin domain-cohesin domain interaction.
U.S. Patent Application Publication Nos. 2009/0155238 and 2011/0016545 disclose enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal β-1,4-xylosidic linkages or endo-β-1,4-glucanase linkages; and/or degrading a linear polysaccharide β-1,4-xylan into xylose. Methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, are also disclosed, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also disclosed. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.
U.S. Patent Application Publication No. 2009/0220480 discloses polypeptides having any cellulolytic activity, e.g., a cellulase activity, an endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a β-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. The disclosed polypeptides can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. Compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of the invention are also disclosed.
There still remains a need for improved degradation of biomass, especially recalcitrant cellulosic biomass. For example, it would be highly beneficial to have bio-engineered, designer cellulosomes showing highly synergistic, improved degradative capabilities.
SUMMARY OF THE INVENTION
The present invention provides bio-engineered multi-enzyme complexes having synergistic enzyme activity comprising xylanases and optionally further comprising additional carbohydrate active enzymes. The combination of different types of enzymatic activities, that work synergistically and in close proximity, may provide a particularly efficient system for degradation of recalcitrant, complex cellulosic biomass.
The present invention discloses for the first time bio-engineered cellulosomes comprising two xylanases having a heterologous dockerin domain, which exhibit enhanced activity, i.e. synergy, of degradation of natural recalcitrant substrates (such as wheat straw) compared to the combined action of the free wild-type enzymes. It is now disclosed that surprisingly, xylanases work synergistically with other xylanases when incorporated into bio-engineered, artificial cellulosomes. It is now disclosed that synergistic activity of xylanases in bio-engineered cellulosomes can be obtained in the case of xylanases that do not naturally include a dockerin domain, and are engineered to include a heterologous dockerin. Further improvement may be achieved when additional carbohydrate active enzymes are added to the complex, including but not limited to cellulases.
The present invention further provides compositions comprising the multi-enzyme complexes and methods for the bioconversion of cellulosic material utilizing same. The present invention further provides cell cultures comprising host cells that produce and secrete the multi-enzyme complexes of the present invention. In some embodiments, a plurality of host cells is present in the culture, each producing at least one component of the multi-enzyme complex.
The present invention further encompasses polypeptides and polynucleotides encoding same comprising components capable of integrating or self-assembling into the bio-engineered complexes of the present invention.
According to one aspect, the present invention provides a bio-engineered multi-enzyme complex comprising:
(i) a scaffold polypeptide comprising a plurality of cohesin domains; and
(ii) a plurality of carbohydrate active enzymes bound to said scaffold polypeptide, each carbohydrate active enzyme comprises a dockerin domain;
(iii) wherein the plurality of carbohydrate active enzymes comprises at least two xylanases;
(iv) wherein at least one of the xylanases comprises a heterologous dockerin domain.
As used herein, “plurality” indicates more than one or at least two.
As used herein, “carbohydrate active enzymes” encompass glycoside hydrolases, polysaccharide lyases and carbohydrate esterases.
As used herein, the term “heterologous”, when referring to a dockerin domain, indicates either a dockerin that is different from the naturally-occurring dockerin of the enzyme, or a dockerin that is introduced into a polypeptide that does not naturally include a dockerin.
Thus, in some embodiments, at least one of the xylanases present in the complex comprises a dockerin domain which is not the naturally-occurring dockerin domain of this enzyme. According to these embodiments, the at least one xylanase is derived from a wild-type sequence that includes a dockerin, but the variant is engineered to include a different dockerin. The different dockerin may originate, for example, from a different microorganism species.
In alternative or additional embodiments, at least one of the xylanases present in the complex does not naturally include a dockerin domain. The phrase “does not naturally include a dockerin domain”, when referring to a carbohydrate active enzyme, refers to a variant of a carbohydrate active enzyme that is derived from a wild-type sequence that does not include a dockerin domain. The wild-type is therefore unable to incorporate into complexes such as the cellulosome. The variant, however, is engineered to include a dockerin domain and is therefore capable of integrating into the enzyme complex of the present invention.
The enzyme complexes of the present invention are multi-functional. For example, the enzyme complexes may be bi-, tri-, quadri-functional, etc.
Typically, the number of cohesin domains introduced into the scaffold polypeptide is determined according to the number of enzymes to be attached thereto.
The cohesin domains of the scaffold polypeptide may be the same or different. As noted above, the cohesin-dockerin interaction is species specific—scaffoldin-borne cohesins of a given species recognize and interact with enzyme-borne dockerins from the same species. It is therefore possible, in some embodiments, to select the cohesin domains such that each cohesin of the scaffold polypeptide would recognize a different dockerin. In a similar way, the enzymes to be incorporated into the multi-enzyme complex would bear divergent dockerins matching the relevant cohesins.
The individual enzymes that constitute the plurality of carbohydrate active enzymes may be the same or different.
In some embodiments, the dockerin domain included in the carbohydrate active enzymes present in the complex is selected from the group consisting of a dockerin derived from Clostridium thermocellum, a dockerin derived from Acetivibrio cellulolyticus, a dockerin derived from Ruminococcus flavefaciens, a dockerin derived from Bacteroides cellulosolvens, a dockerin derived from Archaeoglobus fulgidus and a dockerin derived from Clostridium cellulolyticum. Each possibility represents a separate embodiment of the invention.
In some embodiments, the dockerin domain included in the carbohydrate active enzymes present in the complex is selected from the group consisting of a dockerin derived from Clostridium thermocellum, a dockerin derived from Acetivibrio cellulolyticus, a dockerin derived from Ruminococcus flavefaciens and a dockerin derived from Bacteroides cellulosolvens. Each possibility represents a separate embodiment of the invention.
As noted above, carbohydrate active enzymes are usually characterized by a modular structure, where one or more ancillary or “helper” modules are found in the polypeptide chain in addition to the catalytic module. As used herein, the term “ancillary”, when referring to a module of a carbohydrate active enzyme, refers to a non-catalytic module that is present in the enzyme structure in addition to the catalytic module. The ancillary module may modulate the catalytic activity of the enzyme. A non-limiting example of an ancillary module is a carbohydrate-binding module. In some embodiments, the dockerin domain introduced into the carbohydrate active enzymes present in the multi-enzyme complex of the present invention replaces at least one of the ancillary modules originally found in the enzyme structure. In other embodiment, the dockerin domain is introduced in addition to the original ancillary modules.
The xylanases present in the multi-enzyme complex may be classified in any glycoside hydrolase family that include xylanases, as defined in the Carbohydrate-Active Enzymes (CAZy) server and/or CAZypedia.
In some embodiments, the xylanases present in the multi-enzyme complex are classified in a glycoside hydrolase family selected from the group consisting of family 5, 8, 10, 11, 26 and 43. Each possibility represents a separate embodiment of the invention. In some typical embodiments, the xylanases are classified in a glycoside hydrolase family selected from the group consisting of family 10 and 11.
The individual xylanases present in the complex may be classified in the same or different glycoside hydrolase families.
In some embodiments, at least one of the xylanases present in the complex is derived from Thermobifida fusca xylanases. In additional embodiments, all xylanases present in the complex are derived from T. fusca xylanases. In some embodiments, the T. fusca xylanases are selected from the group consisting of Xylanase 10B (Xyn10B) and Xylanase 11A (Xyn11A). Each possibility represents a separate embodiment of the invention. The amino acid sequences of the wild-type Xyn10B (Accession No. AAZ56824.1) and Xyn11A (Accession No. AAA21480.1) are set forth in SEQ ID NOs: 1 and 3, respectively. The DNA encoding wild-type Xyn10B (Accession No. CP000088.1) and Xyn11A (Accession No. U01242.1) are set forth in SEQ ID NOs: 2 and 4, respectively.
In some embodiments, the xylanases present in the complex are selected from the group consisting of a variant Xyn10B comprising a dockerin derived from C. thermocellum, and a variant Xyn11A comprising a dockerin derived from A. cellulolyticus. Each possibility represents a separate embodiment of the invention. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from C. thermocellum and one cohesin domain derived from A. cellulolyticus. 
In some embodiments, the xylanases present in the complex are selected from the group consisting of a variant Xyn10B comprising a sequence homologous to the sequence set forth in SEQ ID NO: 5 and a variant Xyn11A comprising a sequence homologous to the sequence set forth in SEQ ID NO: 7.
As used herein, the degree of homology between a certain reference sequence and its homologous sequence is such that the different functional modules (for example, catalytic module, substrate binding module) are considered to be the same, according to criteria defined in CAZY database and/or CAZYpedia, as detailed above.
In some embodiments, the xylanases present in the complex are selected from the group consisting of a variant Xyn10B comprising the sequence set forth in SEQ ID NO: 5 and a variant Xyn11A comprising the sequence set forth in SEQ ID NO: 7. Each possibility represents a separate embodiment of the invention.
In some embodiments, the plurality of carbohydrate active enzymes present in the complex further comprises at least one carbohydrate active enzyme selected from the group consisting of a glycoside hydrolase, polysaccharide lyase and carbohydrate esterase, wherein the at least one carbohydrate active enzyme is other than a xylanase. Each possibility represents a separate embodiment of the invention.
In some embodiments, the at least one carbohydrate active enzyme is a cellulase. In some embodiments, more than one cellulase is present in the complex, in addition to the xylanases.
In some embodiments, the cellulases are selected from the group consisting of an endocellulase and exocellulase. Each possibility represents a separate embodiment of the invention.
The cellulases present in the multi-enzyme complex may be classified in any glycoside hydrolase family that include cellulases (also referred to as endoglucanases and exoglucanases), as defined in the Carbohydrate-Active Enzymes (CAZy) server and/or CAZypedia.
In some embodiments, the cellulases are classified in a glycoside hydrolase family selected from the group consisting of family 5, 6, 7, 8, 9, 12, 26, 44, 45, 48, 51, 61, and 74. Each possibility represents a separate embodiment of the invention.
The individual cellulases present in the complex may be classified in the same or different glycoside hydrolase families.
In some embodiments, at least one of the cellulases present in the complex is derived from T. fusca cellulases. In additional embodiments, all cellulases present in the complex are derived from T. fusca cellulases. In some embodiments, the T. fusca cellulases are selected from the group consisting of Cellulase 5A (Cel5A) and Cellulase 48A (Cel48A). Each possibility represents a separate embodiment of the invention. The amino acid sequences of the wild-type Cel5A (Accession No. AAZ54939.1) and Cel48A (Accession No. AAD39947.1) are set forth in SEQ ID NOs: 9 and 11, respectively. The DNA encoding wild-type Cel5A (Accession No. CP000088.1) and Cel48A (Accession No. AF144563.1) are set forth in SEQ ID NOs: 10 and 12, respectively.
In some embodiments, the cellulases present in the complex are selected from the group consisting of a variant Cel5A comprising a dockerin derived from R. flavefaciens, and a variant Cel48A comprising a dockerin derived from B. cellulosolvens. Each possibility represents a separate embodiment of the invention. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from R. flavefaciens and one cohesin domain derived from B. cellulosolvens. 
In some embodiments, the cellulases present in the complex are selected from the group consisting of a variant Cel5A comprising a sequence homologous the sequence set forth in SEQ ID NO: 13 and a variant Cel48A comprising a sequence homologous the sequence set forth in SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention.
In some embodiments, the cellulases present in the complex are selected from the group consisting of a variant Cel5A comprising the sequence set forth in SEQ ID NO: 13 and a variant Cel48A comprising the sequence set forth in SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention.
In some embodiments, the bio-engineered multi-enzyme complex is bi-functional. As used herein, “bi-functional”, when referring to the enzyme complex, indicates a complex with two enzymes. According to these embodiments, the complex comprises two xylanases, each comprising a dockerin domain.
In some embodiments, one of the two xylanases is derived from T. fusca xylanases. In additional embodiments, at least one of the two xylanases is derived from T. fusca xylanases. In yet additional embodiments, both xylanases are derived from T. fusca xylanases. In some embodiments, the T. fusca xylanases are selected from the group consisting of Xyn10B and Xyn11A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the bi-functional complex comprises a variant Xyn10B and a variant Xyn11A, each variant comprises an added dockerin domain.
In some embodiments, the variant Xyn10B comprises a dockerin domain derived from C. thermocellum and the variant Xyn11A comprises a dockerin domain derived from A. cellulolyticus. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from C. thermocellum and a second cohesin domain derived from A. cellulolyticus. 
In some embodiments, the variant Xyn10B comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 7.
In some embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7.
In some embodiments, the bio-engineered multi-enzyme complex is tri-functional. As used herein, “tri-functional”, when referring to the enzyme complex, indicates a complex with three enzymes.
In some embodiments, the tri-functional complex comprises two xylanases and one additional carbohydrate active enzyme selected from the group consisting of a glycoside hydrolase, polysaccharide lyase and carbohydrate esterase, wherein the one additional carbohydrate active enzyme is other than a xylanase. Each possibility represents a separate embodiment of the invention.
In some embodiments, the carbohydrate active enzyme is a cellulase. In some embodiments, the cellulase is an endocellulase. In other embodiments, the cellulase is an exocellulase.
In some embodiments, at least one of the two xylanases and/or the cellulase are derived from T. fusca xylanases and cellulases. In additional embodiments, both xylanases and the one cellulase are derived from T. fusca xylanases and cellulases.
In some embodiments, the T. fusca xylanases are selected from the group consisting of Xyn10B and Xyn11A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the T. fusca cellulase is selected from the group consisting of Cel5A and Cel48A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the tri-functional complex comprises a variant Xyn10B, a variant Xyn11A and a variant Cel5A, each variant comprises an added dockerin domain.
In some embodiments, the variant Xyn10B comprises a dockerin domain derived from C. thermocellum, the variant Xyn11A comprises a dockerin domain derived from A. cellulolyticus and the variant Cel5A comprises a dockerin domain derived from Ruminococcus flavefaciens. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from C. thermocellum, a second cohesin domain derived from A. cellulolyticus and a third cohesin derived from R. flavefaciens. 
In some embodiments, the variant Xyn10B comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 13.
In some embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A comprises the amino acid sequence set forth in SEQ ID NO: 13.
In some embodiments, the bio-engineered multi-enzyme complex is quadri-functional. As used herein, “quadri-functional”, when referring to the enzyme complex, indicates a complex with four enzymes.
In some embodiments, the quadri-functional complex comprises two xylanases and two additional carbohydrate active enzymes selected from the group consisting of a glycoside hydrolase, polysaccharide lyase and carbohydrate esterase, wherein the two additional carbohydrate active enzymes are other than xylanases. Each possibility represents a separate embodiment of the invention.
In some embodiments, the quadri-functional complex comprises two xylanases and two cellulases. In some typical embodiments, the complex comprises two xylanases, one endocellulase and one exocellulase.
