RU2845387C1 - Modified filamentous fungi for production of exogenous proteins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a genetically modified ascomycete filamentous fungus. Said fungus is intended to produce an exogenous protein of interest and contains at least one cell having a reduced expression and/or activity of KEX2 protease, and reduced expression and/or activity of at least 12 additional proteases, wherein at least one cell contains an exogenous polynucleotide encoding the protein of interest. Present invention also relates to a method of producing said fungus, as well as a method of producing at least one protein of interest.

EFFECT: present invention provides an increase in the stability and amount of expressed exogenous proteins, achieved due to low expression and/or activity of KEX2 protease and at least 12 additional proteases.

42 cl, 17 dwg, 5 tbl, 11 ex

Description

AREA OF TECHNOLOGY

The present invention relates to the production of exogenous proteins in genetically modified ascomycetous filamentous fungi, in particular of the species Thermothelomyces heterothallica (formerly Myceliophthora thermophila), having reduced expression or activity of the proteases KEX2 and/or ALP7. The genetically modified ascomycetous filamentous fungi are used for the reliable production of highly stable proteins.

LEVEL OF TECHNOLOGY

Recombinant protein production

Expression and purification of recombinant proteins that have functional post-translational protein modifications such as glycosylation or phosphorylation can only be accomplished using eukaryotic expression systems. Eukaryotic protein expression systems, including mammalian, plant, and fungal cells, have become indispensable for the production of functional eukaryotic proteins.

Thermothelomyces heterothallica (Th. heterothallica) wild-type C1 (until recently named Myceliophthora thermophila, which in turn was renamed from Chrysosporium lucknowense) is a thermotolerant ascomycete filamentous fungus that produces high levels of cellulases, making it attractive for the production of these and other proteins on a commercial scale.

For example, U.S. Patent Nos. 8,268,585 and 8,871,493 issued to the applicant of the present invention disclose a transformation system in the field of filamentous fungal hosts for the expression and secretion of heterologous proteins or polypeptides. Also disclosed is a method for producing large quantities of polypeptides or proteins in an economical manner. The system includes a transformed or transfected strain of a fungus of the genus Chrysosporium, more particularly Chrysosporium lucknowense, and mutants or derivatives thereof. Also disclosed are transformants containing Chrysosporium coding sequences, as well as sequences regulating the expression of Chrysosporium genes.

Wild-type Cl has been deposited under the Budapest Treaty under accession number VKM F-3500 D, deposit date August 29, 1996. Strains with high (HC) and low (LC) cellulase levels have also been deposited, as described, for example, in U.S. Patent No. 8,268,585.

Recently, the applicant of this application has shown that filamentous fungi, especially Th. heterothallica, are very well suited for the production of secondary metabolites. In the international (PCT) application No. PCT/IB2020/051015, it is disclosed that Th. heterothallica is capable of producing cannabinoids and their precursors, in particular, producing cannabigerolic acid (CBGA) and/or cannabigerovaric acid (CBGVA) and their products, including tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabidivaric acid (CBDVA), and its use for the production of said precursors and cannabinoids.

International Publication No. WO/2015/004241 to Landowski et al. discloses filamentous fungal cells deficient in multiple proteases and methods useful for producing heterologous proteins.

Coronavirus

Coronaviruses (CoV) are the largest group of viruses belonging to the order Nidovirales, which includes the families Coronaviridae, Arteriviridae, and Roniviridae. Coronavirinae is one of two subfamilies of the family Coronaviridae, the other being Torovirinae. Coronaviruses are associated with diseases ranging from the common cold to more severe conditions such as severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA coronavirus that causes coronavirus disease 2019 (COVID-19). Coronaviruses are zoonotic, meaning they are transmitted between animals and humans. Common signs of coronavirus infection include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. High concentrations of cytokines have been detected in the plasma of severely ill patients infected with COVID-19. In more severe cases, the infection can cause pneumonia, airway inflammation, severe acute respiratory syndrome, renal failure, and death. Recombinant production of viral proteins can be used as a potential vaccine. The coronavirus spike proteins are considered the main target for vaccine development.

There remains a need for expression systems for mass production of proteins that can be used efficiently and economically in the pharmaceutical industry. In particular, there is a need for improved and robust expression systems that can produce stable antibodies as well as viral antigens for vaccination.

ESSENCE OF THE INVENTION

The present invention provides genetically modified ascomycete filamentous fungi with reduced expression of KEX2 and/or ALP7 proteases, capable of producing a large number of highly stable proteins.

In particular, the present invention provides a strain of CI Thermothelomyces heterothallica as an example of an ascomycete filamentous fungus genetically modified to increase the production of exogenous proteins. In some embodiments, the fungi described herein have been modified to lack fourteen proteases, including KEX2 and ALP7.

Surprisingly, the present invention shows that Th. heterothallica, an example of an ascomycetous filamentous fungus, can be genetically modified to significantly increase the expression and stability of exogenous proteins expressed by T. heterothallica cells compared to previously disclosed fungal strains. The present invention shows that deletion of certain proteases, including KEX2 or ALP7, significantly increases the stability of the expressed proteins.

Furthermore, it is revealed that combined deletion of KEX2 and ALP7 significantly increases the stability and amount of expressed exogenous proteins.

Preferably, in some embodiments, a genetically modified ascomycete filamentous fungus with reduced expression of secreted proteases according to the invention has been developed to produce secreted proteins. Secretion of the expressed proteins and avoidance of fragmented proteins in the medium simplifies the purification procedure and increases protein yield.

The Th. heterothallica C1 system prototype of the present invention was designed to produce a protein of interest by disrupting genes encoding proteases naturally expressed by the fungus. Surprisingly, it was found that deletion of even thirteen or fourteen proteases not only does not disrupt the growth and proliferation rate of the fungi, but at least maintains and even increases the growth rate, making it possible to mass-produce exogenous protein.

Several strains of Th. heterothallica C1 developed by the applicant of the present invention are less sensitive to feedback repression by glucose and other fermentable sugars present in the growth medium as a carbon source than conventional yeast strains as well as most other ascomycete filamentous fungi hosts and, therefore, can tolerate a higher rate of carbon source supply, resulting in high yield production of the fungus.

In addition, some of the Th. heterothallica C1 strains developed by the applicant of the present invention can be grown in fermenters in liquid cultures with a significantly reduced viscosity of the medium compared to most other species of ascomycetous filamentous fungi. Low viscosity cultures of Th. heterothallica C1 are comparable to cultures of S. cerevisiae and other yeast species. The low viscosity may be due to morphological changes in the strain from having long and strongly intertwined hyphae in the original strain(s) to short and less intertwined hyphae in the developed strain(s). Low viscosity of the medium is very advantageous in large-scale industrial production.

According to one aspect, the present invention relates to filamentous fungi genetically modified to produce a protein of interest, wherein the genetically modified filamentous fungi comprise at least one cell having reduced expression and/or protease activity of KEX2 and/or ALP7, wherein the at least one cell comprises at least one exogenous polynucleotide encoding the protein of interest.

According to some embodiments, ALP7 comprises an amino acid sequence that has at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or 100% amino acid sequence identity to Thermothelomyces heterothallica ALP7. According to some embodiments, Thermothelomyces heterothallica ALP7 comprises the amino acids of SEQ ID NO: 13.

According to some embodiments, KEX2 comprises an amino acid sequence that has at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or 100% amino acid sequence identity to KEX2 of Thermothelomyces heterothallica. According to some embodiments, KEX2 of Thermothelomyces heterothallica comprises the amino acids of SEQ ID NO: 14.

According to some embodiments, the modified filamentous fungus comprises at least one cell having reduced expression and/or activity of KEX2 and ALP7.

According to some embodiments, the modified filamentous fungus comprises at least one cell having reduced expression and/or activity of at least one additional protease.

According to some embodiments, the modified filamentous fungus comprises at least one cell having reduced expression and/or activity of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 proteases. Each possible embodiment is a separate embodiment of the invention. According to some embodiments, the modified filamentous fungus comprises at least one cell having reduced expression and/or activity of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 proteases. Each possible embodiment is a separate embodiment of the invention.

According to some embodiments, the at least one additional protease is selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6 and ALP4. Each possible variant represents a separate embodiment of the present invention.

According to some embodiments, the at least one additional protease is selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, ALP5, ALP6, SRP3, SRP5 and SRP8.

According to some embodiments, at least one cell has reduced expression and/or activity of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteases selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, ALPS, ALP6, SRP3, SRP5, SRP8, and SRP10.

