ES2697920B2 - CELLULAS WITH IMPROVED CELLULOLIC ACTIVITY - Google Patents

Description

DESCRIPTION

Cellulases with improved cellulolytic activity

The present invention belongs to the field of enzymes useful for the production of bioproducts, preferably biofuels, and more particularly refers to variants of cellulases comprising linkers resistant to proteolysis and having an improved cellulolytic activity with respect to native cellulases comprising native linkers . Additionally, the invention also relates to the use of said cellulase variants in the production of fermentable sugars and bioproducts from cellulosic material.

STATE OF THE ART

Biofuels are an attractive alternative to fossil fuels and can be obtained through the fermentation of monomeric sugars derived from starch or cellulose and hemicellulose.

Vegetable biomass provides a complete source of potential energy in the form of sugars that can be used for numerous industrial and agricultural processes, and is therefore a significant renewable resource for the generation of fermentable sugars that can result in commercially valuable end products, such like biofuels. However, the enormous potential energy of these carbohydrates is currently underutilized because the sugars are part of complex polymers that are not easily accessible for fermentation.

Any vegetable biomass can be considered as raw material for the production of biofuels, so you can use herbaceous crops, other agricultural remains or even urban solid waste. These materials mainly comprise cellulose and hemicellulose. Once cellulose and hemicellulose are converted into glucose and xylose, respectively by means of an enzymatic hydrolysis process, these compounds are easily fermented by other organisms to ethanol. In this way, how much more complex sugars remain at the end of the hydrolytic process, the lower the yield of ethanol production at the end of the fermentation process. Therefore, an area of research aimed at reducing costs and enhancing the performance of biofuel production processes is focused on improving the efficiency of cellulolytic enzymes, as well as the enzymatic compositions comprising these enzymes that can be used to generate fermentable sugars from biomass .

Due to the complexity of the biomass, its conversion into monomeric sugars involves the action of various types of enzymes with diverse enzymatic activities, which digest cellulose, hemicellulose, as well as other complex polymers present in the biomass. After cellulose, hemicellulose is the second most abundant fraction available in nature. Both cellulose and hemicellulose can be pretreated, mechanically, chemically, enzymatically or in other ways, to increase their susceptibility to hydrolysis. After this pretreatment process a saccharification stage takes place, which is an enzymatic process by which the complex carbohydrates are degraded into their monosaccharide components. The objective of any saccharification technology is to alter or eliminate the structural and compositional impediments to hydrolysis in order to improve the enzymatic hydrolysis rate and increase the yields of fermentable sugars from the biomass, which mainly comprises cellulose and hemicellulose (N. Mosier et al., 2005, Bioresource Technology, 96: 673-686). After this saccharification stage, a fermentation process is carried out.

Cellulolytic enzymes have become biocatalysts due to their complex nature and extensive industrial applications. Nowadays considerable attention is paid to the production of cellulases and the advances in research, especially in the direction of improving the economy of the process of several industries, in order to obtain enzymatic compositions that present a greater activity and better cellulolytic properties.

Individual enzymes have been shown to only partially digest cellulose and hemicellulose and, therefore, the concerted action of all or at least several of the enzymes called "cellulases or cellulolytic enzymes" is needed to complete the conversion of the different complex polymers, specifically , cellulose and hemicellulose, to monomeric sugars Cellulases (1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4) comprise at least three enzymatic activities, endo-beta-glucanases (EC 3.2.1.4), exo-beta-glucanases or cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21), of which their synergistic action in the hydrolysis of cellulose (Woodward, J. 1991, Bioresource Technology Vol 36, pages 67-75). In addition to these three activities nowadays, others of equal importance are recognized, such as xylanases (EC 3.2.1.8), betaxilosidases (EC 3.2.1.37) and polysaccharide mono-oxygenates (family AA9).

The hydrolytic efficiency of a multienzyme complex, formed by a wide variety of cellulolytic enzymes, in the process of cellulose saccharification depends both on the properties of the individual enzymes and on the relationship of each enzyme with respect to the rest, in the complex.

Most cellulases exhibit a multimodular organization comprising at least three modules or domains: a cellulose binding (CBD carbohydrate binding domain, its acronym) and a catalytically active domain (CAD, catalytically active domain , in its acronym in English), both domains being joined by a region of linker or linker highly glycosylated and rich in proline and residues of hydroxyaminoacids (serines and threonines) (Fagerstam et al., 1984, FEBS lett.167: 309-315) ).

It has been described that the glycosylation of the residues of the linker region protects this region from proteolysis, increasing the ratio of enzymes that preserve their CBD (Langsford et al., 1987, FEBS lett.225: 163-167). The sequences of the CBDs of different cellulolytic enzymes are very conserved, on the contrary, the sequences of the linkers apart from not being conserved, present a characteristic and different glycosylation pattern, which confers them particular characteristics. The CBD domain of the cellulases serves as an anchor and the linker region provides the necessary flexibility for the CAD domain to reach the degradation sites. The modular organization of the cellulases is important to increase the synergy between the CAD and CBD domains with their natural substrate and, consequently, to provide cellulose enzymes with greater cellulolytic capacity (Srisodsuk et al., 1993, J.Biol.Chem. 268: 20756-20761). Precisely, most of the efforts in the present technical field have been directed to the generation of cellulases that present a greater cellulolytic capacity and therefore, improve the yield and efficiency of the process of degradation of biomass. In this sense, cellulases with better hydrolytic activity have been designed due to the modification of the modular regions, both of the CBD and CAD regions, and of the linkers that link them. The international patent application WO94 / 07998 describes variants of a cellulase classified in family 45, which comprises a CBD, a catalytically active domain (CAD) and a region connecting the CBD with the CAD (linker), where one or more amino acid residues and / or another CBD have been added, deleted or substituted. has been added at the other end of the CAD. On the other hand, the international patent application WO95 / 16782 refers to the cloning and the high level expression of new truncated cellulase proteins or derivatives thereof in Trichoderma longibrachiatum comprising different catalytic regions with several CBDs. On the other hand, patent US8637293B2 describes variants of cellulase Cbh1 comprising mutations in the catalytic domain and / or an increase in the O-glycosylation region in the binding domains or linkers.

It would, therefore, be useful to have cellulases with improved cellulolytic activity, able to produce fermentable sugars more efficiently, thus improving the overall hydrolytic performance of the enzymatic mixtures containing them.

DESCRIPTION OF THE INVENTION

The present invention describes polynucleotide sequences that code for regions or linker modules that are more resistant to proteolysis than native or wild-type linkers . In general, cellulases, during their production by the cells that synthesize them, can undergo proteolytic processes, preferably in the area of the linker, which separate the carbohydrate and catalytic binding motifs, thus preventing synergy between them, drastically decreasing the cellulolytic activity of each cellulase on its natural substrate, and therefore, reducing the yield and efficiency of the biomass degradation process.

In this sense, the invention describes variants of cellulases to which their native linker has been exchanged for one of the linkers described here, obtaining variants of cellulases comprising linker more resistant to proteolysis than native or wild-type linkers , and which, for therefore, said cellulase variants have a greater cellulolytic activity on their own substrate. Additionally, the use of said variants for hydrolysis of cellulosic material in fermentable sugars is also described, as well as a process for producing fermentable sugars and a process for producing bioproducts, such as ethanol, in which said variants are employed.

Therefore, the present invention represents a solution to the need to provide cellulases with improved cellulolytic activity, useful for the optimization of the step of hydrolysis of cellulosic material in fermentable sugars, thanks to which they comprise a linker more resistant to proteolysis, with respect to the native linker that presents a parental cellulase (native).

The inventors have demonstrated the highest resistance to hydrolysis of the variants of the cellulases comprising the linkers described herein with respect to native cellulases, significantly increasing the yield of the hydrolysis step by using said variants or the enzymatic compositions comprising them. , obtaining a greater amount of monosaccharide sugars released at the end of said hydrolytic stage (mainly glucose), which leads to an increase in the production of the bioproduct, preferably ethanol.