In some embodiments, at least one of the two xylanases and/or at least one of the two cellulases are derived from T. fusca xylanases and cellulases. In additional embodiments, both xylanases and both cellulases are derived from T. fusca xylanases and cellulases.
In some embodiments, the T. fusca xylanases are selected from the group consisting of Xyn10B and Xyn11A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the T. fusca cellulases are selected from the group consisting of Cel5A and Cel48A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the quadri-functional complex comprises a variant Xyn10B, a variant Xyn11A, a variant Cel5A and a variant Cel48A, each variant comprises an added dockerin domain.
In some embodiments, the variant Xyn10B comprises a dockerin domain derived from C. thermocellum, the variant Xyn11A comprises a dockerin domain derived from A. cellulolyticus, the variant Cel5A comprises a dockerin domain derived from R. flavefaciens and the variant Cel48A comprises a dockerin domain derived from Bacteroides cellulosolvens. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from C. thermocellum, a second cohesin domain derived from A. cellulolyticus, a third cohesin derived from R. flavefaciens and a fourth dockerin domain derived from B. cellulosolvens. 
In some embodiments, the variant Xyn10B comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the variant Cel48A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 15.
In some embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the variant Cel48A comprises the amino acid sequence set forth in SEQ ID NO: 15.
In some typical embodiments, the scaffold polypeptide further comprises at least one substrate-binding module. In some embodiments, the substrate-binding module is a carbohydrate-binding module (CBM). In some embodiments, the CBM is a cellulose-binding CBM. In other embodiments, the CBM is a xylan-binding CBM. In some embodiments, the CBM is classified in a CBM family selected from the group consisting of family 1, 2 and 3, as defined in the CAZY server and/or CAZYpedia as detailed above. In some embodiments, the CBM is derived from C. thermocellum CBMs. In some exemplary embodiments, the C. thermocellum CBM is CBM3a.
In some embodiments, the scaffold polypeptide comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 17, 19, 21, 23 and 25. Each possibility represents a separate embodiment of the invention.
In some embodiments, the scaffold polypeptide comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 19, 23 and 25. Each possibility represents a separate embodiment of the invention.
In some typical embodiments, the scaffold polypeptide and each of the carbohydrate active enzymes present in the multi-enzyme complexes of the present invention are non-covalently linked. In additional typical embodiments, they are linked via an interaction between the cohesins present on the scaffold polypeptide and the dockerins present on each of the cellulolytic enzymes.
In other embodiments, the scaffold polypeptide and each of the cellulolytic enzymes are covalently linked. In additional or alternative embodiments, the scaffold polypeptide and each of the cellulolytic enzymes are crosslinked.
According to another aspect, the present invention provides a composition for degrading biomass comprising the multi-enzyme complex of the present invention.
The different components of the multi-enzyme complex of the present invention may be produced from genetically-modified host cells. In some embodiments, each individual component is isolated and the different components are mixed and assembled. In other embodiments, genetically-modified host cells capable of producing and secreting at least one component of the multi-enzyme complex are co-cultured under conditions that allow the secretion and assembly of the different components.
Thus, according to another aspect, the present invention provides a host cell comprising at least one component of the bio-engineered multi-enzyme complex of the present invention.
In some embodiments, a genetically modified host cell is provided, comprising a polynucleotide encoding at least one component of the bio-engineered multi-enzyme complex of the present invention.
In some embodiments, the host cell is selected from a prokaryotic and eukaryotic cell. Each possibility represents a separate embodiment of the invention.
According to a further aspect, the present invention provides a cell culture comprising at least one genetically modified host cell, the host cell comprising at least one component of the multi-enzyme complex of the present invention. The host cells that constitute the culture of the present invention are capable of producing, and typically secreting, at least one component of the multi-enzyme complex of the present invention.
In some embodiments, a cell culture is provided, comprising at least one genetically modified host cell comprising a polynucleotide encoding at least one bio-engineered component of the multi-enzyme complex of the present invention.
In some embodiments, a plurality of different host cells is modified to express and produce at least one component of the multi-enzyme system. According to these embodiments, a plurality of different genetically modified host cells are present in the culture, each comprises at least one component of the multi-enzyme complex.
In other embodiments, a single host cell is genetically modified to express all components of the multi-enzyme complex of the present invention. According to these embodiments, a single host cell is present in the culture, the single host cell comprises all components of the multi-enzyme complex.
The multi-enzyme complexes of the present invention, compositions comprising same and cells cultures producing same may be utilized for the bioconversion of cellulosic material into soluble sugars.
Thus, according to another aspect, the present invention provides a method for bioconversion of cellulosic material into degradation products, the method comprising exposing said cellulosic material to the multi-enzyme complex of the present invention.
In some embodiments, a method for bioconversion of cellulosic material into degradation products is provided, the method comprising exposing said cellulosic material to host cells comprising at least one component of the multi-enzyme system of the present invention.
The degradation products typically comprise mono-, di- and oligosaccharides, including but not limited to glucose, xylose, cellobiose, xylobiose, cellotriose, cellotetraose, arabinose, xylotriose.
According to a further aspect, the present invention provides a system for bioconversion of cellulosic material, the system comprising a multi-enzyme complex of the present invention.
The present invention further provides variant xylanases capable of integrating into the bio-engineered complex of the present invention, and suitable scaffold polypeptides.
Thus, according to another aspect, the present invention provides an isolated polypeptide comprising a variant xylanase, the variant comprises a heterologous dockerin domain.
In some embodiments, the dockerin domain is selected from the group consisting of C. thermocellum, A. cellulolyticus, R. flavefaciens, B. cellulosolvens, A. fulgidus and C. cellulolyticum dockerins. Each possibility represents a separate embodiment of the invention. In some embodiments, the dockerin domain is selected from the group consisting of C. thermocellum, A. cellulolyticus, R. flavefaciens and B. cellulosolvens. Each possibility represents a separate embodiment of the invention.
The variant xylanase may be classified in any glycoside hydrolase family that includes xylanases, as defined in the Carbohydrate-Active Enzymes (CAZy) server and/or CAZypedia.
In some embodiments, the variant xylanase is classified in a glycoside hydrolase family selected from the group consisting of family 5, 8, 10, 11, 26 and 43. Each possibility represents a separate embodiment of the invention. In some typical embodiments, the variant xylanase is classified in a glycoside hydrolase family selected from the group consisting of family 10 and 11.
In some embodiments, the variant xylanase is derived from T. fusca xylanases.
Xylanases from T. fusca does not naturally include a dockerin domain, and the introduction of a dockerin according to embodiments of the present invention enables their utilization and incorporation into enzyme complexes of the present invention.
In some embodiments, the T. fusca xylanases are selected from the group consisting of Xyn10B and Xyn11A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the variant T. fusca xylanase is selected from the group consisting of a variant Xyn10B comprising a dockerin domain derived from C. thermocellum and a variant Xyn11A comprises a dockerin domain derived from A. cellulolyticus. Each possibility represents a separate embodiment of the invention.
In some embodiments, the variant T. fusca xylanase comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant T. fusca xylanase comprises the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, the variant T. fusca xylanase comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant T. fusca xylanase comprises the amino acid sequence set forth in SEQ ID NO: 7.
According to another aspect, the present invention provides an isolated scaffold polypeptide comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from C. thermocellum, a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. 
In some embodiments, the plurality of cohesin domains is consisting of a cohesin derived from C. thermocellum, a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. 
In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23.
According to yet another aspect, the present invention provides an isolated scaffold polypeptide comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from C. thermocellum, a cohesin derived from A. cellulolyticus, a cohesin derived from R. flavefaciens and a cohesin derived from B. cellulosolvens. 
In some embodiments, the plurality of cohesin domains is consisting of a cohesin derived from C. thermocellum, a cohesin derived from A. cellulolyticus, a cohesin derived from R. flavefaciens and a cohesin derived from B. cellulosolvens. 
In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 25.
According to yet another aspect, the present invention provides an isolated scaffold polypeptide comprising at least two cohesin domains, wherein the at least two cohesin domains are selected from the group consisting of:
(i) a cohesin derived from A. cellulolyticus and a cohesin derived from C. thermocellum; 
(ii) a cohesin derived from B. cellulosolvens and a cohesin derived from R. flavefaciens; and
(iii) a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. 
In some embodiments, an isolated scaffold polypeptide is provided, comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from A. cellulolyticus and a cohesin derived from C. thermocellum. In some embodiments, the plurality of cohesin domains is consisting of a cohesin derived from A. cellulolyticus and a cohesin derived from C. thermocellum. In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19.
In some embodiments, an isolated scaffold polypeptide is provided, comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from B. cellulosolvens and a cohesin derived from R. flavefaciens. In some embodiments, the plurality of cohesin domains is consisting of a cohesin derived from B. cellulosolvens and a cohesin derived from R. flavefaciens. In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21.
In some embodiments, an isolated scaffold polypeptide is provided, comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. In some embodiments, the plurality of cohesin domains is consisting of a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17.
According to another aspect, the present invention provides an isolated polynucleotide comprising a sequence encoding a polypeptide of the present invention.
In some embodiments, the isolated polynucleotide comprises a polynucleotide sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 6, 8, 18, 20, 22, 24 and 26. Each possibility represents a separate embodiment of the invention.
According to another aspect, the present invention provides a construct comprising a polynucleotide sequence of the present invention. According to yet another aspect, the present invention provides a host cell comprising a polynucleotide sequence of the present invention. In some embodiments, the cell is selected from a prokaryotic and eukaryotic cell.
These and further aspects and features of the present invention will become apparent from the figures, detailed description, examples and claims which follow.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Schematic representation of the recombinant proteins. The source of the representative module (see key) is indicated as follows: black (A. cellulolyticum), white (C. thermocellum), dots (R. flavefasciens), diagonal stripes (T. fusca) and diamonds (B. cellulosolvens). In the shorthand notation for the engineered enzymes, the numbers 5, 10, 11 and 48 refer to the corresponding GH family (GH5, GH10, GH11 and GH48) of the catalytic module; upper case characters (B, F, T and A) indicate the source of the cohesin module and lower case (b, f t and a) indicate the source of the dockerin module: B. cellulosolvens, R. flavefaciens, C. thermocellum and A. cellulolyticum respectively.
FIG. 2. Electrophoretic mobility of components and assembled complexes on non-denaturing and denaturing gels. A) Equimolar concentrations of the chimeric enzymes (f-5A and 11A-a) and their matching scaffoldin (Scaf.AF) were combined. The single fusion proteins and the mixtures were subjected to non-denaturing PAGE (top panel) and SDS-PAGE (bottom panel). Analysis of the matching components by native PAGE clearly shows their complete or near-complete interaction. B) Equimolar concentrations of the chimeric enzymes (f-5A, b-48A, 11A-XBM-a and 10B-t) and their matching scaffoldin (Scaf.BTAF) were combined. The single fusion proteins and the mixtures were subjected to non-denaturing PAGE (top panel) and SDS-PAGE (bottom panel). Analysis of the matching components by native PAGE clearly shows their complete interaction.
FIG. 3. Specific activities of single dockerin-containing enzymes or combinations thereof. The composition of the complexes and free enzymes systems is indicated at the bottom of the graph. Each enzyme or enzyme combination was assayed at 0.3 μM with 3.5 g/l hatched wheat straw in a 200 μl reaction. Specific activity is defined as μM reducing sugar per min per μM enzyme. Triplicate samples of each reaction mixture were employed, and standard deviations for straw hydrolysis are indicated.
FIG. 4. Comparative degradation of straw by the various complexes and free enzyme systems. The composition of the complexes and free enzymes systems is indicated in symbolic form at the bottom of the graph. White encoded bars represent the free enzyme system; black bars the enzyme-scaffoldin complexes and the dotted bars the enzymes attached to their corresponding individual single-cohesin scaffoldins. Specific activity is defined as μM reducing sugar per min per enzyme. Each reaction was performed in triplicate, and standard deviations for straw hydrolysis are indicated.
FIG. 5. Kinetics studies of hatched-straw hydrolysis by the various complexes and free enzyme systems. The black square curve represents the scaffoldin-bound enzyme complexes (designer cellulosomes), the white triangle curve represents the free enzyme system (lacking CBMs), the white square curve represents the chimeric enzymes attached to single-cohesin scaffoldin (CBM added extraneously) and the black triangle curve represents the wild-type enzymes (containing native CBMs). Triplicates of each reaction were carried out. Error bars are indicated.
FIG. 6. Kinetics studies of hatched-straw hydrolysis by the various complexes and free enzyme systems. The black square curve represents the degradation by a single quadri-functional designer cellulosome complex bearing the four enzymes, the black triangle curve represents the degradation by two bi-functional designer cellulosomes complexes: one bearing the cellulases and the other bearing the xylanases, and the white triangle curve represents the degradation by the free wild-type enzymes. Triplicates of each reaction were carried out. Error bars are indicated.
FIG. 7. Analysis of the influence of scaffoldin-borne versus native (enzyme-borne) XBM on degradation of wheat straw. The composition of the complexes and free enzymes systems is indicated at the bottom of the graph. White encoded bars represent the free enzyme system and black bars the scaffoldin-bound enzyme complexes. Specific activity is defined as μM reducing sugar per min per μM enzyme. Triplicates of each reaction were carried out, and standard deviations for straw hydrolysis are indicated.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to bio-engineered cellulosomes incorporating at least two xylanases and optionally further comprising other carbohydrate active enzymes. These enzymatic complexes may be useful in the process of bioconversion of cellulosic biomass into soluble sugars.
The present invention is based in part on the unexpected discovery that enhanced synergistic enzymatic activity on raw cellulosic material is observed when two xylanases are bio-engineered to be compatible to the cellulosomal mode via inclusion of a heterologous dockerin domain into at least on of them, and then integrated into artificial cellulosomes. As exemplified hereinbelow, enhanced synergistic enzymatic activity is also observed when the two xylanases are further combined with other carbohydrate active enzymes, such as one or two cellulases, in one artificial cellulosome.
While carbohydrate active enzymes from different classes might theoretically be expected to work synergistically when incorporated into bio-engineered cellulosomes, it was unexpectedly found that synergy is observed between two xylanases incorporated into a bio-engineered cellulosome.
DEFINITIONS
As used herein, the term “enzyme” refers to a polypeptide having a catalytic activity towards a certain substrate or substrates.
