WO2017115005A1 - Production of fusion proteins in trichoderma - Google Patents

Abstract

An isolated polypeptide comprising a fusion of a structural protein, e.g. resilin or elastin-like protein, with at least one carbohydrate binding domain and/or with a crosslinking component, e.g. hydrophilin, wherein said polypeptide is produced in a filamentous fungi is provided. Also a nucleic acid sequence encoding such a polypeptide, an expression vector comprising a nucleic acid molecule encoding the polypeptide, an isolated host cell transformed with the expression vector comprising a nucleic acid molecule encoding the polypeptide and a fiber comprising the polypeptide are provided.

Description

PRODUCTION OF FUSION PROTEINS IN TRICHODERMA

FIELD OF THE INVENTION

The invention relates to production of material proteins in filamentous fungi. Especially the invention relates to recombinant production of structural proteins in Trichoderma reesei.

BACKGROUND OF THE INVENTION

Proteins are nature's multiform building blocks, which form a premise for all living functions. Structural proteins are a subgroup of proteins and they have interesting applications in material technology. Biomaterials having novel characteristics have been obtained by combining structural proteins and other biomaterials as well as by mimicking the nature.

The mechanically strong biological structural materials are nanocomposites where the reinforcing hard components are interlinked in balanced ways by soft components which provide sufficient binding for promoted overall strength and stiffness, still allowing energy dissipation upon deformations, such as silk, bone, tendons, mollusk shells, and insect exoskeletons. The choice of suitable components and building blocks is crucial because a binding molecule that binds too strongly may not allow the optimal packing and rearrangements that may be necessary for the overall architecture. In addition, the way that the multiple components are mixed and processed into a material may influence the resulting material properties.

Spider silk is one of the strongest biomaterials known. Dragline silk or major ampullate silk has extraordinary physical properties, such as high tensile strength, toughness and elasticity. The elasticity is circa 35%. Spider dragline silk fiber is constituted of at least two similar proteins called major ampullate spidroin (MaSp) 1 and MaSp2 in N. clavipes and A. diadematus fibroins ADF-3 and ADF-4 in A. diadematus. Both proteins, ADF-3 and -4 belong to the class of MaSp II proteins (major ampullate spidroin II) .

Among the different types of spider silks, draglines are most intensely studied. The island C-terminal domains of spider dragline silk proteins have mainly R-helical structure, and interestingly, both form homodimers. Dimerization of the end domains allows spidroin multimerization independent of the repetitive part. N-terminal domain regulates spidroin assembly, The N-terminal domain is highly soluble, which may be important for spidroin processing, while the C-terminal domain appears to dictate ordered polymerization of the repetitive region.

Spider silk is both biodegradable and biocompatible, which makes it suitable for medical applications. It has various applications, e.g . textile applications and composite materials (parachutes, sails and body armor), medical applications (wound sutures and dressings, membranes, surfaces for cultivated cells, as scaffolds for artificial organs); and as film-forming agents in personal care products (skin and hair care products). However, isolation of silk fibres from the spiders themselves is very demanding. Therefore, heterologous production mainly in E. coli has been applied. There are, however, problems when producing proteins in E. coli, for example protein having disulfide bridges cannot be produced. Machado et al. (2013) disclose silk-elastin-like polymers (SELPs) that are protein- based polymers composed of repetitive amino acid sequence motifs found in silk fibroin (GAGAGS) and mammalian elastin (VPGVG). They developed four novel SELPs, one of them being SELP-59, wherein the elastomer forming sequence poly(VPGVG) was replaced with a plastic-like forming sequence, poly(VPAVG), and combined in varying proportions with the silk motif. They also optimized a simplified production procedure with high yields in E. coli (150 mg/L).

WO 2004/104020 discloses a genetically engineered silk-elastin polymer (SELP47K) that was produced, isolated and purified from E. coli bacteria. Comparative studies of the two major dragline silk proteins of the garden spider Araneus diadematus, ADF-3 and ADF-4, produced in bacteria, revealed that, although their amino acid sequences are rather similar, they display remarkably different solubility and assembly characteristics. While ADF-3 was shown to be soluble even at high concentrations, ADF-4 appeared to be virtually insoluble and self-assembles into filamentous structures under specific conditions.

Production of dragline silk proteins in yeast has been disclosed . Production in Saccharomyces cerevisiae is disclosed f. ex. in patent publication RU 2451023 CI. Production of dragline silk proteins in Pichia pastoris is disclosed in patent publication US 6,268,169 Bl. The methylotrophic yeast Pichia pastoris has also been tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered sizes as a result of gene rearrangements at the time of transformation. Production of spider silk proteins on multiple yeasts has been mentioned in patent publication EP 1919938 Bl, but no experimental data has been presented on any eukaryotic organisms. Patent publication US 20100311645 Al mentions all kinds of hosts, such as Aspergillus, but the only fungal data is the mentioning of the trpC terminator.

WO 2012/104840 discloses a method utilizing cellulose binding domain's (CBD's) ability to form dimers for directing ordered assembly of fibrous proteins such as silk proteins into super-molecular fibrillar structures.

Currently, cost-effective alternatives are needed to replace oil-based polymer materials. Raw materials based on biomolecules are renewable and they have interesting features from the material technological point of view. Especially hybrid materials, wherein different structural components form a coherent whole, are desired, such as silk, nacker, carbon fibre and reinforced concrete. Said materials have incomparable properties compared to their separate components.

Novel efficient production systems are still needed to produce large amounts of material proteins.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to recombinant production of structural proteins, as well as fusion proteins comprising carbohydrate binding domain and/or at least one crosslinking protein in addition to a structural protein in a filamentous fungi. Preferably the protein is produced in T. reesei. The present invention is also directed to recombinant structural proteins and also to fusion proteins comprising carbohydrate binding domain and/or at least one crosslinking protein in addition to a structural protein. The invention is further related to nucleic acids coding for these recombinant structural and fusion proteins, as well as hosts suitable for expressing those nucleic acids. Furthermore, the present invention is directed to a method of producing these material proteins as wells as to the use of these proteins in the field of biotechnology and/or medicine and other industrial fields.

More specifically, the present invention relates to the production of recombinant structural protein based on spider silk proteins in T. reesei. The isolation of spider silk fibres from the spiders themselves is very demanding. Until now, heterologous protein production mainly in E. coli has been applied. Expression of authentic spider silk genes in bacterial hosts is generally inefficient since some parts of the genes contain codons that are not efficiently translated in bacteria. In addition, gene manipulation and amplification by PCR are difficult due to the repetitive nature of silks. At the present invention, the production has been applied for filamentous fungus. Especially, the production has been applied for T. reesei.

The recombinant proteins according to the invention can also be composed of different functional parts, i.e. of structural protein in combination with a carbohydrate binding domain and/or at least one crosslinking protein component. The carbohydrate binding domain part binds strongly to carbohydrates, such as cellulose, while the crosslinking protein is responsible of interlinking and providing suitable energy dissipation. By fusing different proteins and/or proteins and protein domains together bi- and multifunctional proteins are obtained.

