ES2542621A1 - Polypeptides with polysaccharide monooxygenase activity and their use for the production of fermentable sugars (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Polypeptides with polysaccharide monooxygenase activity and their use for the production of fermentable sugars. The invention relates to polypeptides with polysaccharide monooxygenase activity, to a host cell expressing them, preferably a myceliophthora thermophila cell, to an enzymatic composition comprising at least one of said polypeptides, preferably together with other cellulolytic enzymes, to the use of this host cell, of at least one of the polypeptides with polysaccharide monooxygenase activity or of the enzymatic composition for the degradation of cellulosic biomass and to a process for producing bioproducts, preferably bioethanol, comprising the use of said host cell, of at least one of the polypeptides of the invention or of the enzymatic composition of the invention. (Machine-translation by Google Translate, not legally binding)

Description

The invention relates to the field of bioproducts, more particularly, to enzymes with monooxygenase polysaccharide activity and their use, forming part of enzymatic cocktails, for the production of fermentable sugars from cellulosic biomass during the production processes of bioproducts such as

10 bioethanol

PREVIOUS TECHNIQUE

Vegetable biomass provides an abundant source of potential energy in form

15 carbohydrates that can be used for numerous industrial and agricultural processes and, therefore, is an important renewable source for the generation of fermentable sugars. The fermentation of these sugars can produce commercially valuable end products, such as ethanol also called bioethanol.

20 Although the fermentation of sugars to ethanol is relatively straightforward, the efficient conversion of cellulosic biomass into fermentable sugars, such as glucose, is a major challenge. The enormous potential energy of the large amounts of carbohydrates that make up plant biomass is not sufficiently used because sugars are part of complex polymers

25 (polysaccharides, such as cellulose and hemicellulose) and, therefore, are not readily accessible for fermentation. Thus, cellulose can be pretreated, mechanically, chemically, enzymatically or in other ways, to increase its susceptibility to hydrolysis. After this pretreatment process, a saccharification or hydrolysis stage consisting of a

Enzymatic process whereby complex carbohydrates (such as starch or cellulose) are hydrolyzed into their monosaccharide components. The objective of any saccharification technology is therefore to alter or eliminate structural and compositional impediments in order to improve the rate of enzymatic hydrolysis and increase the yields of fermentable sugars obtained from cellulose or

35 hemicellulose (N. Mosier et al., 2005, Bioresource Technology 96, 673--686). After this saccharification stage a fermentation process is carried out. Thus,

The greater the amount of complex sugars that remain at the end of the hydrolytic process, the lower the yield of ethanol production at the end of the fermentation process. Thus, an area of research aimed at reducing costs and improving the performance of biofuel production processes is

5 focused on improving the technical efficiency of hydrolytic enzymes, or in general on improving the efficiency of enzymatic cocktails used to generate fermentable sugars from biomass.

It has been shown that individual enzymes are only able to digest

10 partially cellulose and hemicellulose and, therefore, the concerted action of different classes of enzymes is required to complete their conversion into monomeric sugars. Many more enzymes are required to digest hemicellulose to monomeric sugars than cellulose, including enzymes with xylanase, beta-xylosidase, arabinofuranosidase, mannanase, galactosidase and

15 glucuronidase Other enzymes without glycosyl hydrolase activity, such as acetyl xylan esterase and ferulic acid esterase, may also be involved. Therefore, the enzymatic hydrolysis of polysaccharides for conversion into soluble sugars and, finally, in monomers such as xylose, glucose and other pentoses and hexoses, is cataloged by several enzymes that together are called "cellulases." The

20 cellulases are multienzyme complexes comprising at least three main components, endo- ~ -glucanase (EC 3.2.1.4), exo- ~ -glucanase or cellobiohydrolase (EC 3.2.1.9.1) and ~ -glucosidase (EC 3.2. 1.21), and have been shown to act synergistically in the hydrolysis of cellulose (Woodward, J. 1991, Bioresource Technology Vol. 36, p. 67-75).

25 Microbial cellulases have become focal biocatalysts due to their complex nature and extensive industrial applications (Kuhad R. C. et al., 2011, Enzyme Research, Article ID 280696). Today, considerable attention has been given to current knowledge about cellulose production and

30 the challenges in cellulase research, especially in the direction of improving the process economy of several industries, in order to obtain cellulases with more activity and better properties.

On the other hand, the glycosyl-hydrolase proteins of family 61 (GH61) are known

for over 20 years. The first GH61 described, called CEL 1, was

isolated from Agaricus bisporus in 1992 (Raguz et al., 1992, Gene 119: 183-190). These

GH61 proteins are accessory proteins that contribute to cellulose degradation. The fact that these enzymes act by direct oxidation of cellulose, instead of hydrolysis, has led to its current name: Cu-dependent polysaccharide monooxygenases (Polysaccharide Monooxygenase; PMOs). In comparison with other cellulolytic enzymes, PMOs are relatively small proteins with molecular masses typically between 20 and 50 kDa (Baldrian and Valaskova 2008, FEMS Mierobiology Reviews 32: 501-521; Harris et al., 2010, Biochemistry 49: 3305 -3316). These proteins require two oxygen molecules to produce the breakage of the product and its oxidation. One of these molecules derives from

10 water, the other enters the reaction in the form of molecular oxygen, which is necessary for direct oxidation of the substrate. Therefore, members of this family of enzymes act as Cu monooxygenases that catalyze cellulose rupture by an oxidative mechanism, releasing celodextrins (Langston et al., 201 1, Applied and Environmental Mierobiology 77: 7007-7015).

The hydrolytic efficiency of a multi-enzymatic complex in the process of saccharification of cellulosic material depends on both the properties of individual enzymes and the proportion of each enzyme present in the complex. Therefore, in the context of biofuel production processes, it is

It is necessary to design enzymatic cocktails with improved individual activities, and with the proportion of each one optimized, whose use during the saccharification or hydrolysis stage of cellulosic biomass leads to an improvement in the performance of said stage through an increase in the amount of fermentable sugars obtained at low doses of enzyme. These sugars may be

25 subsequently fermented to produce biofuels, such as bioethaneJ, which would ultimately increase the efficiency and profitability of the entire biofuel production process.

DESCRIPTION OF THE INVENTION

The present invention provides polypeptides with monooxygenase polysaccharide (PMO) activity, also known as glycosyl hydrolases of family 61 or GH61) which have been identified, isolated and characterized from strain C1 of Myceliophthora thermophiJa. As the examples here show

In this invention, these polypeptides have the advantage that they are capable of increasing the saccharification performance of cellulosic biomass, by increasing the amount of fermentable sugars produced at the end of said hydrolytic process, when they are added to the enzymatic cocktails used in the processes of biofuel production.

5 Such polypeptides are monooxygenase enzymes of polysaccharides that are involved in the initial phases of the cellulosic biomass decomposition process up to fermentable sugars, these enzymes being responsible for improving the accessibility of the rest of the enzyme machinery to the substrate. Thus, increase the yield of the hydrolysis process by increasing the amount of

10 simple sugars obtained (mainly glucose) and with it, ultimately, the yield of ethanol production from biomass.

The inventors of the present invention have identified, in the genome of Myceliophthora thermophila, 21 genes encoding enzymes with PMO activity (the 15 amino acid sequences of these 21 mature enzymes are shown in SEO ID NO: 43 to SEO ID NO: 63 ). As can be seen in the examples, these polypeptides were isolated from the fungus, characterized and added to an enzymatic mixture comprising the main cellulolytic enzymes produced by the C1 strain of Myceliophthora thermophila, in order to evaluate their effect on the yield of

20 saccharification of pretreated biomass (peS or pretreated corn stover), showing an increase in the concentration of fermentable sugars (mainly glucose) released in the hydrolytic process compared to an enzymatic cocktail that did not comprise the PMO activity polypeptides of the invention.

The improvement in the yield of the enzymatic mixtures used to produce fermentable sugars from cellulosic material in biofuel production processes, preferably ethanol, is fundamental to ensure its profitability. The supplementation of the enzyme cocktail with these enzymes provided by the invention thus contributes to improving the yield of the

30 hydrolytic process where these cocktails are used.

Therefore, a first aspect of the present invention relates to an isolated polypeptide with monooxygenase polysaccharide activity, hereafter referred to as "polypeptide of the invention", which comprises an amino acid sequence having at least

35 80% identity with an amino acid sequence selected from SEO ID NO: 43 to SEO ID NO: 63.

The polypeptide of the invention can be isolated, preferably from M. thermophila, or recombinantly produced. The pOlipypeptide of the present invention and its variants

or derivatives can be synthesized, for example, but not limited to, in vitro. For example, by solid phase peptide synthesis or by recombinant DNA approaches. The polypeptide of the invention can be produced recombinantly, not only directly but as a fusion polypeptide together with a heterologous polypeptide, which may contain, for example, but not limited to, a signal sequence or other polypeptide having a protease cleavage site. , for example, but not limited to, at the N-terminal end of the protein

10 mature or polypeptide.