As shown in the examples described below, the cellulase variants comprising the linkers of the present invention were expressed in a host fungal cell and the enzyme mixture produced by the resulting strain was evaluated in saccharification experiments of pretreated biomass (PCS). , demonstrating an increase in the yield of the saccharification process, specifically an increase in the concentration of fermentable sugars (glucose) released at the end of the process, in comparison with the same enzyme mixture produced by the control strain that does not express or secrete cellulases comprising the linkers of the invention.

Therefore, a first aspect of the present invention relates to sequences more resistant to proteolysis linkers, regarding linker native or wild, comprising an amino acid sequence having a sequence identity of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with any of the sequences included in the following list: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 SEQ ID NO: 34 and SEQ ID NO: 36.

In a preferred embodiment, the linkers of the invention have a sequence identity of 100% with the sequences selected from the following list: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 SEQ ID NO: 34 and SEQ ID NO: 36

The term "identity" refers to the ratio of nucleic acid or amino acid residues that are identical between two nucleic acid or amino acid sequences being compared.The degree of identity can be determined by the Clustal method, the method of Wilbur-Lipman, the GAG program, which includes GAP, BLAST or BLASTN, EMBOSS Needle and FASTA, In addition, the Smith Waterman algorithm can be used to determine the degree of identity between two sequences.

In the context of the present invention, the term "carbohydrate binding domain" (CBD) refers to the polynucleotide sequence encoding the amino acid sequence capable of carrying out the binding between the cellulolytic enzyme comprising said CBD and its cellulosic substrate. Specifically, said domain is intended to be understood as defined by Peter Tomme et al. "Cellulose-Binding Domains: Classification and Properties" in "Enzymatic Degradation of Insoluble Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1996. This definition classifies more than 120 CBDs in 10 families (IX), showing that CBDs are found in different groups of cellulolytic enzymes, such as endoglucanases, xylanases, mannanases, arabinofuranosidases, acetyl esterases and chitinases.

For the purposes of the present invention, the term "cellulose" refers to a linear polysaccharide comprising hundreds to thousands of D-glucose units linked by beta- (1,4) bonds.This polysaccharide is also known as beta- (1). , 4) glucan.

In the context of the present invention, the term "catalytically active domain" (CAD) refers to the region or sequence of polynucleotides that encodes the amino acid sequence responsible for catalyzing the degradation or hydrolysis of cellulose.

In the context of the present invention, the term "binding domain" or "binding region" or " linker1 ' or " linker region ", used interchangeably throughout the present document refers to the polynucleotide sequence encoding the amino acid sequence that serves as a link between the CBD and CAD domains present in a cellulase. The linkers play an important role at the structural and functional level, either to unite both regions, either to maintain a specific distance from one region with respect to the other and thus allow the cellulolytic activity of the cellulase on its substrate, etc. The linkers also comprise sites for proteolytic cleavage. Thus, the native linker regions present in the native cellulases, during the process of obtaining the variants of the cellulases and / or during the storage phase (between their synthesis and their use) and / or during the enzymatic hydrolysis process, can suffer a proteolytic break, which causes a dissociation between the CAD domain and the CBD domain of the cellulase. The fact that a cellulase loses the CBD domain prevents an efficient recognition and binding thereof to cellulose, which leads to a loss or decrease in the performance of the enzymatic hydrolysis process.

For the purposes of the present invention the term " native linker " or " wild linker " refers to the polynucleotide sequence encoding the amino acid sequence that serves as a link between the CBD and CAD domains present in a native, unmodified cellulase. For the purposes of the present invention, the linkers described herein are linker more resistant to proteolysis than native or wild-type linkers .

For the purposes of the present invention, the terms "processing", "proteolysis", "cellulase processing", or "cellulase proteolysis" can be used interchangeably throughout the present invention and refers to chain breakage peptide of a cellulase by the specific or non-specific proteolytic activity and that gives rise to a modified cellulase that can have a lower cellulolytic activity as a consequence of which during said processing they lose some fragment or domain of their structure responsible for said cellulolytic activity. For the purposes of the present invention, the terms "processing", "proteolysis", "cellulase processing", or "cellulase proteolysis", preferably refer to loss or breakage of the domain or binding region between the CBD domain and the CAD domain. of a cellulase.

For the purposes of the present invention, the terms "more resistant to proteolysis" or "more resistant to processing", both in relation to the linker described here, and to the cellulases themselves comprising said linker, refer to those linker or cellulases that have a minor or even no, proteolytic processing during their synthesis, storage and / or secretion, giving rise to cellulases that show a greater cellulolytic activity against cellulases that undergo cellulolytic processing and as a result of this decreases the synergy between the domains CAD and CBD, preventing the CAD domain from reaching the target sites of cellulolytic degradation. For the purposes of the present invention, the linkers and cellulases described herein show at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, less proteolytic processing than the linker or parental or wild cellulases.

In a preferred embodiment of the present invention, the linker is selected from any of the following: SEQ ID NO: 10, 26 or 30. In a more preferred embodiment even the preferred linker is the linker of SEQ ID NO: 26.

In Table 1 shown below, the nucleotide and peptide sequences of the linkers described in the present invention are shown to have a greater resistance to proteolysis than native or wild-type linkers .

The term "cellulolytic enzyme" or "cellulase", used interchangeably throughout the present invention, refers to a category of enzymes that can degrade complex polymers, such as cellulose and / or hemicellulose (P-1,4-glucan or beta-D-glucosidic bonds) to shorter oligosaccharides, such as, cellobiose and / or glucose and xylobiose and / or xylose, respectively. Within said category of enzymes are preferably found the following groups: 1,4-beta-D-glucan glucanohydrolase ("endoglucanase" or "EG"); 1,4-beta-D-glucan cellobiohydrolase ("exoglucanase", "cellobiohydrolase", or "CBH"), beta-D-glucosidoglucohydrolase ("betaglucosidase", "cellobiase" or "BGL"), endoxylanases or xylanases ("Xyl" "), Beta-xylosidases (" beta-Xyl ") and polysaccharide mono-oxygenates (" PMO ").

The endoglucanases or EGs, break internal bonds and alter the crystalline structure of cellulose, exposing individual cellulose polysaccharide chains ("glucans") .This term refers to a group of cellulase enzymes classified as EC 3.2.1.4. hydrolyze the internal p-1,4 glycosidic bonds of cellulose.

The cellobiohydrolases or CBHs, gradually shorten the glucan molecules, mainly releasing units of cellobiose (a glucose dimer linked in p-1,4 soluble in water), in addition to glucose, celotriose and celotetraose. The term "cellobiohydrolase", as used herein, refers to a protein of class EC 3.2.1.91, which catalyzes the hydrolysis of polymeric cellulose to cellobiose by an exoglucanase activity, sequentially releasing cellobiose molecules from the reducing or non-reducing ends of cellulose.

Beta-glucosidases or BGL, divide cellobiose into glucose monomers. The term "beta-glucosidase", as used herein, refers to a group of cellulase enzymes classified as EC 3.2.1.21. These enzymes catalyze the hydrolysis of sugar oligomers, including, but not limited to, the glucose or cellobiose dimer, with the release of a corresponding sugar monomer, used, but not limited, for the synthesis of ethanol. The enzyme beta-glucosidase acts on the beta- (1,4) bonds that bind to two molecules of glucose or substituted glucose (ie, the disaccharide cellobiose). It is an exocellulase with specificity for a variety of beta-D-glucoside substrates. It catalyzes the hydrolysis of non-reducing terminal residues in beta-D-glucosides with glucose release.

Xylanases or Xyl, catalyze the random hydrolysis of polymeric xylan, polymeric pectin or hemicellulose containing xylose residues which results in the formation of sugar oligomers containing xylose and / or monomeric xylose residues. As used herein, xylanases refer to a group of enzymes (EC 3.2.1.8) that catalyze the endohydrolysis of 1,4-beta-D-xyloside bonds in xylans. This enzyme can also be referred to as endo-1,4-beta-xylanase or 1,4-beta-D-xylan xylanhydrolase.