The present invention specifically discloses molecules that can be used for example to assemble cellulose nanofibrils. The present invention is a part of a project, wherein an objective is to use mechanically strong native nanocellulose as a basis for producing high performance fiber-like materials. An approach is to assemble nanocellulose with selected protein/peptide molecules that are designed to give the fiber product the functional properties. The fusion protein of the present invention can thus be used to modify the material characteristics of nanocellulose. Obtained fusion proteins can be used for producing nanocomposite materials to be used for example in textile applications and composite materials, medical applications; and as film-forming agents in personal care products.

Also a nucleic acid sequence encoding a polypeptide comprising structural protein or combination of a structural protein and a carbohydrate binding protein domain and/or at least one crosslinking component, wherein said polypeptide comprises one or more sequences selected from the group consisting of 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, SEQ ID NO : 36 and SEQ ID NO : 38 is an object of the invention.

Further object of the invention is an expression vector comprising a nucleic acid molecule encoding a polypeptide comprising structural protein or combination of a structural protein and a carbohydrate binding protein domain and/or at least one crosslinking component. An isolated host cell transformed with the expression vector comprising a nucleic acid molecule encoding a polypeptide comprising structural protein or combination of a structural protein and a carbohydrate binding protein domain and/or at least one crosslinking component is still another object of the invention. A fiber comprising the polypeptide comprising structural protein or combination of a structural protein and a carbohydrate binding protein domain and/or at least one crosslinking component is also an object of the invention.

A Further object of the invention is a composition comprising an isolated polypeptide comprising structural protein or combination of a structural protein and a carbohydrate binding protein domain and/or at least one crosslinking component and a cellulose nanofibril; and optionally further comprising a carrier, diluent or excipient. Also the use of the composition comprising an isolated polypeptide comprising at least one carbohydrate binding domain, a structural protein and a crosslinking protein component and a cellulose nanofibril; and optionally further comprising a carrier, diluent or excipient in a nanocomposite material is an object of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic design of the multidomain constructs that were created with combinatorial Golden Braid cloning system and expressed in T. reesei. CBDI; T. reesei cellobiohydrolase I cellulose binding domain, ELP; elastin like polypeptide, synthetic pentapeptide repeat of amino acids VPGVG, CBDII; T. reesei cellobiohydrolase II cellulose binding domain, cmyc; detection tag, strepll; detection tag, Linkl-4; flexible linker, HFBI; Resilin, Drosophila melanogaster resilin, T. reesei hydrophobin I, His, detection tag.

Figure 2. Expression of ELP-CBD constructs in Trichoderma reesei. a) Total protein staining of SDS PAGE gel from day 3 of culture supernatants. b) Anti-ELP immunoblot from day 3 culture supernatants. Arrows point out the proteins of interest. All the samples were analyzed in duplicates. Figure 3. Purification of of ELP-CBD constructs from T. reesei culture supernatant, a) Schematic diagram of the workflow of inverse transitional cycling (ITC). b) Result of ITC purification of various ELP-CBD proteins. All the samples were analyzed in dublicates. The arrowheads poit out the proteins of interest, c) Chromatorgram of VYDAC reverse phase column purification run. d) SDS-PAGE analysis of the chromatrography fractions. Fractions C10-14 were collected and pooled for further analysis.

Figure 4. Rheological behaviour of nanofibrillated cellulose (NFC) in the presence and absence of CBDI-ELP5-CBDII (pJJJ693) protein.

Figure 5. Expression of Resilin-CBD constructs in Trichoderma reesei . a) Anti-StrepII immunoblot of pMILsl24 and pAW116 constructs form day 4 culture supernantants. b) SDS-PAGE total protein stains and anti-StrepII immunoblots of pHYB37 and pHYB38 constructs from day 4 culture supernantants.

SEQUENCE LISTINGS

SEQ ID NO: l Nucleotide sequence of CBHI signal sequence.

SEQ ID NO:2 Amino acid sequence of CBHI signal sequence.

SEQ ID NO:3 Nucleotide sequence of CBHI carrier.

SEQ ID NO:4 Amino acid sequence of CBHI carrier.

SEQ ID NO:5 Nucleotide sequence of Kex2 cleavage site.

SEQ ID NO:6 Amino acid sequence of Kex2 cleavage site.

SEQ ID NO:7 Nucleotide sequence of CBDI.

SEQ ID NO:8 Amino acid sequence of CBDI.

SEQ ID NO:9 Nucleotide sequence of CBDII.

SEQ ID NO: 10 Amino acid sequence of CBDII.

SEQ ID NO: ll Nucleotide sequence of ELP5.

SEQ ID NO: 12 Amino acid sequence of ELP5.

SEQ ID NO: 13 Nucleotide sequence of ELP10.

SEQ ID NO: 14 Amino acid sequence of ELP10.

SEQ ID NO: 15 Nucleotide sequence of ELP20.

SEQ ID NO: 16 Amino acid sequence of ELP20.

SEQ ID NO: 17 Nucleotide sequence of CMYC.

SEQ ID NO: 18 Amino acid sequence of CMYC.

SEQ ID NO: 19 Nucleotide sequence of StrepII.

SEQ ID NO:20 Amino acid sequence of StrepII.

SEQ ID NO:21 Nucleotide sequence of His-tagi. SEQ ID NO 22 Amino acid sequence of His-tagi.

SEQ ID NO 23 Nucleotide sequence of Resilin.

SEQ ID NO 24 Amino acid sequence of Resilin.

SEQ ID NO 25 Nucleotide sequence of Linker 3.

SEQ ID NO 26 Amino acid sequence of Linker 3.

SEQ ID NO 27 Nucleotide sequence of HFBI.

SEQ ID NO 28 Amino acid sequence of HFBI.

SEQ ID NO 29 Nucleotide sequence of Linker 2.

SEQ ID NO 30 Amino acid sequence of Linker 2.

SEQ ID NO 31 Nucleotide sequence of Linker 1.

SEQ ID NO 32 Amino acid sequence of Linker 1.

SEQ ID NO 33 Nucleotide sequence of Linker 4.

SEQ ID NO 34 Amino acid sequence of Linker 4.

SEQ ID NO 35 Nucleotide sequence of Resilin 1st half.

SEQ ID NO 36 Amino acid sequence of Resilin 1st half.

SEQ ID NO 37 Nucleotide sequence of Resilin 2nd half.

SEQ ID NO 38 Amino acid sequence of Resilin 2nd half.

SEQ ID NO 39 Primer sequence 1.

SEQ ID NO 40 Primer sequence 2.

SEQ ID NO 41 Primer sequence 3.

SEQ ID NO 42 Primer sequence 4.

SEQ ID NO 43 Primer sequence 5.

SEQ ID NO 44 Primer sequence 6. DETAILED DESCRIPTION OF THE INVENTION

It was surprisingly found out that structural proteins, as well as fusion proteins comprising carbohydrate binding domain and/or at least one crosslinking protein in addition to a structural protein, can be produced in a filamentous fungi. Preferably said proteins are produced in T. reesei. Conventionally T. reesei is used as a production host of industrial enzymes, such as enzymes for second generation biofuels and degradation of cellulose, but the use in production of structural proteins has not been known. In industrial processes T. reesei is able to secrete more than 100 g/l target protein into the growth media. The present invention relates to a process of producing a desired protein in a filamentous fungal host cell and particularly in a Trichoderma cell, the process comprising obtaining a filamentous fungal or T. reesei host cell comprising a fusion DNA construct according to the invention and culturing the filamentous fungal or T. reesei host cell under suitable conditions which allow the expression and secretion of the desired protein. In some aspects of this embodiment, the desired protein will be recovered .