The polypeptide of the invention may have variants. These variants refer to limited variations in the amino acid sequence, which allow the maintenance of the functionality of the polypeptide. This means that the reference sequence and the sequence of the variant are similar as a whole, and identical in many regions. These variations are generated by substitutions, deletions or additions. These substitutions are conserved amino acids. The conserved amino acids are amino acids that have similar side chains and properties in terms of, for example, hydrophobicity or aromaticity. These substitutions include, but are not limited to, substitutions between glutamic acid (Glu) and aspartic acid (Asp), between lysine (Lys) and arginine (Arg), between asparagine (Asn) and glutamine (Gln), between serine (Ser) and threonine (Thr), and / or among the amino acids that make up the alanine (Ala), leucine (Leu), valine (Val) and isoleucine (lIe) group. Variations can be artificially generated variations, such as by mutagenesis or direct synthesis.

25 These variations do not cause essential modifications in the essential characteristics or properties of the polypeptide. Therefore, peptides or polypeptides whose amino acid sequence is identical or homologous to the sequences described in the present invention are also included within the scope of the present invention.

The term "polysaccharide monooxygenase", "PMO", "Glycosyl hydrolase of family 61" or "GH61" refers to an enzyme that exhibits GH61 or PMO activity, which when included in a saccharification reaction (for example, that in which endoglucanases, beta-glucosidases and cellobiohydrolases are used) results in a greater amount (higher yield) of one or more soluble sugars (eg glucose) compared to the saccharification reaction carried out under them

conditions but in the absence of the GH61 protein. PMO activity can be determined by, for example, indirect oxidative tests that colorimetrically evidence the electron transfer phenomenon using different electron donor and acceptor compounds (Kitt et al., 2012, Biotechno / ogy for

5 Biofuels Vol. 5:79, p. 1-13 or the one shown in Example 3). On the other hand, biomass efficiency could be measured, for example, by combining the PMO polypeptide with cellulase enzymes in a saccharification reaction and determining if there is an increase in glucose yield compared to the same saccharification reaction carried out in the absence. of said polypeptide.

PMOs comprising amino acid sequences that are at least 80% identical to a sequence selected from SEa ID NO: 43 to SEO ID NO: 63 can be obtained from a filamentous fungus, such as, Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Gibberella, Filibasidium, Fusarium, 15 HumicoJa, Magnaporthe, Mucor, Neocallimastix, Neurospora, PaeciJomyces, PeniciIJium, Piromyces, Schizophyllum, TaJaromyces, Thermoascus, Thie / avia, TolypocJadium, Trichoderma or MyceJiophthora. In a more preferred embodiment, the PMO is a PMO of Myceliophthora thermophifa, GibbereJla zeae, Humicola insolens, HumicoJa lanuginosa, Muror miehei, Neurospora crassa, PeniciJlium

20 purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma Jongibrachiatum, Trichoderma reesei, or Trichoderma viride.

In a more preferred embodiment, the polypeptide of the invention comprises an amino acid sequence having at least 81%, 82%, 83%, 84%, 85%,

25 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from between SEO ID NO: 43 to SEO ID NO: 63.

The term "identity" or "percent identity" refers to the waste ratio

30 of nucleic acids or amino acids that are identical between two nucleic acid or amino acid sequences that are being compared. Preferably, "identity" means the ratio of nucleic acid or amino acid residues that are identical between two nucleic acid or amino acid sequences, with respect to the full length of the reference sequence. The degree of

Identity through the Clustal method, the Wilbur-Lipman method, the GAG program, which includes GAP, BLAST or BLASTN, EMBOSS Needle and FASTA. Also I know

You can use the Smith Waterman algorithm in order to determine the degree of identity between two sequences.

For sequence comparison, typically one of the sequences acts as

5 reference sequence with which the "problem" sequences are compared. When a sequence comparison algorithm is used to determine its identity, the reference sequence and the problem sequences are entered into the program, and the parameters thereof are configured. You can use the program parameters that appear by default or be configured, preferably those parameters

10 will be the ones that appear by default. Thus, the sequence comparison algorithm calculates the percentage of identity between the problem sequences and the reference sequence based on the program parameters. Two examples of algorithms that are useful for determining percent sequence identity are BLAST and BLAST 2.0, described in Altschul al. (1997) Nucleic Acids Res 25 (17): 3389-3402 and

15 Altschul al. (1990) J. Mol Biol 215 (3) -403-410, respectively. Preferably, the degree of identity referred to in the present invention is calculated by BLAST. The software to carry out the BLAST analysis is publicly available in the Nationaf Center for Biotechnology Information (NCBI).

In a more preferred embodiment, the polypeptide of the invention comprises an amino acid sequence selected from SEO ID NO: 43 to SEa ID NO:

63. Examples of the polypeptide of the invention comprising an amino acid sequence selected from SEa ID NO: 43 to SEa ID NO: 63, are the polypeptides that correspond to the immature pre-proteins or polypeptides of the mature PMO enzymes. from SEO ID NO: 43 to SEO ID NO: 63. These pre-proteins comprise a signal peptide located at the N-terminal end of the amino acid sequence of the mature enzyme. These pre-proteins are, for example, but not limited to, SEO ID NO: 22 to SEO ID NO: 42. The signal peptide of each of them has been identified and is indicated in the respective sequences

30 polypeptides in the sequence listing.

In an even more preferred embodiment, the polypeptide of the invention consists of an amino acid sequence selected from SEa ID NO: 43 to SEQ ID NO: 63, said sequences correspond to the following mature polypeptides: PMO-04725 35 (SEO ID NO: 43), PMO-06230 (SEO ID NO: 44), PMO-09768 (SEO ID NO: 45), PMO01470 (SEO ID NO: 46), PMO-03248 (SEO ID NO: 47), PMO-00727 (SEO ID NO: 48),

PMO-10391 (SEO ID NO: 49), PMO-10824 (SEO ID NO: 50), PMO-09767 (SEO ID NO: 51), PMO-03778 (SEO ID NO: 52), PMO-04750 (SEO ID NO: 53), PMO-10518 (SEO ID NO: 54), PMO-05022 (SEO ID NO: 55), PMO-05366 (SEO ID NO: 56), PMO02839 (SEO ID NO: 57), PMO-10366 (SEO ID NO: 58), PMO-04874 (SEO ID NO: 59),

5 PMO-08101 (SEO ID NO: 60), PMO-00657 (SEO ID NO: 61), PMO-04859 (SEO ID NO: 62) and PMO-03723 (SEO ID NO: 63).

The term "pre-protein" refers to a polypeptide that includes a signal peptide (or leader sequence) at its amino terminal end. Said signal peptide is cleaved from the

10 pre-protein for a peptidase, thus secreting the mature protein. The secreted portion of the polypeptide is called "mature protein" or "secreted protein." The "signal peptide" is one that directs the polypeptide into the cell towards its secretion pathway.

As the examples of the present invention show, the so-called polypeptides

here as PMO-01470, PMO-04725, PMO-09768 and PMO-06230 were what

greater increases in saccharification capacity provided when added, separately or in synergistic combinations, as a supplement to mixtures

or enzymatic cocktails produced by M. thermophila (see figures 1, 2, 3 and 27).

20 For example, the increase in saccharification capacity produced by the PMO-06230 polypeptide, responsible for an increase of more than 10% in the production capacity of glucose from cellulosic biomass by the enzyme cocktail produced by M. thermophila (Fig. 2 and 27); And for the PMO09768 polypeptide, responsible for a more than 10% increase in production capacity

25 glucose from cellulosic biomass by an enzymatic cocktail produced by M. thermophila or an enzymatic cocktail comprising the main cellulolytic enzymes (beta-glucosidase, cellobiohydrolases and endoglucanase) of M. thermophila (Fig. 27). Therefore, in a more preferred embodiment, the polypeptide of the invention consists of an amino acid sequence selected from SEO ID NO: 43,

30 SEO ID NO: 44, SEO ID NO: 45 or SEO ID NO: 46.

In addition, as shown in the examples of the present invention, PMO-04725 and PMO-06230 have a synergistic effect in improving hydrolysis efficiency when used in combination in an enzyme cocktail comprising the main cellulolytic enzymes (beta). -glucosidase, cellobiohydrolases and endoglucanase) from M. thermophila. Specifically, Fig. 3 shows that the mixture

of both PMOs in said cocktail yields more glucose than the sum of the effect of improving the hydrolysis of both enzymes used separately in the same cocktail. Therefore, in an even more preferred embodiment, the polypeptide of the invention consists of the amino acid sequence SEO ID NO: 43 or SEO ID NO: 44.

The term "increase" or "increase" as used in the present invention refers to the increase in the yield of a reaction product, for example, of a fermentable sugar, produced when a particular component present during the reaction (such as a PMO polypeptide of the invention) causes increased production

10 of the product compared to a reaction carried out under the same conditions and with the same substrate but in the absence of the component in question.

Due to the degeneracy of the genetic code, in which several nucleotide triplets give rise to the same amino acid, there are several sequences of

15 nucleotides that give rise to the same amino acid sequence. Therefore, another aspect of the invention relates to an isolated polynucleotide, hereinafter "polynucleotide of the invention", which encodes at least one polypeptide of the invention. Another aspect of the invention relates to an isolated polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of the invention.