Beta-xylosidases are enzymes with 4-beta-D-xylan xylohydrolase activity catalyzing the reaction from the xylose oligomers, including xylobiose, finally releasing D-xylose. As used herein, betaxilosidases refers to a group of enzymes (EC 3.2.1.37) which catalyzes the hydrolysis of 1,4-beta-D-xylans, to eliminate successive D-xylose residues from the non-reducing ends. This enzyme can also be referred to as xylan 1,4-beta-xylosidase, 1,4-beta-D-xylan xylohydrolase, exo-1,4-beta-xylosidase or xylobiase.

PMOs are metalloproteins with endocellulolytic activity that act with a mechanism different from that performed by endoglucanases, since they break the cellulose chains by oxidation of their glucose monomers in carbons 1, 4 and / or 6. The terms "polysaccharide monooxygenase" , "PMO", "Glycosyl hydrolase of the family 61" or "GH61" or "AA9", all of them used to denominate cellulases of PMO type, refer to a group of enzymes, originally classified within the family of proteins GH61, since they exhibit GH61 or PMO activity, and that being included in a saccharification reaction results in a greater amount (higher yield) of one or more soluble sugars (eg, glucose) compared to the saccharification reaction carried to under the same conditions but in the absence of the GH61 protein.The members of this family of enzymes act as copper monooxygenases that catalyze the breakdown of cellulose chains by an oxidative mechanism at the level of several carbons (C1, C4 and / or C6), releasing cellodextrins (Langston et al Applied and Environmental Microbiology, 2011, 77: 7007-7015 ).

Another of the objects described in the present invention refers to a cellulase comprising at least one CBD, at least one CAD and at least one linker that confers a greater resistance to proteolysis as described in the present invention, with respect to the native or wild linkers of a native parental cellulase, unmodified.

For the purposes of the present invention, the terms "cellulase of the invention" or "cellulase variant of the invention" are used interchangeably.

The term "variant", as used herein, refers to an enzyme that is derived from a native enzyme by one or more deletions, insertions and / or substitutions of one or more amino acids and, therefore, has a sequence different from that of the native enzyme. As used herein, the term "cellulase variant" means a polypeptide having cellulolytic activity, preferably produced by an organism expressing a nucleotide sequence coding for a native cellulase that has been modified to encode said cellulase variant. Said modified nucleotide sequence is obtained by human intervention by modification of the nucleotide sequence encoding a native cellulase. The term "modification" means in this document any modification of the amino acid or nucleic acid sequence of a native cellulase.

The term "native cellulase" refers to a cellulolytic enzyme or its preprotein, expressed by a microorganism and comprising its unmodified natural sequence. Preferably, the native cellulase enzyme referred to in the present invention is expressed by a filamentous fungus, more preferably by a fungus belonging to the genus Myceliophthora, even more preferably by a fungus of the species Myceliophthora thermophila.

In a preferred embodiment the cellulase of the invention is selected from any of the following list: endoglucanases, beta-glucosidases, cellobiohydrolases, beta-xylosidases, xyloglucanases, polysaccharide monooxygenases, xylanases and arabinofuranosidases. In a more preferred embodiment, the cellulase of the invention is selected from a cellobiohydrolase or a polysaccharide monooxygenase.

In another preferred embodiment, the cellulase of the invention, as described herein, comprises any of the linkers selected from the following list: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 SEQ ID NO: 34 and SEQ ID NO: 36. In another preferred embodiment, the cellulase of the invention comprises at least one of the linker selected from any of the following: SEQ ID NO: 10, SEQ ID NO: 26 or SEQ ID NO: 30. In another more preferred embodiment, the cellulase of the invention as described herein comprises the linker of the sequences SEQ ID NO: 26 or SEQ ID NO: 30. In another even more preferred embodiment, the cellulase of the invention as described herein comprises the linker of the sequence SEQ ID NO: 26.

In another preferred embodiment, the cellulase of the invention is a CBH, preferably a Cbh1, comprising any of the linkers selected from the following list: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO : 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 SEQ ID NO: 34 and SEQ ID NO: 36. In another preferred embodiment, Cbh1 comprises at least one of the linkers selected from any of the following: SEQ ID NO: 10, SEQ ID NO: 26 or SEQ ID NO: 30. In another more preferred embodiment, the Cbh1 comprises the linker of SEQ ID NO: 26 or SEQ ID NO: 30. In an even more preferred embodiment, Cbh1 comprises the linker of SEQ ID NO: 26.

In another preferred embodiment, the cellulase of the invention is a CBH, preferably a Cbh1 comprising at least one of the linkers described in the present invention. In a still more preferred embodiment, the Cbh1 described in the present invention comprises a sequence of amino acids having a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or 100%, with the Cbh1 of SEQ ID NO: 38, and comprising at least one of the linkers described in the present invention.

In another more preferred embodiment, the cellulase of the invention is a PMO, preferably a PMO-06230, comprising any of the linkers selected from the following list: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 SEQ ID NO: 34 and SEQ ID NO: 36. In another preferred embodiment, the PMO-06230 comprises at least one of the linker selected from any of the following: SEQ ID NO: 10, SEQ ID NO: 26 or SEQ ID NO: 30. In another more preferred embodiment, the PMO-06230 comprises the linker of SEQ ID NO. : 26 or SEQ ID NO: 30. In an even more preferred embodiment, PMO-06230 comprises the linker of SEQ ID NO: 26.

In another preferred embodiment, the cellulase of the invention is a PMO, preferably a PMO-06230 comprising at least one of the linkers described in the present invention. In a still more preferred embodiment, the PMO-06230 described in the present invention comprises a sequence of amino acids having a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, with PMO-06230 of SEQ ID NO: 40, and comprising at least one of the linkers described herein invention.

In another more preferred embodiment, the cellulase with linker more resistant to proteolysis, with respect to the linker of the native cellulases, as described in the present invention are selected from any of those described in Table 2.

For purposes of the present invention, the term "isolated nucleic acid molecule", "nucleotide sequence", "nucleic acid sequence" or "polynucleotide", refers to a nucleic acid molecule (polynucleotide) that has been extracted from its natural environment (ie, it has been subjected to human manipulation) and may include DNA, RNA or DNA or RNA derivatives, including cDNA The nucleotide sequence of the present invention may or may not be chemically or biochemically modified, and can be obtained artificially by means of cloning, amplification and selection or synthesis procedures.The nucleic acid sequence of the invention can encode the mature polypeptide or a preprotein consisting of a signal peptide attached to the mature enzyme that will then have to be processed.

The nucleotide sequence of the present invention may also comprise other elements, such as introns, non-coding sequences at the 3 'and / or 5' ends, ribosome binding sites, etc. This nucleotide sequence may also include coding sequences for additional amino acids that are useful for the purification or stability of the encoded peptide.

When applied to a protein / polypeptide, the term "isolated" indicates that the protein is in a condition different from its native environment. In a preferred form, the isolated protein is substantially free of other proteins, originated by the cell. It is preferred to provide the protein in a form greater than 40% pure, more preferably in a form greater than 60% pure. Even more preferably it is preferred to provide the protein in a highly purified form, ie, more than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.

The term "isolated protein / polypeptide" can be alternatively referred to as "purified protein / polypeptide".

Another of the objects described in the present invention relates to an isolated nucleic acid sequence coding for linkers or for the cellulase comprising at least one of said linker as described in the present invention.

In a preferred embodiment, the nucleic acid sequences encoding the linkers of the invention are shown in Table 1. In the same manner, the nucleotide sequences that code for the cellulases of the invention that comprise at least one of the linker plus resistant to proteolysis than the native or wild-type linkers described herein, are shown in Table 2.

Table 2. native cellulases and cellulases variants resistant to proteolysis linker described herein.

The term "complementary nucleic acid sequence" of a nucleic acid sequence encoding the linkers or cellulases of the invention refers to the nucleic acid sequence of the complementary strand to which it codes for the linkers and cellulases of the invention. It will be appreciated that a double-stranded DNA encoding a given amino acid sequence comprises a single-stranded DNA and its complementary strand, which has a sequence that is complementary to the single-stranded DNA.

Another object described in the present invention thus relates to an isolated nucleic acid sequence complementary to the nucleic acid sequences of the linkers and cellulases described in the present invention.