As used herein, the term “variant” refers to a protein which differs from an unaltered, wild-type protein due to one or more amino acid substitutions introduced into the amino acid sequence and/or due to the inclusion of sequences/domains not included in the wild-type protein.
The terms “wild type” and “unaltered sequence” are used interchangeably and as used herein refer to the naturally occurring DNA/protein sequence.
As used herein, the term “derived from”, when referring to a dockerin or cohesin domain, refers to a variant that has been modified without adversely affecting its ability to recognize the matching cohesin/dockerin, respectively. Typically, the recognition site of the relevant counterpart, also referred to as the binding site, is maintained. When referring to an enzyme, the term “derived from” indicates a variant that has been modified without adversely affecting its catalytic activity. Typically, the catalytic domain is maintained. A derivative generally retains the properties or activity observed in the wild-type to the extent that the derivative is useful for similar purposes as the wild-type form.
As used herein, the term “gene” has its meaning as understood in the art. In general, 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).
As used herein, the term “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.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
As used herein, the term “DNA construct” refers to an artificially assembled or isolated nucleic acid molecule which comprises the gene of interest.
As used herein, the term “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. One exemplary type of vector is a “plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another exemplary type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced.
As used herein, 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 5 under suitable conditions.
As used herein, the terms “transformation” refers to the introduction of foreign DNA into cells. The terms “transformants” or “transformed cells” include the primary transformed cell and cultures derived from that cell regardless to the number of transfers. All progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants.
Multi-Enzyme Complexes and Compositions
According to one aspect, the present invention provides a bio-engineered multi-enzyme complex comprising:
(i) a scaffold polypeptide comprising a plurality of cohesin domains; and
(ii) a plurality of carbohydrate active enzymes bound to said scaffold polypeptide, each carbohydrate active enzyme comprises a dockerin domain;
(iii) wherein the plurality of carbohydrate active enzymes comprises two xylanases;
(iv) wherein at least one of the xylanases comprises a heterologous dockerin domain.
As noted above, the different carbohydrate active enzymes, as well as the different carbohydrate-binding modules. are classified into classes and families according to a standard classification system. Information about the classification system is available on the Carbohydrate-Active Enzymes (CAZy) server and CAZypedia database. Along with the classification system, a unifying scheme for designating the different catalytic modules and the different carbohydrate active enzymes was suggested and has been widely adopted. A catalytic module is designated by its enzyme class and family number. For example, a glycoside hydrolase having a catalytic module classified in family 10 is designated as “GH10”. An enzyme is designated by the type of activity, the family it belongs to and typically an additional letter. For example, a cellulase from a certain organism having a catalytic module classified as family 5 glycoside hydrolase, which is the first reported GH5 cellulase from this organism, is designated as “Cel5A”.
Carbohydrate active enzymes according to embodiments of the present invention encompass glycoside hydrolases, polysaccharide lyases and carbohydrate esterases.
As used herein, the term “glycoside hydrolases” refers to enzymes that hydrolyze glycosidic bonds between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. The glycoside hydrolases may catalyze the hydrolysis of O-, N- and/or S-linked glycosides. The glycoside hydrolases are sometimes referred to as glycosidases and glycosyl hydrolases. Non-limiting examples of glycoside hydrolases include a cellulase, xylanase, α-L-arabinofuranosidase, cellobiohydrolase, β-glucosidase, β-xylosidase, β-mannosidase, mannanase.
Information about glycosidic bonds and other types of bonds found in carbohydrate molecules, can be found, for example, in M. L. Sinnott (2007) Carbohydrate Chemistry and Biochemistry: Structure and mechanism, 1st edition, Royal Society of Chemistry.
As used herein, the term “polysaccharide lyases” refers to a group of carbon-oxygen lyases that catalyze the breakage of a carbon-oxygen bond in polysaccharides leading to an unsaturated product and the elimination of an alcohol. Typically, polysaccharide lyases cleave uronic acid-containing polysaccharide chains via a β-elimination mechanism, to generate an unsaturated hexenuronic acid residue and a new reducing end. Non-limiting examples of polysaccharide lyases include pectate lyase, alginate lyase.
As used herein, the term “carbohydrate esterases” refers to enzymes that hydrolyze carbohydrate esters. Typically, “carbohydrate esterases” catalyze the de-O or de-N-acylation of substituted saccharides. Non-limiting examples of carbohydrate esterases include acetylxylan esterase, pectin methyl esterase, pectin acetyl esterase, ferulic acid esterases.
The enzyme complexes of the present invention are multi-functional. For example, the enzyme complexes may be bi-, tri-, quadri-functional, etc.
As noted above, mixtures of free carbohydrate active enzymes may show synergistic activity. Further enhancement of the observed synergy (i.e., enhanced synergy) may be obtained when the enzymes are incorporated or self-assembled into multi-enzyme complexes, such as the multi-enzyme complexes of the present invention. Typically, the multi-enzyme complexes of the present invention exhibit enhanced synergy compared to the combined action of the free enzymes.
Thus, multi-enzyme complexes of the present invention typically provide enhanced synergy for the degradation of cellulosic biomass.
Typically, the number of cohesin domains introduced into the scaffold polypeptide is determined according to the number of enzymes to be attached thereto. The cohesin domains of the scaffold polypeptide may be the same or different.
In some embodiments, the cohesin domains are selected such that each cohesin of the scaffold polypeptide would recognize a different dockerin. In a similar way, the enzymes to be incorporated into the multi-enzyme complex would bear divergent dockerins matching the relevant cohesins.
The individual enzymes that constitute the plurality of carbohydrate active enzymes may be the same or different.
In some embodiments, at least one additional enzyme of the plurality of carbohydrate active enzymes present in the multi-enzyme complex comprises a heterologous dockerin domain.
In some embodiments, the dockerin domain included in the carbohydrate active enzymes present in the complex is selected from the group consisting of a dockerin derived from Clostridium thermocellum, a dockerin derived from Acetivibrio cellulolyticus, a dockerin derived from Ruminococcus flavefaciens, a dockerin derived from Bacteroides cellulosolvens a dockerin derived from Archaeoglobus fulgidus and a dockerin derived from Clostridium cellulolyticum. Each possibility represents a separate embodiment of the invention.
In some embodiments, the dockerin domain included in the carbohydrate active enzymes present in the complex is selected from the group consisting of a dockerin derived from Clostridium thermocellum, a dockerin derived from Acetivibrio cellulolyticus, a dockerin derived from Ruminococcus flavefaciens and a dockerin derived from Bacteroides cellulosolvens. Each possibility represents a separate embodiment of the invention.
Exemplary amino acid sequences of dockerin domains that can be used according to embodiments of the present invention are set forth in SEQ ID NOs: 27 (from C. thermocellum), 29 (from A. cellulolyticus), 31 (from R. flavefaciens) and 33 (from B. cellulosolvens). The sequences of the polynucleotides encoding them are set forth in SEQ ID NOs: 28, 30, 32 and 34, respectively.
In some embodiments, the dockerin domain introduced into the carbohydrate active enzymes present in the multi-enzyme complex of the present invention replaces at least one of the ancillary modules originally found in the enzyme structure. In other embodiment, the dockerin domain is introduced in addition to the original ancillary modules.
The xylanases present in the multi-enzyme complex may be classified in any glycoside hydrolase family that include xylanases, as defined in the Carbohydrate-Active Enzymes (CAZy) server (www.cazy.org) and/or CAZypedia (www.cazypedia.org).
In some embodiments, the xylanases present in the multi-enzyme complex are classified in a glycoside hydrolase family selected from the group consisting of family 5, 8, 10, 11, 26 and 43. Each possibility represents a separate embodiment of the invention. In some typical embodiments, the xylanases are classified in a glycoside hydrolase family selected from the group consisting of family 10 and 11.
Non-limiting examples of additional xylan-degrading enzymes that can be incorporated into the multi-enzyme complex include β-xylosidase, for example β-xylosidase classified in glycoside hydrolase family selected from the group consisting of family 3, 30, 39, 43, 52, 54.
The individual xylanases present in the complex may be classified in the same or different glycoside hydrolase families.
In some embodiments, all xylanases present in the multi-enzyme complex belong to the same family. In other embodiments, the xylanases present in the complex belong to different families.
In some embodiments, at least one of the xylanases present in the complex is derived from Thermobifida fusca xylanases. In additional embodiments, all xylanases present in the complex are derived from T. fusca xylanases.
T. fusca is an aerobic thermophilic soil bacterium with strong cellulolytic activity. This actinomycete produces six different cellulases that have been well studied. T. fusca also has the ability to grow on xylan and it produces several enzymes involved in xylan degradation, such as xylanases, β-xylosidase, α-L arabinofuranosidase and acetylesterases.
In some embodiments, the T. fusca xylanases are selected from the group consisting of Xylanase 10B (Xyn10B) and Xylanase 11A (Xyn11A). Each possibility represents a separate embodiment of the invention.
T. fusca xylanases 11A and 10B are the most abundant xylanases produced by this organism during growth on xylan. They both function as endoxylanases but differ in structure: Xyn11A contains catalytic module followed by a C-terminal family-2 CBM, whereas Xyn10B lacks a CBM.
As exemplified hereinbelow, T. fusca xylanases can be engineered to include a dockerin domain and can then be incorporated into enzyme complexes of the present invention.
In some embodiments, the xylanases present in the complex are selected from the group consisting of a variant Xyn10B comprising a dockerin derived from C. thermocellum, and a variant Xyn11A comprising a dockerin derived from A. cellulolyticus. Each possibility represents a separate embodiment of the invention. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from C. thermocellum and one cohesin domain derived from A. cellulolyticus. 
In some embodiments, the xylanases present in the complex are selected from the group consisting of a variant Xyn10B comprising a sequence homologous to the sequence set forth in SEQ ID NO: 5 and a variant Xyn11A comprising a sequence homologous to the sequence set forth in SEQ ID NO: 7.
In some embodiments, the xylanases present in the complex are selected from the group consisting of a variant Xyn10B comprising the sequence set forth in SEQ ID NO: 5 and a variant Xyn11A comprising the sequence set forth in SEQ ID NO: 7. Each possibility represents a separate embodiment of the invention.
In some embodiments, the xylanases present in the complex are selected from the group consisting of a variant Xyn10B consisting of the sequence set forth in SEQ ID NO: 5 and a variant Xyn11A consisting of the sequence set forth in SEQ ID NO: 7. Each possibility represents a separate embodiment of the invention.
In some embodiments, the plurality of carbohydrate active enzymes present in the complex further comprises at least one carbohydrate active enzyme selected from the group consisting of a glycoside hydrolase, polysaccharide lyase and carbohydrate esterase, wherein the at least one carbohydrate active enzyme is other than a xylanase. Each possibility represents a separate embodiment of the invention.
Carbohydrate active enzymes that participate in the degradation of hemicelluloses, a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, are sometimes referred to as hemicellulases. Non-limiting examples of such carbohydrate active enzymes include cellulases, xylanases, mannanases, α-L-arabinofuranosidases, ferulic acid esterases, acetyl-xylanesterases, α-D-glucuronidases, β-xylosidases, 3-mannosidases, β-Glucosidases, acetyl-mannanesterases, α-galactosidase, -α-L-arabinanase, β-galactosidase.
In some embodiments, the at least one carbohydrate active enzyme which is further included in the complex is a hemicellulase other than a xylanase. In some exemplary embodiments, the at least one carbohydrate active enzyme is selected from the group consisting of α-L-arabinofuranosidases, β-xylosidase and β-glucosidase. Each possibility represents a separate embodiment of the invention.
In some embodiments, the at least one carbohydrate active enzyme is a cellulase. In some embodiments, more than one cellulase is present in the complex, in addition to the xylanases.
In some embodiments, the cellulases are selected from the group consisting of an endocellulase and exocellulase. Each possibility represents a separate embodiment of the invention. Endocellulases and exocellulases are sometimes referred to as “endoglucanases” and “exoglucanases”, respectively.
In some embodiments, the multi-enzyme complex comprises both endocellulases and exocellulases. In other embodiments, only one type of cellulase is present in the complex.
In some embodiments, the ratio between exocellulases and endocellulases in the complex is other than 1:1 ratio. In some embodiments, the exocellulases-to-endocellulases ratio is greater than 1.
The use of scaffold polypeptides bearing several copies of a certain cohesin matching the exocellulases dockerin may be used to modify exocellulases to-endocellulases ratios within the bio-engineered multi-enzyme complex.
The cellulases present in the multi-enzyme complex may be classified in any glycoside hydrolase family that include cellulases (also referred to as endoglucanases and exoglucanases), as defined in the Carbohydrate-Active Enzymes (CAZy) server and/or CAZypedia.
In some embodiments, the cellulases are classified in a glycoside hydrolase family selected from the group consisting of family 5, 6, 7, 8, 9, 12, 26, 44, 45, 48, 61, and 74. Each possibility represents a separate embodiment of the invention.
The individual cellulases present in the complex may be classified in the same or different glycoside hydrolase families.
In some embodiments, at least one of the cellulases present in the complex is derived from T. fusca cellulases. In additional embodiments, all cellulases present in the complex are derived from T. fusca cellulases. In some embodiments, the T. fusca cellulases are selected from the group consisting of Cellulase 5A (Cel5A) and Cellulase 48A (Cel48A). Each possibility represents a separate embodiment of the invention.
In some embodiments, the cellulases present in the complex are selected from the group consisting of a variant Cel5A comprising a dockerin derived from R. flavefaciens, and a variant Cel48A comprising a dockerin derived from B. cellulosolvens. Each possibility represents a separate embodiment of the invention. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from R. flavefaciens and one cohesin domain derived from B. cellulosolvens. 
In some embodiments, the cellulases present in the complex are selected from the group consisting of a variant Cel5A comprising a sequence homologous the sequence set forth in SEQ ID NO: 13 and a variant Cel48A comprising a sequence homologous the sequence set forth in SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention.
In some embodiments, the cellulases present in the complex are selected from the group consisting of a variant Cel5A comprising the sequence set forth in SEQ ID NO: 13 and a variant Cel48A comprising the sequence set forth in SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention.
In some embodiments, the cellulases present in the complex are selected from the group consisting of a variant Cel5A consisting of the sequence set forth in SEQ ID NO: 13 and a variant Cel48A consisting of the sequence set forth in SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention.
In some embodiments, the bio-engineered multi-enzyme complex is bi-functional. As used herein, “bi-functional”, when referring to the enzyme complex, indicates a complex with two enzymes. According to these embodiments, the complex comprises two xylanases, each comprising a dockerin domain.
In some embodiments, one of the two xylanases is derived from T. fusca xylanases. In additional embodiments, both xylanases are derived from T. fusca xylanases. In some embodiments, the T. fusca xylanases are selected from the group consisting of Xyn10B and Xyn11A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the bi-functional complex comprises a variant Xyn10B and a variant Xyn11A, each variant comprises an added dockerin domain.
In some embodiments, the variant Xyn10B comprises a dockerin domain derived from C. thermocellum and the variant Xyn11A comprises a dockerin domain derived from A. cellulolyticus. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from C. thermocellum and a second cohesin domain derived from A. cellulolyticus. 
In some embodiments, the variant Xyn10B comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 7.