The terms "nucleotide sequence", "nucleotide sequence", "nucleic acid",

"oligonucleotide" and "polynucleotide" are used interchangeably herein and are

refer to a polymeric form of nucleotides of any length that may be

or not, chemically or biochemically modified. They refer, therefore, to any

25 polyiribonucleotide or polydeoxyribonucleotide, both single stranded and double stranded. The polynucleotide of the invention can therefore be DNA, RNA, or derivatives of both DNA and RNA, including cDNA. The polynucleotide of the invention can be obtained artificially by conventional cloning and selection methods, or by sequencing. The polynucleotide, in addition to the

The coding sequence may carry other elements, such as, but not limited to, introns, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, or stabilizing sequences. These polynucleotides can additionally include coding sequences for additional amino acids that may be useful, for example, but not limited to increasing the stability of the peptide.

35 generated from it or allow a better purification of it.

Nucleic acid sequences encoding the polypeptides of the invention can, for example, be designed based on the amino acid sequences provided in the present invention. The nucleotide sequences encoding the immature PMO polypeptides described in the present invention consist,

5 preferably, in: PMO-04725 (SEO ID NO: 1), PMO-06230 (SEO ID NO: 2), PMO09768 (SEO ID NO: 3), PMO-01470 (SEO ID NO: 4), PMO-03248 (SEO ID NO: 5), PMO-00727 (SEO ID NO: 6), PMO-l 0391 (SEO ID NO: 7), PMO-l 0824 (SEO ID NO: 8), PMO-09767 (SEO ID NO : 9), PMO-03778 (SEO ID NO: 10), PMO-04750 (SEO ID NO: 11), PMO-l0518 (SEO ID NO: 12), PMO-05022 (SEO ID NO: 13), PMO- 05366

10 (SEO ID NO: 14), PMO-02839 (SEO ID NO: 15), PMO-l0366 (SEO ID NO: 16), PMO04874 (SEO ID NO: 17), PMO-08101 (SEO ID NO: 18) , PMO-00657 (SEO ID NO: 19), PMO-04859 (SEO ID NO: 20) and PMO-03723 (SEO ID NO: 21). Sequence

Nucleotide coding for the signal peptide of each of the immature PMOs has been identified and is indicated in the respective sequences of the sequence listing.

The polynucleotide of the invention can be introduced into a gene construct, for example, into a cloning vector or expression vector, to allow its replication.

or its expression Preferably, said vector is an appropriate vector for expression.

20 and purification of the polypeptide of the invention. Therefore, another aspect of the invention relates to a gene construct comprising at least one of the polynucleotides of the invention, hereafter referred to as the "gene construct of the invention".

The term "gene construct" as used herein refers to a nucleic acid molecule, both single stranded and double stranded, that is isolated from a gene that occurs naturally or that is modified to contain nucleic acid segments of a way that could not exist in nature. The term "nucleic acid construct" or "gene construct" is synonymous with the term

"Expression cassette" when the nucleic acid construct contains the control sequences required for the expression of the polynucleotide of the invention. Thus, the genetic construct of the invention may further comprise one or more control or regulatory sequences of gene expression, such as promoter sequences, leader sequences, transcription terminator sequences, sequences.

35 polyadenylation, signal sequences, etc.

The term "control sequences" includes all the components that are necessary or advantageous for the expression of the polynucleotide of the present invention. Every control sequence may be of the same or different origin as the polynucleotide of the invention. Such control sequences include, but are not limited to, a sequence. 5 leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator sequence. How minimum, control sequences include a signal peptide sequence, and more preferably also a promoter, and transcription termination signals and the translation. Control sequences can also be provided with linkers with

10 in order to introduce specific restriction sites that facilitate the binding of the control sequences with the coding region of the polypeptide of the invention. Appropriate control sequences for the expression of a polynucleotide in eukaryotic cells are known in the state of the art.

As used herein, the term "promoter" refers to a nucleotide sequence, generally "upstream" or "upstream" of the transcription start point, which is capable of initiating transcription in a cell. This term includes, for example, but not limited to, constitutive promoters, specific cell-type promoters and inducible or repressible promoters. In general, the

20 control sequences depend on the origin of the host cell.

In a preferred embodiment, the gene construct of the invention is an expression vector. An "expression vector" is a linear or circular DNA molecule that comprises at least one polynucleotide of the invention, and that binds in a manner

Operative to additional nucleotides that are provided for expression. Said vector comprising the polynucleotide of the invention can be introduced into a host cell such that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector.

The term "operably linked" refers to a configuration in which a control sequence is placed in a suitable position with respect to the polynucleotide coding sequence of the invention, such that the control sequence directs the expression of said polynucleotide.

35 When creating the expression vector, the coding sequence is located in the vector such that it is operably linked to the control sequences suitable for its expression. Thus, the expression vectors referred to in the present invention comprise the polynucleotide of the invention, a promoter, and transcription and translation termination signals. The various nucleic acids and control sequences described herein can be linked together to produce

5 a recombinant expression vector that may include one or more convenient restriction sites to allow insertion or replacement of the polypeptide of the invention at said sites.

The expression vector referred to in the present invention can be any

10 vector (for example, a plasmid or virus) that can be conveniently subjected to a recombinant DNA process and can produce expression of the polynucleotide of the invention. The choice of the vector will normally depend on the compatibility of the vector with the host cell into which it is to be introduced. The expression vector can be a plasmid, a cosmid, a fa90, a virus,

15 an artificial bacterial chromosome (BAC), an artificial yeast chromosome (YAC),

or similar.

The vectors can be closed linear or circular plasmids. The vector can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication. for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into the host cell, integrates into the genome and replicates

25 together with the chromosome (s) in which it has been integrated. In addition, a single vector or plasmid 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 can be used.

The vectors used in the present invention preferably contain one or more selectable markers that allow easy selection of transformed, transfected, transduced cells, or the like. A selectable marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, prototrophy to auxotrophs and the like. Selectable markers

35 for use in a filamentous fungus host cell include, but is not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidid) -5'-phosphate decarboxylase), pyr5, pyr4, cysC (sulfate adenyltransferase), and lrpC (anthranilate synthase), as well as their equivalents.

The vectors referred to in the present invention preferably contain an element (s) that allows the integration of the vector into the genome of the cell host or autonomous replication of the vector in the cell regardless of genome For integration into the genome of the host cell, the vector can be based on the polynucleotide sequence of the invention or any other

10 element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences to direct integration by homologous recombination into the genome of the host cell at a precise location (s) of 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 mediates autonomous replication that works in a cell. He

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 fungus cell are AMA1 and ANS1.

More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the production of gene products / s. An increase in the number of copies of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including with the polynucleotide an amplifiable selectable marker gene, where

The cells containing the amplified copies of the selectable marker gene, and thus, additional copies of the polynucleotide, can be selected by culturing the cells in the presence of the appropriate selection agent.

The methods used to join the elements described above to construct the recombinant expression vectors referred to in the present invention are well known to those skilled in the art.

In a more preferred embodiment, the gene construct of the invention comprises a polynucleotide encoding the pOlipypeptide consisting of SEO ID NO: 43 and another polynucleotide encoding the polypeptide consisting of SEa ID NO: 44.

The gene construct of the invention can be introduced into a host cell competent to carry out the expression of the polypeptide of the invention. Therefore, another aspect of the invention relates to a host cell comprising the gene construct of the invention, "host cell of the invention".

The "host cell", as used herein, includes any type of cell that is susceptible to transformation, transfection, transduction, and the like, with the gene construct of the invention. The host cell can be eukaryotic, such as a mammalian, insect, plant or fungus cell. In a preferred embodiment, the host cell is a filamentous fungus cell. The filamentous fungi are

15 generally characterized by presenting a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. In a more preferred embodiment, the filamentous fungus host cell is an Aeremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Corio / us, Cryptoeoeeus, FiJibasidium, Fusarium, Gibberella, Humieola, Magnaporthe cell,

20 Die, Myeeliophthora, Neoeallimastix, Neurospora, Paeeilomyees, Penieillium, Phaneroehaete, Phlebia, Piromyees, Pleurotus, Sehizophyllum, Talaromyees, Thermoaseus, Thielavia, Tolypoeladium, Trametes, or Triehoderma. In a more preferred embodiment, the filamentous fungus host cell is a cell of Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonieus,

25 Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae. In another more preferred embodiment, the filamentous fungus host cell is a Fusarium baetridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium pseudogram, Fusarium pseudogram, Fusarium

30 Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotriehioides, Fusarium sulphureum, Fusarium lorulosum, Fusarium tricholhecioides, or Fusarium venenalum. In another more preferred embodiment, the filamentous fungus host cell is a Bjerkandera adusta cell, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis

35 gilveseens, Cernoporiopsis pannoeinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Gibberella zeae, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myce , Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, T richoderma

5 Jongibrachiatum, Trichoderma reesei, or Trichoderma viride. In one embodiment yet more preferred, the host cell of the invention is any strain of the species MyceJiophthora thermophiJa. In an even more preferred embodiment, the cell Host of the invention is strain C1 of MyceJiophthora thermophila.

It will be understood that for the aforementioned species, the invention encompasses the perfect and imperfect states, and other taxonomic equivalents, for example, the anamorphs, with respect to 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, MyceJiophthora thermophila is equivalent to Chrysosporium

15 Jucknowense.

The host cell of the invention therefore comprises at least one

polynucleotide of the invention recombinantly introduced by the

genetic construction of the invention. Such polynucleotides can encode the

20 mature polypeptide or a pre-protein consisting of a signal peptide bound to the mature enzyme that will have to be further processed in order to produce the mature PMO enzyme.