The nucleic acid sequence of the invention can be included in a genetic construct, preferably in an expression vector. Said genetic construct may further comprise one or more gene expression regulatory sequences, such as promoters, terminators, etc. Therefore, in another aspect, the invention provides a genetic construct comprising the nucleic acid sequence of the invention or the nucleic acid sequence complementary thereto, hereinafter "gene construct of the invention". In a preferred embodiment, said gene construct is an expression vector.

The term "gene construct" or "nucleic acid construct" as used herein refers to a functional unit necessary for the transfer or expression of a nucleotide sequence or gene of interest, in this document, the nucleic acid sequence of the invention as described, and regulatory sequences, including, for example, a promoter, operably linked to the sequence encoding the protein. Therefore, for the purposes of the present invention, the gene construct refers to a double-stranded nucleic acid molecule, which is isolated from a natural nucleic acid or which is artificially modified to contain segments of nucleic acids. The expression "nucleic acid construct" is synonymous to the expression "expression cassette" when the nucleic acid construct contains the control sequences required for the expression of the coding sequence.

The term "expression vector", also known as "expression construct" or "plasmid", refers to a DNA molecule, linear or circular, which comprises the nucleic acid sequence of the invention and which is operatively linked to segments additional that allow the transcription of the encoded peptide. Generally, a plasmid is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cell transcription and translation machinery. The plasmid is often genetically engineered to contain regulatory sequences that act as enhancer and promoter regions and that lead to efficient transcription of the gene carried in the vector expression. The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA and, therefore, protein. Expression vectors are basic tools of biotechnology and the production of proteins, such as enzymes. The expression vector of the invention is introduced into a host cell such that the vector is maintained as a chromosomal integrator or as an extrachromosomal self-replicating vector.

The term "recombinant expressed" or "recombinantly expressed" used herein in relation to the expression of a polypeptide or protein is defined according to the standard definition of the art. The recombinant expression of a protein is generally carried out using an expression vector as described above.

Examples of expression vectors are plasmids, phages, cosmids, phagemids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), human artificial chromosomes (HAC) or viral vectors, such as adenovirus, retrovirus or lentivirus.

The gene constructs of the present invention encompass an expression vector, wherein the expression vector can be used to transform a suitable host or host cell so that the host can express the cellulase variants comprising the linkers described in the invention. Methods for the recombinant expression of proteins in fungi and other organisms are well known in the art and numerous expression vectors are available or can be constructed using routine procedures.

The term "control sequences" is defined herein to include all components that are necessary or advantageous for the expression of the nucleic acid sequence of the present invention. Said control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence and a transcription terminator. At a minimum, the control sequences include a promoter and transcription and translation termination signals. The control sequences can be provided with linkers in order to introduce specific restriction sites that facilitate the binding of the control sequences to the coding region of the nucleic acid sequence of the present invention. The term "operatively linked" indicates in the present document a configuration in which a control sequence is placed in a suitable position with respect to the nucleic acid sequence of the present invention, in such a way that the control sequence directs the expression of the nucleic acid sequence of the present invention.

The expression vector of the invention can be an autonomous replication vector, ie a vector that exists as an extrachromosomal entity, whose replication is independent of the replication of the chromosome, for example a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome . The vector can contain any means to guarantee self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) in which it has been integrated.

In addition, a single vector or plasmid can be used, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon.

The vectors used in the present invention preferably contain one or more selectable markers that allow easy selection of transformed, transfected, transduced or similar cells. A selectable marker is a gene product that provides resistance to a biocide or a virus, to heavy metals, prototrophy to auxotrophs and the like. Selectable markers for use in a host cell of a filamentous fungus include, but are not limited to, AmdS (acetamidase), ArgB (ornithine carbamoyltransferase), Bar (phosphinothricin acetyltransferase), Hph (hygromycin phosphotransferase), NiaD (nitrate reductase), PyrG ( orotidine-5'-phosphate decarboxylase), CysC (sulfate adenyltransferase), and TrpC (anthranilate synthase), as well as equivalents thereof.

The vectors used in the present invention preferably contain one or more elements that allow the integration of the vector into the genome of the host cell or the autonomous replication of the vector in the cell regardless of the genome. For integration into the host cell genome, the vector may depend on the nucleic acid sequence of the present invention or on any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional sequences of nucleotides to direct integration by homologous recombination in the genome of the host cell at one or more precise locations (s) on the chromosome (s).

For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator that participates in the autonomous replication that works in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that allows a plasmid or vector to replicate in vivo. Examples of useful origins of replication in a filamentous fungal cell are AMA1 and ANS1 (Verdoes et al., 2007, Ind. Biotechnol., 3: 48-57).

In the host cell, more than one copy of the nucleic acid sequence of the present invention can be inserted to increase the production of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including a selectable marker gene amplifiable with the polynucleotide, where the cells containing amplified copies of the marker gene selectable and, therefore, additional copies of the polynucleotide, can be selected by culturing the cells in the presence of the appropriate selectable agent. The methods used to ligate the elements described above to construct the recombinant expression vectors referred to in the present invention are well known to one skilled in the art.

In another aspect, the invention provides a host cell comprising the gene construct of the invention, hereinafter referred to as "host cell of the invention". Therefore, said host cell expresses the variant of the cellulase of the invention, comprising at least one linker more resistant to proteolysis than a native or wild-type linker . The "host cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction and the like with the gene construct of the invention. The host cell may be eukaryotic, such as a mammalian, insect, plant or fungal cell. In a preferred embodiment, the host cell is a filamentous fungal cell. Filamentous fungi are generally characterized by a micellar wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. In a more preferred embodiment, the filamentous fungal host cell is an Acremonium cell , Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a more preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori cell , Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae. In another more preferred embodiment, the filamentous fungal host cell is a Bjerkandera adusta cell , Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Gibberella zeae, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride. In another even more preferred embodiment, the host cell of the invention is any strain of the Myceliophthora thermophila species . In an even more preferred embodiment, the host cell of the invention is strain C1 of the species Myceliophthora thermophila.

It will be understood that, for the species mentioned above, the invention encompasses both perfect and imperfect states and other taxonomic equivalents, for example anamorphs, regardless of the name of the species by which they are known. Those skilled in the art will readily recognize the identity of suitable equivalents. For example, Myceliophthora thermophila is equivalent to Chrysosporium lucknowense .

The term "expression" includes any step involved in the production of the strongest linker to proteolysis than native or variant cellulases comprising the toughest linkers to proteolysis of the invention linker that cellulases with linker native, comprising , but is not limited to, transcription, posttranscriptional modification, translation, post-translational modification, and secretion, in the case of the variants of the cellulases of the invention.

In another preferred embodiment, the host cells of the invention are characterized in that they exhibit overexpression of at least one of the cellulase variants of the invention and / or of at least one homologous and / or heterologous cellulase, as described below. of the invention.

For purposes of the present invention, the terms "expression enhancement" or "overexpression" can be used interchangeably throughout the present document and refer to any form of expression that is additional or greater than the original level of expression in a parent cell or wild. For the purposes of the present invention, overexpression, in increasing order of preference is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, compared to expression in the parental or wild-type host cells. Methods for increasing the expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. The isolated nucleic acids can also serve as promoters or enhancers by being able to be introduced at an appropriate position in a non-heterologous form of a polynucleotide in order to up-regulate the expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters can be altered in vivo by mutation, deletion, and / or substitution, or isolated promoters can be introduced into a polynucleotide sequence that codes for a gene of interest to control expression thereof. For the purposes of the present invention, these terms are intended to encompass the increase in the expression of both homologous and heterologous enzymes. In some embodiments, the increase in expression includes a high transcription rate and / or a gene level also high compared to the rate of homologous transcription of said gene. In some other embodiments, a heterologous gene is introduced into a host cell to induce an increase in the expression of a gene encoding a homologous enzyme. In some embodiments, the heterologous gene is a gene that has been modified to increase the expression of the gene product. In some embodiments, the term also encompasses the secretion of the polypeptide from a cell.

For purposes of the present invention, the terms "endogenous" or "homologous" refer to both genes and proteins, which occur naturally in a host cell, ie, without any human intervention. Additionally, these terms also refer to those same genes or proteins that once isolated from the organism can be reintroduced (transgen) by genetic engineering.