According to some embodiments, the modified filamentous fungus comprises at least one cell having reduced expression and/or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, and ALP7. According to some embodiments, the modified filamentous fungus comprises at least one cell having reduced expression and/or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, and KEX2. According to some embodiments, the modified filamentous fungus further comprises at least one cell having reduced expression and/or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, ALP7, and KEX2.

According to some embodiments, the filamentous fungus is further modified to produce proteins with N-glycans similar to proteins of humans, domestic animals, and other mammals. According to some embodiments, the filamentous fungus comprises a deletion or disruption of the alg3 gene such that the fungus cannot produce a functional alpha-1,3-mannosyltransferase. According to some additional or alternative embodiments, the filamentous fungus comprises a deletion or disruption of the alg11 gene such that the fungus cannot produce a functional alpha-1,2-mannosyltransferase. According to yet another additional or alternative embodiment, the filamentous fungus is modified to overexpress flippase. Overexpression of flippase can be achieved by overexpressing an endogenous fungal flippase or by expressing a heterologous flippase.

According to certain additional or alternative embodiments, the filamentous fungus further comprises expression of heterologous GlcNAc transferase 1 (GNT1) and GlcNAc transferase 2 (GNT2). In some embodiments, GNT1 comprises a heterologous Golgi localization signal.

According to some embodiments, the protein of interest is selected from the group consisting of an antigen, an antibody, an enzyme, a vaccine, and a structural protein.

According to some embodiments, the protein of interest is a secreted protein. According to certain embodiments, the protein of interest has a leader or signal peptide. According to other embodiments, the protein of interest is an intracellular protein.

According to some embodiments, the protein of interest comprises two or more repeating protein or protein fragment sequences.

According to some embodiments, the protein of interest is fused to a tag. According to some embodiments, the tag is a C-terminal or N-terminal tag. According to some embodiments, the tag is selected from the group consisting of chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), FLAG tag, Spytag tag, C-tag, ALFA tag, V5 tag, Myc tag, HA tag, Spot tag, T7 tag, NE tag, and polyhistidine (poly(His)) tag. According to some embodiments, the tag is Spytag. According to some embodiments, the tag is a C-tag.

In some embodiments, the protein of interest is an antibody or a fragment thereof. In some embodiments, the antibody is IgG4 or IgG1. In further embodiments, the antibody is a bi- or multi-specific antibody. In certain embodiments, the antibody or fragment thereof is a neutralizing antibody against coronavirus.

According to some embodiments, the protein of interest is anticalin.

According to some embodiments, the protein of interest is fused to an FC protein.

According to some embodiments, the protein of interest is an antigen.

According to some embodiments, the protein of interest is a component of an infectious agent. According to some embodiments, the protein of interest is a component of fungi, bacteria, or viruses. According to some embodiments, the protein of interest is a viral component.

According to some embodiments, the viral component is an epidemic virus. According to certain exemplary embodiments, the viral components are coronavirus, influenza virus, hepatitis B, hepatitis C, papillomavirus, HIV, human T-cell leukemia virus 1 (HTLV-1), or Epstein-Barr virus (EBV).

According to some embodiments, the protein of interest is an influenza virus protein. According to certain embodiments, the protein of interest is hemagglutinin (HA) or a fragment thereof. According to certain embodiments, the protein of interest comprises a transmembrane domain (TMD) of hemagglutinin. According to particular embodiments, the protein of interest is of the H1N1 influenza subtype.

According to some embodiments, the hemagglutinin protein produced is secreted.

In some embodiments, the viral component is a coronavirus. In some currently exemplary embodiments, the coronavirus is SARS-CoV-2 (COVID-19).

According to some embodiments, the protein of interest is the spike protein. According to some embodiments, the protein of interest comprises a receptor binding domain (RBD) sequence of the SARS-CoV-2 spike protein or a fragment thereof. According to some embodiments, the protein of interest comprises the RBD of the SARS-CoV-2 spike protein. According to some embodiments, the protein of interest consists of the RBD of the SARS-CoV-2 spike protein. According to certain embodiments, the protein of interest comprises the receptor binding motif (RBM) of the SARS-CoV-2 spike protein. According to particular embodiments, the RBD or a fragment thereof is fused to a Spytag. According to certain embodiments, the protein of interest comprises two, three, or four repeats of the RBD or a fragment thereof. According to further embodiments, the protein of interest is a nucleocapsid. According to some embodiments, the protein of interest is the S2 fragment of the SARS-CoV-2 spike protein.

According to some embodiments, the protein of interest is a viral antigen fused to an Fc region. According to certain embodiments, the Fc is fused to the N-terminus of the antigen. According to other embodiments, the Fc is fused to the C-terminus of the antigen.

In some embodiments, the protein of interest is Fc-RBD. In other embodiments, the protein of interest is RBD-Fc.

According to some embodiments, the protein of interest comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, and SEQ ID NO: 57.

According to some embodiments, the protein of interest is insulin. According to further embodiments, the protein of interest is fibrinogen.

According to some embodiments, the protein of interest is a therapeutic protein.

According to some embodiments, the protein of interest is a Rift Valley fever virus (RVFV) vaccine protein antigen.

According to some embodiments, the protein of interest is a fusion protein consisting of two different antigens. According to some embodiments, the protein of interest is a fusion protein consisting of two components of different viral antigens. According to some embodiments, the viral antigens are coronavirus and influenza virus.

According to some embodiments, the viral antigen is fused to an MHCII targeting sequence. According to some embodiments, the viral antigen and the MHCII targeting sequence are connected via a linker.

In some embodiments, the label is a site-specific fluorescent label for peptides/proteins.

According to some embodiments, the genetically modified ascomycetous filamentous fungus produces an exogenous protein in an increased amount compared to the amount produced in a corresponding non-genetically modified parental ascomycetous filamentous fungus cultivated under similar conditions. According to some embodiments, the genetically modified ascomycetous filamentous fungus is capable of producing at least 2 times more exogenous protein compared to the parental strain.

According to some embodiments, the genetically modified ascomycetous filamentous fungus is capable of increasing the amount of secreted exogenous protein in a nutrient medium by at least 1.5, 2, 5, or 10 times compared to the original ascomycetous filamentous fungus. According to certain embodiments, the secreted protein is an intact protein.

According to some embodiments, the genetically modified ascomycetous filamentous fungus is capable of increasing the amount of intracellular exogenous protein in fungal cells by at least 1.5, 2, 5, or 10 times compared to the original ascomycetous filamentous fungus.

According to some embodiments, the exogenous protein produced by the genetically modified ascomycetous filamentous fungus has increased stability compared to the corresponding protein produced by the original strain of ascomycetous filamentous fungus cultivated under similar conditions.

According to some embodiments, the genetically modified ascomycetous filamentous fungus grows at a higher rate compared to a corresponding original strain of ascomycetous filamentous fungus cultivated under similar conditions.

A polynucleotide encoding a protein of interest may form part of a DNA construct or expression vector.

According to some embodiments, at least one exogenous polynucleotide is a DNA construct or expression vector, further comprising at least one regulatory element functional in said ascomycete filamentous fungus. According to some embodiments, the regulatory element is selected from the group consisting of a regulatory element endogenous to said fungus and a regulatory element heterologous to said fungus.

According to some embodiments, the ascomycete filamentous fungus is a member of the Pezizomycotina group.

According to some embodiments, the ascomycete filamentous fungus is of a genus selected from the group consisting of Thermothelomyces, Myceliophthora, Trichoderma, Aspergillus, Penicillium, Rasamsonia, Chrysosporium, Corynascus, Fusarium, Neurospora, and Talaromyces.

According to some embodiments, the ascomycete filamentous fungus is a species selected from the group consisting of Thermothelomyces heterothallica (also called Myceliophthora thermophila), Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma viride, Rasamsonia emersonii, Penicillium chrysogenum, Penicillium verrucosum, Sporotrichum thermophile, Corynascus fumimontanus, Corynascus thermophilus, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venenatum, Neurospora crassa, and Talaromyces piniphilus.

According to some embodiments, the ascomycetous filamentous fungus is a strain of Thermothelomyces heterothallica comprising an rDNA sequence that has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO: _2O .

According to some embodiments, the ascomycetous filamentous fungus is Thermothelomyces heterothallica C1.

In one aspect, the present invention provides a method for producing a fungus capable of producing a protein of interest, comprising engineering the fungus to have inhibited or reduced expression and/or activity of KEX2 and/or ALP7.

According to some embodiments, the method comprises transforming at least one fungal cell with at least one exogenous polynucleotide.

According to a further aspect, the present invention provides a method for producing a fungus capable of producing a protein of interest, comprising transforming at least one fungal cell with at least one exogenous polynucleotide, wherein said at least one cell has reduced expression and/or protease activity of KEX2 and/or ALP7.