The host cell of the invention expresses at least one of the PMO polypeptides

25 of the invention, or any combination thereof, so that these are functional, and is capable of secretaries to the extracellular environment. The term "functional" means that expressed enzymes retain their ability to oxidize cellulose. This activity can be measured by any suitable procedure known in the state of the art to evaluate PMO activity, preferably

30 by means of the procedure described below in the examples of the present invention.

The term "expression" includes any stage involved in the production of the polypeptide of the invention that includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

When the host cell of the invention is Myceliophfhora thermophiJa, preferably M. thermophiJa strain C1, said cell overexpresses at least one of the PMO polypeptides of the invention, since by understanding the gene construct of the invention, it contains more copies of the polynucleotide of the invention. invention of which

5 comprises the genome of the non-transformed cell. Expression use is understood as of PMO "the expression of the PMO polypeptide of the invention above the levels of expression of said polypeptide in the same cell not transformed with the gene construct of the invention.

The expression of the polypeptide of the invention can be carried out in the host cell of the invention by any method known in the art, such as the transformation of a suitable host cell with at least one polynucleotide of the invention, or the genetic construction of the invention, and the culture of the transformed host cell under conditions that induce the

Expression of said polynucleotide in order to obtain the secreted enzyme.

The host cell can be cultured in a suitable nutrient medium, solid or liquid, for the production of PMO, using methods well known in the art. For example, the cell can be cultured by flask culture with agitation, and small-scale or large-scale fermentation (which includes continuous, batch or batch fermentation, discontinuous or fedbatch feeding, or solid state) carried out in a laboratory or industrial bioreactor in a suitable medium and under conditions that allow to express and / or isolate the PMO. The cultivation takes place in a suitable nutrient medium comprising carbon sources

25 and nitrogen and inorganic salts, using methods known in the art. If PMO is secreted in the nutrient medium, it can be recovered directly from the medium.

The expressed PMO can be detected using procedures known in the

30 specific techniques for polypeptides. These detection procedures may include the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate.

The resulting PMO can be recovered using procedures known in the art. For example, PMO can be recovered from the nutrient medium by conventional procedures that include, but are not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

The PMO's produced in the present invention can be purified by a

A variety of methods known in the art that include, but are not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatophocalization, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), solubility differential (for example, precipitation in ammonium sulfate), SoS-PAGE, or extraction, in order to obtain

10 a substantially pure PMO that can be included in an enzymatic composition together with other cellulolytic enzymes.

The host cell of the invention can express at least one of the polypeptides of the invention, or any combination thereof, for example

15 but not limited to, polypeptides consisting of SEO ID NO: 43 and SEO ID NO: 44. Likewise, said cell can in turn express one or more other cellulolytic enzymes, native or recombinant.

Another aspect of the invention relates to an enzymatic composition comprising

At least one of the polypeptides of the invention, hereafter referred to as "composition of the invention". This composition of the invention may further comprise other enzymatic activities, such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, such as endoglucanases, betaglucosidases and / or cellobiohydrolases; chitinase, cutinase, cyclodextrin

25 glucosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, betagalactosidase, glucoamylase, alpha-glucosidase, haloperoxidase, invertase, lacasa, lipase, mannosidase, oxidase, reductase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phylasease, polyase, transaminase, polyamase, transaminase, polyase, transferase or xylanase, or any of its combinations. The additional enzyme (s) are

30 can (n) produce, for example, by a microorganism belonging to the genus Aspergillus, such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumiga tus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae; Fusarium, such as Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,

35 Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium pseudograminearum, Fusarium reticulatum, Fusarium roseum,

Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium loruloseum, Fusarium tricholhecioides, or Fusarium venenatum; Gibberella, such as Gibberella zeae; Humicola, such as Humicola inso / ens or Humicola lanuginosa; Trichoderma, such as Trichoderma harzianum, Trichoderma koningii, Trichoderma

5 Jongibrachiatum, Trichoderma reesei, or Trichoderma viride; or Myceliophthora, such as Myceliophthora thermophila.

In a preferred embodiment, the composition of the invention further comprises other cellulolytic enzymes. The term "cellulolytic enzymes" also known as "cellulases" refers to a class of enzymes capable of hydrolyzing cellulose (bonds of 3-1,4-glucan or 3-o-glucosides) or hemicellulose to oligosaccharides shorter, cellobiose and / or glucose. Examples of cellulolytic enzymes are, but are not limited to, endoglucanases, beta-glucosidases, cellobiohydrolases, polysaccharide monooxygenases (other than those described in the present invention), beta15 xylosidases, endoxyglucanases or endoxylanases. Thus, in a more preferred embodiment, these cellulolytic enzymes are selected from the list consisting of endoglucanases, beta-glucosidases, cellobiohydrolases, polysaccharide monooxygenases, beta-xylosidases, endoxylanases, endoxyglucanases, or any combination thereof. These cellulolytic enzymes can be derived from the cell

20 host of the invention or other microorganisms producing cellulolytic enzymes other than the host cell of the invention. They can also be produced naturally or recombinantly.

In a preferred embodiment, the composition of the invention comprises a polypeptide.

25 of the invention consisting of the amino acid sequence SEa ID NO: 43 and a polypeptide of the invention consisting of the amino acid sequence SEO ID NO: 44.

Preferably, the composition of the invention comprises at least one polypeptide of the invention, more preferably the pOlipypeptide consisting of SEO ID NO: 43 30 and the polypeptide consisting of SEO ID NO: 44, and other cellulolytic enzymes derived from the host cell of the invention. In an even more preferred embodiment, the composition of the invention is an enzymatic mixture expressed by the host cell of the invention. In an even more preferred embodiment, the composition of the invention is an enzymatic mixture obtained by the cell.

Host of the invention, preferably M. thermophila of strain C1.

The term "endoglucanase" or "EG" refers to a group of cellulase enzymes

classified as E.C. 3.2.1.4. These enzymes hydrolyse the ~ 1, 4 internal glucosidic bonds of cellulose.

5 The term "cellobiohydrolase" refers to a protein that catalyzes the hydrolysis of Cellulose to cellobiose through an exoglucanase activity, releasing sequentially cellobiose molecules from the reducing or non-reducing ends of cellulose

or the celooligosaccharides.

10 The term "beta-glucosidase", as used herein, refers to an enzyme that catalyzes the hydrolysis of a sugar dimer, including, but not limited to cellobiose, with the release of a corresponding sugar monomer, used , but not limited, for the synthesis of ethanol. The enzyme beta-glucosidase acts on the bridges \ 31-> 4 that bind two molecules of glucose or substituted glucose (it is

15 say, the cellobiose disaccharide). It is an exocellulase with specificity for a variety of beta-D-glycoside substrates. It catalyzes the hydrolysis of non-reducing terminal residues in beta-D-glycosides with glucose release.

The term "endoxylanase" refers to an enzyme that catalyzes the endohydrolysis of 1, 4-beta-D-xylosidic bonds in xylanes.

The term "p-xylosidase" refers to a protein that hydrolyzes 1,4-¡-3-D-xylo-oligomers short to xylose.

The term "endoxyglucanase" refers to a specific xyloglycan enzyme, capable of catalyzing the solubilization of xyloglycan in oligosaccharides but which does not show substantial cellulolytic activity.

In another preferred embodiment, the composition of the invention comprises at least one

30 of the polypeptides of the invention, more preferably the polypeptide consisting of SEO ID NO: 43 and the polypeptide consisting of SEO ID NO: 44, and a cellulolytic mixture consisting of: endoglucanase, beta-glucosidase, cellobiohydrolase I and cellobiohydrolase 11. More preferably, these cellulolytic enzymes are derived from M. Ihermophila.

The composition of the invention can be prepared according to the procedures known in the art and can be in liquid form or a dry composition. For example, the composition may be in the form of a granulate or a microgranulate. The enzymes to be included in the composition may

5 stabilize according to the procedures known in the art.

As indicated above, the host cell of the invention expresses

at least one PMO polypeptide of the invention, which is capable of oxidizing cellulose

when it is secreted to the extracellular environment. This host cell is capable of

10 secrete this enzyme (s) to the medium together with other cellulolytic enzyme / s produced naturally or recombinantly, thus being useful for optimizing the hydrolysis stage of the biomass in fermentable sugars.

Therefore, another aspect of the invention relates to the use of the host cell of the invention, of at least one polypeptide of the invention, or of the composition of the invention, for the degradation of cellulosic biomass.

The term "biomass" means the biodegradable fraction of products, residues and residues of biological origin from agriculture (including substances

20 plants, 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 waste of paper. In a preferred embodiment, the biomass is straw or the organic solid waste fraction

25 urban. In a more preferred embodiment, the biomass is plant biomass, more preferably selected from the list consisting of: biomass rich in fermentable sugars, such as sugar cane; starch biomass, for example, grains or wheat straw; or corn or corn straw or corn grain or corn fiber; or grains or barley straw; or grains or sorghum straw. Biomass can also be,

30 rice, grass, branches, etc.