For the purposes of the present invention, the term "heterologous" refers to a nucleic acid that is derived from a different species or, if derived from the same species, a nucleic acid that is substantially modified from its native form. For example, a promoter that is operatively linked to a heterologous structural gene belongs to a different species from which the structural gene was originally obtained as long as it originates from a deliberate human intervention. In case of belonging to the same species, one or several heterologous genes must be substantially modified from their original form. A heterologous protein can originate from a different species, or from the same species as long as it originates from a deliberate human intervention.

As used herein, the term "recombinant" refers to a polynucleotide or polypeptide that does not occur naturally in a host cell. In some embodiments, "recombinant cells" express genes that are not found in identical form within the native or wild (i.e., non-recombinant) form of the cell and / or express native genes that would otherwise be expressed , diminished or canceled due to deliberate human intervention. The recombinant cells contain at least one recombinant polynucleotide or polypeptide. A nucleic acid construct comprising the nucleic acid itself and the elements necessary for its expression, the nucleic acid (eg, a polynucleotide), cell or polypeptide are referred to herein as "recombinant" when it is of unnatural, artificial or processed origin .

For the purposes of the present invention, the term "decrease", "reduction", "deletion", "inhibition", "deletion", "silencing", "deletion", refer to a decrease in the expression level of a gene and / or secretion of the protein with respect to the original level of expression of the same gene and / or secretion of the protein in the parental or wild type genotype. For the purposes of the present invention, the decrease, elimination, reduction, suppression, inhibition, deletion, silencing or inhibition of the expression and / or secretion in increasing order of preference is at least 10%, 20%, 30%, %, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, reduced by comparison with expression in the parental or wild-type host cells. A person skilled in the art knows the different tools and routine techniques for the elimination, reduction, deletion, deletion, silencing or inhibition of the expression of a gene or protein. For the purposes of the present invention, serve as an example, the cloning of a target gene or genes in the form of inverted repetition (in part or totally), loss, substitution or blocking of genetic material resulting in a complete or partial interruption of the sequence of the DNA that makes up the gene, alterations of the promoter or any other reduction of the level of transcription, alteration of the expression of regulatory proteins, gene silencing (dsRNA, siRNA, etc.), modification of the sequence of initiation of the translation, alteration of the reading frame, if any changes in secretion (signal peptide alterations, etc.), mutagenesis, etc. In some embodiments, gene silencing is preferred. In other embodiments, deletion or partial removal of the gene is preferred. In other embodiments, complete or nearly complete deletion of the gene sequence is preferred.

A person skilled in the art knows the different tools and routine techniques for the elimination, reduction, suppression or inhibition of the secretion of a protein. For the purposes of the present invention, serve as an example the directed or random genetic modification of the signal sequences (signal peptide) that allow the secretion of a protein or the directed or random genetic modification of the secretion system of the host cell itself same. Genetic modifications may consist of loss of regions, modifications, substitutions, integrations, alterations, silencing, alteration of the reading frame, etc.

For the purposes of the present invention, the term "secretion" refers to the transport of a protein from the interior of the cell to the exterior For the purposes of the present invention, the term secretion refers, preferably to the secretion of enzymes with cellulolytic activity , that by effect of this transport appear in the enzymatic composition produced by said cell.

For the purposes of the present invention, the terms "host or wild type host cell" or " wild type host cell " may be used interchangeably and refer to that host cell that has not been modified to express the cellulase variants of the invention . Preferably, the parent or wild type cell of the present invention is Myceliophtora thermophila, more preferably M. thermophila C1.

The cellulase variant of the invention has a greater resistance to proteolysis thanks to its sequence comprising at least one of the linkers described in the present invention. The cellulase variant of the invention does not lose the CBD domain keeping its activity intact even in conditions where the native cellulase loses said CBD domain. An enzymatic composition comprising at least one of the cellulase variants of the invention is more effective than an enzymatic composition where the cellulases are native, undergo proteolysis and lose the CBD domain. Therefore, an enzymatic composition comprising at least one of the cellulase variants described in the present invention, improves the performance of the hydrolysis step of the cellulosic material in fermentable sugars in the processes for the production of a bioproduct, preferably ethanol, with respect to an enzymatic composition comprising native cellulases. Additionally, an enzymatic composition comprising at least one of the cellulase variants of the invention exhibits greater stability against the conditions of the hydrolytic stage and / or during the time from its production to its use.

Therefore, in another aspect of the invention there is provided an enzymatic composition comprising at least one of the cellulase variants of the invention, hereinafter known as "enzyme composition of the invention". In a preferred embodiment, the enzyme composition of the invention further comprises other cellulases.

It should be understood that the cellulase variant of the invention can be combined with one or more of the cellulolytic enzymes described herein or with any other enzyme available and suitable for producing a multienzyme composition intended for saccharification of cellulosic biomass, which can be both a homologous or heterologous cellulase. One or more components of the multienzyme composition (apart from the enzymes described in the present invention) can be obtained or derived from a microbial, plant or other source or combination thereof, and will contain enzymes capable of degrading the cellulosic material.

This composition of the invention may further comprise other enzymatic activities, such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulases such as endoglucanases, beta-glucosidases and / or cellobiohydrolases, polysaccharide monooxygenases, cellobiohydrolases, chitinase, cutinase, cyclodextrin glucosyltransferase, deoxyribonuclease. , esterase, alfagalactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, reductase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, protease, ribonuclease, transglutaminase, or xylanase, or any of its combinations. The additional enzyme (s) can be produced, for example, by a microorganism belonging to the genus Aspergillus, such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans. , Aspergillus niger, Aspergillus oryzae or Aspergillus terreus; Fusarium, such as Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium pseudograminearum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum; Gibberella, such as Gibberella zeae; Humicola, such as Humicola insolens or Humicola lanuginosa; Talaromyces, such as Talaromyces muroii, Talaromyces aculeatus or Talaromyces atroviride; Trichoderma, such as Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride; Penicillium, such as Penicillium brasilianum, Penicillium canescens, Penicillium chrysogenum, Penicillium decumbens, Penicillium ethinulatum, Penicillium funiculosum, Penicillium janthinellum, Penicillium pinophilum or Penicillium purpurogenum or Myceliophthora, such as Myceliophthora thermophila.

In a preferred embodiment, the enzyme composition of the invention further comprises the host cell of the invention.

The composition of the invention can be prepared according to methods known in the art and can be in liquid form or be a dry composition. Enzymes to be included in the composition can be stabilized according to procedures known in the art.

Another aspect described in the invention refers to the use of the host cell of the invention or of the composition of the invention, for the degradation of biomass.

The term "biomass" refers in the present invention to the biodegradable fraction of products, residues and residues of biological origin from agriculture (including plant substances, such as crop residues, and animal substances), forest industries (such as timber resources) and related industries that include fisheries and aquaculture, as well as the biodegradable fraction of industrial and urban waste, such as urban solid waste or paper waste, and energy crops In a preferred embodiment, the biomass is straw or the fraction organic solid urban waste In a more preferred embodiment, the biomass is vegetable biomass, more preferably selected from the list consisting of: biomass rich in fermentable sugars, such as sugarcane, starch biomass, for example, grains of cereal, corn straw, wheat straw, barley straw, sorghum straw, sugar cane straw, weeds, trunks, branches and leaves.

The host cell or the composition of the present invention can be used to produce, from plant biomass, monosaccharides, disaccharides and polysaccharides as chemical raw materials or from fermentation for the production of ethanol, plastics, or other products or intermediates.

The host cell of the present invention can be used as a source of the cellulase variants of the invention and of other polypeptides having cellulase activity, in saccharification or degradation processes or hydrolysis and fermentation of lignocellulosic material.

Therefore, in a preferred embodiment, the enzyme composition of the invention is an enzyme composition obtained (secreted) by the host cell of the invention. This composition can be obtained by culturing the host cell of the invention under conditions suitable for the production and secretion of cellulolytic enzymes.