By "recombinant proteins" are meant here proteins that are not natural products of an organism.

By "fusion protein" is meant a fusion of at least two parts obtained from separate proteins into a single molecule. Such a fusion protein that is made by combining different parts of unrelated proteins can also be called a chimeric protein. Two basic forms of recombinant structural proteins or fusion proteins comprising structural proteins and/or carbohydrate binding domain and/or one or more crosslinking proteins are produced in the present invention. First form of proteins is secreted. This protein form comprises fusion to a well secreted carrier CBHI. The carrier is then cleaved off in the secretory pathway. By "secretable protein" or "secreted protein" is meant here a protein that is secreted outside of the host cell. The second form of proteins is intracellular protein comprising endoplasmic reticulum (ER) retention signal HDEL. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the his-asp- glu-leu (HDEL) and lys-asp-glu-leu (KDEL) are used for retention of proteins in yeast and animal ER.

Recombinant structural or fusion protein according to the invention is produced both as intracellular protein particles and by secreting proteins to growth media. The yield of intracellular fusion protein is preferably 100 - 1000 mg/l, such as around 500 mg/l.

The term "signal sequence" or "signal peptide" refers to a sequence of amino acids at the N-terminal portion of a protein, which facilitates the secretion of the mature form of the protein outside the cell. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process. Preferably the signal sequence is encoded by a Trichoderma or Aspergillus gene which encodes a CBH. More preferably the signal sequence is encoded by a Trichoderma gene which encodes a CBHI. In further embodiments, the promoter and signal sequence of the heterologous exo-endo cellulase fusion construct are derived from the same source. In some embodiments, the signal sequence is a Trichoderma cbhl signal sequence that is operably linked to a Trichoderma cbhl promoter. In further embodiments the signal sequence has the amino acid sequence of SEQ ID NO : 2 or an equivalent sequence or a sequence having at least 95% identity thereto.

Certain species of fungi, in particular filamentous fungi Trichoderma reesei and Aspergillus niger, are commonly used in biotechnological industry for protein production. The recombinant proteins, either heterologous or homologous, are typically produced under the regulation of promoters of abundantly expressed genes encoding secreted proteins in the fungi, e. g. the promoter of cbhl of T. reesei and the promoter gla of A. niger. T. reesei and A. niger produce homologous hydrolases very efficiently into the culture medium, but the yields of heterologous proteins produced are typically much lower compared to those of homologous proteins. Especially, the proteins originating from distant species e. g . mammalian proteins are produced at a very low level (Archer and Peberdy, 1997, Penttila, 1998). As reasons for the low yields have been suggested inefficient translation and translocation of the polypeptide into the secretory pathway, hindrances in folding and transport of the protein, and low transcript levels of the heterologous gene due to proteases. A "host" denotes here any protein production host selected or genetically modified to produce efficiently a desired product and being useful for protein production for e.g. analytical, medical or industrial use.

The recombinant protein or the fusion protein can be produced in a fungal or yeast host, selected from the group comprising Trichoderma spp, Aspergillus spp. Neurospora spp., Fusarium spp., Penicillium spp., Humicola spp., Tolypocladium geodes, Kluyveromyces spp., Pichia spp., Hansenula spp., Candida spp., Yarrowia spp, Schizosaccharomyces ssp., Saccharomyces spp. and Schizophyllum spp. Preferably the recombinant protein or the fusion protein is produced in a filamentous fungi. Most preferably the recombinant protein or the fusion protein is produced in Trichoderma host.

The host cell of the invention is a filamentous fungus. It is advantageous to use a host cell of the invention in recombinant production of a polypeptide of interest. The cell may be transformed with the DNA construct encoding the polypeptide of interest, conveniently by integrating the DNA construct in one or more copies into the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA construct into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described in the examples below in connection with the different types of host cells.

There has been interest in using yeast and fungal cells to express more complex proteins. Recently, filamentous fungal cells, such as Trichoderma fungus cells, that can stably produce heterologous proteins, and preferably at high levels of expression, have been developed. Preferably, the expression level of several proteases is reduced or eliminated in filamentous fungal cells. The filamentous fungal cell has preferably total protease activity reduced to 49% or less, preferably 31% or less, of the total protease activity of the corresponding parental filamentous fungal cell in which the proteases do not have the reduced activity. Preferably, the filamentous fungal cell comprises mutations that reduce or eliminate the corresponding protease activity of at least three genes encoding endogenous proteases each comprise. According to one preferred embodiment of the invention T. reesei is used as a host for high level production of recombinant and fusion proteins. Suitable hosts are such as disclosed by publications WO/2013/102674 and WO/2015/004241. Protease deficient host strains can thus be used for the production of the proteins according to the invention. T. reesei has an ability to produce several to tens of grams of protein per litre. One advantage of the present invention is that it is possible to produce a large amount of proteins in T. reesei cost-effectively. By the method of the invention, the proteolytic activities of metalloprotease, alkaline protease, serine protease of the subtilisin type and optionally serine protease of the kexin subfamily are significantly reduced, thereby improving the stability and increasing the yield of susceptible protein products synthesized by the host cell of the invention. More specifically, by the method of the invention, the host cell is genetically modified within structural and/or regulatory regions encoding or controlling the metalloprotease, alkaline, serine protease of the subtilisin type and optionally serine protease of the kexin subfamily protease genes.

Therefore, another aspect of the invention provides a method of producing proteins in a host cell of the invention, including heterologous polypeptides, in which the method comprises introducing into said host cell a nucleic acid sequence encoding the protein product of interest, cultivating the host cell in a suitable growth medium, followed by recovery of the protein product. Thus, the host cell of the invention must contain structural and regulatory genetic regions necessary for the expression of the desired product. The nature of such structural and regulatory regions greatly depends on the product and the host cell in question. The genetic design of the host cell of the invention may be accomplished by the person skilled in the art using standard recombinant DNA technology for the transformation or transfection of a host cell (vide, e.g., Sambrook et al., inter alia).

In the scope of the present invention are filamentous fungal cells, such as Trichoderma fungal cells having reduced or no detectable activity of at least three proteases. These proteases include, without limitation, aspartic proteases, trypsin-like serine proteases, subtilisin proteases, glutamic proteases, metalloproteases and sedolisin proteases.

Fifteen aspartic proteases have been identified in Trichoderma fungal cells: pepl (tre74156), pep2 (tre53961), pep3 (trel21133), pep4 (tre77579), pep5 (tre81004), pep6 (tre68662), pep7 (tre58669), pep8 (trel22076), pep9 (tre79807), pepIO (tre78639), pepll (trel21306), pepl2 (trel 19876), pepl3 (tre76887), pepl4 (trel08686) and pepl6 (trel 10490). One trypsin-like serine protease has been identified in Trichoderma fungal cells: tspl (tre73897).

Seven subtilisin proteases have been identified in Trichoderma fungal cells: slpl (tre51365); slp2 (trel23244); slp3 (trel23234); slp5 (tre64719), slp6 (trel21495), slp7 (trel23865), and slp8 (tre58698).