According to some embodiments, the method comprises transforming at least one fungal cell with at least two exogenous polynucleotides encoding different proteins.

According to some embodiments, the method further comprises engineering the fungus with inhibited or reduced expression and/or activity of at least one protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6 and ALP4 in at least one cell.

According to some embodiments, the method further comprises engineering the fungus with inhibited or reduced expression and/or activity of at least two different proteases selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6 and ALP4.

According to some embodiments, inhibiting the expression of a protease comprises deleting or damaging an endogenous gene encoding the protease.

According to some embodiments, the ascomycete filamentous fungus is of a genus within the group Pezizomycotina.

According to some embodiments, the ascomycete filamentous fungus is of a genus selected from the group consisting of Thermothelomyces, Myceliophthora, Trichoderma, Aspergillus, Penicillium, Rasamsonia, Chrysosporium, Corynascus, Fusarium, Neurospora, and Talaromyces.

According to some embodiments, the ascomycete filamentous fungus is a species selected from the group consisting of Thermothelomyces heterothallica (or Myceliophthora thermophila), Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma viride, Rasamsonia emersonii, Penicillium chrysogenum, Penicillium verrucosum, Sporotrichum thermophile, Corynascus fumimontanus, Corynascus thermophilus, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venenatum, Neurospora crassa, and Talaromyces piniphilus.

According to some embodiments, the ascomycete filamentous fungus is a strain of Thermothelomyces heterothallica comprising an rDNA sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 20.

According to some embodiments, the ascomycetous filamentous fungus is Thermothelomyces heterothallica C1.

According to another aspect, the present invention provides a method for producing at least one protein of interest, comprising culturing a genetically modified fungus as described herein in a suitable medium; and isolating at least one protein product.

According to some embodiments, the isolating step comprises isolating the protein from the culture medium, from the fungal mass, or from both.

According to some embodiments, the protein is isolated from the nutrient medium. According to a particular embodiment, at least 50%, 60%, 70%, 80%, 90%, or 95% of the protein is secreted.

According to some embodiments, the medium comprises a carbon source selected from the group consisting of glucose, sucrose, xylose, arabinose, galactose, fructose, lactose, cellobiose, glycerol, and any combination thereof.

According to some embodiments, culturing a genetically modified fungus in a suitable medium provides for the production of a protein of interest in an increased amount compared to the amount produced in a corresponding non-genetically modified, non-genetically modified original fungal strain cultivated under similar conditions.

According to some embodiments, the corresponding original fungus is of the same species as the genetically modified fungus. According to some embodiments, the corresponding original fungus is isogenic with respect to the genetically modified fungus.

According to another aspect, the present invention relates to a protein of interest obtained by any of the methods described herein.

According to one aspect, the present invention relates to a protein of interest obtained by a method comprising culturing a genetically modified fungus as described herein in a suitable medium; and isolating the protein of interest.

The protein of interest is as described above.

In some embodiments, the protein of interest is a coronavirus antigen. In some embodiments, the protein of interest is a coronavirus spike protein. In some embodiments, the protein of interest comprises a coronavirus RBD sequence or a fragment thereof. In some embodiments, the protein of interest comprises a receptor binding motif (RBM) sequence of a coronavirus spike protein.

The present invention further provides a composition comprising two or more different proteins of interest obtained by any of the methods described herein.

According to certain embodiments, the composition comprises at least two different coronavirus antigens, wherein said antigens comprise sequences of different coronavirus variants.

It should be clearly understood that the scope of the present invention covers homologues, analogs, variants and derivatives, including shorter and longer polypeptides, proteins and polynucleotides, as well as analogs of polypeptides, proteins and polynucleotides with one or more amino acid or nucleic acid substitutions, as known in the art, provided that such variants and modifications must retain the activity of the protein or enzymes described herein.

It should be understood that any combination of each of the aspects and embodiments disclosed herein is expressly included within the disclosure of the present invention.

Other objects, features and advantages of the present invention will be apparent from the following description and drawings.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1. Shows Western blot analysis of C-tag detection in 24-well plate cultures of C1 transformants producing either RBD-C-tag (left panel) or RBD-Spytag-C-tag (right panel).

Fig. 2. Production of RBD-C-tag and RBD-Spytag-C-tag in C1 protease-deficient strains with deletion of 12-14 protease genes. The highest production of RBD proteins was observed in strains DNL155 and DNL159 with deletion of kex2. One of three parallel clones with both RBD-C-tag and RBD-Spytag-C-tag grew poorly and thus produced lower levels of protein.

Fig. 3A-3B. C-tag affinity purification of RBD-C-tag from a bioreactor culture of C1 strain M4169. Stained SDS gel (Fig. 3A) and Western analysis (Fig. 3B) of samples from different stages of purification are shown. Start = Initial sample after clarification, diluted 1:5 in gel; Start Flow = Flow at the beginning of sample loading, diluted 1:5 in gel; End Flow = Flow at the end of sample loading, diluted 1:5 in gel; Fr4-Fr9 = elution fractions. Note that the migration of eluate samples before dialysis is not normal due to the high concentration of MgCl ₂ .

Fig. 4. Schematic description of line C1.

Figure 5 shows antibody addition experiments with different protease-deficient strains. C1 protease deletion strains were cultured in 24-well culture plates for 4 days. For addition experiments, antibodies were incubated in culture supernatants, samples were taken at different times (0 h, 3 h, d/d, and d/d) and analyzed by Western blotting. Separate antibodies were used to detect heavy and light chains. 270 ng of mAb were loaded into each lane. The control was 200 ng.

Figure 6 shows antibody addition experiments with strains deficient in various of the 13 proteases. Strains deficient in C1 protease were grown in 24-well culture plates for 4 days. For addition experiments, antibodies were incubated in culture supernatants, samples were taken at different times (0 h, 3 h, d/d (next day), and d/d (every other day)) and analyzed by Western blotting. Each lane was loaded with 270 ng of mAb. Control was 200 ng.

Fig. 7 shows experiments with the addition of fibrinogen to strains deficient in various of the 13 proteases. Strains with a deletion of protease C1 were grown in 24-well culture plates for 4 days. For the addition experiments, fibrinogen protein was incubated in the culture supernatants. Samples were taken from the samples at different times (0 h, 3 h, pm, and pm) and analyzed by Western blotting. Polyclonal antibodies to fibrinogen (to all fibrinogen chains) were used for detection. 240 ng of fibrinogen were loaded into each lane. The control was 200 ng.

Fig. 8 shows experiments with addition of Fc-FGF21 to strains deficient in various of the 13 proteases. Strains deficient in C1 protease were grown in 24-well culture plates for 4 days. For addition experiments, Fc-FGF21 was incubated in culture supernatants. Samples were taken from samples at different times (0 h, 4 h, pm, and pm) and analyzed by Western blotting. Two antibodies (anti-Fc and anti-FGF21) were used for detection. Each lane was loaded with 240 ng of Fc-FGF21. The control was 200 ng.

Fig. 9 shows the addition (left panel) and expression (right panel) of mAbs in 12 protease-deficient strains compared to 13 protease-deficient strains, as indicated.

Figure 10 shows mAb expression in 12- and 13-protease deficient strains. The mAb expression construct was transformed into a 13-protease deletion strain and a kex2 deletion strain. Transformants were grown in 24-well plates and the resulting mAbs were analyzed by Western blotting. The same mAbs expressed in the original 12-protease deletion strain and in the 13xΔalp7 deletion strain are shown as controls.

Fig. 11 shows the production of rvfv antigenic protein under the bgl promoter by the 14-protease deficient strain dnl 155 and the 13-protease deficient strain, as indicated.

Fig. 12A-12B show that RBD-Spytag and RBD-SpyTag conjugates with SpyCatcher HBsAg HPV generate trimers and/or dimers. Fig. 12A - Western blot. Fig. 12B - SDS-PAGE.

Fig. 13A-13F show the binding of soluble and conjugated RBD to hACE-2 as determined by indirect ELISA. Fig. 13A is a schematic representation of the binding of anti-RBD antibody CR3022 to the RBD-ST:SC-HBsAg HPV particle and detection by labeled goat anti-human IgG alpha-alkaline phosphatase (ALP) conjugate. Fig. 13B is a Detection of different RBD lots with or without the HPV particle. Fig. 13C-13D is a schematic representation of the binding of RBD-ST:SC-HBsAg HPV to hACE (13C) and control (13D). Fig. 13E-13F. ELISA results of hACE binding to HPV-RBD in the conjugated protein (13E) or to HPV alone (13F).

Fig. 14A-14B. Western analysis of C1 transformants producing RBD-Fc (Fig. 14A) or Fc-RBD (Fig. 14B) fusion protein. The parental strains used for production are shown. Strain DNL155 is shown as a negative control. Lanes numbered 1-12 correspond to individual transformants.