The host cell can be cultured in a suitable nutrient medium, solid or liquid, for the production of the cellulase variants of the invention, and of the entire enzymatic composition of the invention, using well-known methods in The technique. For example, the cell can be grown by shake flask culture, and small-scale or large-scale fermentation (including continuous, batch, fed-batch , or solid state fermentation ) carried out in a laboratory or industrial bioreactor in a suitable medium and under conditions that allow expressing and / or isolating the variant or composition. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using the procedures known in the art. If the variant is secreted, along with other cellulolytic enzymes in the nutrient medium, they can be recovered directly from the medium.

The cellulase variants of the invention expressed, together with other expressed cellulolytic enzymes, can be detected using methods known in the art specific for polypeptides. These detection methods may include the use of specific antibodies, the formation of a product of the enzyme, or the disappearance of a substrate from the enzyme.

The cellulase variants of the resulting invention, together with the rest of cellulolytic enzymes secreted by the host cell, can be recovered using methods known in the art. For example, they can be recovered from the nutrient medium by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

The cellulase variants produced in the present invention, along with other cellulolytic enzymes secreted by the host cell, can be purified by a variety of methods known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing, and exclusion by molecular size), electrophoretic procedures (eg, preparative isoelectric focusing), differential solubility (eg, ammonium sulfate precipitation), SDS-PAGE, or extraction, in order to obtain the substantially pure enzymes that can be included in an enzymatic composition.

The degradation or hydrolysis of the cellulose material into fermentable sugars, a process also known as "saccharification", by means of the cellulase variants of the invention, the host cell of the invention or the composition of the invention, can be accompanied after a process of fermentation in which the sugars Fermentable obtained are used in order to finally obtain a bioproduct such as bioethanol.

The term "bioproduct" or "biobased products" refers to products with high added value that can be obtained by chemical transformation of sugars or by fermentation of said sugars with different microorganisms Fermentative micro-organisms, within the scope of the present invention, include, yeasts, bacteria, fungi, preferably filamentous fungi, microalgae and combinations of any of the foregoing, The fermentative microorganisms mentioned above may be wild type microorganisms or wild type or recombinant microorganisms, Microorganisms included in the present invention include microalgae. , defined as eukaryotic microorganisms comprising a chloroplast or plastid, and which are optionally capable of carrying out photosynthetic processes, or prokaryotic microorganisms that are capable of carrying out photosynthetic processes. oautotrophs that perform photosynthesis to obtain energy. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, mirobes such as, for example, Volvox. Microalgae include cells such as Chlorella, Dunaliella and Prototheca. Microalgae also include other microbial photosynthetic organisms, such as Agmenellum, Anabaena, and Pyrobotrys.

Other fermentative microorganisms can be selected from any of the following list: Bacillus thermoglucosidaisus, Clostridium butyricum, Clostridium acetobutylicum, Clostridium beijerinckii, Corynebacterium glutamicum, Enterobacter aerogenes, Escherichia coli, Geobacillus themoglucosidasius, Klebsiella oxytoca, Lactobacillus sp. Leunoscoc mesenteroides, Thermoanaerobacter BG1L1, Thermoanaerobacter ethanolicus, Thermoanaerobacter mathranii, Thermoanaerobacter thermosaccharolyticum, Zymobacter palmae, Zymomonas mobilis Candida arabinofermentans, Candida boidinii, Candida diddensis, Candida fermentans, Chrysosporium lucknowense, Candida pastoris, Candida shehatae, Candida sonorensis, Candida tropicalis, Hansenula anomalous, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces marxianus, Pichia pastoris, Pichia stipitis, Saccharomyces cerevisiae, Saccharomyces bulderi, Saccharomyces barnetti, Saccharomyces exiguus, Saccharomyces diastaticus, Saccharomyces uvarum or Schizosaccharomyces pombe, Pseudomonas, Streptomyces, or mixtures thereof.

These and other microorganisms can provide, by fermentation of different sugars, bioproducts, among which the following can be mentioned in a non-limiting manner: alcohols, organic acids, alkanes, alkenes, aromatics, aldehydes, ketones, triglycerides, fatty acids, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, pharmaceuticals, and their combinations. Non-limiting examples of alcohols are ethanol, methanol, butanol, hexanol, octanol, decanol, dodecanol, 1,3-butanediol (1,3-diol), propanol, isopropanol, ethylene glycol, propanediol, butanediol, glycerol, erythritol, xylitol, sorbitol , 1-alcohol, and combinations thereof. Non-limiting examples of organic acids are citric acid, acetic acid, itaconic acid, lactic acid, glutamic acid, succinic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, gluconic acid, levulinic acid, beta-ketoacid, betacetoalcohol, beta-hydroxy acid and combinations thereof. Non-limiting examples of ketones are acetone; gases such as hydrogen or carbon dioxide; hydrocarbons such as alkanes, alkenes or alkynes; nitrogenous substances such as amines, amide, nitro compounds or nitriles; halides; amino acids such as glutamic acid, aspartic acid, methionine, lysine, glycine, arginine, threonine, phenylalanine, tyrosine, and combinations thereof; antibiotics such as penicillin or tetracyclines; vitamin such as riboflavin, vitamin B12 or beta-carotene; fatty acids such as dodecanoic acid, trans-A2 fatty acids or palmitic acid; and other products such as ethylene, glycerol, 1,3-propane-diol, beta-lactam, cephalosporins, trans or furan fatty acids and industrial enzymes.

Ethanol can be produced by the enzymatic degradation of the biomass and the conversion of the liberated saccharides into ethanol. This type of ethanol is often called bioethanol. It can be used as a fuel additive or extender in mixtures of less than 1% up to 100% (a substitute for fuel).

In a more preferred embodiment, the bioproduct is biofuel. The term "biofuel", as used herein, refers to a mixture of organic substances, which can be used as a fuel and is obtained using the fermentable biomass as starting material Examples of biofuels include, but are not limited to, ethanol or bioethanol and biodiesel In a more preferred embodiment, the biofuel is bioethanol.

The term "bioethanol" refers to an alcohol prepared by fermentation, often from fermentable biomass such as carbohydrates produced in sugar or starch cultures such as corn or sugarcane.

Therefore, in another aspect, the present invention relates to a process for producing fermentable sugars from cellulosic biomass, hereinafter "first method of the invention", comprising:

a) Incubate biomass, preferably pre-treated biomass, with the variant of the cellulase of the invention, or with the host cell of the invention, or with the composition of the invention, and

b) Recover the fermentable sugars obtained after the incubation of stage (a).

Frequently a biomass pretreatment procedure is required to increase the access of the enzymes to their substrates and the consequent effective hydrolysis. The pretreatment uses various techniques, including, but not limited to chemical and / or mechanical treatments, such as the explosion of fiber with ammonium, treatment with diluted acid and explosion with steam at elevated temperatures to alter the structure of the biomass cellulose and make cellulose more accessible. The use of the host cell of the invention or of the enzymatic composition of the invention in the methods of the present invention is advantageous because high temperatures are not required in the biomass pretreatment process.

The term "fermentable sugar" as used herein, refers to simple sugars, such as glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose or fructose, among others.

Another aspect described in the present invention relates to a process for producing a bioproduct from biomass, hereinafter referred to as the "second method of the invention", which comprises:

a) Incubate biomass, preferably pre-treated biomass, with the host cell of the invention or with the composition of the invention, b) Ferment the fermentable sugars obtained after the incubation step (a) with at least one fermenting microorganism, and

c) Recover the bioproduct obtained after the fermentation of stage (b).

Before (i.e. in step (a)) and / or simultaneously with the fermentation of step (b), the biomass, preferably pretreated biomass, is hydrolysed to degrade cellulose and hemicellulose into sugars and / or oligosaccharides. The solids content during the hydrolysis can be, but without limitation, comprised between 5-40% of the total weight, preferably between 10-40% of the total weight, more preferably between 15-25% of the total weight. The hydrolysis is carried out as a process in which the biomass, preferably pre-treated biomass, is incubated with the host cell of the invention or with the composition of the invention containing cellulases and thus form the hydrolysis solution. The suitable process time, temperature and pH conditions can be easily determined by one skilled in the art. Preferably, said hydrolysis is carried out at a temperature between 25 ° C and 60 ° C, preferably between 40 ° C and 60 ° C, specifically around 50 ° C. The process is preferably carried out at a pH in the range of 3-8, preferably pH 4-6, especially around pH 5. Preferably, the hydrolysis is carried out in a time comprised between 12 and 144 hours, preferably between 16 and 120 hours , more preferably between 24 and 96 hours, even more preferably between 32 and 72 hours.