In some embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7.
In some embodiments, the variant Xyn10B is consisting of the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A is consisting of the amino acid sequence set forth in SEQ ID NO: 7.
In some embodiments, a multi-enzyme complex is provided, wherein the plurality of carbohydrate active enzymes consists of a variant Xyn10B and a variant Xyn11A, each comprises a dockerin domain. In some particular embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5 and the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7.
In some embodiments, the bio-engineered multi-enzyme complex is tri-functional. As used herein, “tri-functional”, when referring to the enzyme complex, indicates a complex with three enzymes.
In some embodiments, the tri-functional complex comprises two xylanases and one carbohydrate active enzyme selected from the group consisting of a glycoside hydrolase, polysaccharide lyase and carbohydrate esterase, wherein the one carbohydrate active enzyme is other than a xylanase. Each possibility represents a separate embodiment of the invention.
In some embodiments, the carbohydrate active enzyme is a cellulase.
In some embodiments, the cellulase is an endocellulase. In other embodiments, the cellulase is an exocellulase.
In some embodiments, at least one of the two xylanases and/or the cellulase are derived from T. fusca xylanases and cellulases. In additional embodiments, both xylanases and the one cellulase are derived from T. fusca xylanases and cellulases.
In some embodiments, the T. fusca xylanases are selected from the group consisting of Xyn10B and Xyn11A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the T. fusca cellulase is selected from the group consisting of Cel5A and Cel48A. In some embodiments, the T. fusca cellulase is Cel5A.
In some embodiments, the tri-functional complex comprises a variant Xyn10B, a variant Xyn11A and a variant Cel5A, each variant comprises an added dockerin domain.
In some embodiments, the variant Xyn10B comprises a dockerin domain derived from C. thermocellum, the variant Xyn11A comprises a dockerin domain derived from A. cellulolyticus and the variant Cel5A comprises a dockerin domain derived from Ruminococcus flavefaciens. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from C. thermocellum, a second cohesin domain derived from A. cellulolyticus and a third cohesin derived from R. flavefaciens. 
In some embodiments, the variant Xyn10B comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 13.
In some embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A comprises the amino acid sequence set forth in SEQ ID NO: 13.
In some embodiments, the variant Xyn10B is consisting of the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A is consisting of the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A is consisting of the amino acid sequence set forth in SEQ ID NO: 13.
In some embodiments, a multi-enzyme complex is provided, wherein the plurality of carbohydrate active enzymes consists of a variant Xyn10B, a variant Xyn11A and a variant Cel5A, each comprises a dockerin domain. In some particular embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5, the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7 and the variant Cel5A comprises the amino acid sequence set forth in SEQ ID NO: 13.
In some embodiments, the bio-engineered multi-enzyme complex is quadri-functional. As used herein, “quadri-functional”, when referring to the enzyme complex, indicates a complex with four enzymes.
In some embodiments, the quadri-functional complex comprises two xylanases and two carbohydrate active enzymes selected from the group consisting of a glycoside hydrolase, polysaccharide lyase and carbohydrate esterase, wherein the two carbohydrate active enzymes are other than xylanases. Each possibility represents a separate embodiment of the invention.
In some embodiments, the quadri-functional complex comprises two xylanases and two cellulases. In some typical embodiments, the complex comprises two xylanases, one endocellulase and one exocellulase.
In some embodiments, at least one of the two xylanases and/or at least one of the two cellulases are derived from T. fusca xylanases and cellulases. In additional embodiments, both xylanases and both cellulases are derived from T. fusca xylanases and cellulases.
In some embodiments, the T. fusca xylanases are selected from the group consisting of Xyn10B and Xyn11A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the T. fusca cellulases are selected from the group consisting of Cel5A and Cel48A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the quadri-functional complex comprises a variant Xyn10B, a variant Xyn11A, a variant Cel5A and a variant Cel48A, each variant comprises an added dockerin domain.
In some embodiments, the variant Xyn10B comprises a dockerin domain derived from C. thermocellum, the variant Xyn11A comprises a dockerin domain derived from A. cellulolyticus, the variant Cel5A comprises a dockerin domain derived from R. flavefaciens and the variant Cel48A comprises a dockerin domain derived from Bacteroides cellulosolvens. According to these embodiments, the scaffold polypeptide comprises one cohesin domain derived from C. thermocellum, a second cohesin domain derived from A. cellulolyticus, a third cohesin derived from R. flavefaciens and a fourth dockerin domain derived from B. cellulosolvens. 
In some embodiments, the variant Xyn10B comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the variant Cel48A comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 15.
In some embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the variant Cel48A comprises the amino acid sequence set forth in SEQ ID NO: 15.
In some embodiments, the variant Xyn10B is consisting of the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant Xyn11A is consisting of the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant Cel5A is consisting of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the variant Cel48A is consisting of the amino acid sequence set forth in SEQ ID NO: 15.
In some embodiments, a multi-enzyme complex is provided, wherein the plurality of carbohydrate active enzymes consists of a variant Xyn10B, a variant Xyn11A, a variant Cel5A and a variant Cel48A, each comprises a dockerin domain. In some particular embodiments, the variant Xyn10B comprises the amino acid sequence set forth in SEQ ID NO: 5, the variant Xyn11A comprises the amino acid sequence set forth in SEQ ID NO: 7, the variant Cel5A comprises the amino acid sequence set forth in SEQ ID NO: 13 and the Cel48A comprises the amino acid sequence set forth in SEQ ID NO: 15.
The scaffold polypeptide of the present invention is typically a non-catalytic peptide structure which has the ability to have a plurality of enzymes attached thereto.
In some typical embodiments, the scaffold polypeptide further comprises at least one substrate-binding module. In some embodiments, the substrate-binding module is a carbohydrate-binding module (CBM). In some embodiments, the CBM classified in a CBM family selected from the group consisting of family 1, 2 and 3. In some embodiments, the CBM is derived from a thermophilic bacterium. In other embodiments, the CBM is derived from a bacterium other than a thermophilic bacterium. In some embodiments, the CBM is derived from C. thermocellum CBMs. In some exemplary embodiments, the C. thermocellum CBM is CBM3a.
In some particular embodiments, the scaffold polypeptide comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 17, 19, 21, 23 and 25. Each possibility represents a separate embodiment of the invention.
In some particular embodiments, the scaffold polypeptide comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 19, 23 and 25. Each possibility represents a separate embodiment of the invention.
In some typical embodiments, the scaffold polypeptide and each of the cellulolytic enzymes present in the multi-enzyme complexes of the present invention are non-covalently linked. In additional typical embodiments, they are linked via an interaction between the cohesins present on the scaffold polypeptide and the dockerins present on each of the cellulolytic enzymes.
“Non-covalent bond” or “non-covalently bound” or “non-covalently linked” refer to a molecular interaction which is not the result of a covalent bond. A non-covalent bond includes, for example, hydrophobic attraction, hydrophilic attraction, van der Waals interaction, ionic interaction or any other equivalent molecular interaction which does not involve the formation of a covalent bond.
In other embodiments, the scaffold polypeptide and each of the cellulolytic enzymes are covalently linked. In additional or alternative embodiments, the scaffold polypeptide and each of the cellulolytic enzymes are crosslinked.
The different components of the multi-enzyme complexes of the present invention may be produced by recombinant methods from genetically-modified host cells. The different components may also be prepared by synthetic methods. Exemplary procedures are described below.
In some embodiments, a composition is provided, the composition comprising the multi-enzyme complex of the present invention.
Host Cells and Cell Culture
The different components of the multi-enzyme complex of the present invention may be produced from genetically-modified host cells. In some embodiments, the host cells are grown, each individual component is expressed and isolated, and the different components are mixed and assembled. In other embodiments, genetically-modified host cells capable of producing and secreting at least one component of the multi-enzyme complex are co-cultured under conditions that allow the secretion and assembly of the different components. According to these embodiments, the different components are engineered to include a suitable signal sequence to enable their secretion.
Thus, according to another aspect, the present invention provides a host cell comprising at least one component of the bio-engineered multi-enzyme complex of the present invention.
In some embodiments, a host cell is provided, wherein the host cell is genetically modified to produce at least one component of the multi-enzyme system of the present invention.
In some embodiments, a genetically modified host cell is provided, comprising a polynucleotide encoding at least one bio-engineered component of the multi-enzyme complex of the present invention. In some embodiments, the host cell comprises a combination of polynucleotides that encode more than one component of the multi-enzyme system of the present invention.
In some embodiments, a genetically modified host cell is provided, capable of producing at least one component of the bio-engineered multi-enzyme system of the present invention.
According to a further aspect, the present invention provides a cell culture comprising at least one genetically modified host cell, the host cell comprising at least one component of the multi-enzyme system of the present invention. The host cells that constitute the culture of the present invention are capable of producing and secreting at least one component of the multi-enzyme system of the present invention.
In some embodiments, a cell culture is provided, comprising at least one genetically modified host cell, the at least one genetically modified host cell comprising a polynucleotide encoding at least one bio-engineered component of the multi-enzyme complex of the present invention.
In some embodiments, a plurality of different host cells is modified to express and produce at least one component of the multi-enzyme system. According to these embodiments, a plurality of different genetically modified host cells are present in the culture, each comprises at least one component of the multi-enzyme complex. For example,
In some embodiments, each host cell is modified to produce one component of the multi-enzyme system, for example a dockerin-containing enzyme or a scaffold protein. According to these embodiments, the culture comprises a plurality of different types of host cells, each type is modified in a different way and produces a different component of the multi-enzyme complex.
The individual host cells may be from the same or different species.
In some embodiments, a single host cell is modified to produce more than one component of the multi-enzyme system.
In other embodiments, a single host cell is genetically modified to express all components of the multi-enzyme complex of the present invention. According to these embodiments, a single host cell is present in the culture, the single host cell comprises all components of the multi-enzyme complex.
Any host cell known in the art for the production of recombinant proteins may be used for the present invention.
In some embodiments, the host cell is a prokaryotic cell. Representative, non-limiting examples of appropriate prokaryotic hosts include bacterial cells, such as cells of Escherictahia coli and Bacillus subtilis. 
In other embodiments, the host cell is a eukaryotic cell. In some exemplary embodiments, the host cell is a fungal cell, such as yeast. Representative, non-limiting examples of appropriate yeast cells include Saccharomyces cerevisiae and Pichia pastoris. In additional exemplary embodiments, the host cell is a plant cell.
Genes encoding the different complex components may be cloned into the selected host cells by cloning methods known in the art. Exemplary procedures for cloning and expressing recombinant proteins in host cells are described below.
In the case of secreted proteins, secretion of the different components to the medium may be confirmed, for example by Western blotting. For example, the different components may be engineered to include a certain tag, such as a His-tag, thereby allowing their probing by a suitable antibody.
Host cells secreting the different components are co-cultured and the correct assembly of the multi-enzyme complex may be confirmed, for example by using non-denaturing gel electrophoresis mobility assay. An exemplary procedure is exemplified herein below.
The appropriate culturing conditions that allow expression and secretion of the recombinant proteins are determined according to the particular host cells. Determination of the suitable conditions is within the capabilities of a person skilled in the art.
The activity of transformed host cells or cell cultures, as well as the multi-enzyme complexes may be assayed. For example, cellulase activity can be determined for example, using Avicel as the substrate as is well known to a person skilled in the art and is exemplified herein below. The activity of the secreted components may be assayed on complex substrates, such as wheat straw, as also exemplified herein below.
Methods and Systems for Bioconversion of Cellulosic Material
The multi-enzyme complexes of the present invention, compositions comprising same and cells cultures producing same may be utilized for the bioconversion of cellulosic material into degradation products.
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.
Thus, according to another aspect, the present invention provides a method for bioconversion of cellulosic material into degradation products comprising exposing said cellulosic material to the multi-enzyme complex of the present invention.
According to a further aspect, the present invention provides a system for bioconversion of cellulosic material comprising a multi-enzyme complex of the present invention.
Multi-enzyme complexes 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 multi-enzymes of the invention may be recycled.
By relieving end-product inhibition of endoxylanases and exo/endo-glucanases (such as xylobiose and cellobiose), it may be possible to further enhance the hydrolysis of the cellulosic material.
Polypeptides and Polynucleotides
The present invention further provides variant xylanases capable of integrating into the bio-engineered complex of the present invention, and suitable scaffold polypeptides.
The variant xylanases of the present invention are modified to include a heterologous dockerin. Thus, the variant xylanases are chimeric polypeptides, comprising a xylanase catalytic module and a heterologous dockerin domain.
According to another aspect, the present invention provides an isolated polypeptide comprising a variant xylanase, the variant comprises a heterologous dockerin domain.
In some embodiments, the dockerin domain is selected from the group consisting of C. thermocellum, A. cellulolyticus, R. flavefaciens, B. cellulosolvens A. fulgidus and C. cellulolyticum dockerins. Each possibility represents a separate embodiment of the invention. In some embodiments, the dockerin domain is selected from the group consisting of C. thermocellum, A. cellulolyticus, R. flavefaciens and B. cellulosolvens. Each possibility represents a separate embodiment of the invention.
In some embodiments, the variant xylanase is classified in a glycoside hydrolase family selected from the group consisting of family 5, 8, 10, 11, 26 and 43. Each possibility represents a separate embodiment of the invention. In some typical embodiments, the variant xylanase is classified in a glycoside hydrolase family selected from the group consisting of family 10 and 11.
In some embodiments, the variant xylanase is derived from T. fusca xylanases.
Xylanases from T. fusca does not naturally include a dockerin domain, and the introduction of a dockerin according to embodiments of the present invention enables their utilization and incorporation into enzyme complexes of the present invention.
In some embodiments, the T. fusca xylanases are selected from the group consisting of Xyn10B and Xyn11A. Each possibility represents a separate embodiment of the invention.
In some embodiments, the variant T. fusca xylanase is selected from the group consisting of a variant Xyn10B comprising a dockerin domain derived from C. thermocellum and a variant Xyn11A comprises a dockerin domain derived from A. cellulolyticus. Each possibility represents a separate embodiment of the invention.