Fig. 15. - Western blot analysis with C-label detection in 24-well plate cultures of C1 transformants producing recombinant αMHCII-Cal07 antigen under the control of either the endogenous C1 bgl8 promoter or the synthetic AnSES promoter in transformants derived from strains DNL155 and M3599. The mobility of the target protein in the gel is consistent with its predicted size of 87 kDa. In addition, an endogenous background C1 protein of 70 kDa reactive with the antibody is present in all samples derived from the parent strain DNL155.

Fig. 16A-16C. Affinity purification of C-labeled αMHCII-Cal07 from a bioreactor culture of the CI strain M4540. SDS-stained gel (Fig. 16A) and Western blot (Fig. 16B) of samples from different stages of purification are shown. Input = Initial sample after clarification, diluted 1:10 in the gel; Flow = Flow at the beginning of loading the sample, diluted 1:10 in the gel; Wash = Flow during column wash. Note that the migration of the eluate samples before dialysis is not normal due to the high concentration of MgCl2. Fig. 16C. SDS-PAGE and Western blot analysis of the αMHCII-Cal07 sample after dialysis, compared to the reference protein. Fig. 17 - Western blot results of a 24-well plate culture of C1 transformants producing RBD variants. Yellow is the superposition of the signals of the detection agents for both RBD_ (red signal) and the C-tag (green signal). UK denotes RBD_B.1.1.7-UK, SA - RBD_B.1.351-SA, and BR (Brazil) RBD_1.1.28.1(P.1)-BR. The sample designated as Wuhan is obtained from the M4169 C1 strain producing the Wuhan RBD (Example 4).

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

The present invention provides an alternative, highly efficient system for producing large quantities of proteins. The system of the present invention is based in part on the filamentous fungus Thermothelomyces heterothallica C1 and specific strains thereof, which have previously been developed as a natural biological factory for the production of protein, as well as a secondary metabolite. These strains exhibit a high growth rate while maintaining a low culture viscosity and are thus very well suited for continuous growth in fermentation cultures at volumes of up to 100,000-150,000 liters or more. The present invention, in some embodiments, provides genetically modified fungi having reduced expression and/or activity of KEX2 and/or ALP7.

Definitions

Ascomycete filamentous fungi, as defined herein, refer to any strain of fungi belonging to the group Pezizomycotina. Pezizomycotina includes, but is not limited to, the following groups:

Sordariales, including genera:

Thermothelomyces (including species: heterothallica and thermophila),

Myceliophthora (including the species lutea and unnamed species),

Corynascus (including the species fumimontanus),

Neurospora (including the species crassa); Hypocreales, including the genera:

Fusarium (including graminearum and venenatum species),

Trichoderma (including species reesei, harzianum, longibrachiatum and viride);

Onygenales, including genera:

Chrysosporium (including the species lucknowense); Eurotiales, including the genera:

Rasamsonia (including the species emersonii),

Penicillium (including verrucosum species),

Aspergillus (including species funiculosus, nidulans, nigem oryzae), Talaromyces (including species piniphilus (formerly Penicillium funiculosum)); It should be understood that the above list is not definitive and is intended to provide a non-exhaustive list of industrially significant species of ascomycetous filamentous fungi.

Some species of ascomycete filamentous fungi may exist outside the Pezizomycotina, and this group does not include the Saccharomycotina, which contains the most widely known non-mycelial industrially important genera such as Saccharomyces, Komagataella (including the species formerly called Pichia pastoris), Kluyveromyces, or Taphrinomycotina, which contains some other commonly known non-mycelial industrially important genera such as Schizosaccharomyces.

All of the above taxonomic categories are defined according to the NCBI Taxonomy Browser (ncbi.nlm.nih.gov/taxonomy) on the filing date of the patent application.

It should be understood that fungal taxonomy is in constant flux, and the names and hierarchical position of taxa may change in the future. However, a specialist in the field will be able to determine with certainty whether a particular fungal strain belongs to the group defined above.

According to some embodiments, the genus of filamentous fungi is selected from the group consisting of Myceliophthora, Thermothelomyces, Aspergillus, Penicillium, Trichoderma, Rasamsonia, Chrysosporium, Corynascus, Fusarium, Neurospora, Talaromyces, etc. According to some embodiments, the fungus is selected from the group consisting of Myceliophthora thermophila, Thermothelomyces thermophila (formerly M. thermophila), Thermothelomyces heterothallica (formerly M. thermophila and heterothallica), Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, Penicillium chrysogenum, Penicillium verrucosum, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma viride, Chrysosporium lucknowense, Rasamsonia emersonii, Sporotrichum thermophile, Corynascus fumimontanus, Corynascus thermophilus, Fusarium graminearum, Fusarium venenatum, Neurospora crassa and Talaromy piniphilus.

In particular, the present invention provides the C1 strain of Thermothelomyces heterothallica as a model ascomycete filamentous fungus capable of producing a large number of stable proteins.

The terms "Thermothelomyces" and its species "Thermothelomyces heterothallica and thermophila" are used herein in the broadest sense known in the art. Descriptions of the genus and its species are given, for example, in Marin-Felix Y (2015. Mycologica 107(3): 619-632 doi.org/10.3852/14-228) and van den Brink J et al. (2012, Fungal Diversity 52(1): 197-207). As used herein, the terms "CT" or "Thermothelomyces heterothallica C1" or Th.heterothallica C1 or C1 all refer to the strain C1 of Thermothelomyces heterothallica.

It is noted that the above-mentioned authors (Marin-Felix et al., 2015) proposed a subdivision of the genus Myceliophthora based on differences in optimal growth temperature, conidiospore morphology, and details of the sexual reproduction cycle. According to the proposed criteria, C1 clearly belongs to the newly established genus Thermothelomyces, which contains thermotolerant species previously belonging to Myceliophthora, rather than to the genus Myceliophthora, which still contains non-thermotolerant species. Since C1 can form ascospores with some other Thermothelomyces (formerly Myceliophthora) strains with opposite mating type, C1 is best classified as Th. heterothallica strain C1 rather than Th.

It should also be taken into account that fungal taxonomy has also been in a state of flux in the past, so the current names listed above may be preceded by various older names other than Myceliophthora thermophila (van Oorschot, 1977. Persoonia 9(3):403), which are now considered synonyms. For example, synonyms of Thermothelomyces heterothallica (Marin-Felix et al., 2015. Mycologica, 3:619–63) include Corynascus heterotchallicus, Thielavia heterothallica, Chrysosporium lucknowense and thermophile, and Sporotrichium thermophile (Alpinis 1963. Nova Hedwigia 5:74).

It is furthermore expressly understood that the present invention encompasses any strain comprising a ribosomal DNA (rDNA) sequence that exhibits 99% or greater homology with SEQ ID NO: 20, and all such strains are considered conspecific with Thermothelomyces heterothallica.

In particular, the term Th. heterothallica C1 strain includes genetically modified substrains derived from a wild-type strain that have been mutated using random or targeted approaches, such as using UV mutagenesis, or by deletion of one or more endogenous genes. For example, a C1 strain may refer to a wild-type strain modified to delete one or more genes encoding an endogenous protease. For example, C1 strains encompassed by the present invention include strain UV18-25, Deposit No. VKM F-3631 D; strain NG7C-19, Deposit No. VKM F-3633 D; and strain UV13-6, Deposit No. VKM F-3632 D. Other C1 strains that can be used in accordance with the description of the present invention include strain HC UV18-100f, Deposit No. CBS141147; strain HC UV18-100f, deposition number CBS141143; strain LC W1L#100I, deposition number CBS141153; and strain LC WIUI00I, deposition number CBS141149, and derivatives thereof.

It should be clearly understood that the principles of the present invention encompass mutants, derivatives, progeny and clones of T. heterothallica C1 strains, provided that these derivatives, progeny and clones, when genetically modified in accordance with the description of the present invention, are capable of producing at least one protein product according to the present invention.

It should be clearly understood that the term "derivative" when applied to a fungal line includes any parental fungal line with modifications that positively affect product yield, efficiency or effectiveness, or affect any trait that improves the fungal derivative as a tool for producing a desired protein. As used herein, the term "progeny" refers to an unmodified or partially modified descendant of the parental fungal line, such as a cell by cell. The term "parental strain" refers to a corresponding fungal strain that does not have reduced expression or activity of a specific protease of the invention.

According to one aspect of the present invention, a genetically modified filamentous fungus is provided for producing a protein of interest, wherein the genetically modified filamentous fungus comprises at least one cell with reduced or abolished expression and/or activity of the KEX2 and/or ALP7 protease and at least one additional protease, and said filamentous fungus comprises at least one cell containing at least one exogenous polynucleotide encoding the protein of interest.