The hydrolysis (step (a)) and the fermentation (step (b)) can be carried out simultaneously (SSF process) or sequentially (SHF process). According to the invention, the hydrolyzed biomass, and preferably pretreated, is fermented by at least one fermenting microorganism capable of fermenting fermentable sugars, such as glucose, xylose, mannose and galactose directly or indirectly in the desired fermentation product. The fermentation is preferably carried out in a time comprised between 8 and 96 hours, preferably between 12 and 72, more preferably between 24 and 48 hours. In another preferred embodiment, the fermentation is carried out at a temperature between 20 ° C and 40 ° C, preferably from 26 ° C to 34 ° C, in particular around 32 ° C. In another preferred embodiment, the pH is from 3 to 6 units, preferably from 4 to 5. A yeast of the species Saccharomyces cerevisiae, both wild and genetically modified, is preferred for ethanolic fermentation. Strains that are resistant to high levels of ethanol are preferred, up to, for example, 5 or 7% vol. of ethanol or more, such as 100% vol. of ethanol.

The term "fermentor or fermentation" as used herein, refers to a process of biological transformation produced by the activity of some microorganisms in which sugars such as glucose, fructose, and sucrose are converted to ethanol.

The microorganisms used in this way are fermentation-promoting microorganisms, such as yeasts, preferably S. cerevisiae.

The term "recovery" as used herein, refers to the collection of fermentable sugars obtained after the incubation of step (a) of the first process of the invention or of the bioproduct obtained after the fermentation of step (b) of the second method of the invention. Recovery can be accomplished by any method known in the art, including mechanical or manual.

In a preferred embodiment of the second method of the invention, the bioproduct is biofuel, more preferably bioethanol.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be given to them by one skilled in the art to which this invention pertains. In the practice of the present invention methods and materials similar or equivalent to those described herein can be used. Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Photograph of an SDS-PAGE gel showing the loss of the CBD domain of type I cellobiohydrolase by proteolytic processing of the linker, decreasing its size over time. (A) Enzymatic composition at the start of the production of Cbh1 (72 hours). (B) Enzymatic composition at the end of the production of Cbh1 (120 hours). The molecular weight marker is included in the first lane. Cbh1-CBD (-) refers to the Cbh1 that has lost the CBD and Cbh1-CBD (+) refers to the intact Cbh1 enzyme.

Figure 2. Graph showing the performance of an enzymatic composition where the Cbh1 enzyme has lost CBD (Cbh1-CBD (-)) versus another enzyme composition where the Cbh1 enzyme is intact (Cbh1-CBD (+)). The yield drop with the Cbh1 enzyme without CBD is around 20%.

Figure 3. Scheme of the plasmid pBase-5K-4 where the nucleotide sequences coding for the cellulase variants described in the present invention have been cloned.

Figure 4. Photograph of an SDS-PAGE demonstrating the greater stability of the Cbh1 variants of the invention (B and C) versus the native Cbh1 enzyme (A). At the end of production the native cellulase Cbh1 has lost its CBD domain by linker proteolysis , while the Cbh1 cellulases of the invention (B and C) remain intact. The molecular weight marker is included in the first lane.

Figure 5. Analysis of saccharification performance by analyzing the cellobiohydrolase activity of enzymatic compositions obtained from strains of M. thermophila comprising the variants of Cbh1 of SEQ ID NO: 66 and SEQ ID NO: 70 of the invention, with respect to the respective strain of parent M. thermophila expressing the native Cbh1 of SEQ ID NO: 38. The control is reifered to an enzyme composition obtained from strain of a strain of M. thermophila that does not express Cbh1.

EXAMPLES

The invention will now be illustrated by means of tests that demonstrate the effectiveness of the objects of the invention.

Example 1. Obtaining variants of the cellulases of the invention.

As previously indicated throughout the present document, during the production process and / or during the storage phase (between the production and its use) and / or during the enzymatic hydrolysis process, the linker region of the cellulases it can undergo proteolysis or processing, which causes a dissociation between the CAD domain and the CBD domain. The fact that a cellulase loses the CBD domain prevents its efficient recognition and binding to cellulose, which leads to a loss or decrease in the yield of enzymatic hydrolysis. During the production of the enzymatic composition generated by Myceliophthora thermophila C1 (Visser et al., 2011, Ind. Biotechnol 7: 214-223) an enzymatic mixture is generated that varies as said production progresses. As seen in the Figure 1, in the case of cellobiohydrolase type I (Cbh1), the band corresponding to the protein with its CBD domain disappears throughout the production process of the enzymatic composition. In an early phase of production (72 hours), the predominant form of Cbh1 is that which contains the CBD domain. Throughout production, Cbh1 undergoes proteolysis and dissociates from the CBD domain, predominating at the end of production (120 hours) the form without CBD.

Additionally, the release of fermentable sugars from the enzymatic compositions comprising the Cbh1 enzyme with its intact CBD domain (lane A, Figure 1) and the enzyme composition comprising the Cbh1 enzyme after losing the CBD domain (lane B, Figure one). As a substrate for enzymatic hydrolysis, pretreated corn stover, or PCS, was used.Pretreatment was done by steam explosion (Keller et al., 1998, Appl. Biochem. Biotechnol., 70-72: 137 -148), and its compositional analysis was carried out according to the procedures described by NREL in "Standard Biomass Analytical Procedures" (http://www.nrel.gov/biomass/analytical_procedures.htmL). Before use in hydrolysis, the biomass was neutralized by adjusting to a pH of 5.5. For the enzymatic hydrolysis process, 100 ml ISO bottles were used with 20 g of the 20% (w / w) reaction mixture of total solids and supplemented with 12 mg protein per g of glucan of each enzymatic composition. The bottles with the mixture were incubated for 72 h at 50 ° C with an agitation at 150 rpm in an orbital incubator with a diameter of 25 mm (Infors HT). Once the process was carried out, the glucose content in the samples resulting from the hydrolyzate (slurry) was analyzed with the GOPOD method (K-GLUC Kit, Megazyme). As seen in Figure 2, the yield of the sugar release process of the enzyme composition comprising the Cbh1 enzyme without the CBD domain (Cbh1-CBD (-)) is 20% lower than the yield of the sugar release process of the enzymatic composition comprising the Cbh1 enzyme with its intact CBD domain (Cbh1-CBD (+)).

Taking into account the previous results, we proceeded to design a cellulase that presented the modified linker region, with respect to the parental cellulase. By way of example and without being limiting, it has been taken as an example to design variants of cellulases comprising the linkers described in the present invention and which are more resistant to hydrolysis than the native linkers of the cellulases, to the sequence Cbh1 cellulase nucleotide SEQ ID NO: 37, which codes for the cellulase of SEQ ID NO: 38. Therefore, in order to obtain variants of cellulases more resistant to the hydrolysis of the breakage of the CBD domain, we started with cellulase Cbh1, which was removed from its native linker and replaced by at least one of the linkers described in the present invention that have a greater resistance to hydrolysis than the native linkers , giving rise to all the variants of the cellulase Cbh1 described in Table 2, as they comprise each of the linkers described herein.

Example 2. Transformation of M. thermophila with the variants of the invention:

Each of the Cbh1 variants comprising the linkers described in the present invention (Table 2) was synthesized in vitro, the recognition sites being eliminated for the main restriction enzymes without altering their peptide sequence. Briefly, said variants were synthesized in vitro in the plasmid pBase-5K-4 (Figure 3), which contains the cellulase promoter sequence itself Cbh1 (Pcbh1), corresponding to a region of 1796 bp upstream of the cbhl gene of M. thermophila (cbhl, NCBI Accession number XP_003660789.1). This expression plasmid also contains the terminator of the cbh1 gene which corresponds to a region of 1014 bp downstream of the cbh1 gene . As a selection marker, it contains the pyr4 gene (NCBI Accession number XP_003660657.1) which codes for a functional decarboxylase of orotidine-5'-monophosphate, whose expression allows the complementation of the auxotrophy of uridine in the corresponding auxotrophic strain of M. thermophila C1 (pyr4-). The expression vector map pBase-5k-4 is shown in Figure 3.