Two glutamic proteases have been identified in Trichoderma fungal cells: gapl (tre69555) and gap2 (trel 06661). Sedolisin-like protease is typically a tppl protease. Other sedolisin-like proteases include, without limitation, sed2 (Tre70962), sed3 (Tre81517), or sed5 (Trel 11838), and homologs thereof.

Five metalloproteases have been identified in Trichoderma fungal cells: mpl (trel22703), mp2 (trel22576), mp3 (tre4308), mp4 (tre53343), mp5 (tre73809).

There exist also other proteases in Trichoderma fungal cells. Sep proteases are serine proteases belonging to the S28 subtype, for example Trichoderma reesei sepl 124051. In addition, two aminopeptidases have been identified in Trichoderma fungal cells: ampl (tre81070) and amp2 (trel08592).

Thus, in the present invention, the expression level of the at least three proteases is reduced or eliminated. In certain embodiments, genes encoding the three proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain embodiments that may be combined with the preceding embodiments, the three protease encoding genes are pepl, tspl, and si pi . In other embodiments, the three protease encoding genes are gapl, si pi, and pepl.

More preferably there is the deletion of 4, 5, 6, 7, 8, 9 proteases. Most preferably of 10 proteases.

On the most preferred embodiment the Trichoderma or closely related species fungal cell has ten protease encoding genes, each of which comprise a mutation that reduces the corresponding protease activity, and the ten protease encoding genes with such mutation are pepl, slpl, gapl, gap2, pep4, pep3, pep5, pep2, sepl, slp8. In such embodiment, the cell may further comprise an additional mutation that reduces or eliminates the protease activity of tspl.

The identification of certain proteases in Trichoderma reesei and corresponding T. reesei cells with deficient protease activity have been described in Examples 1-23 of WO 2013/102674 and Examples 1-38 of WO/2015/004241 which contents are incorporated herein by reference.

An isolated host cell transformed with the expression vector comprising a nucleic acid molecule encoding a polypeptide comprising a structural protein is one aspect of the invention. Another aspect of the invention is a fusion proteins comprising carbohydrate binding domain and/or at least one crosslinking protein in addition to said structural protein.

According to one aspect of the invention, the "structural protein" is selected from the group consisting of resilin, resilin-like proteins, elastin, elastin-like protein, collagen, abductin, byssus, flagelliform silk, dragline silk, gluten high molecular weight subunit, titin, fibronectin, leminin, gliadin, glue polypeptide, ice nucleating protein, keratin, mucin, , and a mixture thereof. More preferably the elastic protein is resilin or elastin and comprises any amino acid sequences selected from the group of amino acid sequences SEQ ID NO: 24, 12, 14, 16. Most preferably the structural protein is resilin. Specific examples of structural proteins according to the invention are silk-elastin-like polymer SELP and engineered ADF4 (eADF4(C16)), which is composed of 16 repeats of module C. The multimerization of amino acid consensus motifs (modules A, Q, and C) derived from the repetitive part of the natural proteins ADF3 and ADF4 lead to the engineered proteins eADF3 and eADF4.

Resilin is an insect structural protein, which exhibits rubber-like elasticity characterized by low stiffness, high extensibility, efficient energy storage, and exceptional resilience and fatigue lifetime. Resilin is able to effectively absorb and discharge mechanical energy. It is a natural extracellular matrix protein and is hydrophilic. Resilin possesses conformational flexibility, repeated contraction/extension cycles in diverse functions (e.g., sound, wing motion). Resilin can be used in protein polymers with tailored materials functions. Resilin contains repetitive hydrophilic sequence (e.g. repeats of GRPSDSYGAPGGGN in fruit fly resilin) that are crosslinked between Tyr residues.

The repetitive part of ADF4 is generally composed of a single conserved repeat unit displaying only slight variations. In engineered ADF4 (eADF4(C16)) these variations were combined and one consensus module termed C has been designed, which was multimerized to obtain the rep-protein Ci6. The engineered ADF4(C16) spider silk protein is mimicking the sequence of the dragline silk protein ADF4 of the spider A. diadematus. The purified monomeric ADF4(C16) strongly interacts with hydrophobic surfaces and particles of poorly water-soluble substances. Based on this effect ADF4(C16) could be used for colloidal stabilization of hydrophobic particles in aqueous environment. Poorly water-soluble substances can also be encapsulated in ADF4(C16) microbeads during self-assembly. From the soluble ADF4(C16) monomers assembly of nanofibers, microbeads and films is possible.

Silk-elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Because of the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. Various hybrid copolymers have been designed that have a similar molecular weight (55 kDa) but different silk to elastin block ratios or different number of silk blocks. One of hybrid copolymers is SELP-59-A with composition (S5E9)9. Also under the scope of the present invention are ELP's, SLP's, CLP's, SLPL's, SLPF's and SELP's families of genetically engineered protein polymers consisting of silk-like blocks, elastin-like blocks, collagen-like blocks, laminin-like blocks, fibronectin-like blocks and the combination of silk-like and elastin-like blocks. The ELP's, SLP's, CLP's, SLPL's, SLPF's and SELP's can be produced in va rious block lengths and compositional ratios. Generally, blocks include groups of repeating amino acids making up a peptide sequence that occurs in a protein. Genetically engi neered proteins are qualitatively distinguished from sequential polypeptides found in nature in that the length of their block repeats can be g reater (up to several hundred amino acids versus less than ten for sequential polypeptides) and the sequence of their block repeats can be almost infinitely complex. Silk-elastin-like polymers (SELPs) are a new class of bioinspired, biologically synthesized copolymers, composed of alternating silk and elastin blocks. A number of novel SELPs consisting of multiple blocks of the silkworm silk consensus sequence GAGAGS in various combinations with a variant (VPAVG) of the natural mammalian elastin repetitive sequence block VPGVG have recently been synthesized and produced SELP-59 which contains five blocks of the silk motif, GAGAGS and nine blocks of the elastin-like sequence.

Silk fibroin and spider silk protein contain amorphous hydrophilic reg ions forming β turns (e.g . GPGXX repeats) and helical structures (e.g . GGX repeats) alternating with β-sheet-rich hydrophobic regions (Ala-rich) that form noncovalent crosslinks.

Structural proteins according to the present invention may comprise repeats of elastic and compact amorphous hydrophobic domains (e.g . repeats of VPGVG) alternating with crosslinked (via Lys residues) hyd rophilic domains.