According to an aspect of the present invention, a genetically modified filamentous fungus is provided for producing a heterologous protein, wherein the genetically modified filamentous fungus comprises at least one cell with reduced or abolished expression and/or activity of KEX2 and at least one additional protease, and said filamentous fungus comprises at least one cell containing at least one exogenous polynucleotide encoding a heterologous protein.

According to an aspect of the present invention, a genetically modified filamentous fungus is provided for producing a heterologous protein, wherein the genetically modified filamentous fungus comprises at least one cell with reduced or abolished expression and/or activity of ALP7 and at least one additional protease, and said filamentous fungus comprises at least one cell containing at least one exogenous polynucleotide encoding a heterologous protein.

According to an aspect of the present invention, a genetically modified filamentous fungus is provided for producing a heterologous protein, wherein the genetically modified filamentous fungus comprises at least one cell with reduced or abolished expression and/or activity of the proteases ALP7, KEX2 and at least one additional protease, and said filamentous fungus comprises at least one cell containing at least one exogenous polynucleotide encoding a heterologous protein.

According to some embodiments, at least one cell has reduced or abolished expression and/or activity of 13 proteases, one of which is KEX2. According to some embodiments, at least one cell has reduced or abolished expression and/or activity of 13 proteases, one of which is ALP7. According to some embodiments, at least one cell has reduced or abolished expression and/or activity of 14 proteases, including KEX2 and ALP7.

The terms "protein" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acids, without reference to the specific length of the product; thus, peptides, oligopeptides, and polypeptides are included within this definition.

As used herein, the term "protein of interest" refers to a protein that is desirably expressed at high levels in filamentous fungi. Such proteins include, but are not limited to, antibodies, enzymes, substrate binding proteins, structural proteins, antigens, and the like.

According to some embodiments, the ascomycete filamentous fungus comprises at least one cell with reduced or abolished expression and/or activity of KEX2 and at least one other protease.

According to some embodiments, the ascomycetous filamentous fungus comprises at least one cell with reduced or abolished expression and/or activity of at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen proteases.

According to some embodiments, the genetically modified filamentous fungus does not express KEX2. According to some embodiments, the genetically modified filamentous fungus does not express ALP7.

According to some embodiments, the genetically modified filamentous fungus does not express ALP1. According to some embodiments, the genetically modified filamentous fungus does not express PEP4. According to some embodiments, the genetically modified filamentous fungus does not express ALP2. According to some embodiments, the genetically modified filamentous fungus does not express PRT1. According to some embodiments, the genetically modified filamentous fungus does not express SRP1. According to some embodiments, the genetically modified filamentous fungus does not express ALP3. According to some embodiments, the genetically modified filamentous fungus does not express PEP1. According to some embodiments, the genetically modified filamentous fungus does not express MTP2. According to some embodiments, the genetically modified filamentous fungus does not express PEP5. According to some embodiments, the genetically modified filamentous fungus does not express MTP4. According to some embodiments, the genetically modified filamentous fungus does not express PEP6. According to some embodiments, the genetically modified filamentous fungus does not express ALP4.

According to specific embodiments, the ascomycetous filamentous fungus comprises at least one cell with reduced or abolished expression and/or activity of at least one additional protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6 and ALP4. Each possible variant represents a separate embodiment of the present invention.

According to an aspect of the present invention, a genetically modified ascomycetous filamentous fungus is provided for producing a protein of interest, wherein the genetically modified filamentous fungus comprises at least one cell comprising exogenous polynucleotides encoding the protein of interest, wherein said genetically modified ascomycetous filamentous fungus does not express or expresses a reduced amount of KEX2 and/or ALP7, and at least one additional protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6 and ALP4.

According to some embodiments, the filamentous fungus does not express or expresses a reduced amount of KEX2, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4.

According to some embodiments, the filamentous fungus does not express or expresses a reduced amount of ALP7, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4.

According to some embodiments, the filamentous fungus does not express or expresses a reduced amount of KEX2, ALP7, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4.

According to an aspect of the present invention, a genetically modified ascomycetous filamentous fungus is provided for producing a viral antigen, wherein the genetically modified filamentous fungus comprises at least one cell comprising exogenous polynucleotides encoding a viral antigen, and said genetically modified ascomycetous filamentous fungus does not express or expresses a reduced amount of KEX2, ALP7, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6 and ALP4.

According to some embodiments, the viral antigen is a Rift Valley fever virus (RVFV) vaccine antigen protein.

According to an aspect, the present invention relates to a genetically modified ascomycetous filamentous fungus for producing a receptor-binding domain (RBD) of the SARS-CoV2 spike domain, wherein the genetically modified filamentous fungus comprises at least one cell comprising exogenous polynucleotides encoding the RBD, wherein said genetically modified ascomycetous filamentous fungus does not express or expresses a reduced amount of KEX2, ALP7, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTB2, PEP5, MTP4, PEP6 and ALP4.

The kex2 gene, also known as qds1, srb1, and vmn45, encodes the KEX2 protease or KEXIN. The KEX2 protease is a serine peptidase. The amino acid sequence of Thermothelomyces heterothallica KEX2 is shown in SEQ ID NO: 14.

According to some embodiments, KEX2 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 14.

The amino acid sequence of Thermothelomyces heterothallica ALP7 is shown in SEQ ID NO: 13.

According to some embodiments, ALP7 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13.

The alp1 gene encodes alkaline protease 1. ALP1 is a secreted alkaline protease that allows the assimilation of protein substrates. The amino acid sequence of Thermothelomyces heterothallica ALP1 is shown in SEQ ID NO: 1.

According to some embodiments, ALP1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1.

The pep4 gene (synonyms: pho9, pral, yscA) is an aspartic peptidase. The amino acid sequence of Thermothelomyces heterothallica PEP4 is presented in SEQ ID NO: 2.

According to some embodiments, PEP4 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2.

The amino acid sequence of Thermothelomyces heterothallica ALP2 is shown in SEQ ID NO: 3.

According to some embodiments, ALP2 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3.

The amino acid sequence of Thermothelomyces heterothallica PRT1 is shown in SEQ ID NO: 4.

According to some embodiments, PRT1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4.

The amino acid sequence of SRP1 of Thermothelomyces heterothallica is shown in SEQ ID NO: 5.

According to some embodiments, SRP1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5.

The amino acid sequence of Thermothelomyces heterothallica ALP3 is shown in SEQ ID NO: 6.

According to some embodiments, ALP3 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6.

The amino acid sequence of Thermothelomyces heterothallica PEP1 is shown in SEQ ID NO: 7.

According to some embodiments, PEP1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7.

The amino acid sequence of Thermothelomyces heterothallica MTP2 is shown in SEQ ID NO: 8.

According to some embodiments, MTP2 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8.

The amino acid sequence of Thermothelomyces heterothallica PEP5 is shown in SEQ ID NO: 9.

According to some embodiments, PEP5 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9.

The amino acid sequence of Thermothelomyces heterothallica MTP4 is shown in SEQ ID NO: 10.

According to some embodiments, MTP4 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10.

The amino acid sequence of Thermothelomyces heterothallica PEP6 is shown in SEQ ID NO: 11.

According to some embodiments, PEP6 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11.

The amino acid sequence of Thermothelomyces heterothallica ALP4 is shown in SEQ ID NO: 12.

According to some embodiments, ALP4 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12.

The amino acid sequence of Thermothelomyces heterothallica ALP5 is shown in SEQ ID NO: 15.

According to some embodiments, ALP5 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15.

The amino acid sequence of Thermothelomyces heterothallica ALP6 is shown in SEQ ID NO: 16.

According to some embodiments, ALP6 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16.

The amino acid sequence of SRP3 of Thermothelomyces heterothallica is shown in SEQ ID NO: 17.

According to some embodiments, SRP3 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17.

The amino acid sequence of SRP5 of Thermothelomyces heterothallica is shown in SEQ ID NO: 18.

According to some embodiments, SRP5 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18.

The amino acid sequence of SRP8 of Thermothelomyces heterothallica is shown in SEQ ID NO: 19.

According to some embodiments, SRP8 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 19.

According to some embodiments, the protein of interest is fused to a tag. According to some embodiments, the tag is a C-terminal or N-terminal tag. According to some embodiments, the tag is selected from the group consisting of chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), FLAG tag, Spytag tag, C-tag, ALFA tag, V5 tag, Myc tag, HA tag, Spot tag, T7 tag, NE tag, and polyhistidine (poly(His) tag. According to some embodiments, the tag is Spytag. According to some embodiments, the tag is a C-tag.