Each of the plasmids generated comprising sequences encoding variants CBH1 enzyme the linker described comprising herein, were transformed and amplified in electrocompetent cells XL1BlueMRF E. coli following the protocol described by the manufacturer (Stratagene ).

Each of the plasmids amplified in E. coli containing the Cbh1 variants with the linkers described in the present invention, under the control of the Pcbh1 promoter and with the selection marker pyr4, were transformed into M. thermophila C1 pyr4 (-) cbhI (-) (Verdoes et al., 2007, Ind. Biotechnol.3 : 36-47). A deleted strain in the cbh1 gene (NCBI Accession number XP_003660789.1) has been used for the purpose of obtain an enzyme composition comprising the variant CBH1 with linker described herein, and do not understand the native cbh1.

The nucleotide sequences coding for each of the Cbh1 variants comprising the different linkers described in the present invention were introduced into the M. thermophila host cell using the protoplast transformation method (US7399627B2). The transformants obtained were plated on agar plates without uridine supplementation. After 5 days of incubation at 35 ° C, the obtained prototrophic transformants (expressing pyr4) were analyzed in high throughput screening format (US7794962B2) in 96-well plates. Those transformants which showed clear expression of the variant CBH1 linkers were produced flask scale (Verdoes et al, 2007, Ind Biotechnol 3:... 36 47); Visser et al., 2011, Ind. Biotechnol. 7: 214-223). The presence of the Cbh1-CBD (+) or Cbh1-CBD (-) protein was analyzed in SDS-PAGE gel at the end of the procedure for the production of the enzymatic compositions.

As shown in Figure 4, some of the variants showed greater resistance to proteolysis (Figure 4 lanes B and C) with respect to the native Cbh1 enzyme that under the same conditions has lost its CBD domain as a result of proteolysis (Figure 4 lane A). The native enzyme has greater sensitivity to proteolysis than the Cbh1 variants of SEQ ID NO 66 (Figure 4, lane B) and of SEQ ID NO 70 (Figure 4, line C) comprising the linker SEQ ID NO 26 and SEQ ID NO. 30 respectively, which implies a drastic reduction of its cellulolytic activity with respect to the more resistant variants described in the present invention, and therefore, lower efficiency of the biomass degradation process.

Results similar to those shown in Figure 4 with the Cbh1 variants of sequences SEQ ID NO: 66 and 70, were obtained for the rest of Cbh1 variants described in the present invention.

Example 3. Evaluation of the Cbh1 variants of SEQ ID NOs: 66 and 70 comprising the linkers SEQ ID Nos: 26 and 30 respectively, in comparison with the native Cbh1 enzyme of M. thermophila C1 (SEQ ID NO: 38).

Enzymatic compositions comprising the Cbh1 variants with linker more resistant to proteolysis, as described in the present invention, and which are obtained as described in Example 2, were analyzed to verify that an enzymatic composition comprising said variants of Cbh1 has a better saccharification yield with respect to an enzymatic composition comprising the native Cbh1. For this, the cellobiohydrolase activity of the native enzymatic compositions and those containing the variants of the invention were measured using the substrate Avicel (microcrystalline cellulose). For this assay of cellobiohydrolase activity the mixtures of the enzymatic reaction (1 ml final volume) were prepared with 200 pL of sodium acetate buffer (pH 5.0, 200 mM), 10 mg of Avicel, and 50 pg of the enzyme composition. This mixture was incubated at 50 ° C for 120 minutes at 1400 rpm shaking. The reaction was stopped by incubating the mixture for 10 min at 99 ° C. Subsequently, the samples were centrifuged for 5 min at 4000xg. To measure the concentration of glucose produced in the enzymatic reaction, the enzymatic method GOPOD (Glucose oxidase / peroxidase) (GOPOD kit, Megazyme) was used according to the manufacturer's specifications. One unit of hydrolysis activity on Avicel was defined as the amount of enzyme equivalent to the release of 1 pmol of cellobiose per minute. The protein concentration of the enzymatic compositions was quantified by the BCA AppliChem kit (Ref. A7787), against a bovine gamma globulin standard, after treatment of the sample with the "Compat-Able Protein Assay Preparation Reagent Set" kit (Thermo Scientific Ref . 23215) ", both according to the manufacturer's specifications.

The results obtained show, as seen in Figure 5, that the enzymatic compositions comprising the Cbh1 variants with the linkers of the invention, specifically the Cbh1 variants of sequences SEQ ID NO: 66 and 70, show a an increase of 18% in the yield of the cellulose degradation process, with respect to the yield obtained with the enzymatic composition containing the native Cbh1 enzyme of SEQ ID NO: 38. Therefore, said results confirm that an enzymatic composition comprising the cellulase variants with the linkers described in the present invention show greater resistance to hydrolytic processing and loss of the CBD region and as a consequence the efficiency and efficiency of the biomass degradation process is increased.

Claims (22)

1. Linker comprising an amino acid sequence of SEQ ID NO: 26. 2. Cellulase comprising at least one cellulose binding domain (CBD), at least one catalytically active domain (CAD) and at least one linker region , which binds the CBD and CAD domains, according to claim 1. 3. Cellulase according to claim 2 wherein the cellulase is a cellobiohydrolase or a polysaccharide monooxygenase. 4. Cellulase according to any of claims 2 to 3 which is selected from SEQ ID NO: 66 and SEQ ID NO: 78. 5. Isolated nucleic acid sequence encoding the linker according to claim 1, or the cellulase according to any of claims 2 to 4. 6. Isolated nucleic acid sequence complementary to the nucleic acid sequence according to claim 5. 7. Gene construct comprising the nucleic acid sequence according to any of claims 5 or 6. 8. Gene construct according to claim 7, wherein the gene construct is an expression vector. 9. Host cell comprising the gene construct according to any of claims 7 or 8, or the cellulase according to any of claims 2 to 4. 10. Host cell according to claim 9, wherein said cell is Myceliophthora thermophila C1. 11. Enzymatic composition comprising a cellulase according to any of claims 2 to 4. 12. Enzymatic composition of claim 11, further comprising other cellulases. 13. Enzymatic composition according to claim 12, wherein the other cellulases are selected from the list consisting of: endoglucanases, beta-glucosidases, cellobiohydrolases, beta-xylosidases, xyloglucanases, polysaccharide monooxygenases, xylanases, arabinofuranosidases, and any combination thereof. 14. Enzymatic composition according to any of claims 11 to 13, further comprising the cell according to any of claims 9 or 10. 15. Enzymatic composition according to any of claims 11 to 14 obtained by the cell according to claims 9 to 10. 16. Use of the host cell according to any of claims 9 or 10, or of the enzymatic composition according to any of claims 11 to 15, for the degradation of the cellulosic biomass. 17. Use according to claim 16, for the degradation of the biomass in a production process of a bioproduct. 18. Use according to claim 17, wherein the bioproduct is biofuel. 19. Use according to claim 18, wherein the biofuel is bioethanol. 20. Process for producing fermentable sugars from cellulosic biomass, comprising: a) Incubation cellulosic biomass with the cellulase according to any of claims 2 to 4, with the host cell according to any of claims 9 or 10, or with the enzymatic composition according to any of claims 11 to 15, and b) Recover the fermentable sugars obtained after incubation in stage (a). 21. Procedure for producing a bioproduct from cellulosic biomass comprising: a) Incubating cellulosic biomass with the cellulase according to any of claims 2 to 4, with the host cell according to any of claims 9 or 10, or with the enzymatic composition according to any of claims 11 to 15, and ) Fermenting the fermentable sugars obtained after the incubation of stage (a) with at least one fermenting microorganism, and c) Recovering the bioproduct obtained after fermentation in stage (b). 22. The process according to claim 21, wherein the bioproduct is ethanol.