The polypeptide of the invention, as well as the host cell or the composition of the present invention, can be used in the production of monosaccharides, disaccharides and polysaccharides as chemical or fermentation raw materials, from

35 biomass for the production of ethanol, butanol, plastics, alkanes, alkenes, or other products or intermediates.

The host cell of the present invention can be used as a source of the

pOlipypeptide of the invention, and other cellulolytic enzymes, in a process of

fermentation with biomass.

5 The predominant polysaccharide in the primary cell wall of plant biomass is cellulose, the second most abundant is hemicellulose, and the third, depending on the The biomass in question can be pectin. The secondary cell wall, produced after the cell has stopped growing, it also contains polysaccharides and is reinforced by polymeric lignin covalently

10 crosslinked with hemicellulose. Cellulose is a homopolymer of anhydrocellulose and is thus a linear beta- (1-4) -D-glucan, while hemicellulose includes a variety of compounds, such as xii years, xyloglucans, arabinoxylans, and mannans in branched structures complex with a range of substituents. Although generally polymorphic, cellulose is found in plant tissue

15 mainly as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses normally bind by hydrogen bonds to cellulose, as well as to other hemicelluloses, which helps stabilize the cell wall matrix. The polypeptides of the invention can be used together with the rest of cellulolytic enzymes to degrade the cellulose component of the substrate of the substrate.

20 biomass

Degradation or hydrolysis of biomass to fermentable sugars, known process

also as "saccharification", by means of the polypeptide of the invention, the cell

host of the invention or the composition of the invention, may be followed by a

The fermentation process in which the fermentable sugars obtained are used in order to finally obtain a bioproduct such as bioethanol.

Thus, another preferred embodiment of this aspect of the invention relates to the use of at least one pOlipypeptide of the invention, of the host cell of the invention or of the composition of the invention, for the degradation of biomass in a Production process of a bioproduct.

The term "bioproduct" or "biobased products" refers to those materials, chemicals and energy derived from renewable biological resources. Examples 35 of these bioproducts are, but are not limited to, hydrocarbon compounds in their different forms, such as aliphatic compounds (saturated, unsaturated, cyclic)

or aromatic, such as alkanes, alkenes, alkynes, cyclic forms of these compounds

or aromatic hydrocarbons; oxygenated substances such as alcohols (such as ethanol, butanol, sorbitol), ethers, aldehydes, ketones or carboxylic acids; substances nitrogenated as amines, amides, nitro compounds or nitriles; substances 5 halogenated as halides; organic acids (such as lactic acid, acrylic acid, acetic acid, succinic acid, glutamic acid, citric acid or propionic acid). He term "bioproducts" also includes any combination of the compounds described above, compounds further derived from the compounds described above by any type of physical, chemical or

Biological, polymers of the compounds described above, compounds described above substituted by any group or functional element in one more of its bound and branched forms of the compounds described above.

Ethanol can be produced by enzymatic degradation of biomass and

15 conversion of the saccharides released 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 fuel substitute).

In a more preferred embodiment, the bioproduct is biofuel. The term

"Biofuel", as used herein, refers to a hydrocarbon, or one of its mixtures, which can be used as fuel and is obtained using fermentable biomass as a starting material. Examples of biofuels include, but are not limited to, ethanol or bioethanol, butanol or biobutanol and biodiesel. In a more preferred embodiment, the biofuel is bioethanol.

The term "bioethanol" refers to an alcohol prepared by fermentation, to

from fermentable biomass such as carbohydrates produced in crops

of sugar or starch such as corn or sugarcane.

In another aspect, the present invention relates to a process for producing fermentable sugars, referred to herein as "first process of the invention", comprising:

a) incubating cellulosic biomass, preferably pretreated biomass, with the composition of the invention, and b) recovering the fermentable sugars obtained after the incubation of step (a).

Frequently a biomass pretreatment process is required to increase the access of enzymes to their substrates and consequent effective hydrolysis. Pretreatment uses various techniques, which include, but are not limited to chemical treatment (e.g., explosion of the fiber with ammonium or exposure to a

5 solvent), physical treatment (for example, steam explosion at high temperatures), mechanical treatment (for example, crushing or grinding), treatment biological, or any combination thereof, to alter the biomass structure cellulosic and make cellulose more accessible.

10 The term "fermentable sugar" as used herein refers to simple sugars (monosaccharides, disaccharides and short oligosaccharides), such as glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose or fructose, among others. A fermentable sugar is any that can use or ferment a microorganism.

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

a) incubating cellulosic biomass, preferably pretreated biomass, with the composition of the invention, 20 b) fermenting the fermentable sugars obtained after the step of incubation of step (a) with at least one fermenting microorganism, and c) recovering the bioproduct obtained after of stage fermentation

(b)

The term "fermenter 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 into ethanol. The microorganisms used in this way are fermenting microorganisms that have fermentation capacity, such as yeasts of the genera

30 Saccharomyces, Pichia or Kluyveromyces, preferably Saccharomyces cerevisiae, both natural and genetically modified strains for the conversion of pentoses.

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 fermentation.

of step (b) of the second process of the invention. Recovery can be performed by any procedure known in the art, including mechanics or manuals.

In some embodiments, the first and / or second process of the invention preferably comprises a pretreatment process before step (a). In general, a pretreatment process will result in the cellulosic material components being more accessible for later stages or more digestible by enzymes after treatment in the absence of hydrolysis. He

Pretreatment may be a chemical, physical, mechanical or biological pretreatment, or any mixture thereof.

Before (ie in stage (a »and / or simultaneously with the fermentation of the stage

(b) of the second method of the invention, biomass, preferably biomass

Pretreated, it is hydrolyzed to degrade cellulose and hemicellulose into sugars and / or oligosaccharides. The solids content during hydrolysis may be, but not limited to, between 10-30% of the total weight, preferably between 1525% of the total weight, more preferably between 18-22% of the total weight. Hydrolysis is performed as a process in which biomass, preferably pretreated biomass,

20 is incubated with at least one polypeptide of the invention, with the host cell of the invention or with the composition of the invention and thus form the hydrolysis solution. The appropriate process time, temperature and pH conditions can easily be determined by one skilled in the art. Preferably, said hydrolysis is carried out at a temperature between 25 oC and 60 oC, preferably between 40 oC and 60 oC,

25 specifically around 50 oC. The process is preferably performed at a pH in the range of 4-6, preferably pH 4.5-5.5, especially around pH 5.2. Preferably, the hydrolysis is carried out in a time 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) of the second method of the invention) can be carried out simultaneously (SSF process) or sequentially (SHF process). According to the invention, hydrolyzed, and preferably pretreated, biomass 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 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 oC and 40 oC, preferably from 26 oC to 34 oC, in particular around 32 oC. In other

5 preferred embodiment, the pH is 3 to 6 units, preferably 4 to 5. Preferred for the ethanolic fermentation a yeast of the species Saccharomyces cerevisiae, preferably strains that are resistant to high levels of ethanol, up to, by example, 5 or 7% in vol. of ethanol or more, such as 10-15% in vol. of ethanol

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

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or

15 steps For those skilled in the art, other objects, advantages and features of the invention will be derived 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.

20 DESCRIPTION OF THE FIGURES

Figure 1. Production of glucose in biomass hydrolysis process led to carried out by different enzymatic mixtures. The yields of the complete enzyme mixture produced by M. were compared.

25 Ihermophila (Complete cocktail, CC), the mixture of purified enzymes (endoglucanase, cellobiohydrolase 1, cellobiohydrolase 11 and Betaglucosidase) called minimum cocktail (CM), and said minimum cocktail supplemented with the enzyme PMO-06230. Dosages were made in mg of protein per 9 of cellulose or glucan. Analytical methods for the determination of glucose, cellulose and protein are described.

30 in Example 6.

Figure 2. Supplementation of the complete cocktail produced by M_ thermophila with different amounts of PMO-06230. The yields of the complete enzyme mixture produced by M. were compared.

35 Ihermophila (CC) and supplemented with different doses of purified enzyme PMO-06230. The full cocktail dose controls allow the comparison of the performance of the whole cocktail and it supplemented with the same dose. The experimental conditions in which there was addition of enzyme PMO-06230 are indicated in the figure with weft bars. Dosages were made in mg of protein per 9 of cellulose or glucan. Analytical methods for the determination of

5 glucose, cellulose and protein are described in Example 6.

Figure 3. Synergistic effect between different polysaccharide monooxygenases.

The yields of the purified enzyme mixture (endoglucanase,

cellobiohydrolase 1, cellobiohydrolase 11 and betaglucosidase) called minimal cocktail

10 (CM) And of said minimum cocktail supplemented with the enzymes PMO-04725, PMO06230 and a mixture of both. Dosages were made in mg of protein per g of cellulose or glucan. The analytical methods for the determination of glucose, cellulose and protein are described in Example 6.

15 Figure 4. Expression vector pBASES-K4. The vector carries Pcbh1 as promoter sequence, Tcbh1 as termination sequence and pyr4 as a selection marker in fungi. The propagation in E. coli was selected by kanamycin resistance. Ndel and EcoRI or EcoRV were the restriction sites chosen to clone the PMOs.

Figure 5. Plasmid for overexpression of PMO-01470.

Figure 6. Plasmid for overexpression of PMO-03248.