As used herein, the term "tag" refers to an amino acid sequence that is commonly fused to or included in another amino acid sequence in the art to a) facilitate purification of the entire amino acid sequence or polypeptide, b) improve expression of the entire amino acid sequence or polypeptide, and/or c) facilitate detection of the entire amino acid sequence or polypeptide.

The term "C-tag" is well known in the art and refers to a 4 amino acid affinity tag: EPEA (glutamic acid-proline-glutamic acid-alanine), which can be fused to the C-terminus of any recombinant protein. The tag provides high affinity and selectivity when used for purification purposes.

The term "Spytag" is well known in the art and refers to a short peptide that covalently binds to the SpyCatcher protein. The sequence of Spytag is Ala-His-Ile-Val-Met-Val-Asp-Ala-Tyr-Lys-Pro-Thr-Lys.

The term "Strep-Tag" is used herein as is known in the art and refers to a method for purifying and detecting proteins using affinity chromatography. The method is based on binding to Strep-Tactin.

The term "glutathione S-transferase (GST)" is used herein as it is known in the art and is based on the strong binding affinity of the GST protein for glutathione (GSH). The GST tag is often used to separate and purify proteins containing a GST fusion protein. The tag is 2 ₂ O amino acids long.

The term "FLAG tag" is used herein as is known in the art and refers to a polypeptide protein tag that can be added to a protein using recombinant DNA technology. It is one of the most specific tags, representing an artificial antigen to which specific high-affinity monoclonal antibodies have been developed and can thus be used to purify proteins using affinity chromatography.

The term "ALFA tag" is used herein as is known in the art and refers to an epitope tag specifically recognized by a nanobody that can be used for detection and purification.

The V5 tag is a short peptide tag for protein detection and purification. The V5 tag can be fused/cloned to recombinant protein and detected by ELISA, flow cytometry, immunoprecipitation, immunofluorescence, and Western blotting using antibodies and nanobodies.

The term "Myc tag" is used herein as is known in the art and refers to a short peptide tag derived from the c-myc gene that can be recognized by specific antibodies.

"HA tag" is used herein as is known in the art and refers to a peptide derived from the human influenza hemagglutinin (HA) molecule corresponding to amino acids 98-106. This tag is used to facilitate detection, isolation and purification of the protein of interest.

The "Spot Tag" is a 12 amino acid peptide tag recognized by a single-domain nanoantibody (sdAb). The tag can be used for a variety of applications including immunoprecipitation, affinity purification, immunofluorescence, and super-resolution microscopy.

The term "T7 tag" is used herein as is known in the art and refers to an epitope tag consisting of an 11-residue peptide encoded by the leader sequence of gene 10 of bacteriophage T7.

The term "NE tag" is used herein as is known in the art and refers to a synthetic peptide tag (NE tag) developed as an epitope tag for detection, quantification and purification of recombinant proteins. This peptide tag consists of eighteen hydrophilic amino acids.

The term "poly(His) tag" or "polyhistidine tag" is known in the art and refers to an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as a hexahistidine tag, 6xHis tag, and His6 tag. The short peptide can be bound by metal ions such as divalent nickel or cobalt.

According to some embodiments, the filamentous fungus is further modified to produce proteins with N-glycans similar to proteins of humans, domestic animals, and other mammals. According to some embodiments, the filamentous fungus comprises a deletion or damage to the alg3 gene, such that the fungus cannot produce a functional alpha-1,3-mannosyltransferase. According to some embodiments, the filamentous fungus comprises a deletion or damage to the alg11 gene, such that the fungus cannot produce a functional alpha-1,2-mannosyltransferase. According to some embodiments, the filamentous fungus is characterized by overexpression of an endogenous flippase or expression of a heterologous flippase.

According to some embodiments, the filamentous fungus further comprises expression of heterologous GlcNAc transferase 1 (GNT1) and GlcNAc transferase 2 (GNT2). In some embodiments, GNT1 comprises a heterologous Golgi localization signal. In some embodiments, heterologous GNT1 and GNT2 are of animal origin.

According to some embodiments, the protein of interest is an antigen. According to some embodiments, the protein of interest is the spike protein. According to some embodiments, the protein of interest comprises a receptor binding domain (RBD) sequence of the SARS-CoV-2 spike protein, or a fragment thereof. According to some embodiments, the protein of interest is the RBD of the SARS-CoV-2 spike protein. According to certain embodiments, the protein of interest comprises the receptor binding motif (RBM) of the SARS-CoV-2 spike protein. According to some embodiments, the protein of interest comprises a glycoprotein binding domain (GBD) sequence of the SARS-CoV-2 S protein. According to certain embodiments, the RBD or a fragment thereof is fused to a Spytag. According to some embodiments, the RBD or a fragment thereof is fused to a C-tag. According to additional embodiments, the RBD is fused to the Fc of the antibody. According to certain embodiments, the protein of interest comprises two, three, or four repeats of the RBD or a fragment thereof.

The coronavirus antigen sequence can be manipulated according to any known or discovered coronavirus variant. For example, the sequence can be manipulated according to the sequence described in Rambaut et al. nCoV-2019 Genomic Epidemiology, December 2020 (https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563), Tegally, H. et al. 2020 (https://www.medrxiv.org/content /10.1101/ ₂ O ₂ O. 21.12. ₂ O24 8640vl), or Faria NR, et al. 2020 (https://virological.org/t/genomic-characterisation-of-an-emergentsars-cov-2-lineage-in-manaus-preliminary-findings/586). The present invention encompasses amino acid sequences that are substantially homologous to amino acid sequences based on any of the sequences defined in this application. The terms "sequence identity" and "sequence homology" are considered synonymous in this specification.

There are many established algorithms available for aligning two amino acid sequences. Typically, one sequence serves as a reference sequence to which test sequences can be compared. The sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) compared to the reference sequence based on specified program parameters. Amino acid sequence alignment for comparison can be performed, for example, using computer-implemented algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA) or the BLAST and BLAST 2.0 algorithms.

When compared, the identity may exist over a region of sequences of at least 10 amino acid residues in length (e.g., at least 15, ₂₀ , 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 685 amino acid residues in length, e.g., up to the full length of the reference sequence). Each possible variant represents a separate embodiment of the present invention.

The term "exogenous" as used herein refers to a polynucleotide or protein that is not naturally expressed in a fungus (e.g., a heterologous polynucleotide from another species). An exogenous polynucleotide may be stably or transiently introduced into a fungus to produce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule.

As used herein, the term "heterologous" includes a sequence that has been inserted into a fungus and is not naturally occurring in said fungi.

The terms "DNA construct", "expression vector", "expression construct" and "expression cassette" are used to refer to an artificially assembled or isolated nucleic acid molecule that includes a nucleic acid sequence encoding a protein of interest and that is assembled such that the protein of interest is functionally expressed in a target host cell. An expression vector typically contains appropriate regulatory sequences operably linked to the nucleic acid sequence encoding the protein of interest. An expression vector may further include a nucleic acid sequence encoding a selectable marker.

The terms "polynucleotide", "nucleic acid sequence" and "nucleotide sequence" are used herein to refer to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA) and modified forms thereof as a single unit or as a component of a larger construct. A nucleic acid sequence may be a coding sequence, i.e., a sequence that codes for an end product in a cell, such as a protein.

A sequence (such as a nucleic acid sequence and an amino acid sequence) that is "homologous" to a reference sequence refers herein to a percent identity between the sequences, wherein the percent identity is at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%. Each possible variant represents a separate embodiment of the invention. Homologous nucleic acid sequences include variants related to codon usage and degeneracy of the genetic code.

The nucleic acid sequences encoding the proteins of the present invention may be optimized for expression. Examples of such sequence modifications include, but are not limited to, altering the G/C content ratio to more closely approximate that typically observed in filamentous fungi.

The phrase "codon optimization" refers to the selection of appropriate DNA nucleotides for use in a structural gene or fragment thereof that approximates the codon usage of the organism of interest and/or the process of modifying a nucleic acid sequence to enhance expression in host cells of interest by replacing at least one codon (e.g., one or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell, while maintaining the native amino acid sequence. Different species exhibit particular preferences for certain codons for a particular amino acid. Codon preference (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend on, among other things, the properties of the codons being translated and the availability of certain transfer RNA (tRNA) molecules. The prevalence of selected tRNAs in a cell typically reflects the codons most frequently used in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Thus, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified to use statistically preferred or statistically favorable codons in the organism.

Sequence identity can be determined using a nucleotide/amino acid sequence comparison algorithm known in the art.