It is generally preferred to select the dragline silk proteins of orb-web spiders (Araneidae and Araneoids) . More preferably the dragline proteins are derived from one or more of the following spiders : Arachnura higginsi, Araneus circulissparsus, Araneus diadematus, Argiope picta, Banded Garden Spider (Argiope trifasciata), Batik Golden Web Spider (Nephila antipodiana), Becca ri's Tent Spider (Cyrtophora becca rii), Bird-dropping Spider (Celaenia excavata), Black-and-white Spiny Spider (Gasteracantha kuhlii), Black-and-yellow Garden Spider (Argiope aurantia), Bolas Spider (Ordgarius furcatus), Bolas Spiders - Magnificent Spider (Ordgarius magnificus), Brown Sailor Spider (Neoscona nautica), Brown-Legged Spider (Neoscona rufofemorata), Capped Black-Headed Spider (Zygiella calyptrata), Common Garden Spider (Parawixia dehaani), Common Orb Weaver (Neoscona oxancensis), Crab-like Spiny Orb Weaver (Gasteracantha cancriformis (elipsoides)), Curved Spiny Spider (Gasteracantha arcuata), Cyrtophora moluccensis, Cyrtophora parnasia, Dolophones conifera, Dolophones turrigera, Doha's Spiny Spider (Gasteracantha doriae), Double-Spotted Spiny Spider (Gasteracantha mammosa), Double-Tailed Tent Spider (Cyrtophora exanthematica), Aculeperia ceropegia, Eriophora pustulosa, Flat Anepsion (Anepsion depressium), Four-spined Jewel Spider (Gasteracantha quadrispinosa), Garden Orb Web Spider (Eriophora transmarina), Giant Lichen Orbweaver (Araneus bicentenarius), Golden Web Spider (Nephila maculata), Hasselt's Spiny Spider (Gasteracantha hasseltii), Tegenaria atrica, Heurodes turrita, Island Cyclosa Spider (Cyclosa insulana), Jewel or Spiny Spider (Astracantha minax), Kidney Garden Spider (Araneus mitificus), Laglaise's Garden Spider (Eriovixia laglaisei), Long-Bellied Cyclosa Spider (Cyclosa bifida), Malabar Spider (Nephilengys malabarensis), Multi-Coloured St Andrew's Cross Spider (Argiope versicolor), Ornamental Tree-Trunk Spider (Herennia ornatissima), Oval St. Andrew's Cross Spider (Argiope aemula), Red Tent Spider (Cyrtophora unicolor), Russian Tent Spider (Cyrtophora hirta), Saint Andrew's Cross Spider (Argiope keyserlingi), Scarlet Acusilas (Acusilas coccineus), Silver Argiope (Argiope argentata), Spinybacked Orbweaver (Gasteracantha cancriformis), Spotted Orbweaver (Neoscona domiciliorum), St. Andrews Cross (Argiope aetheria), St. Andrew's Cross Spider (Argiope Keyserlingi), Tree-Stump Spider (Poltys illepidus), Triangular Spider (Arkys clavatus), Triangular Spider (Arkys lancearius), Two-spined Spider (Poecilopachys australasia), Nephila species, e.g. Nephila clavipes, Nephila senegalensis, Nephila madagascariensis and many more (for further spider species, see also below). Most preferred, the dragline proteins are derived from Araneus diadematus.

In addition to the structural protein, the recombinant fusion protein according to the present invention can further comprise one or more of the following proteins or subunits: one or two carbohydrate binding domains (such as cellulose binding domains CBHI and/or CBHII) and/or a crosslinking protein such as hydrophobin (HFBI) capable to hydrophobic interactions.

In an embodiment the carbohydrate binding part of the polypeptide is selected from the group consisting of CBHI and CBHII. The carbohydrate binding protein part can thus comprise an amino acid sequence SEQ ID NO : 8 (CBHI). On the other hand the crosslinking protein can comprise an amino acid sequence SEQ ID NO : 10 (CBHII).

The DNA construct according to the present invention can comprise sequences encoding structural proteins and in addition sequences encoding one or several carbohydrate binding domains and/or a sequence encoding a crosslinking protein. In addition, the DNA construct may comprise other sequences such as protease sequences, sequences for specific linker regions, tag sequence and a CBHI signal sequence. The carbohydrate binding modules CBMs were initially classified as cellulose binding domains (CBDs), based on the initial discovery of several modules that bind cellulose. A CBM is defined as a contiguous amino acid sequence within a carbohydrate-active enzyme with a discrete fold having carbohydrate binding activity. To date, more than 300 putative sequences in more than 50 different species have been identified, and the binding domains have been classified into 43 different families based on amino acid sequence, binding specificity, and structure. CBMs have also been found in several polysaccharide-degrading enzymes other than cellulases and xylanases. In T. reesei, CBMs have been identified in hemicellulase, endomannanase, and acetylxylanesterase.

CBDs are used for cellulose binding and for anchoring, such as anchoring of enzyme to substrate. With coupling of two CBDs together using a specific linker a double CBD (DCBD) is obtained. One can obtain a molecular motif with a modularly adjustable affinity to cellulose without altering the residue-level interactions. In a preferred embodiment of the invention, the CBD domain or domains is/are selected from the group consisting of CBHI and CBHII.

A "crosslink" according to the present invention is a bond that links one polymer chain to another, i.e. crosslinking occurs between different polymer chains. The bonds can be for example non-covalent, covalent, or ionic bonds. The crosslinking interactions of the present invention result in non-covalent interactions. "Polymer chains" refer here to synthetic polymers or natural polymers (such as proteins).

Proteins can naturally contain crosslinks generated by enzyme-catalyzed or spontaneous reactions. Such crosslinks are important in generating mechanically stable structures such as hair, skin and cartilage. Disulfide bond formation is one of the most common crosslinks, but isopeptide bond formation is also common. Proteins can also be cross-linked artificially using small-molecule crosslinkers.

Crosslinks can be formed by chemical reactions that are initiated by heat, pressure, change in pH, or radiation. For example, mixing of an unpolymerized or partially polymerized resin with specific chemicals called crosslinking reagents results in a chemical reaction that forms cross-links. Cross-linking can also be induced in materials that are normally thermoplastic through exposure to a radiation source, such as electron beam exposure gamma-radiation, or UV light.

Crosslinking reaction according to the present invention can be enzyme-catalyzed or a spontaneous reaction or formed by chemical reactions. "Cross-linking component" is selected from the group consisting of hydrophobin (HFB), avidin protein, biotin protein and also of proteins containing coiled-coil interaction domains such as cohesin, cohibin, condensing, monopolin, myosin, tropomyosin and intermediate filament proteins, Cross-linking interaction may occur for example as between biotin and avidin.

HFBs are small, secreted proteins of about 70 to 150 amino acids which occur in filamentous fungi, for example Trichoderma reesei or Schizophyllum commune. They usually have eight cysteine residues. HFBs can be isolated from natural sources, but can also be obtained by means of genetic engineering methods. In the context of the present invention, the term "hydrophobin" is intended to include all polypeptides belonging to the classes of hydrophobins, including HFBI, HFBII, HFBIII, SRHI, SC3, HGFI and other polypeptides that have resemblance in properties or sequence to said polypeptides.

According to one embodiment of the invention, the hydrophobins include polypeptides comprising amino acid sequences, which have at least 40% similarity at the amino acid sequence level to the mentioned HFBI, HFBII, HFBIII, SRHI, SC3 and HGFI. The evel of similarity can be also higher, preferably at least 50%, more preferably at least 60%), particularly at least 80%>, and most suitably at least 90%> .

HFBs may be organized in a water-insoluble form on the surface of various fungal structures, such as e.g. aerial hyphae, spores, fruiting bodies. The genes for HFBs could be isolated from ascomycetes, deuteromycetes and basidiomycetes. HFBs identified to date are generally classed as either class I or class II. Both types have been identified in fungi as secreted proteins that self-assemble at hydrophobilic interfaces into amphipathic films. Assemblages of class I HFBs are generally relatively insoluble whereas those of class II HFBs (HBF II) readily dissolve in a variety of solvents.

HFB possesses hydrophobic interactions and is able adhere or become organized on surfaces. HFB is used for hydrophobic interactions, binding and purification.