In some embodiments, the variant T. fusca xylanase comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant T. fusca xylanase comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the variant T. fusca xylanase is consisting of the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, the variant T. fusca xylanase comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant T. fusca xylanase comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variant T. fusca xylanase is consisting of the amino acid sequence set forth in SEQ ID NO: 7.
The variant xylanases may be utilized for the bio-conversion of cellulosic material. In some embodiments, a composition is provided, comprising the variant xylanase of the present invention.
The variant xylanases they may be incorporated into multi-enzyme complexes, such as the bio-engineered complexes described above. Thus, there is provided a bio-engineered multi-enzyme complex comprising a variant xylanase of the present invention. In some embodiments, there is provided a bio-engineered cellulosome comprising a variant xylanase of the present invention.
There is further provided a method for the bio-conversion of cellulosic material into degradation products comprising exposing said cellulosic material to a variant xylanase of the present invention.
There is further provided a system for bioconversion of cellulosic material comprising a variant xylanase of the present invention.
According to another aspect, the present invention provides an isolated scaffold polypeptide comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from C. thermocellum, a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. 
It is to be understood the plurality of cohesin domains present in scaffold polypeptides of the present invention may be arranged in any order in the scaffold polypeptide.
In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the isolated scaffold polypeptide is consisting of the amino acid sequence set forth in SEQ ID NO: 23.
According to yet another aspect, the present invention provides an isolated scaffold polypeptide comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from C. thermocellum, a cohesin derived from A. cellulolyticus, a cohesin derived from R. flavefaciens and a cohesin derived from B. cellulosolvens. 
In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the isolated scaffold polypeptide is consisting of the amino acid sequence set forth in SEQ ID NO: 25.
According to yet another aspect, the present invention provides an isolated scaffold polypeptide comprising at least two cohesin domains, wherein the at least two cohesin domains are selected from the group consisting of:
(i) a cohesin derived from A. cellulolyticus and a cohesin derived from C. thermocellum; 
(ii) a cohesin derived from B. cellulosolvens and a cohesin derived from R. flavefaciens; and 
(iii) a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. 
In some embodiments, an isolated scaffold polypeptide is provided, comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from A. cellulolyticus and a cohesin derived from C. thermocellum. In some embodiments, the plurality of cohesin domains is consisting of a cohesin derived from A. cellulolyticus and a cohesin derived from C. thermocellum. In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the isolated scaffold polypeptide is consisting of the amino acid sequence set forth in SEQ ID NO: 19.
In some embodiments, an isolated scaffold polypeptide is provided, comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from B. cellulosolvens and a cohesin derived from R. flavefaciens. In some embodiments, the plurality of cohesin domains is consisting of a cohesin derived from B. cellulosolvens and a cohesin derived from R. flavefaciens. In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the isolated scaffold polypeptide is consisting of the amino acid sequence set forth in SEQ ID NO: 21.
In some embodiments, an isolated scaffold polypeptide is provided, comprising a plurality of cohesin domains, wherein the plurality of cohesin domains comprises a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. In some embodiments, the plurality of cohesin domains is consisting of a cohesin derived from A. cellulolyticus and a cohesin derived from R. flavefaciens. In some embodiments, the isolated scaffold polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the isolated scaffold polypeptide is consisting of the amino acid sequence set forth in SEQ ID NO: 17.
The scaffold polypeptides of the present invention may be utilized for the bio-conversion of cellulosic material. In some embodiments, a composition is provided, comprising a scaffold polypeptide of the present invention.
In some embodiments, a bio-engineered multi-enzyme complex is provided, comprising a scaffold polypeptide of the present invention. In some embodiments, there is provided a bio-engineered cellulosome comprising a scaffold polypeptide of the present invention.
There is further provided a system for bioconversion of cellulosic material comprising a scaffold polypeptide of the present invention.
According to another aspect, the present invention provides an isolated polynucleotide comprising a sequence encoding a polypeptide of the present invention.
In some particular embodiments, the isolated polynucleotide comprises a polynucleotide sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 6, 8, 18, 20, 22, 24 and 26. Each possibility represents a separate embodiment of the invention.
According to another aspect, the present invention provides a construct comprising a polynucleotide sequence of the present invention.
According to yet another aspect, the present invention provides a host cell comprising a polynucleotide sequence of the present invention. In some embodiments, the cell is selected from a prokaryotic and eukaryotic cell.
The variant polypeptides disclosed herein may be produced by recombinant or chemical synthetic methods. For example:
Recombinant Expression:
The variant polypeptides 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.
DNA sequences encoding wild type polypeptides may be isolated from any cell 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)). For example, a DNA encoding the wild-type polypeptide may be amplified from genomic DNA of an appropriate microorganism (for example, a bacterium) producing them by polymerase chain reaction (PCR) using specific primers, constructed on the basis of the nucleotide sequence of the known wild type sequence. Suitable techniques are well known in the art, described for example in U.S. Pat. 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, Aug. 1987, Pages 474-478.
The isolated polynucleotide encoding the wild type polypeptide may be cloned into a vector, such as the pET28a plasmid.
Upon isolation and cloning of the polynucleotide encoding the wild type polypeptide, 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, 151:119-123; Ishii et al., Methods Enzymol. 1998, 293:53-71); cassette mutagenesis (see for example, Kegler-Ebo et al., Nucleic Acids Res. 1994 May 11; 22(9):1593-1599); recursive ensemble mutagenesis (see for example, Delagrave et al., Protein Engineering 1993, 6(3):327-331), and gene site saturation mutagenesis (see for example, U.S. Pat. Application No. 2009/0130718). Methods are also well known for introducing multiple mutations into a polynucleotide (see for example, Michaelian et al., Nucleic Acids Res. 1992, 20:376; Dwivedi et al., Anal. Biochem. 1994, 221:425-428; Bhat Methods Mol. Biol. 1996, 57:269-277; Meetei et al., Anal. Biochem. 1998, 264:288-291; Kim et al., Biotechniques 2000, 28:196-198; and International patent Application Publication Nos. WO 03/002761A1 and WO 99/25871). For example, introduction of two and/or three mutations can be performed using commercially available kits, such as the QuickChange site-directed mutagenesis kit (Stratagene).
An alternative method to producing a polynucleotide with a desired sequence is the use of a synthetic gene. A polynucleotide encoding a desired polypeptide 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 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.
In the case of a fusion protein, different polynucleotides may be ligated to form one polynucleotide. For example, different polynucleotides may be ligated into linearized pET21a.
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.
Representative, non-limiting examples of appropriate hosts include 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, for example, include, but are not limited to, pET, pK233, pT7 and lambda pSKF. Other expression vector systems are based on betagalactosidase (pEX); maltose binding protein (pMAL); and glutathione S-transferase (pGST).
The proteins may be deigned to include a tag, for example, a 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. For example, in the case of E. coli, it 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.
Upon transformation of a suitable host cell, and propagation under conditions appropriate for protein expression, the polypeptide may be identified in cell extracts of the transformed cells. Transformed hosts expressing the polypeptide may be identified by analyzing the proteins expressed by the host, for example, 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 the desired polypeptide.
The desired polypeptides 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. The polypeptides of the invention may be produced as fusion proteins, attached to an affinity purification tag, such as a His-tag, in order to facilitate their rapid purification.
The isolated polypeptide may be analyzed for its various properties, for example specific activity, using methods known in the art, some of them are described hereinbelow.
Conditions for carrying out the aforementioned procedures as well as other useful methods are readily determined by those of ordinary skill in the art (see for example, Current Protocols in Protein Science, 1995 John Wiley & Sons).
Synthetic Production:
The 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, Ill., pp. 11-12). 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). In addition, a number of available 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 431A 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.
As is readily apparent to those of skill in the art, the codon used in the polynucleotide for encoding a particular amino acid 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. Accordingly, it will be evident.
EXAMPLES
Example 1
Construction And Expression of Recombinant Proteins
Methods
Cloning of Wild-Type Enzymes:
Plasmids encoding wild-type enzymes pCel5A, pXyn11A, pXyn10B, pCel48A were cloned as described in Irwin et al. 1994 Appl. Environ. Microbiol. 60:763-770; Irwin et al. 2000 Eur. J. Biochem. 267:4988-97; Kim et al. 2004 Can. J. Microbiol. 50:835-843. The sequences of the resulting polynucleotides and corresponding polypeptides are set forth in the following SEQ ID NOs:
Cel5A cloned wild-type: DNA—SEQ ID NO: 64; amino acids—SEQ ID NO: 65.
Xyn11A cloned wild-type: DNA—SEQ ID NO: 66; amino acids—SEQ ID NO: 67.
Xyn10B cloned wild-type: DNA—SEQ ID NO: 68; amino acids—SEQ ID NO: 69.
Cel48A cloned wild-type: DNA—SEQ ID NO: 84; amino acids—SEQ ID
NO: 85.
Cloning of Chimeric Enzymes:
The recombinant pf-5A was engineered as reported in Caspi et al. 2009 Appl. Environ. Microbiol. 75:7335-7342. The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 14 and 13, respectively. The b-48A chimera was cloned as described in Caspi et al. 2008 J. Biotechnol. 135:351-357. The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 16 and 15, respectively.
The 11A-a chimera, i.e., the catalytic module of Xyn11A attached directly to a dockerin from Acetivibrio cellulolyticus ATCC 33288, but lacking the family-2 xylan-binding CBM (XBM), was constructed from pXyn11A using primers:
SEQ 1N NO: 35:
5′CTGCCGCTAGCATGCACCATCACCATCACCACGCCGTGACCTCC AACGAGACC-3′ (NheI site in boldface), and SEQ 1N NO: 36:
5′-TCCCAAGAGCTCCGTCGAACTAGTGCCACCGCCACCGGGGGGG TTGCC-3′ (SpeI and SacI sites in boldface) for cloning the catalytic module, and primers:
SEQ 1N NO: 37:
5′ATGTATACTAGTAAATTTATATATGGTGATGT-3′ (SpeI site in boldface) and SEQ 1N NO: 38:
5′-TACCAAGAGCTCTTATTCTTCTTTCTCTTCAACAG-3′ (SacI site in boldface) for amplification of the A. cellulolyticus dockerin B.
The two modules were ligated (T4 DNA Ligase: Fermentas UAB Vilnius, Lithuania) into NheI-SacI (New England Biolabs Inc., Beverly, Mass.) linearized pET21a (Novagen Inc. Madison, Wis.) to form the p11A-a. The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 70 and 71, respectively.
The 11A-XBM-a chimera, i.e., the catalytic module of Xyn11A together with its adjacent XBM attached to a dockerin from A. cellulolyticus ATCC 33288, was
constructed using primers: Primer (SEQ 1N NO: 39) 5′-TATCCGGAGCTCGTTGGCGCTGCAGGACACCG-3′ (SacI site in boldface) was used to clone the full-length Xyn11A (together with the forward Xyn11A primer used above), and primers for cloning A. cellulolyticus dockerin B were designed:
SEQ 1N NO: 40:
5′ TTATTCGAGCTCACAGCAACTACAACACCAACTACAACACCAAC TACAACACCAACGCCTAAAT—3′ (SacI site in boldface) and SEQ 1N NO: 41: 5′ TTATTCGAGCTCACAGCAACTACAACACCAACTACAACACCAAC TACAACACCAACGCCTAAAT-3′ (XhoI site in boldface). The two PCR products were then ligated into NheI-XhoI linearized pET21a to form the p11A-XBM-a. The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 8 and 7, respectively.
The 10B-t chimera, i.e., the catalytic module of Xyn10B attached directly to a dockerin from C. thermocellum, was cloned as follows: the DNA encoding the C. thermocellum dockerin S (from Cel48S) was amplified from the genomic DNA (strain YS) using primers: SEQ 1N NO: 42:
5′-TTATTCACTAGTACATATAAAGTACCTGGTACTCC-3′ and SEQ IN NO: 43:
5′-TTATTCCTCGAGTTAGTTCTTGTACGGCAATGTATC-3′ (SpeI and XhoI sites in boldface). The Xyn10B catalytic module was cloned from the Xyn10B plasmid using primers: SEQ 1N NO: 44:
5′CATATTGCTAGCCATCACCATCACCATCACGGACCGGTCCACGA CCATCATCCC-3′ and SEQ 1N NO: 45:
5′ TTATTCCTCGAGTTATTAACTAGTACAGTGATCGTGCTTGGGGCC C-3′ (NheI, SpeI and XhoI sites in boldface).
The two modules were then ligated into NheI-XhoI linearized pET21a to form the p10B-t. The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 6 and 5, respectively. All enzymes constructs were designed to contain a His-tag for subsequent purification.
Cloning of Chimeric Scaffoldins:
Scaffoldins were assembled from modules (cohesins, dockerins, CBM) cloned from different genomic DNAs. The following primers represent the homologous gene sequences only and were used with different restriction sites, either NcoI, KpnI, BamHI or Xhoi, depending on the desired construct. Cohesin F (cohesin 1 from Ruminococcus flavefaciens strain 17 scaffoldin B) was amplified using: SEQ 1N NO: 46:
5′-CGCCGGTGGTTTATCCGCTGTG-3′ and SEQ 1N NO: 47:
5′-TTAATGGTGATGGTGATGGTGAACAATGATAGCGCCATCAGT-3′. The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 72 and 73, respectively.
Cohesin A (cohesin 3 from A. cellulolyticus scaffoldin C) was cloned from genomic DNA using SEQ 1N NO: 48: 5′-ATTTACAGGTTGACATTGGAAGT-3′ and SEQ 1N NO: 49: 5′-GATGCAATTACCTCAATTTTTCC-3′. The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 74 and 75, respectively.
Cohesin B (cohesin 3 from Bacteroides cellulosolvens scaffoldin B) was amplified using SEQ 1N NO: 86: 5′ CCATGGCGGGGAAAAGTTCACCAG 3′ (five random bases were added before the restriction site in each primer) and SEQ 1N NO: 87: 5′ GGTACCTTAGTTACAGTAATGCTTCC 3′ primers (NcoI and KpnI sites in boldface type). The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 76 and 77, respectively.
CBM-T (CBM3a and cohesin 3 from the cellulosomal scaffoldin subunit Clostridium thermocellum YS) was cloned from C. thermocellum YS genomic DNA using: SEQ 1N NO: 50: 5′-GACAAACACACCGACAAACACA-3′ and SEQ 1N NO: 51: 5′-CTATATCTCCAACATTTACTCCAC-3′. The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 80 and 81, respectively. The sequence of the DNA encoding Cohesin T (cohesin 3 from Clostridium thermocellum CipA scaffoldin) and the resulting polypeptide are set forth in SEQ ID NOs: 78 and 79, respectively.
The different modules were assembled in the linearized pET28a plasmid to form the chimeric scaffoldins.
pScaf.T, pScaf.F and pScaf-A were cloned as described in Haimovitz et al. 2008 Proteomics 8:968-979. The amino acid sequences of the resulting polypeptides and the DNA encoding them are set forth in the following SEQ ID NOs:
Scaf-T DNA—SEQ ID NO: 52; Scaf-T amino acids—SEQ ID NO: 53.
Scaf-B DNA—SEQ ID NO: 54; Scaf-B amino acids—SEQ ID NO: 55.
Scaf-F DNA—SEQ ID NO: 56; Scaf-F amino acids—SEQ ID NO: 57.
Scaf-A DNA—SEQ ID NO: 58; Scaf-A amino acids—SEQ ID NO: 59.
To form the dockerin-containing pf-XBM, the XBM was amplified from the Xyn11A plasmid using the following primers: SEQ 1N NO: 60:
5′-AAATAAGGTACCTACCAGCGGCGGTGGAAACCCC-3′ (KpnI site in boldface) and SEQ 1N NO: 61:
5′-AAATTACTCGAGCTAGTTGGCGCTGCAGGACA-3′ (XhoI site in boldface), and ligated to linearized pET28a together with the R. flavefasciens ScaB dockerin, cloned from genomic DNA using: SEQ 1N NO: 62:
5′TGATCCATGGCACACCATCACCATCACCATGCACCATCACCCGG CACAAAGC-3′ (BamHI site in boldface) and SEQ 1N NO: 63:
5′-ATGCTTGGTACCGCTTGAGGAAGTGTGATGAGTTCAA-3′ (KpnI site in boldface). The sequences of the resulting polynucleotide and corresponding polypeptide are set forth in SEQ ID NOs: 82 and 83, respectively. Other recombinant scaffoldins were cloned as described in Irwin et al. 1994 Appl. Environ. Microbiol. 60:763-770; Irwin et al. 2000 Eur. J. Biochem. 267:4988-97; Haimovitz et al. 2008 Proteomics 8:968-979. The sequences of the resulting polynucleotides and corresponding polypeptides are set forth in SEQ ID NOs:
Scaf-AF: DNA—SEQ ID NO: 18; amino acids—SEQ ID NO: 17.
Scaf-BF: DNA—SEQ ID NO: 22; amino acids—SEQ ID NO: 21.
Scaf AT: DNA—SEQ ID NO: 20; amino acids—SEQ ID NO: 19.
Scaf-ATF: DNA—SEQ ID NO: 24; amino acids—SEQ ID NO: 23.
Scaf-BTFA: DNA—SEQ ID NO: 26; amino acids—SEQ ID NO: 25.
PCR reactions were performed using ABgene Reddymix ×2 (Advanced Biotechnologies Ltd., Epsom, UK), DNA samples were purified using a HiYieldTM Gel/PCR Fragments Extraction Kit (Real Biotech Corporation, RBC, Banqiao City, Taiwan).
Protein Expression and Purification:
Cel5A, Xyn11A, Xyn10B were prepared as described in Irwin et al. 1994 Appl. Environ. Microbiol. 60:763-770; Irwin et al. 2000 Eur. J. Biochem. 267:4988-97; Kim et al. 2004 Can. J. Microbiol. 50:835-843. The f-5A chimera was expressed as reported in Caspi et al. 2009 Appl. Environ. Microbiol. 75:7335-7342.
The 11A-a, 11A-XBM-a, 10B-t and f-XBM plasmids were expressed in E. coli BL21 (λDE3) pLysS cells and purified on a Ni column, as reported in Caspi et al. 2006 Biocat. Biotransform. 24:3-12. Cel48A and the b-48A chimera were expressed as reported in Caspi et al. 2008 J. Biotechnol. 135:351-357. Scaffoldins were expressed and purified on phosphoric acid swollen cellulose 7.5 mg m-1-1 pH 7 (PASC) according to the previously described methodology Haimovitz et al. 2008 Proteomics 8:968-979. Protein expression and purification was carried out by transforming BL21 (XDE3) pLysS cells with the designated plasmids and growing them on Luria-Bertani medium at 37° C. to an A600 ̃1. Isopropyl thio-β-D-galactoside (IPTG) was added to a final concentration of 0.1 mM. Following induction, cells were incubated for 3 h at 37° C., centrifuged, sonicated, and the supernatant fluids were incubated with amorphous cellulose for 1-2 h at 4° C. to allow binding of the CBM-Coh. The amorphous cellulose was washed three times with TBS, pH 7.4, containing 1M NaCl and three times with TBS. The protein was eluted with 1% v/v triethylamine, and neutralized with MES buffer.
Purity of the recombinant proteins was tested by SDS-PAGE on 12% acrylamide gels. The concentration of each purified protein was estimated by absorbance (280 nm) based on the known amino acid composition of the protein using the Protparam tool. Proteins were stored in 50% (v/v) glycerol at −20° C.
Results
The recombinant proteins are shown schematically in FIG. 1. Four different T. fusca enzymes were used: two xylanases, Xyn11A and Xyn10B, and two cellulases, the family-5 endoglucanase, Cel5A and the family-48 exoglucanase Cel48A. Cel5A and Cel48A are typical free (non-cellulosomal) enzymes that both contain a family-2 cellulose-binding CBM. Xyn11A contains a CBM from the same family, which shows binding specificity for both cellulose and xylan. Xyn10B lacks a CBM.
Dockerins from different specificities were used to replace the CBM or XBM of the native enzymes, generating f-5A, b-48A and 11A-a, or added in the C-terminal of the xylanases generating 11A-XBM-a and 10B-t.f-5A is a recombinant cellulose-hydrolyzing enzyme consisting of two fused modules: a catalytic module of the family-5 endoglucanase Cel5A from T. fusca, and a dockerin from Ruminococcus flavefaciens (Ding et al. 2001 J. Bacteriol. 183:1945-1953). b-48 was designed to contain the catalytic module of the T. fusca exoglucanase Cel48A ligated with a dockerin from the Bacteroides cellulosolvens ScaA scaffoldin.
Two recombinant forms of Xyn11A were designed: one, 11A-XBM-a, in which dockerin B from Acetivibrio cellulolyticus was appended at the C-terminus of the original Xyn11A thus retaining the original catalytic module and xylan-binding CBM (XBM), and a second, 11A-a, in which XBM was replaced by the same A. cellulolyticus dockerin. The resultant fusion protein is identical to 11A-XBM-a, but now lacks the XBM. 11A-a is employed as a crucial control, in order to assay the importance of the XBM module in the enzymatic activity of the enzyme alone or within a complex. XBM alone was also examined for its contribution to activity; therefore the dockerin of scaffoldin A from Ruminococcus flavefaciens (Ding et al. 2001 J. Bacteriol. 183:1945-1953) was fused to the XBM module at the N-terminus of the protein. In order to integrate Xyn10B into an enzymatic complex, the dockerin from exoglucanase Cel48S of Clostridium thermocellum (Wang et al. 1993 J. Bacteriol. 175:1293-1302) was fused at its C-terminus resulting in 10B-t.ScafAF has two cohesins of divergent specificity, allowing the possibility of binding two different dockerin-containing proteins selectively. The specific modules that comprise the construct are as follows: cohesin 3 from A. cellulolyticus scaffoldin C (designated A) (Xu et al. 2003 J. Bacteriol. 185:4548-4557), CBM3a from C. thermocellum, which binds strongly to cellulose (Morag et al. 1995 Appl. Environ. Microbiol. 61:1980-1986), and cohesin 1 from R. flavefaciens scaffoldin B (designated F) (Ding et al. 2001J. Bacteriol. 183:1945-1953). Scaf.AF allows the specific incorporation of the previously described enzymes (either 11A-XBM-a or 11A-a and f-5A), and will direct the complex to the substrate via the CBM.
Scaf.AT also has 2 different cohesins and a cellulose-binding CBM. A. cellulolyticus cohesin 3 (A, as specified above) will interact specifically with enzymes carrying the matching dockerin, i.e., 11A-XBM-a or 11A-a. At the C-terminus, T-cohesin 3 from the CipA C. thermocellum scaffoldin (Yaron et al. 1995 FEBS Lett. 360:121-124)—will bind with the dockerin S-containing enzyme, 10B-t.
Scaf.ATF, includes all three above-described cohesin types together with the cellulose-binding CBM. This 3-cohesin scaffoldin enables the integration of the two xylanases, 10B-t and 11A-XBM-a (or 11A-a), and endoglucanase f-5A. Scaf-BF has two cohesins of divergent specificity, allowing the possibility of binding two different dockerin-containing proteins selectively. The specific modules that comprise the construct are as follows: cohesin from B. cellulosolvens ScaB, CBM3a from C. thermocellum, which binds strongly to cellulose (described above), and cohesin 1 from R. flavefaciens scaffoldin B (designated F, described above). Scaf.BF allows the specific incorporation of the previously described enzymes (b-48A and f-5A), and will direct the complex to the substrate via the CBM. Scaf.BTFA includes all four above-described cohesin types together with the cellulose-binding CBM. This 4-cohesin scaffoldin enables the integration of the two xylanases, 10B-t and 11A-XBM-a, and the two cellulases f-5A and b-48A. All purified recombinant proteins showed a single major band on SDS-PAGE and in each case their mobility was consistent with their molecular mass.
Example 2
Interaction Between Scaffoldin and Enzymes
Methods:
Affinity-Based ELISA:
The matching fusion-protein procedure of Barak et al. 2005J. Mol. Recogit. 18:491-501; and Caspi et al. 2006 Biocat. Biotransform. 24:3-12. was followed to determine cohesin-dockerin specificity.
For example, for the analysis of dockerin-containing xylanases:
Analysis of Divergent Dockerins Using Immobilized Cohesins:
MaxiSorp ELISA plates (Nunc A/S, Roskilde, Denmark) were coated overnight at 4° C. with predetermined concentrations (designated below) of the desired CBM-Coh (100 μl/well) in 0.1M sodium carbonate (pH 9). The following steps were performed at room temperature with all reagents at a volume of 100 μl/well. The coating solution was discarded and blocking buffer (TBS, 10 mM CaCl2, 0.05% Tween 20, 2% BSA) was added (1 h incubation). The blocking buffer was discarded, and incremental concentrations of the desired XynDoc constructs, diluted in blocking buffer, were added. After a 1 h incubation period, the plates were washed three times with wash buffer (blocking buffer without BSA), and the primary antibody preparation (rabbit anti-xylanase T-6 antibody, diluted 1:10,000 in blocking buffer) was added. Following another 1 h incubation period, the plates were washed three times with wash buffer and the secondary antibody preparation (HRP-labeled anti-rabbit antibody diluted 1:10,000 in blocking buffer) was added. After another 1 h incubation, the plates were again washed (four times) with wash buffer and 100 μl/well TMB+Substrate-Chromogen were added. Color formation was terminated upon addition of 1M H2SO4 (50 μl/well), and the absorbance was measured at 450 nm using a tunable microplate reader. Absorbance was plotted as a function of XynDoc concentration, usually resulting in a sigmoidal (dose-response) curve. For presentation of results in bar graph form, two alternative and complementary methods were used: (i) the pEC50 was determined for the binding curve of the test XynDoc constructs and compared with that of the XynDoc standard; and (ii) the ‘reference concentration’ of a XynDoc standard that generates a maximum response was employed for comparison of the level of response produced by other test XynDoc constructs at that concentration. In the latter case, the data can be normalized as a percentage (relative binding) of maximum response by the reference XynDoc. The two methods produced very similar results.
Analysis of Divergent Cohesins Using Immobilized Dockerins:
The assay for divergent cohesins was essentially the converse of that described above for the divergent dockerins: instead of immobilizing a cohesin construct, a suitable dockerin construct was substituted. The following modifications were then introduced: coating was performed with 20 nM of the desired XynDoc construct, the desired CBM-Coh(s) was diluted to concentrations of 10 μM to 10 nM, and rabbit anti-CBM antibody (diluted 1:10,000) was used as the primary antibody preparation. Subsequent steps were performed as described in the previous section.
Non-Denaturing PAGE:
A differential mobility assay on non-denaturing gels was used to check the full interaction between scaffoldin and enzymes. In a 30 μl reaction (in which 15 μl of Tris Buffer Saline pH 7.4 (TBS) buffer, supplemented with 10 mM CaCl2 and 0.05% Tween 20), 4-8 μg of each protein were added in an equimolar manner. The 1.5 ml tubes were incubated 1.5 h at 37° C. Sample buffer (7.5 in the absence of SDS) was added to 15 μl of the reaction mixture, and the samples were loaded onto non-denaturing gels (4.3%-stacking/9%-separating phase). A parallel SDS-PAGE gel (10%) was performed on the remaining 15 μl sample.
Results
The specificity of the cohesins for the chimeric dockerin-bearing enzymes was examined semi-quantitatively by a sensitive enzyme-linked affinity assay in microtiter plates. All of the cohesins in each scaffoldin specifically bound their respective dockerin and did not bind (or bound very poorly) other non-matching dockerin-bearing molecules. The scaffoldin-borne cohesins bound their matching dockerins just as efficiently as the individual single-cohesin scaffoldins, indicating that the binding capabilities of the scaffoldins were reliable and selective. All specific cohesin-dockerin interactions, for each scaffoldin, were of similar intensity, thus indicating that similar amounts of protein were bound in each well, suggesting a molar equivalent of 1:1 scaffoldin (cohesin): dockerin ratio. For each chimeric designer cellulosome, complex formation was tested by non-denaturing PAGE. Denaturing PAGE was used as a control for sample content verification. Stoichiometric mixtures of the enzymes and the scaffoldin resulted in a single band with altered mobility (band strengthened and shifted) thus indicating that complete or near-complete complexation was achieved in all cases (an example is given in FIG. 2).
Example 3
Binding to Insoluble Polysaccharides
Method
Insoluble xylan was prepared by boiling oat-spelt xylan (Sigma Chem. Co., St. Louis, Mo.) for 30 min in distilled water and recovering the residue by centrifugation; this was followed by 3 washes with distilled water, and its dry weight was determined. Microcrystalline cellulose (Avicel) was purchased from FMC Biopolymer (Philadelphia, Pa., USA). The binding of each protein to insoluble polysaccharides (insoluble xylan from oat spelt and microcrystalline cellulose) was determined qualitatively using SDS-PAGE. Pure protein (10 μg for xylan binding assays and 5 μg for cellulose binding experiments in 50 mM citrate buffer pH 6.0, 12 mM CaCl2, 2 mM EDTA) was mixed with 0.5 mg of insoluble xylan or 10 mg of microcrystalline cellulose, in a final volume of 100 μl. Tubes were incubated on ice for 1 h with gentle mixing before being centrifuged at 14000 rpm for 1 min, and the supernatant fluids (containing unbound protein) were carefully removed. The polysaccharide pellet was then washed once by resuspending in 100 μl of the same buffer and centrifugation before resuspending in 60 μl of SDS-containing buffer and boiled for 10 min to dissociate any bound protein. BSA was used as negative control to ensure specificity of binding. Bound and unbound fractions were analyzed by SDS-PAGE using a 12% polyacrylamide gel.
Results
The majority of the family-10B enzyme was able to bind to insoluble xylan this was due to the inherent binding capacity of the catalytic module only, since the protein does not include any XBM. The same result was obtained for 11A-a in which the XBM module was replaced by the dockerin module—the enzyme was found both in the bound and unbound fractions. The Xyn11A, 11A-XBM-a, and f-XBM proteins were located in the bound fractions, suggesting that the dockerin module in 11A-XBM-a did not disturb the binding function of the XBM and that the XBM alone is able to bind xylan. Upon mixing the proteins with microcrystalline cellulose, Xyn11A and 11A-XBM-a were found exclusively in the bound fractions; thus the cellulose-binding ability of the protein is maintained in 11A-XBM-a, and has not been affected by the dockerin module. f-XBM was found in approximately equal portions in both fractions, suggesting that the binding function to Avicel reflects the combined action of the entire protein (catalytic module and XBM), and the lack of the catalytic module results in a weakened ability to bind the substrate. Indeed, 11A-a, which lacks the XBM, was also found in both fractions (the major part was found in the bound fraction) reinforcing the hypothesis that the protein needs its catalytic module together with its XBM to achieve full substrate-binding capacity. The Xyn10B and 10B-t enzymes bound cellulose very weakly, indicating a low but measurable cellulose-binding activity associated with the family 10 catalytic module. As expected, more than  ̃95% of the BSA negative control was found in the unbound fraction. These results are in perfect accord with previous publications (Irwin et al. 1994 Appl. Environ. Microbiol. 60:763-770; Kim et al. 2004 Can. J. Microbiol. 50:835-843), in which the binding capacity of the wild-type Xyn11A and Xyn10B enzymes was investigated. Xyn11A was found to bind strongly to insoluble xylan and cellulose, and a weak ability to bind insoluble xylan binding was demonstrated for Xyn10B. In previous binding experiments to microcrystalline cellulose, Cel5A exhibited an ability to bind to cellulose whereas f-5A failed to bind cellulose due to the lack of CBM2 (Caspi et al. 2009 Appl. Environ. Microbiol. 75:7335-7342).
Example 4
Enzymatic Activity of Free Xylanases on Xylans
Method
Xylanase activity was determined quantitatively by measuring the reducing sugars released from xylan by the dinitrosalicyclic acid (DNS) method (Ghose et al. 1987 Pure. Appl. Chem. 59:257-268; Miller et al. 1959 Anal. Biochem. 31:426-428). A typical assay mixture consisted of 100 μl buffer (50 mM citrate buffer pH 6.0, 12 mM CaCl2, 2 mM EDTA) with enzyme (0-10 nM). The reaction was commenced by adding 100 μl of 2% xylan (birchwood, beechwood or oat spelt from Sigma Chem. Co, St. Louis Mo.), suspended in 50 mM citrate buffer, pH 6.0, and the reaction was continued for 20 min at 50° C. The reaction was stopped by transferring the tubes to an ice-water bath, 100 μl of the supernatant were then added to 150 μl DNS reagent, and the tubes were boiled for 10 min, after which absorbance was measured at 540 nm. Dockerin-containing enzymes were subjected to 1.5-h incubation (37° C., in the absence of substrate) in the presence of equimolar concentrations of scaffoldin, prior to assay.
Results
All recombinant xylanases were tested for xylan degradation on a variety of xylan substrates. Three different substrates were used to test the degradation activity of the transformed xylanases: birchwood xylan, beechwood xylan and oat spelt xylan. The characteristics (composition and properties) of xylans from different origins were previously investigated by Hespell et al. 1995 Appl. Environ. Microbiol. 61:3042-3050. Birchwood xylan is more than 90% soluble in water and is composed of a high percentage of neutral sugars (87.7% mainly xylose residues, small amounts of glucose and traces of arabinose and galactose can be found) and 10.2% of hexuronic acids. The ratio of sugars in beechwood xylan is comparable to birchwood xylan, but they differ in their relative content of hexuronic acids (less than 3% found in beechwood xylan) and in their water solubility: beechwood xylan is approximately 95% insoluble in water. Oat spelt xylan is a mixture of a high percentage of xylose (84%) and some arabinose, glucose and galactose, its water solubility varies greatly depending on temperature and extent of centrifugation. The family-11A enzymes were more effective in the degradation of xylans than the family-10B enzymes. On each substrate the enzymatic activities of the wild-type enzyme and its derivatives were very close, suggesting that the addition of the dockerin module had very little effect on the structural conformation of the enzymes.
Table 1 below summarizes the specific activity values obtained for all the tested chimeras and wild-type constructs. Oat spelt xylan was the most efficiently degraded substrate for all the enzymes. For the 11A enzymes, 11A-a had similar specific activity on all three substrates, which was lower than those of the XBM-containing enzymes, i.e., Xyn11A and 11A-XBM-a (which had comparable specific activities). Deletion of the XBM module may have a negative impact on its activity on these substrates, suggesting that the XBM-targeting role is important even for easily degraded substrates like purified xylan. Interestingly, the specific activities observed for 10B-t were higher than those of wild-type Xyn10B on all substrates; so the addition of the dockerin module may have allowed better access of the catalytic module towards its substrate. Xyn10B exhibited the lowest activity on beechwood xylan compared to the other substrates. The solubility properties of the xylan may thus play a role in degradation by this enzyme: the more insoluble the substrate, the more difficult for the enzyme to degrade it. Enzymatic activities of Cel5A and f-5A on a variety of cellulosic substrates were reported earlier (Caspi et al. 2009 Appl. Environ. Microbiol. 75:7335-7342).