The term "coding sequence" is used herein to refer to a sequence of nucleotides beginning with a start codon (ATG) and containing any number of codons, except stop codons, and a stop codon (TAA, TGA, TAA), that encodes a functional polypeptide.

Any coding sequence or amino acid sequence referred to herein also encompasses truncated sequences that lack 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons or amino acids from any portion of the sequence. Shortened variants of coding sequences or amino acid sequences can be identified using a nucleotide/amino acid sequence comparison algorithm as known in the art.

Any coding sequence or amino acid sequence disclosed herein also encompasses fusion sequences that comprise, in addition to the coding sequence disclosed herein or a truncated version of this sequence as defined above, other sequences. Fused sequences may be sequences disclosed herein and other sequences. Fused coding sequences or amino acid sequences may be identified using a nucleotide/amino acid sequence comparison algorithm known in the art.

The DNA sequences are assembled into expression cassettes, selection cassettes and further into DNA constructs and/or expression vectors using conventional molecular biological approaches, using restriction endonucleases and ligases, Gibson assembly or yeast recombination. In addition, the above synthesis can be carried out by DNA synthesis service providers. As is known in the art, the same result can be achieved in several different ways.

The DNA sequences are assembled into expression cassettes connecting 5' regulatory regions (promoters), a coding sequence, and 3' regulatory regions (terminators), as described below and as known in the art. Any combination of these three sequences can result in the formation of a functional expression cassette.

The list of terminators includes, but is not limited to, the terminators of the Th. heterothallica genes encoding the uncharacterized protein G2QF75 (XP_003664349); polyubiquitin homolog (G2QHM8, XP_003664133); uncharacterized protein (G2QIA5, XP_003664731); beta-glucosidase (G2QD93, XP_003662704); elongation factor 1-alpha (G2Q129, XP_003660173); chitinase (G2QDD4, XP_003663544), phosphoglycerate kinase (PGK) (Uniprot G2QLD8), glyceraldehyde-3-phosphate dehydrogenase (GPD) (G2QPQ8), phosphofructokinase (PFK) (G2Q605); or triosephosphate isomerase (TPI) (G2QBR0); actin (ACT) (G2Q7Q5); cbhl (GenBank AX284115) or β-glucosidase 1 bgl1 ( _{XM_003662656} ). Exogenous terminators include the Aspergillus nidulans gpdA terminator.

5'-Regulatory regions (promoters) are generally defined as a region up to _2,000 base pairs in length preceding the start codon of the coding sequence of the gene they regulate, provided that the preceding region is non-coding.

3'-Regulatory regions (terminators) are practically defined as a region up to 300 base pairs long located after the end codon of the coding sequence of a gene, provided that the subsequent region is not coding.

DNA sequences are also assembled into selectable marker cassettes, which are expression cassettes in which the coding sequence codes for a gene that provides a selective advantage when present in the transformed strain. Such an advantage could be the use of a new carbon or nitrogen source, resistance to a toxic substance, etc.

Deletion of the proteases disclosed herein can be accomplished as is known in the art. In some embodiments, the deletion is accomplished by transformation of suitable DNA constructs. DNA constructs used for targeted transformation consist of (a) a suitable vector that allows the DNA construct to be maintained in a particular host, (b) zero, one or more expression cassettes in either direction, (c) a selectable marker cassette in either direction, and (d) sequences identical to selected regions of the target genomic DNA (also called targeting regions). These components are arranged such that two targeting regions span any expression cassettes and the selectable marker cassette, such that upon homologous recombination between the targeting regions and the two identical regions of the genomic DNA, the sequence between the targeting regions of the DNA construct is inserted into the chromosome and replaces the sequence originally present in the chromosome. Using this principle, genes can be removed from or inserted into the genome. By placing a sequence following the selectable marker cassette that is identical to the sequence immediately preceding the selectable marker cassette, the marker can be recycled, as is known in the art.

The term "regulatory sequences" refers to DNA sequences that control the expression (transcription) of coding sequences, such as promoters, enhancers, and terminators.

The term "promoter" refers to a regulatory DNA sequence that controls or directs the transcription of another DNA sequence in vivo or in vitro. Typically, a promoter is located 5' of the sequence to be transcribed (i.e., preceding, or upstream of it). Promoters may be derived entirely from a native source or may consist of different elements derived from different promoters found in nature or even contain synthetic nucleotide segments. Promoters may be constitutive (i.e., promoter activation is not regulated by an inducing agent and, therefore, the rate of transcription is constant) or inducible (i.e., promoter activation is regulated by an inducing agent or environmental conditions). Promoters may also restrict transcription to a particular developmental stage or to a particular morphologically distinct part of an organism. In most cases, the precise boundaries of regulatory sequences are not fully defined, and in some cases cannot be fully defined, so that several variants of DNA sequences may have the same promoter activity.

The term "terminator" refers to another regulatory DNA sequence that regulates transcription termination. The termination sequence is operably linked to the 3' end of the nucleic acid sequence being transcribed.

The terms "C1 promoter" and "C1 terminator" refer to promoter and terminator sequences suitable for use in C1, i.e. capable of driving gene expression in C1.

However, as is known to those skilled in the art, the choice of promoters and terminators may not be critical, and similar results can be obtained with different promoters and terminators that provide similar or identical gene expression.

The term "operably linked" means that the selected nucleic acid sequence is located in proximity to a regulatory element (promoter, enhancer and/or terminator) that allows the regulatory element to regulate the expression of the selected nucleic acid sequence.

The present invention discloses the production of a protein of interest using genetically modified strains of Th. heterothallica C1. As described above, filamentous fungi of other species having similar endogenous pathways for producing precursors can also be used.

According to some embodiments, the polynucleotides of the invention are designed based on the amino acid sequence of a protein to be produced using codons of a filamentous fungus. According to some embodiments, the filamentous fungus belongs to the group Pezizomycotina. According to some embodiments, the filamentous fungus belongs to a group selected from the group consisting of Sordariales, Hypocreales, Onygenales and Eurotiales, including the genera and species described above in the Definitions section. According to some exemplary embodiments, the fungus is Th. heterothallica. According to these embodiments, the polynucleotides of the invention are polynucleotides identified in Th. heterothallica or homologues thereof. According to some currently exemplary embodiments, the fungus is Th. heterothallica C1.

According to some exemplary embodiments, Th. heterothallica C1 is derived from strain UV18-#100.

Each DNA construct or expression vector or a plurality thereof comprises regulatory elements that control transcription of polynucleotides in at least one cell of the fungus. The regulatory element may be a regulatory element endogenous to the fungus, in particular, to Th. heterothallica C1, or exogenous to the fungus.

According to some embodiments, the regulatory element is selected from the group consisting of a 5' regulatory element (collectively referred to as a promoter) and a 3' regulatory element (collectively referred to as a terminator), even though these nucleotide sequences may contain additional regulatory elements not classified as promoter or terminator sequences in the strict sense.

According to some embodiments, the DNA construct or expression vector comprises at least one promoter operably linked to at least one polynucleotide comprising a coding sequence operably linked to at least one terminator. According to some embodiments, the promoter is an endogenous fungal promoter, in particular a Th. heterothallica promoter. According to further or alternative embodiments, the promoter is heterologous to a fungus, in particular Th. heterothallica. According to some embodiments, the terminator is an endogenous fungal terminator, in particular Th. heterothallica. According to further or alternative embodiments, the terminator is heterologous to a fungus, in particular Th. heterothallica.

According to some exemplary embodiments, the DNA constructs comprise synthetic regulatory elements, referred to as a "synthetic expression system" (SES), substantially as described in International Patent Application (PCT) Publication No. WO 2017/144777.

According to some embodiments, the polynucleotide is stably integrated into at least one chromosomal locus of at least one cell of the genetically modified fungus. According to some embodiments, the polynucleotide is stably integrated into a specific site on the fungal chromosomes. According to some embodiments, the polynucleotide is stably integrated into a random site on the chromosome. According to some embodiments, the polynucleotide can be included in a targeted or random manner as 1, 2 or more copies in 1, 2 or more chromosomal loci.

According to some alternative embodiments, the polynucleotide is transiently expressed using extrachromosomal expression vectors, as known to those skilled in the art.

According to some embodiments, culturing a genetically modified fungus in a suitable medium results in the production of a protein of interest in an increased amount compared to the amount produced in a corresponding original fungus cultivated under similar conditions.

According to some exemplary embodiments, the present invention relates to a genetically modified fungus Th. heterothallica C1, which allows the production of a protein of interest. According to these embodiments, such a genetically modified fungus Th. heterothallica C1 comprises at least one cell having reduced expression and/or activity of KEX2 and/or ALP7 and at least one additional protease.