In a preferred embodiment the crosslinking protein component of the polypeptide is selected from the group consisting of HFB I and II. In another embodiment of the present invention avidin and/or biotin is used instead of HFB. In still another embodiment chemical crosslinking is used. The structural protein is separated from the other proteins in the construct body with one (when said structural protein is in the either end of the construct) or two (when said structural protein is in the middle of the construct) linker regions. Generally, linkers of lengths between 0 - 100 -mers can be used. The linker regions provide modularly tailored binding to the adjacent protein. A short linker tethers the domains close to each other and can promote cooperative binding, because the effective concentration of this domain pair becomes locally high, thus leading to a high probability of binding. Correspondingly, for longer linkers, a less efficient tethering leads to lower affinity. Linker sequence can be for example serine- and threonine- rich sequence.

During protein secretion in a fungal cell, certain proteins are cleaved by KEX2, a member of the KEX2 or "kexin" family of serine peptidase, KEX2 is a highly specific calcium-dependent endopeptidase that cleaves the peptide bond that is immediately C-terminal to a pair of basic amino acids (i.e., the "KEX2 site") in a protein substrate during secretion of that protein. KEX2 proteins generally contain a cysteine residue near the histidine residue of its active site and are inhibited by p-mercuribenzoate. KEX2 is encoded by the yeast gene KEX2. KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately 5' to the KEX2 site.

The Strep tag^® system is a method which allows the purification and detection of proteins by affinity chromatography. The "Strep-tag" is a synthetic peptide consisting of eight amino acids (WSHPQFEK). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin, a specifically engineered streptavidin, and can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. The short peptide tag has negligible effect on the recombinant protein due to its chemically balanced amino acid composition. Also other tag sequences such as FLAG, HA and MYC tag can be used in N- or C-terminal end of the protein produced.

In one embodiment of the present invention genetic fusion protein of the present invention produced in T. reesei is composed of two cellulose binding domains (CBDs) (CBHI and CBHII), of a structural protein (such as resilin) and of a crosslinking protein (such as hydrophobin).

An isolated polypeptide according to the invention comprises at least a structural protein but may also comprise further proteins or protein domains such as carbohydrate binding domains and one or more crosslinking protein components. A nucleic acid sequence encoding a polypeptide comprising at least one carbohydrate binding domain, a structural protein and a crosslinking protein component, wherein said polypeptide is selected from the group consisting of SEQ ID NO : l to SEQ ID NO : 38, is also one aspect of the present invention. Also an expression vector comprising a nucleic acid molecule encoding a polypeptide comprising the polypeptide comprising at least one carbohydrate binding domain, a structural protein and a crosslinking protein component is one aspect of the invention.

In one aspect of the invention the filamentous fungal cells according to the invention are used for the expression of fusion polypeptides. Thus one further aspect of the invention relates to a method for recombinant production of material protein polypeptide in a filamentous fungal host cell of the invention, comprising the steps:

(a) introducing into the host cell a nucleic acid sequence encoding the fusion polypeptide;

(b) cultivating the cell from (a) in a suitable growth medium under conditions conducive for expression of the fusion polypeptide; and

(c) isolating the protein product.

The fusion proteins according to the present invention can be used for example for modulating the properties of nanocellulose material . The fusion protein of the present invention can be used to modify the material characteristics of nanocellulose. Especially, the obtained fusion proteins can be used for producing nanocomposite materials. By "hybrid fibers" are meant here fibers containing engineered or recombinant protein in complex with nanocellulose.

A general molecular strategy to achieve composite structures is to design crosslinking proteins. The idea of crosslinking of materials using bifunctional proteins has been presented for example for improving the properties of paper using a recombinant protein consisting of two CBDs linked together or alternatively using one CBD linked to a starch-binding protein.

One aspect of the present invention is a fiber comprising the polypeptide comprising at least one carbohydrate binding domain, a structural protein and a crosslinking protein component. Still one object of the invention is a composition comprising an isolated polypeptide comprising at least one carbohydrate binding domain, a structural protein and a crosslinking protein component, a recombinant protein comprising the isolated polypeptide; a cellulose nanofibril; and optionally further comprising a carrier, diluent or excipient.

Use of the composition comprising the polypeptide comprising at least one carbohydrate binding domain, a structural protein and a crosslinking protein component in a nanocomposite material is one aspect of the invention. The present invention further discloses nanocomposites in which nanofibrillar cellulose (NFC) is used as a "hard" reinforcing component and the proteins according to invention produced in T. reesei function as a "soft" adhesive matrix with the intention to achieve in-depth tunable interactions between the NFCs and to achieve sequential sacrificial bonds. NFC has a high aspect ratio, being typically several micrometers in length and 5-20 nanometers in width. It is obtained from softwood pulp by mechanical integration. We have previously shown that engineered proteins with specific binding functions can be used as an adhesive matrix, allowing to tune the plastic behavior of NFC composites and in combination with graphene also affecting the stiffness and strength.

An interaction between cellulose nanofibrils is achieved by combining different structural and fusion proteins according to the invention, which are produced in T. reesei with nanofibrils.

A dilute hydrogel (solid content 1.64%) of nanofibrillar cellulose can be used as a starting material. The cellulose can be mechanically disintegrated by ten passes through a M7115 Fluidizer (Microfluidics Corp., U.S.A.) essentially according to previous reports (Paakko et al 2007). Bacterial cellulose (Nata de Coco), which had a solid content of 2.55 g/l, can be used for the binding instead of nanofibrillated cellulose as it can be more readily separated from the dispersion for the binding assay.

The first embodiment of the invention comprises an isolated polypeptide comprising a fusion of a structural protein with at least one carbohydrate binding domain and/or with a crosslinking component, wherein said polypeptide is produced in a filamentous fungi. The second embodiment of the invention comprises the polypeptide according to the first embodiment, wherein it comprises a structural protein and at least one carbohydrate binding domain. The third embodiment of the invention comprises the polypeptide according to the first embodiment, wherein it comprises a structural protein and a crosslinking component.

The fourth embodiment of the invention comprises the polypeptide according to the first embodiment, wherein it comprises a structural protein and at least one carbohydrate binding domain and a crosslinking component.

The fifth embodiment of the invention comprises the polypeptide according to the first embodiment, wherein the filamentous fungi is Trichoderma reesei. The sixth embodiment of the invention comprises the polypeptide according to the fifth embodiment, wherein Trichoderma reesei cells have reduced or no detectable activity of at least three endogenous proteases.

The seventh embodiment of the invention comprises the polypeptide according to the fifth embodiment, wherein Trichoderma reesei cells have reduced or no detectable activity of ten endogenous proteases.

The eighth embodiment of the invention comprises the polypeptide according to the first embodiment, wherein the structural protein is selected from the group consisting of resilin, elastin, elastin-like protein, collagen, abductin, byssus, flagelliform silk, dragline silk, gluten high molecular weight subunit, titin, fibronectin, leminin, gliadin, glue polypeptide, ice nucleating protein, keratin, mucin, and a mixture thereof.

The ninth embodiment of the invention comprises polypeptide according to any of the first to fourth embodiments, wherein the structural protein is resilin and comprises an amino acid sequence SEQ ID NO: 24.