25 Figure 7. Plasmid for overexpression of PMO-00727.

Figure 8. Plasmid for overexpression of PMO-09768.

Figure 9. Plasmid for overexpression of PMO-10391.

Figure 10. Plasmid for overexpression of PMO-10824.

Figure 11. Plasmid for overexpression of PMO-09767.

Figure 12. Plasmid for overexpression of PMO-03778.

Figure 13. Plasmid for overexpression of PMO-06230. Figure 14. Plasmid for overexpression of PMO-04750. 5 Figure 15. Plasmid for overexpression of PMO-10518. Figure 16. Plasmid for overexpression of PMO-05022. Figure 17. Plasmid for overexpression of PMO-05366. 10 Figure 18. Plasmid for overexpression of PMO-02839. Figure 19. Plasmid for overexpression of PMO-04725. 15 Figure 20. Plasmid for overexpression of PMO-10366. Figure 21. Plasmid for overexpression of PMO-04874. Figure 22. Plasmid for overexpression of PMO-08101.

Figure 23. Plasmid for overexpression of PMO-00657. Figure 24. Plasmid for overexpression of PMO-04859. 25 Figure 25. Plasmid for overexpression of PMO-03723. Figure 26. Separation and identification of the main cellulases and PMOs of the invention by polyacrylamide gel electrophoresis (SOS-PAGE, 7.5%).

A. Main cellulases produced by M. thermophila. Lane 1: Molecular weight marker 30; Lane 2: full cocktail; Lane 3: minimum cocktail of purified cellulases from

M. thermophila. B. Monoxygenase of purified polysaccharides (PMOs). Lane 1: Molecular weight marker; Lane 2: PMO-04725; Lane 3: PMO-06230; Lane 4: PMO-09768; Lane 5: PMO-01470; Lane 6: PMO-04750.

Figure 27. Effect on saccharification on ground biomass of supplementation

of the minimum cocktail and the complete cocktail of M. thermophila with the different

PMO

The yields of the whole enzyme mixture (striped bars) were compared.

5 produced by M. thermophila (CC) and the mixture of purified enzymes (endoglucanase, cellobiohydrolase 1, cellobiohydrolase II and betaglucosidase) called minimum cocktail (CM) represented by empty bars; both with and without Supplementation with 2 mg protein / g glucan from different purified PMOs. The Analytical methods for the determination of glucose, cellulose and protein, are

10 described in Example 6.

EXAMPLES

Example 1. Comparative functional evaluation of the mixture of purified enzymes 15 (minimum cocktail) and the complete enzyme mixture (complete cocktail) produced by M.

lhermophila

The enzymatic mixture produced by M. thermophila (complete cocktail) was obtained according to the protocol described by Verdoes et al., 2007, (Ind. Biolechnol. 3 (1 »and Visser 20 al., 2011, (Ind. Biolechnol. , 7 (3 », using for this the development of an industrial enzyme production platform based on the organism M. thermophila C1 by Dyadic Netherlands. This enzymatic mixture or complete cocktail (CC) was used as a source for the purification of the different Enzymes that, mixed in the same original proportion, constituted the so-called minimum cocktail (CM).

Following the protocol subsequently described in Example 4, an endoglucanase enzyme (4-Beta-D-glucan 4-glucanhydrolase, EC 3.2.1.4), two cellobiohydrolase or exo-Beta-Glucanase enzymes (EC 3.2.1.9.1) was purified ) and a Beta-glucosidase enzyme (EC 3.2.1.21).

30 These enzymes act synergistically in cellulose hydrolysis, being primarily responsible for the release of glucose in the biomass hydrolysis process (Ekperigin, M.M., 2007, African Journal of Biotechnology. 6). However, recent work has shown the involvement of other non-hydrolytic auxiliary enzymes in the production of glucose from lignocellulosic materials, highlighting

Among them are the lyso enzymes monooxygenase polysaccharides (PMOs), previously called GH61 (Zifcakova et al., 2012, Fungal Ecology, 5). In the present invention, these enzymes have been identified in the genome of M. Ihermophila (see Example 2), purified and evaluated in the biomass hydrolysis process described later in Example 6. This same evaluation procedure in ground biomass hydrolysis was used to compare performance

5 of the complete enzyme cocktail produced by M. thermophila, the mixture of purified cellulases (minimum cocktail), and this same minimum cocktail supplemented with purified PMO enzyme. The results obtained are shown in Figure 1. The PMO enzyme used was identified by mass spectrometry as PMO-06230 (SEO ID NO: 44) of M thermophi / a.

10 Supplementation of 8.64 mg / g of minimum cocktail with 2 mg / g of PMO-06230 achieved glucose production comparable to that of 10 mg / g of complete cocktail. This result showed the implication of these oxygen enzymes in the efficient production of glucose by hydrolysis of cellulosic material.

15 Moreover, supplementation of the complete cocktail with this enzyme PMO-06230 resulted in very significant increases in glucose production in biomass hydrolysis assays. This is reflected in Figure 2, which compares glucose production by the complete cocktail at different doses and this same cocktail

20 supplemented with increasing amounts of PMO-06230.

Therefore, supplementation of the enzyme mixture produced by M. thermophila with purified PMO enzyme results in a significantly higher yield in biomass hydrolysis. For example, 10 mg / g of complete cocktail supplemented with 4

25 mg / g of PMO-06230 produces approximately a 17% increase in glucose release compared to 14 mg / g of complete cocktail, in the process of hydrolysis of ground biomass described in Example 6 (Fig. 2).

Also as shown in Figure 3, these enzymes have activities

30 complementary and synergistic. The controls without the addition of PMO and dose with the addition of each of the enzymes separately allow to conclude that the mixture of both PMO yields more glucose (30 g / kg) than the sum of both enzymes separately (23g / kg ; 10 g / kg for PMO-04725 and 13 g / kg for PMO-06230) reaching the value of the complete enzyme mixture produced by M. thermophiJa

35 (CC)

These results show the indisputable role of these PMO enzymes in the hydrolysis of biomass and their application in the improvement of the currently available enzyme mixtures.

5 Example 2. Identification of coding sequences for polysaccharide monooxygenases in M. thermophila.

Based on the results of Example 1, it was decided to overexpress all of the genes coding for possible monooxygenase polysaccharide (PMOs) of the 10 M. thermopMla genome for evaluation in the improvement of cellulolytic mixtures. The identification of possible sequences coding for PMOs was carried out by searching for proteins with conserved regions of the monooxygenase polysaccharide type, commonly referred to as family glycosyl hydrolases 61. Thus, 21 sequences were identified in the genome of M. thermophila. In table 1

15 shows the identification numbering corresponding to each sequence of nucleotides and proteins.

Table 1. Identification of the DNA or protein sequences corresponding to each of the PMOs located in the genome of M. thermophila C1. twenty

Acid

Protein sequence of the Protein sequence of the

Denomination

nucleic immature enzyme mature enzyme

SEO ID NO

SEO ID NO

SEO ID NO

PMO-04725 1 22 43 PMO-06230 2 23 44 PMO-09768 3 24 45 PMO-01470 4 25 46 PMO-03248 5 26 47 PMO-00727 6 27 48 PMO-10391 7 28 49 PMO-10824 8 29 50 PMO- 09767 9 30 51 PMO-03778 10 31 52 PMO-04750 11 32 53 PMO-10518 12 33 54 PMO-05022 13 34 55 PMO-05366 14 35 56 PMO-02839 15 36 57 PMO-10366 16 37 58 PMO-04874 17 38 59 PMO-08101 18 39 60

PMO-006S7

19 40 61

PMO-048S9

twenty 41 62

PMO-03723

twenty-one 42 63

Example

3. Overexpression from the sequences codifiers for polysaccharide

monooxygenases in M. thermophila.

s

To carry out the overexpression of the 21 sequences identified in the example

2

be amplified each a of them by PCR using genomic AON of M.

Ihermophila C1 as a mold. The length of the nucleotide sequence (in pairs of

bases, pb) as well

how the weight predicted molecular protein of pre-protein and protein

10

Matures (in KOa) corresponding are shown in Table 2.

Table 2. Length of the AON sequence and predicted molecular sizes of preproteins and mature proteins of each of the PMOs.

Molecular Weight Length of Molecular Weight of Enzyme sequence of mature protein pre-protein

nucleotides (bp) (KDa) (KDa) PMO-0472S 924 26.9 2S, 1 PMO-06230 1316 32.2 30, S PMO-09768 749 24.4 22.7 PMO-01470 1449 31, 5 29.7 PMO-03248 1085 24.0 22.0 PMO-00727 1008 21.5 19.9 PMO-10391 938 24.1 22, S PMO-l 0824 926 24.1 22, S PMO-09767 1029 24.2 22.6 PMO-03778 1034 25.5 23.6 PMO-04750 902 24.8 23.4 PMO-108518 847 24.3 23.0 PMO-OS022 1241 31.4 29.9 PMO-05366 815 26.0 24.0 PMO-02839 1320 23.7 21.8 PMO-10366 1149 28.4 26.1 PMO-04874 1029 34.9 33.0 PMO-08101 1023 35.3 33.2 PMO-00657 1017 35.2 32.9

PMO-04859 737 26.5 24.5 PMO-03723 1395 46.8 44.5

Each genomic sequence encoding PMOs was amplified using the oligonucleotides indicated in Table 3.