TABLE 1







Specific activities* of recombinant enzymes on various xylans












Substrate
Xyn10B
10B-t
Xyn11A
11A-a
11A-XBM-a















Birchwood xylan
89.9
136
430
337
425


Oat spelt xylan
125
152
449
342
433


Beechwood xylan
40.3
97.4
438
345
445





*Katal/mol enzyme






Example 5
Enzymatic Activity on Hatched Wheat Straw
Method
Hatched wheat straw (0.2-0.8 mm) provided by Valagro (Poitiers, France) was treated as described previously (Fierobe et al. 2005 J. Biol. Chem. 280:16325-16334; Tabka et al. 2006 Enzyme Microb. Technol. 39:897-902): the crude substrate was incubated in distilled water under mild stirring for 3 h at room temperature, vacuum filtered on 2.7-μm glass filter, resuspended in water, and incubated for 16 h under mild stirring at 4° C. The suspension was filtered and washed three times with water, and a sample was dried at 100° C. overnight for estimation of dry weight.
A typical assay mixture consisted of 100 μl of buffer (50 mM citrate buffer pH 6.0, 12 mM CaCl2, 2 mM EDTA) and hatched wheat straw 3.5 g/l. The concentration of the enzymes added was 0.2 μM (0.3 μM for xylanases only). For assays described in “A” and “B” below the reactions were incubated for 17 hours at 50° C. For assays described in “C” and “D” below the reaction mixtures were incubated at 50° C. After 1, 4, 7 and 20 hours of substrate degradation, the reactions were stopped by transferring the tubes to an ice-water bath, then after a centrifugation step (5 min at 14000 rpm), 100 μl of the supernatant were added to 150 μl DNS reagent and the tubes were boiled for 10 min, finally absorbance was measured at 540 nm (Miller et al, 1959). Dockerin-containing enzymes were subjected to 1.5-h incubation (37° C., in the absence of substrate) in the presence of equimolar concentrations of scaffoldin, prior to assay for binding interaction.
All assays were performed in triplicate.
Results
A. Enzymatic Assays of Free Enzymes on Hatched Wheat Straw
Each of the dockerin-containing enzymes and their combinations were tested for activity on hatched wheat straw (FIG. 3). 10B-t and f-5A exhibited very weak activity on straw, whereas 11A-XBM-a showed higher levels of hydrolysis. Synergism was demonstrated for the following combinations: 10B-t+11A-XBM-a, 11A-XBM-a+f-5A and the three enzymes together (f-5A+11A-XBM-a+10B-t), with respective activity enhancements of 1.8, 3.1 and 4.6 fold (compared to the theoretical total of the individual activities). Notably, synergism was observed within all the combinations containing 11A-XBM-a, suggesting that 11A-XBM-a attacks the straw substrate in such a manner that it allows the other enzymes access to their specific sites on the complex substrate. Xyn10B proved to be less active than Xyn11A during initial degradation of the different xylans, but the enzyme seemed to contribute to the complete conversion of xylan into xylobiose, as reported earlier (Kim et al., 2004). The same synergistic action may also occur in the degradation of a more complex substrate, like straw, which could involve divergent cleaving mechanisms and would explain why Xyn10B (or its chimeric derivative, 10B-t) cannot achieve substantial levels of degradation by itself, but contributes to the reaction when combined with 11A-XBM-a. This hypothesis could explain the synergistic activities observed for the combinations of enzymes, i.e., the two-enzyme system 11A-XBM-a+f-5A and the three-enzyme mixture f-5A+11A-XBM-a+10B-t. Interestingly, the combination of 10B-t+f-5A reaction did not show improved activity. Xyn10B and Cel5A (or their chimeric derivatives) may have difficulty in accessing their target substrate within the complex matrix of the straw composite, and require the association with an additional enzyme such as Xyn11A to degrade straw efficiently. The same experiment was carried out with the wild-types enzymes and equivalent results were obtained (data not shown), thus indicating that the presence of dockerins in the enzymes do not substantially affect the overall action of these enzymes on the crude substrate. Additional trials were carried out in subsequent experiments only with the enzyme combinations that showed clear synergistic activity. Several concentrations of the enzyme combinations were tested in order to ensure that 0.3 mM enzyme provide linear reaction for the given time points (data not shown). Previous results were confirmed: as the three-enzyme system Cel5A+Xyn11A+Xyn10B (or f-5A+11A-XBM-a+10B-t) appeared to be more effective than the enzyme pair, Cel5A+Xyn11A (or f-5A+11A-XBM-a), which was more effective than Xyn11A+Xyn10B (11A-XBM-a+10B-t). The same trend was evident for the entire range of experimental data.
Kinetics studies also proved that the reaction is still in the linear part of the curve after 16-18 hours of enzymatic action (data not shown).
B. Enzymatic assays of tri-functional designer cellulosomes on hatched Wheat Straw
The combination of cellulase Cel5A with xylanase Xyn11A was examined in three different modes: (i) the free enzyme mixture, (ii) the designer cellulosome and (iii) the individually targeted enzyme system. Designer cellulosomes were constructed by mixing the desired dockerin-containing enzymes with the chimeric scaffoldins bearing the appropriate matching cohesins. The targeting effect was assessed by attaching the individual enzymes to a single-cohesin (CBM-containing) scaffoldin construct. The specific activities of the enzyme mixtures in each of the three modes were compared, and the results are shown in FIG. 4. As free enzymes (cellulase Cel5A and xylanase Xyn11A), the wild-type enzymes appeared to have a better specific activity than the dockerin-bearing enzymes—both f-5A+11A-a and f-5A+11A-XBM-a (FIG. 4A). This can be explained by the cellulose-binding CBM2 in Cel5A, which targets the cellulase to the cellulose substrate, leading to more efficient degradation. The combination of f-5A+11A-XBM-a is also more active than f-5A+11A-a, suggesting that the lack of XBM has a negative influence on the capacity of the enzymes to degrade straw. The addition of the matching single cohesin-bearing CBM to the dockerin-containing enzymes, improved their specific activity, by returning the cellulose-targeting feature to the enzyme. In fact, in the case of the f-5A+11A-XBM-a combination, the wild-type enzyme activity was almost fully recovered by restoration of the targeting function. In both cases, incorporation of the enzymes into designer cellulosomes served to increase the activity substantially. The resultant enhancement in enzyme activity can be attributed to the proximity effect between the enzymes in the designer cellulosome. Although f-5A+11A-a was still less efficient than the wild-type enzymes, the combination of f-5A+11A-XBM-a gave a 1.5-fold enhancement compared to the wild-type T. fusca enzymes, thus demonstrating the impact of assembling the enzymes together into a designer cellulosome complex. This also suggests that the XBM in the xylanase 11A provides a major contribution to the overall degradation of the complex cellulosic substrate.
Similar results were also obtained for the combination of the two xylanases as well as for the three-enzyme system (FIGS. 4B and 4C). In the free state, wild-type enzymes always degraded straw more efficiently than the dockerin-bearing chimeras, due to the fact that in most cases the dockerin replaces the CBM, which has an important influence (substrate targeting) on the activity. Reactions involving 11A-a showed very weak activities, demonstrating once again the crucial role of the XBM.
Connecting each enzyme to its matching single-cohesin scaffoldin improved activity in each case, confirming the CBM effect. Furthermore, as observed in the combined Xyn11A+Cel5A interaction, placing the enzymes in close proximity via the scaffoldin unit had a significant positive impact on the specific activity. Likewise, complexation of the xylanases (11A-XBM-a+10B-t) and the three-enzyme system (11A-XBM-a+10B-t+f-5A) provided activity enhancements of approximately 1.6 and 1.5 fold, respectively, relative to the wild-type enzymes. Nevertheless, complexes including 11A-a showed markedly reduced levels of activity compared to that of the wild-type enzymes.
Interestingly, parallel experiments using soluble and insoluble xylan as substrates showed no apparent difference between free or complexed enzymes (data not shown), thus reinforcing the theory that designer cellulosomes are advantageous on complex substrates relative to free enzymes.
C. Enzymatic Assays of Quadri-Functional Designer Cellulosomes on Hatched Wheat Straw
The activity of the quadri-functional complex on straw is shown in FIG. 5. Control kinetics with the corresponding free enzymes systems (wild-type enzymes or cellulosomal-like enzymes) and the corresponding four single enzyme attached to the cohesin-CBM were performed. Compared with the designer cellulosome, the various samples were found to be less active on straw, indeed the close proximity within the four types of enzymes induced a drastic increase in the enzymatic activity of the complex, especially after 4 hours of degradation. Organizing the enzymes into designer cellulosomes is, in that case more than a success. As observed in FIG. 5, the various samples were found to be less active on wheat straw in comparison with the designer cellulosome complex, which exhibited a  ̃2.4 fold enhancement compared to the other enzyme mixtures after 20 h of degradation. No significant difference was observed between straw degradation by single-cohesin scaffoldin bearing enzymes, the chimeric enzymes and the wild-types enzymes after 7 hours of degradation.
D. Evaluation of the Proximity Effect Between Cellulases and Xylanases
The two described chimeric xylanases 10B-t and 11A-a were incorporated into an appropriate scaffoldin and the two chimeric cellulases, f-5A and b-48A were integrated in a second scaffoldin. Using this approach, it was possible to estimate the differences in substrate degradation between the mixture of the two scaffoldins (cellulase- or xylanase-containing) and a third scaffoldin bearing the four enzymes (FIG. 6). In this manner, the proximity effect between cellulases and xylanases was evaluated by comparing the hydrolysis rates of each reaction. Interestingly, a significant improvement in enzymatic activity was demonstrated in the case of the four-enzyme scaffoldin compared to the mixture of the two distinct scaffoldins ( ̃2 fold enhancement). At a lower level, the mixture of the two distinct designer cellulosome systems also provided an enhanced rate of degradation compared to the wild-type enzymes ( ̃1.5 fold enhancement).
Example 6
Sugar Identification and Analysis
Methods
Analysis of sugar content was performed using a high-performance anion exchange chromatography (HPAEC) system equipped with a PA1 column (Dionex, Sunnyvale, Calif., USA). Reaction mixtures were loaded onto the column and eluted with NaOH (200 mM). Sugar concentrations were determined by integration of the chromatographic peaks, based on arabinose, xylose, xylobiose, xylotriose and cellobiose standards. Low levels of arabinose and xylose were observed in blanks (double-distilled water); these values were deducted in all the samples.
Xylose concentrations were confirmed by a D-xylose assay kit purchased from Megazyme (Wicklow, Ireland); glucose (absence thereof) was determined using a Glucose Assay Kit GAGO20 (Sigma-Aldrich); in both cases, according to the manufacturer's instructions.
Results
Sugar concentration and identification was performed using known concentrations of standards, and the relative amounts were calculated via integration of the identified peaks in the given samples. Combinations of free and scaffoldin-borne enzymes were applied to samples of hatched wheat straw, and the degradation products analyzed. Various quantities of arabinose, cellobiose, xylose, xylobiose and xylotriose were found in the samples (Tables 2 and 3). In accordance with the findings described in FIG. 3, Xyn10B and Cel5A were essentially inactive on the wheat straw substrate. In contrast, Xyn11A alone produced significant amounts of xylotriose, xylobiose and xylose, as well as arabinose but not cellobiose or glucose (Table 2), indicating its specificity for xylan. Many xylanases exhibit residual activity towards L-arabinose, due to the structural similarities between a-L-arabinofuranoside and b-D-xylopyranoside. The addition of Xyn10B or Cel5A appeared to drive the interaction more to completion, as no xylotriose was evident in these samples. The presence of Cel5A in the reaction mixtures resulted in significant quantities of cellobiose production, which was absent in samples lacking the cellulase. Incorporation of the dockerin-containing enzyme derivatives into chimeric scaffoldins served to enhance the levels of disaccharides and arabinose at the expense of xylose.
As seen on Table 3, incorporation of the dockerin-containing enzyme derivatives into two chimeric scaffoldins served to enhance the levels of cellobiose and xylotriose at the expense of xylobiose and arabinose relative to wild-type enzymes degradation. However, the levels of all sugars were higher in the quadri-functional designer cellulosomes in accordance with reducing sugars levels described in FIG. 6.