The tenth embodiment of the invention comprises polypeptide according to any of the first to fourth embodiments, wherein the structural protein is elastin-like-polypeptide (ELP) and where the amino acid sequence is selected from the group consisting of sequences SEQ ID NO: 12, 14, 16. The eleventh embodiment of the invention comprises polypeptide according to first, second or fourth embodiment, wherein the carbohydrate binding domain is selected from the group consisting of CBHI and CBHII.

The twelfth embodiment of the invention comprises polypeptide according to eleventh embodiment, wherein the carbohydrate binding domain comprises an amino acid sequence SEQ ID NO : 8 (CBDI). The thirteenth embodiment of the invention comprises polypeptide according to the eleventh embodiment, wherein the carbohydrate binding domain comprises an amino acid sequence SEQ ID NO : 10 (CBDII).

The fourteenth embodiment of the invention comprises polypeptide according to first, second or fourth embodiment, wherein there are two or more carbohydrate binding domains.

The fifteenth embodiment of the invention comprises polypeptide according to first, third or fourth embodiment, wherein the crosslinking component is selected from the group consisting of hydrophobin (HFB) protein, avidin protein, biotin, cohesin (dockerin pair), cohibin, condensing, monopolin, myosin, tropomyosin and intermediate filament proteins.

The sixteenth embodiment of the invention comprises polypeptide according to fifteenth embodiment, wherein the crosslinking component is hydrophobin and comprises an amino acid sequence SEQ ID NO: 28.

The seventeenth embodiment of the invention comprises polypeptide according to any of the first to sixteenth embodiments, wherein said polypeptide further contains a HDEL signal for the endoplasmic reticulum (ER) retention.

The eighteenth embodiment of the invention comprises a nucleic acid sequence encoding a polypeptide according to any of the first to seventeenth embodiments, wherein said polypeptide comprises on or more sequences selected from the group consisting of 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, SEQ ID NO : 36 and SEQ ID NO : 38. The nineteenth embodiment of the invention comprises an expression vector comprising a nucleic acid molecule encoding a polypeptide comprising the polypeptide according to any one of first to seventeenth embodiments.

The twentieth embodiment of the invention comprises an isolated host cell transformed with the expression vector according to nineteenth embodiment.

The twenty-first embodiment of the invention comprises a fiber comprising the polypeptide according to any of first to seventeenth embodiments.

The twenty-second embodiment of the invention comprises a composition comprising an isolated polypeptide comprising according to any of first to seventeenth embodiments;

a cellulose nanofiber; and

optionally further comprising a carrier, diluent or excipient. The twenty-third embodiment of the invention comprises use of the composition according to the twenty-second embodiment in a nanocomposite material.

EXAMPLES

EXAMPLE 1. Construction of vectors for elastin-like-protein- and resilin-CBD fusion expression in Trichoderma reesei (see Figure 1).

Expression cassettes were created with Golden Braid modular combinatorial cloning strategy by using plasmids pDGBa l, a2, Ω1, and Ω2 as destination vectors (Sarrion- Perdigones et al., 2013). First, synthetic genes omitting internal restriction sites for Bsal, BtgZI and BsmBI encoding gene fragments for CBHI integration flanks, CBHI promoter, CBHI terminator, CBHI secretion carrier, hygromycin selection cassette and elastin-like-polypeptide / resilin CBD fusions were designed, synthesized and cloned to pUC57 plasmid. To organize the genes in desired order in the final expression constructs, gene fragments included overlapping junctions to be released by Bsal or BsmBI in the Golden Braid restriction-ligation reactions. The combinatorial cloning was performed at three levels. First reaction assembled promoter with coding region and terminator to pDGBla. At second level two parallel reactions were performed to assemble promoter-coding region- terminator together with 5' integration flank (in pDGBQl) and hygromycin selection cassette with 3' integration flank (in pDGBQ2). At third level the pieces from second level were combined to pDGBa l for final integration cassette (Figure la). For transformation, linear integration fragments were released with Bsal digestion and purified from agarose gels.

Example 2. Trichoderma transformation and protein expression analysis of CBD-ELP constructs (see Figure 2)

Protoplasts from T. reesei strain M658 carrying deletions for ten extracellular proteases M658 (page 170-172, patent WO2013102674) (Landowski et al., 2013) and mus53 deletion for enhanced homologous integration (Steiger et al., 2010) were transformed as described previously (Penttila et al., 1987). Transformants were selected with hygromycin and purified through single spore cultures. The pure cultures were sporylated on potato dextrose agar plates to be stored as spore suspensions at -80°C in the presence of 20% glycerol. The expression constructs aimed integration to CBHI locus to replace the endogenous cellobiohydrolase I gene. The locus was confirmed with primer pairs (SEQ ID NO : 39 G CTGTTCCTAC AG CTCTTTC & SEQ ID NO :40 AGCCGCACGGCAGC) and (SEQ ID NO: 41 GGTTGACTTACTCCAGATCG & SEQ ID NO:42 AGTCGTTTACCCAGAATGC) for 5' and 3' integration, respectively. To confirm dislocation of the endogenous chbl gene primer pair (SEQ ID NO:43 CAACTCAG ATCCTCCAG G AG AC & SEQ ID NO: 44 GCTCGTTGCCAGAGTAACTAC) was used. Trichoderma strains transformed with pJJJ690-695 and pHYB19-30 expression constructs were cultivated on 24-well plates in 1% (w/v) yeast extract, 2% cellobiose, 1% sorbose, l lOmM KH2P04, 38mM Na2S04, lOOmM PIPPS, 2.4mM Mg2S04, 4.1mM CaCI2, Trichoderma trace elements (Penttila et al., 1987) and 38 mM di-ammonium hydrogen citrate, pH 4.5 for 3 days on a humidity controlled rotary shaker (800 rpm) at 28°C. Culture supernatants were collected by centrifugation and analysed on SDS- PAGE gels. The gels were detected either for total protein (Gel Code Blue Stain, Pierce) or after blotting to nitrocellulose filters with immunodetection. Shortly, the membranes were blocked with 5% non-fat milk in TBS buffer (50 mM Tris, 150 mM NaCI, pH 7.4) and then probed with anti-ELP rabbit serum (1 : 1000 in TBS) followed by anti-rabbit- AP secondary antibody (1 : 1400 in TBS, BioRad, 170-6518) and chromogenic detection with NBT/BCIP. Example 3. Purification CBDI-ELP5-CBDII (pJJJ693) protein.

The CBDI-ELP5-CBDII protein was first concentrated from pJJJ693 strain culture supernatant by inverse transitional cycling (Urry, 1997). The culture supernatant was supplemented with 4.5M NaCI at room temperature and centrifuged at 10 OOOx g, RT to collect the protein precipitate pellet. The pellet was resuspended with cold double distilled water (DDW) and the resuspension was clarified with centrifugation (10 OOOx g). For further purification, the buffer was exchanged to DDW+0.1% trifluoro acetic acid with gel filtration (EconoPac, 10DG, BioRad). Sample was fed to VYDAC semi prep C4 reverse phase column (Grace) and eluted with acetonitrile gradient in 0.5ml fractions with AKTA Explorer liquid chromatograph. Fractions (C10-14) containing the elastin-like-polypeptide-CBD fusion protein (pJJJ593) were pooled and aspired for TFA and acetonitrile, freeze dried and stored in RT desiccator. Example 4. Rheology analysis of CBDI-ELP5-CBDII protein (pJJJ693) with nanocellulose.