Table 3. Oligonucleotides used to amplify each of the genes.

PMO encoders. The cut site is found in the direct oligonucleotides

Nde [yen [os reverses e [EcoR [or EcoRV site.

O [igonuc [direct eotid O [igonuc [reverse eotid]

Gen

SEQ ID NO SEQ ID NO

PMO-04725 64 65 PMO-06230 66 67 PMO-09768 68 69 PMO-01470 70 71 PMO-03248 72 73 PMO-00727 74 75 PMO-l 0391 76 77 PMO-l 0824 78 79 PMO-09767 80 81 PMO-03778 82 83 PMO-04750 84 85 PMO-l 0518 86 87 PMO-05022 88 89 PMO-05366 90 91 PMO-02839 92 93 PMO-l 0366 94 95 PMO-04874 96 97 PMO-08101 98 99 PMO-00657 100 101 PMO-04859 102 103 PMO-03723 104 105

The amplification was performed using iProof High-Fidelity DNA Polymerase (BioRad) by a denaturation cycle at 98 ° C for 30 seconds followed by 35

98 ° C cycles for 10 seconds, 59 ° C for 40 seconds, 72 ° C for 30 seconds and an end cycle of 72 ° C for 10 minutes. The amplified DNA fragments were digested with restriction enzymes Nde 1 and EcoRI in all cases except in the case of PMO-03248 which was digested with Ndel and EcoRV. Each of the DNA fragments was cloned into the expression vector pBASE5K4 previously digested with the same restriction enzymes as the corresponding DNA fragment. The expression vector pBASE5K4 contains the promoter sequence of the cellobiohydrolase 1 gene (Pcbh1), which corresponds to a region of 1796 bp upstream of the cellobiohydrolase 1 gene (cbh1, NCBI Accession number 10 XP_003660789.1) of M Thermophila C1. The expression vector pBASE5K4 also contains the terminator sequence of the M. thermophila C1 cbh1 gene (Tcbh1, which corresponds to a region of 1014 bp downstream of cbh1) and the pyr4 gene as a selection marker (NBCI Access number XP _003666633.1) from the same strain. The pyr4 gene encodes an orotidine-5-phosphate decarboxylase and its expression allows the complementation of uridine auxotrophy in the corresponding auxotrophic M. thermophila C1 host strain (pyr4-). The expression vector pBASE5K4 is shown in Figure 4. The vector and inserts were ligated and the products of the junction were transformed into electro-competent cells of Escherichia coN XL 1 Blue MRF following the protocol provided by the manufacturer (Stratagene).

The recombinant plasmids obtained are shown in Figures 5 to 25.

Plasmids that sing each of the coding sequences for PMOs under the Pcbh1 and pyr4 promoter as a selection marker, were transformed into M. Ihermophila e1 (Verdoes et al., 2007, Ind. Bioleehno !. 3 (1)), the host strain 25 previously used in other high performance selections in M. thermophila. The DNA was introduced into the host strain using a protoplast transformation procedure (US7399627B2). The host strain is auxotrophic (pyr4) so the transformants were plated on agar plates without uridine supplementation. After 5 days of incubation at 35 ° C the transformants were analyzed

30 resulting prototrophs (which express the pyr4 gene). The host strain used as a receptor produces a complete enzyme cocktail that contains, among others, cellobiohydrolase, endoglucanase and beta-glucosidase activity.

The objective of the selection was to identify the PMO activities produced within the complete enzyme cocktail generated by M. thermophila C1 that improved glucose release performance using biomass as a substrate. Therefore the

Transformant assay was based on the cocktail production of each of the

transformants and their evaluation in terms of glucose release from

plant biomass

5 The transformants obtained were inoculated in 96-well microtiter plates (MTP) to carry out their evaluation by fermentation and subsequent biomass hydrolysis (US7794962B2). For the glucose release test from biomass, it was previously processed as adaptation to the test in MTPs. The biomass was suspended at a final concentration of 100 gil in sodium citrate buffer

10 0.1M pH 5.0 with 0.052% sodium azide. After the suspension, it was mixed on a magnetic stirrer for 30 minutes, the pH was adjusted to 5.0 and an additional 30 minutes was kept under stirring. Stirring was maintained during the dispensing of the biomass suspension in the microplates for testing. PMO activity and improvement of saccharification performance were evaluated from 25 IJI of

15 the culture supernatants of each transformant with 75 JI of biomass (prepared as described previously) for 72 hours at 50 ° C and 850 rpm in a 3mm orbit incubator with rotating orbit. The plates were sealed with aluminum adhesive sheets to prevent evaporation. After 72 hours, the saccharification mixture of the MTPs was transferred to MTPs with filter and centrifuged 1 min at 1800xg and 4 ° C. The

The amount of glucose released was measured by GOPOD test (K-Gluc, Megazyme) following the manufacturer's specifications.

Example 4. Purification of Polysaccharide Monoxygenases (PMOs) and cellulases from

M. thermophila.

The enzymatic mixture produced by M. thermophifa was obtained according to the protocol described by Verdoes et al., 2007, (lnd. Biotechnol. 3 (1 »and Visser et al., 2011, Ind. Biotechnol., 7 (3) , using the industrial enzyme production platform based on the organism M. lhermophiJa C1 This enzymatic mixture was the

30 source of purification of the main cellulases, cellobiohydrolases (Cbh), endoglunase (Eg) and Beta-Glucosidase (Bgl) and of all polysaccharide oxygenases (PMOs) that were functionally evaluated.

The PMOs were purified using a sequential chromatographic process. The original sample (enzymatic mixture produced according to the above description) was subjected to a pre-treatment process, consisting of a centrifugation at 16000

rpm for 40 min at 5 oC. The pellet was discarded and the supernatant was filtered with sterile 0.45 IJm nylon filters (VWR). The resulting sample was desalted in 15 mL fractions, using a HiPrep Desalting Sephadex G-25 column (GE Helthcare) desalting column, using 100 mM equilibration buffer

5 Tris-HCI buffer, pH 7.0 Y at a flow rate of 10 mL min · 1. With this desalting process, in addition to reducing the saline content, the pigments in the sample were eliminated in large percentage, a loss that facilitates the subsequent purification process of the different enzymes.

10 With this desalinated sample, the purification process of the different PMOs began. First, an ion exchange chromatography was performed using the HiLoad 26/10 Q-Sepharose HP column (GE Helthcare) column, using 100 mM Tris-HCI buffer, pH 7.0, as an elution buffer as equilibration buffer. 100 mM Tris-HCI buffer, pH 7.0, 1M NaCI and a flow rate of 4 mL min-1 • The

Sample elution was performed by applying a 0-30% NaCI salt gradient. Fractions for purification of the different PMOs were selected for their size, looking for proteins with a molecular mass of less than 50 kDa (Table 2). The fractions selected from the first chromatographic step were subjected to a second step using a hydrophobic chromatography. The samples were

20 conditioned with (1 M NH4 hS04, filtered using 0.45 IJm sterile filters (VWR) and applied on a HiLoad 26/10 Phenyl-Sepharose High Performance column equilibrated with 100 mM Phosphate-sodium buffer, pH 7.0, 1M (NH4) 2S04 After the sample was applied, the column was washed with the equilibration buffer and the different retained proteins eluted by applying a 100-0% saline gradient.

25 (NH4) 2S04. Chromatography was followed at all times by performing polyacrylamide gels (12%). Fractions containing samples with proteins of sizes between 20--45 kDa were tested with the enzymatic methods described below to measure endoglucanase, cellobiohydrolase or Beta-Glucosidase activity to exclude fractions containing these

30 enzymes Subsequently, the fractions were concentrated using 10 kDa vivaspin ultracentrifugation and concentration columns (Amicon) and applied in a

or several rounds through the HiLoad 26/600 Superdex 75 pg molecular exclusion column, using 100 mM sodium phosphate equilibration buffer, pH 7.0. This purification procedure allowed to isolate with a higher degree of purity

35 to 90% different PMOs.

For the purification of the PMO-04750 after passing through the hydrophobic column, a group of fractions was selected, desalted using the HiPrep Desalting Sephadex G-25 column (GE Helthcare) desalting column, as a 100 mM Tris equilibration buffer -HCI buffer, pH 7.0 And a flow rate of 10 mL min-1 • 5 This new sample was passed through the HiLoad 26/10 ion exchange column QSepharose HP column (GE Helthcare), using as equilibration buffer 100 mM Tris-HCI, pH 7.0, as elution buffer 100 mM Tris-HCI buffer, pH 7.0, 1M NaCI and a flow rate of 4 mL min-1_ A set of non-retained column fractions were concentrated and applied to a HiLoad molecular exclusion column

10 26/600 Superdex 75 pg using 100 mM Phosphate Sodium buffer, pH 7.0.