TABLE 2







Sugar concentration (in mmoles/g substrate), obtained by


HPLC analysis following digestion of hatched wheat straw for


17 hours by various enzyme combinations.a












Enzyme







combination
Arabinose
Xylose
Xylobiose
Cellobiose
Xylotriose





Xyn11A
20.3 ± 0.4
49.3 ± 0.5
 7.9 ± 0.9
n.d.
41.7 ± 1.2


Xyn10B
n.d.b
n.d.
n.d.
n.d.
n.d.


Cel5A
n.d.
n.d.
n.d.
n.d.
n.d.


Xyn11A +
23.6 ± 0.5
42.7 ± 0.0
24.6 ± 1.2
n.d.
n.d.


Xyn10B


Scaf(11A-
26.7 ± 0.2
34.7 ± 0.2
33.7 ± 0.2
n.d.
n.d.


XBM-a +


10B-t)


Xyn11A +
21.5 ± 0.4
41.5 ± 0.3
24.4 ± 1.1
20.6 ± 1.3
n.d.


Cel5A


Scaf(11A-
24.2 ± 0.0
  20 ± 0.5
32.4 ± 2.0
22.2 ± 0.5
n.d.


XBM-a +


f-5A)


Xyn11A +
24.3 ± 0.2
  48 ± 0.0
20.2 ± 1.7
21.8 ± 1.4
n.d.


Xyn10B +


Cel5A


Scaf(11A-
  31 ± 0.3
33.3 ± 0.1
24.8 ± 1.3
27.3 ± 0.7
n.d.


XBM-a +


10B-t +


f-5A)





aAbsence of glucose was confirmed by using a glucose assay kit. Values for xylose were corroborated using a xylose assay kit. An unidentified peak, present only after enzymatic treatments, eluted at ~3.9 min (between the xylose and xylobiose peaks), suggesting a monosaccharide, more likely a modified monosaccharide.


bn.d., not detected.


cScaf indicates that the designated chimeric enzymes are complexed to the scaffoldin













TABLE 3







Sugar concentration (in mmoles/g substrate), obtained by HPLC analysis


following digestion of hatched wheat straw for 17 hours by various enzyme


combinations.a












Enzyme







combination
Arabinose
Xylose
Xylobiose
Cellobiose
Xylotriose





Xyn11A +
45.7 ± 0.4
45.7 ± 1.7
68.6 ± 2.1
137.1 ± 3.9
10.9 ± 1  


Xyn10B +


Cel5A + Cel48A


Scaf (11A-XBM-
42.8 ± 3  
34.2 ± 2  
45.7 ± 5.5
168.6 ± 5.6
27.1 ± 1.8


a + 10B-t) +


Scaf (f-5A + b-


48A)


Scaf (11A-XBM-
45.7 ± 1.1
51.4 ± 0.8
111.4 ± 2.6 
  180 ± 2.8
30.6 ± 1.5


a + 10B-t + f-5A + b-


48A)





aAbsence of glucose was confirmed by using a glucose assay kit. Values for xylose were corroborated using a xylose assay kit. An unidentified peak, present only after enzymatic treatments, eluted at ~3.9 min (between the xylose and xylobiose peaks), suggesting a monosaccharide, more likely a modified monosaccharide.


bScaf indicates that the designated chimeric enzymes are complexed to the scaffoldin






Yield Calculations:
Fierobe and colleagues (Fierobe et al. 2005 J. Biol. Chem. 280:16325-16334) analyzed the wheat straw composition after sulfuric acid treatment. The washed straw was found to contain 3.3 mmol of acid-extractable reducing sugars/g of dry matter using the Park and Johnson method (Park et al. 1949 J. Biol. Chem. 181:149-151). Quantification of glucose by high performance liquid chromatography analysis and the glucose oxidase method indicated that the substrate contains approximately 40% cellulose (2.3 mmol of glucose/g of dry matter). The content of xylose was found to be 0.8 mmol/g of dry matter, whereas the amount of arabinose was around 0.1 mmol/g. Accordingly, reaction yields after 17 h comprised about 8.2% and 9.6% for the bi- and tri-enzyme designer cellulosome system, respectively (versus the corresponding yields, 4.9% and 6.3%, of the wild-type enzymes). Accordingly, reaction yield after 20 h comprised about 10.3% for the quadri-functional designer cellulosome system versus the corresponding yield 4.2% for the wild-type enzymes). Since the straw substrate was only physically pretreated, this yield corresponds to a high-quality hydrolysis.
Example 7
Disposition of the XBM
In order to examine whether the importance of the XBM resides in the structural conformation of Xyn11A or reflects the mere presence of that particular CBM in the complex, a scaffoldin was designed that would include XBM together with a dockerin. The dockerin of R. flavefaciens was attached to the N-terminal end of the XBM from Xyn11A, in order to effect its physical separation from the catalytic module. Three complexes were tested for examination of the extrinsic contribution of the XBM to straw degradation (FIG. 7): (i) 11A-a+10B-t, as a negative control for an XBM-lacking system, (ii) 11A-XBM-a+10B-t as a positive control for XBM-containing system, and (iii) the designer cellulosome (11A-a+10B-t+f-XBM). The results clearly demonstrate that the structural conformation of 11A-XBM-a is responsible for the enhancement of activity and synergism between the two enzymes. Thus, function of the XBM is dependent on its presence in the native enzyme, since independent addition of the dockerin-fused XBM to higher-order designer cellulosomes had little or no effect on the activity on wheat straw. Both free systems and scaffoldin-bound designer cellulosome systems remained in the same range of efficiency as that of the 10B-t+11A-a system, which did not contain XBM.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed chemical structures and functions may take a variety of alternative forms without departing from the invention.

## Claims

1. A bio-engineered multi-enzyme complex comprising:
(i) a scaffold polypeptide comprising a plurality of cohesin domains of divergent specificities and at least one substrate-binding module; and
(ii) a plurality of carbohydrate active enzymes bound to said scaffold polypeptide, each carbohydrate active enzyme comprises a divergent dockerin domain matching one of the plurality of cohesin domains of the scaffold polypeptide;

wherein the plurality of carbohydrate active enzymes comprise at least two different xylanases, wherein at least one of the two different xylanases is a non-cellulosomal xylanase bio-engineered with a heterologous dockerin domain.

2. The bio-engineered multi-enzyme complex of claim 1, wherein the heterologous dockerin domain replaces at least one of the ancillary modules originally found in the enzyme structure; or wherein the heterologous dockerin domain is introduced in addition to the original ancillary modules.

3. The bio-engineered multi-enzyme complex of claim 1, wherein the dockerin domain is selected from the group consisting of a dockerin derived from Clostridium thermocellum, a dockerin derived from Acetivibrio cellulolyticus, a dockerin derived from Ruminococcus flavefaciens, a dockerin derived from Bacteroides cellulosolvens, a dockerin derived from Archaeoglobus fulgidus and a dockerin derived from Clostridium cellulolyticum.

4. The bio-engineered multi-enzyme complex of claim 1, wherein the non-cellulosomal xylanase is a Thermobifida fusca xylanase.

5. The bio-engineered multi-enzyme complex of claim 1, wherein the plurality of carbohydrate active enzymes further comprises at least one carbohydrate active enzyme selected from the group consisting of a glycoside hydrolase, polysaccharide lyase and carbohydrate esterase, wherein the at least one carbohydrate active enzyme is other than a xylanase.

6. The bio-engineered multi-enzyme complex of claim 5, wherein the at least one carbohydrate active enzyme is a cellulase classified in a glycoside hydrolase family selected from the group consisting of: family 5, 6, 7, 8, 9, 12, 26, 44, 45, 48, 51, 61, and 74.

7. The bio-engineered multi-enzyme complex of claim 6, wherein at least one of the cellulases present in the complex is a T. fusca cellulase.

8. The bio-engineered multi-enzyme complex of claim 1, wherein the complex is selected from the group consisting of: tri-functional and quadri-functional.

9. A composition for degrading biomass comprising the multi-enzyme complex of claim 1.

10. A genetically modified host cell comprising polynucleotides encoding a plurality of components of the bio-engineered multi-enzyme complex of claim 1, wherein the plurality of components comprise at least two different xylanases, wherein at least one of the two different xylanases is a non-cellulosomal xylanase bio-engineered with a heterologous dockerin domain.

11. A method for bioconversion of cellulosic material into degradation products, the method comprising exposing said cellulosic material to the multi-enzyme complex of claim 1.

12. A system for bioconversion of cellulosic material, the system comprising a multi-enzyme complex of claim 1.

13. The bio-engineered multi-enzyme complex of claim 1, wherein the at least two different xylanases are non-cellulosomal xylanases, each bio-engineered with a heterologous dockerin domain.

14. The bio-engineered multi-enzyme complex of claim 13, wherein the two non-cellulosomal xylanases are T. fusca xylanases.