Nanocellulose water dispersion (UPM-Kymmene Corporation, Finland) with solid content of 1.70% was processed by mechanical disintegration of bleached birch kraft pulp by 6 passes through a M7115 Fluidizer (Microfluidics Corp.) (Paakko et al., 2007). Rheological measurements of ultrasonicated NFC-CBD-ELP5-CBD mixtures (0.2% NFC, 0.1% protein in 50mM Sodium acetate with 50mM NaCI, pH 5.0) were carried out at room temperature (22 °C) with a rheometer (AR-G2, TA instruments, UK) equipped with cross-hatched plate-plate geometry. The diameter of the plates was 30 mm. The viscoelastic properties of the NFC-protein mixtures were determined in small deformation oscillation mode of the rheometer using 2.05 ml sample volume and 1 mm gap. Time sweep (frequency 0.1 Hz, strain 0.1 %; linear region) was run for 20 h followed by amplitude and frequency sweeps. Evaporation was prevented by a solvent trap.

Example 5. Trichoderma transformation and protein expression analysis of CBD- resilin constructs

Transformation procedure of resilin strains followed the protocol described above) Trichoderma strains transformed with pMILsl24, pAW116, pHYB37 and pHYB38 expression constructs were cultivated on 24-well plates in 4% (w/v) lactose, 2% spent grain extract, l lOmM KH2P04, 38mM Na2S04, lOOmM PIPPS, 2.4mM Mg2S04, 4.1mM CaCI2, Trichoderma trace elements (Penttila et al., 1987) and 38 mM di- ammonium hydrogen citrate, pH 4.5 for 4 days on a humidity controlled rotary shaker (800 rpm) at 28°C. Culture supernatants were collected by centrifugation and analysed on SDS-PAGE gels. The gels were detected either for total protein (Gel Code Blue Stain, Pierce) or after blotting to nitrocellulose filters with immunodetection. Shortly, the membranes were blocked with 5% non-fat milk in TBS buffer (50 mM Tris, 150 mM NaCI, pH 7.4) and then probed (pMILsl24, pAW116) with streptactin-AP conjugate (1 : 2000 in TBS, IBA Gmbh) and chromogenic detection with NBT/BCIP or (pHYB37 and pHYB38) with Rabbit anti-Strep-tag polyclonal antibody (1 : 2000 in TBS, Abeam, ab76949) followed by Goat anti Rabbit IRDye 680RD (1 : 30000 in TBS, Li-cor) and Odyssey infrared imaging system (Li-cor) .

REFERENCES

Landowski, C, Huuskonen, A., Saarinen, J., Westerholm-Parvinen, A., Kanerva, A., Natunen, J., Hanninen, A., Salovuori, N., Pentilla, M., and Saloheimo, M. 2013. Protease Deficient Filamentous Fungal Cells and Methods of Use Thereof.

Machado et al. High level expression and facile purification of recombinant silk-elastin- like polymers in auto induction shake flask cultures. AMB Express 2013, 3 : 11. Paakko, M., Ankerfors, M., Kosonen, H., Nykanen, A., Ahola, S., Osterberg, M., Ruokolainen, J., Laine, J., Larsson, P. T., and Ikkala, O. 2007. Enzymatic hydrolysis combined with mechanical shearing and high-pressure homogenization for nanoscale cellulose fibrils and strong gels. Biomacromolecules 8 : 1934-1941. Penttila, M., Nevalainen, H., Ratto, M., Salminen, E., and Knowles, J. 1987. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61 : 155-164.

Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J ., Granell, A., and Orzaez, D. 2013. GoldenBraid 2.0 : a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162 : 1618-1631.

Steiger, M. G., Vitikainen, M., Uskonen, P., Brunner, K., Adam, G., Pakula, T., Penttila, M., Saloheimo, M ., Mach, R. L, and Mach-Aigner, A. R. 2010. A transformation system for Hypocrea (Trichoderma) that favors homologous integration and that uses reusable bidirectionally-selectable markers. Appl. Environ. Microbiol.

Claims

1. An isolated polypeptide comprising a fusion of a structural protein with at least one carbohydrate binding domain and/or with a crosslinking component, wherein said polypeptide is produced in Trichoderma reesei.

2. The polypeptide according to claim 1, wherein it comprises a structural protein and at least one carbohydrate binding domain.

3. The polypeptide according to claim 1, wherein it comprises a structural protein and a crosslinking component.

4. The polypeptide according to claim 1, wherein it comprises a structural protein and at least one carbohydrate binding domain and a crosslinking component.

5. The polypeptide according to claim 1, wherein Trichoderma reesei cells have reduced or no detectable activity of at least three endogenous proteases.

6. The polypeptide according to claim 1, wherein Trichoderma reesei cells have reduced or no detectable activity of ten endogenous proteases.

7. The polypeptide according to claim 1, wherein the structural protein is selected from the group consisting of resilin, elastin, elastin-like protein, collagen, abductin, byssus, flagelliform silk, dragline silk, gluten high molecular weight subunit, titin, fibronectin, leminin, gliadin, glue polypeptide, ice nucleating protein, keratin, mucin, and a mixture thereof.

8. The polypeptide according to any of claims 1 to 4, wherein the structural protein is resilin and comprises an amino acid sequence SEQ ID NO: 24.

9. The polypeptide according to any of claims 1 to 4, wherein the structural protein is elastin-like-polypeptide (ELP) and where the amino acid sequence is selected from the group consisting of sequences SEQ ID NO: 12, 14, 16.

10. The polypeptide according to claim 1, 2 or 4, wherein the carbohydrate binding domain is selected from the group consisting of CBHI and CBHII.

11. The polypeptide according to claim 10, wherein the carbohydrate binding domain comprises an amino acid sequence SEQ ID NO: 8 (CBDI).

12. The polypeptide according to claim 10, wherein the carbohydrate binding domain comprises an amino acid sequence SEQ ID NO: 10 (CBDII).

13. The polypeptide according to claim 1, 2 or 4, wherein there are two or more carbohydrate binding domains.

14. The polypeptide according to claim 1, 3 or 4, wherein the crosslinking component is selected from the group consisting of hydrophobin (HFB) protein, avidin protein, biotin, cohesin (dockerin pair), cohibin, condensing, monopolin, myosin, tropomyosin and intermediate filament proteins.

15. The polypeptide according to claim 14, wherein the crosslinking component is hydrophobin and comprises an amino acid sequence SEQ ID NO : 28.

16. The polypeptide according to any of the claim 1 to 15, wherein said polypeptide further contains a HDEL signal for the endoplasmic reticulum (ER) retention.

17. A nucleic acid sequence encoding a polypeptide according to any of claims 1 to 16, wherein said polypeptide comprises on or more sequences selected from the group consisting of 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, SEQ ID NO: 36 and SEQ ID NO: 38.

18. An expression vector comprising a nucleic acid molecule encoding a polypeptide comprising the polypeptide according to any one of claims 1 to 16.

19. An isolated host cell transformed with the expression vector according to claim 18.

20. A fiber comprising the polypeptide according to any of claims 1 to 16.

21. A composition comprising an isolated polypeptide comprising according to any of claims 1 to 16; a cellulose nanofiber; and

optionally further comprising a carrier, diluent or excipient.

22. Use of the composition according to claim 21 in a nanocomposite material.