A purification procedure analogous to the previous one was applied for the purification of the main M. thermophila cellulases subsequently used for the formulation of the minimum cocktail, enzymatic mixture on which the functional characterization of the different PMOs was carried out by a process of Enzymatic hydrolysis on ground biomass. For the purification of the main cellulolytic enzymes, that is, Cellobiohydrolases (Cbh), Endo-Glucanase (Eg) and BetaGlucosidase (Bgl), the enzymatic activity of each of them was followed in the 20 different fractions obtained. Additionally all were identified by mass spectrometry. All activities were determined under the enzymatic hydrolysis process conditions (pH 5.5 and 50 OC). The B91 activity was determined using p-Nitrofenyl-Beta-D-Glucopyranoside (pNGP, Sigma) as a substrate, following the appearance of the p-nitrophenol product at 410 nm_ The reaction mixture (1 mL final volume) contained 100 I- Imol of sodium acetate buffer (pH 5_0), 100 IJg pNGP (0.33 IJmol) and an appropriate amount of purified enzyme_ This mixture was incubated at 50 oC for 10 min_ The amount of p-nitrophenol released was measured at ~ 10 (E 410 = 15.2 mM-1 cm -1) after stopping the reaction with 100 I-Ig sodium carbonate. A unit of enzymatic activity is defined as the amount of enzyme that is capable of hydrolyzing 1 IJmol p-nitrophenol per minute. The Cbh activity of the different cellobiohydrolases, Cbhl and Cbhll (both contain cellulose binding domains) was measured as the production capacity of cellobiose using crystalline cellulose substrate (Avicel). The reaction mixture (1 mL final volume) contained 100 µMmol of sodium acetate buffer (pH 5.0), 10 mg Avicel and the appropriate amount of purified enzyme, and was incubated at 50 ° C for 120 min_ The amount of cellobiose produced during this reaction was determined by HPLC (Agilent

Technologies, 1200 Series) using a refractive index detector (RID) and a Aminex HPX-87 H column. An enzyme activity unit was defined as the amount of enzyme that is capable of producing 1 I-Imol of cellobiose per minute. The Endoglucanase activity was determined using azo-carboxymethyl cellulose (Azo5 CMC, S-ACMC, Megazyme). The enzymatic method used was an adaptation of the procedure described by the substrate provider, in which the test conditions at enzymatic hydrolysis (pH 5.5 and 50 OC). Activity Endoxylanase was determined using wheat arabinoxylan, "azo-wheat arabionoxylan "(Azo-WAX Megazyme). Again, the enzymatic method used was

10 an adaptation of the procedure described by the substrate supplier, in which the test conditions were adjusted to those of enzymatic hydrolysis (pH 5.5 and 50 OC).

The total protein content of the samples with purified enzymes was determined by the BCA method (The Thermo Scientific Pierce BCA Protein Assay Kit). All purified enzymes were identified by mass spectrometry (MALOI-TOF) and sequencing.

The main purified enzymes of M. thermophila (Bgl, Cbhl, Cbhll and Eg) were mixed in the proportions in which they are in the complete cocktail for the so-called minimum cocktail. A comparison between the complete and minimum cocktails is shown in Figure 26 A, where it is appreciated that both samples contain the same amount of total protein (20 I-Ig) and are applied on a 7.5% polyacrylamide gel (SOS-PAGE ). The different purified PMOs (8-10 ~ g) were applied in the same type of gels (Figure 26 B). The differences in electrophoretic mobility with the predicted molecular mass (Table 2) are due to the fact that the fungal extracellular enzymes are subject to glycosylation (post-translational addition of N-glycans and 0glycans), this being the most frequent post-translational modification in this type of protein (Oeshpande el al., 2008, Glycobiology 18 (8)), which significantly alters its molecular mass. These analyzes indicated that purity

The electrophoretic samples were suitable for the studies in which they were used.

Example 5. Determination of oxygenase activity for the detection of PMOs.

For the detection and lack of characterization of the oxygen polysaccharide enzymes of purified polysaccharides, a method for determining oxygenase activity was carried out. This method was based on the ability to reduce artificial oxygen acceptors of external electrons such as cytochrome C. The oxidized and reduced cytochrome C have different absorption coefficients at 550 nm, so its reduction can be measured along the time as an increase

5 of the signal at this wavelength.

For the cytochrome C reductase assay, a reaction mixture (0.5 mL final volume) containing 50 µmol of cytochrome C, 100 µmol of cellobiose, 0.6 nmol of cellobiose dehydrogenase CDH1 of MyceJiophthora thermophila, and an amount was performed

10 appropriate purified enzyme; everything was prepared in 100 mM sodium phosphate buffer pH 7.0. The reaction was followed spectrophotometrically at 550 nm at room temperature for 1 min. As an extinction coefficient to calculate the cytochrome C reductase activity units, 21000 mM-1 cm -1 was used.

15 Table 4 shows the oxygenase activity values determined by this method for some of the purified PMOs.

Table 4. Specific cytochrome C reductase activity of different purified PMOs.

Enzyme (PMO) Activity (U / g)

PMO-04725

12.8

PMO-06230

10.0

PMO-01470

13.7

PMO-09768

14.4

Example 6. Functional characterization of the different PMOs in the enzymatic hydrolysis process.

25 As a substrate for enzymatic hydrolysis, pretreated corn bagasse (or PCS) biomass was used. Pretreatment was performed using a steam explosion system described by Nguyen et al., 1998, Appl. Biochem Biotechnol 70-72, and its compositional analysis was carried out according to the procedures described by NREL in "Standard Biomass Analytical Procedures"

30 (http://www.nrel.gov/biomass/analytical..procedures.htmL). In this analysis it was obtained that the representation of xylan and xylose in biomass was 4, 06% and 11.11% (w / w dry weight) respectively, and that of glucan and glucose 12.24% and 3.61% (w / w dry weight) respectively. In order to be used in hydrolysis and the homogenization of the mixture, the biomass was previously neutralized, adjusting to a pH of 5.5, lyophilized, ground and sieved with a mesh of

5 200 ~ m.

For the enzymatic hydrolysis process, 10 ml tubes were used with 3 g of the reaction mixture consisting of: 631.6 IJg of 95% ground biomass (w / w dry weight), 8.64 mg protein per g of Minimum purified cocktail glucan as described above, 2 mg protein / g glucan of the corresponding purified PMO and the volume of water necessary to adjust the reaction to a proportion of 20% (w / w) total solids. The tubes with the mixture were incubated in a horizontal position for 72 h at 50 oC with a stirring of 150 rpm in a 25 mm diameter orbital incubator (Infors HT). Once the process is done, the glucose content in the

15 samples resulting from the hydrolyzate (slurry) were analyzed with the GOPOD method (KGLUC Kit, Megazyme).

The effect of purified PMOs on the performance of enzymatic hydrolysis was therefore studied by adding them to the mixture of 4 purified enzymes (8gl, Cbhl, 20 Cbhll and Eg), or minimum cocktail (CM), or to the enzyme cocktail produced by M. thermophila

or full cocktail (CC). The results obtained with a series of selected PMO enzymes are shown in Figure 27, where it can be seen that the addition to the minimum cocktail or to the complete cocktail of all the PMOs analyzed meant an increase in the capacity of saccharification with respect to the control, this is, to the

25 glucose production by mixing the cellulases or minimum cocktail and by the full cocktail. The results with PMO-01470, PMO-06230 and PMO-09768 were especially significant, being responsible for a -50% increase in the release of glucose from ground biomass with respect to the control value of the minimum cocktail and full cocktail.

Claims (12)

1. Gene construct comprising a polynucleotide encoding the polypeptide consisting of SEQ ID NO: 43, and another polynucleotide encoding polypeptide 5 consisting of SEQ ID NO: 44. 2. Gene construct according to claim 1, wherein said gene construct is an expression vector. 3. Host cell comprising the gene construct according to any one of claims 1 or 2. 4. Host cell according to claim 3, wherein the host cell is Myceliophthora thermophila C1. fifteen 5. Enzymatic composition comprising a polypeptide consisting of the amino acid sequence SEQ ID NO: 43 and another polypeptide consisting of the amino acid sequence SEQ ID NO: 44 and, preferably, another cellulolytic enzyme selected from the list consisting of: endoglucanase, beta-glucosidase, Cellobiohydrolase, beta-xylosidase, endoxylanase, endoxyglucanase, monooxygenase polysaccharide or any combination thereof. 6. Enzymatic composition according to claim 5, wherein said composition is an enzymatic mixture expressed by the cell according to any one of claims 3 or 4.

7.

Use of the host cell according to any of claims 3 or 4, or of

the composition according to any of claims 5 or 6, for degradation

of cellulosic biomass.

30

8.

Use according to claim 7, wherein the degradation of cellulosic biomass has

place in a production process of a bioproduct.

9.

Use according to claim 8, wherein the bioproduct is a biofuel.

10.

Use according to claim 9, wherein the biofuel is bioethanol. 41

11. Process for producing fermentable sugars comprising the following Steps: a) incubating cellulosic biomass with the composition according to any of the 5 claims 5 or 6, and b) recover the fermentable sugars obtained after the incubation of step (a). 12. Method for producing a bioproduct from cellulosic biomass comprising the following steps: a) incubating cellulosic biomass with the composition according to any of claims 5 or 6, b) fermenting fermentable sugars obtained after incubation of step (a) with at least one fermenting microorganism, and 15 c) recovering the bioproduct obtained after fermentation of step (b). 13. The method according to claim 12, wherein the bioproduct is a biofuel. The method according to claim 13, wherein the biofuel is bioethanol.