According to certain embodiments, a suitable medium for culturing genetically modified fungi comprises a carbon source selected from the group consisting of glucose, sucrose, xylose, arabinose, galactose, fructose, lactose, cellobiose and glycerol. According to some embodiments, the carbon source is obtained from waste from the production of ethanol or other bioproducts from starch, sugar beet and sugar cane, such as molasses containing fermentable sugars, starch, lignocellulosic biomass containing polymeric carbohydrates such as cellulose and hemicellulose.

According to some currently exemplary embodiments, the fungus is Th. heterothallica C1. According to some embodiments, the Th. heterothallica C1 strain is selected from the group consisting of strain UV18-25, Deposit No. VKM F-3631 D; strain NG7C-19, Deposit No. VKM F-3633 D; and strain UV13-6, Deposit No. VKM F-3632 D. Additional strains that can be used are strain HC UV18-100f, Deposit No. CBS141147; strain HC UV18-100f, Deposit No. CBS141143; LC strain W1L#100I, Deposit No. CBS141153; and LC strain W1L#100I, deposition number CBS141149, and derivatives thereof. Each possible variant represents a separate embodiment of the present invention.

According to another aspect, the present invention provides a method for producing a fungus capable of producing an exogenous protein of interest, comprising transforming at least one fungal cell with at least one polynucleotide encoding the protein of interest, wherein said at least one fungal cell has reduced expression and/or activity of KEX2 and/or ALP7 and at least one additional protease.

According to some embodiments, the method further comprises deleting, inhibiting or reducing the expression of KEX2 or ALP7. According to some embodiments, the method further comprises deleting, inhibiting or reducing the expression of at least one protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4.

The terms "reduced expression" or "inhibited expression" of a protein, particularly a protease, as described herein, are used interchangeably and include, without limitation, deletion or damage to the gene that encodes said protein.

The terms "reduced activity" or "inhibited activity" of a protein, particularly a protease, as described herein, are used interchangeably and further include post-translational modifications that result in a decrease or elimination of protein activity.

Any method known in the art for transforming filamentous fungi with a polynucleotide encoding a protein of interest may be used in accordance with the description of the present invention.

The fungus and polynucleotides are as described above.

According to another aspect, the present invention relates to a method for producing an exogenous protein, comprising culturing a genetically modified fungus, in particular the Th. heterothallica C1 fungi of the present invention, in a suitable medium; and isolating the protein products.

According to some embodiments, the method includes culturing genetically modified fungi as described herein, each expressing a different protein of interest. According to some embodiments, the fungi express antigens of different coronavirus variants.

According to some embodiments, the medium contains a carbon source selected from the group consisting of glucose, sucrose, xylose, arabinose, galactose, fructose, lactose, cellobiose and glycerol. According to some embodiments, the carbon source is waste obtained during the production of ethanol or other bioproducts from starch, sugar beet and sugar cane, such as molasses containing fermentable sugars, starch, lignocellulosic biomass containing polymeric carbohydrates such as cellulose and hemicellulose.

According to some embodiments, the exogenous protein is purified from the fungal growth medium.

According to other embodiments, the exogenous protein is extracted from the fungal mass. Any method known in the art for extracting and purifying proteins from plant tissues can be used.

According to another aspect, the present invention relates to an exogenous protein produced by a genetically modified fungus, in particular the genetically modified Th. heterothallica C1 of the present invention.

According to some embodiments, the exogenous protein product is a coronavirus antigen. According to some embodiments, the antigen is a full-length coronavirus spike protein. According to some embodiments, the antigen comprises the RBD sequence of the coronavirus spike protein or a fragment thereof. According to some embodiments, the RBD or fragment thereof is directly or indirectly fused to a Spytag. According to some embodiments, the antigen is attached to a SpyCatcher protein. The following examples are provided to more fully illustrate some embodiments of the present invention. However, they should in no way be construed as limiting the scope of the invention. One skilled in the art will readily be able to propose many changes and modifications based on the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1: Deletion of the C1 alp7 gene

The alp7 C1 protease gene was deleted from a C1 protease deletion strain in which 12 proteases had previously been deleted. The alp7 deletion cassette was constructed in two parts as two separate plasmids. The marker fragments in the two plasmids overlap each other and it is planned that this region will undergo homologous recombination in C1 between the plasmids simultaneously with recombination of fragments of the 5' and 3' flanking regions with genomic DNA on either side of the alp7 gene. Recombination between the selectable marker fragments makes the marker gene functional and allows transformants to grow under selection conditions. The deletion cassette also contains a direct repeat sequence of the 5' flanking region to remove the pyr4 marker. The sequences of the deletion construct plasmids are shown in SEQ ID NOs: 21 and 22. The pMYT0936 5' plasmid contained a fragment of the 5' flanking region of alp7 for integration (positions 9-1025 of SEQ ID NO: 21) and half of the pyr4 marker (positions 1033-2812 of SEQ ID NO: 21). The pMYT0937 3' plasmid contained the second half of the pyr4 marker (positions 17-1273 of SEQ ID NO: 22), the direct repeat sequence (positions 1282-1781 of SEQ ID NO: 22) and a fragment of the 3' flanking region of alp7 for integration (positions 1790-2759 of SEQ ID NO: 22).

Fragments of the alp7 flanking region and the direct repeat were amplified from C1 genomic DNA and cloned into backbone vectors containing the pyr4 marker derived from plasmid pSR426 by Gibson cloning using the NEBbuilder™ HiFi DNA Assembly Kit (New England Biolabs) according to the manufacturer's instructions. Both parts of the deletion construct were excised from the plasmids and co-transformed into the CI strain DNL146, which has 12 deletions of the protease genes, by the protoplast/PEG method as described by Visser, V.J., et al. (Industrial Biotechnology 2011, 7, 214-223).

Transformed colonies growing on pyr4 selective medium plates were streaked again on the same selective medium. Identification of correct transformants was performed by PCR. Mycelium from streak cultures of transformants was dissolved in ₂₀ mM NaOH and incubated at 100°C to lyse the cells. 1-2 μl of this solution was used as a template for PCR using the Phire Plant PCR™ kit (Thermo Fisher). The oligonucleotide primers used in this PCR are listed in Table 1. Integration of the deletion construct into the alp7 locus was confirmed by two PCR reactions. Integration at the 5' end of the gene was confirmed by a reaction with the primers shown in SEQ ID NOs: 25 and 26. A 1233 bp fragment was detected in the alp7 locus using the primers shown in SEQ ID NOs: 26 and 27. was amplified, indicating successful integration into the alp7 locus. Integration at the 3' end of alp7 was confirmed with the primers shown in SEQ ID NOs: 27 and 28. A 1748 bp fragment was amplified, indicating successful integration into the alp7 locus. Loss of the alp7 gene was demonstrated by reaction with the primers shown in SEQ ID NOs: 29 and 30. A 569 bp fragment was not amplified, indicating deletion of the alp7 gene.

Transformants positive for both the integration PCR reactions and for the loss of the alp7 open reading frame were further analyzed by quantitative PCR with the primers shown in SEQ ID NOs: 31 and 32 to demonstrate complete deletion of the alp7 gene from the transformants tested. One clone of transformant C1, positive for the integration of the deletion cassette into the alp7 locus and negative for the alp7 gene by quantitative PCR, was stored at -80°C and assigned the strain number DNL150.

Example 2: Deletion of the C1 kex2 gene

The C1 protease gene kex2 was deleted from a C1 protease deletion line strain in which 12 proteases had been deleted previously. The deletion cassette for kex2 was constructed in two parts as two separate plasmids and functions in C1 transformation in a similar manner to the alp7 deletion cassette (described above). The deletion cassette also contains a direct repeat sequence of the 5' flanking region for removal of the pyr4 marker. The sequences of the deletion construct plasmids are shown in SEQ ID NOs: 23 and 24. The 5' region plasmid pMYT0997 contained a fragment of the kex2 5' flanking region for integration (positions 9-1058 of SEQ ID NO: 23) and half of the pyr4 marker (positions 1033-2812 of SEQ ID NO: 23). The 3' region of pMYT0998 plasmid contained the second half of the pyr4 marker (positions 17-1273 of SEQ ID NO: 24), the direct repeat sequence (positions 1281-1782 of SEQ ID NO: 24) and a fragment of the 3' flanking region of kex2 for integration (positions 1791-2690 of SEQ ID NO: 24).

Fragments of the flanking regions of kex2 and the direct repeat were amplified from C1 genomic DNA and cloned into pyr4 marker-containing backbone vectors derived from pSR426 by Gibson cloning using the NEBbuilder™ HiFi DNA Assembly Kit (New England Biolabs) according to the manufacturer's instructions. Both parts of the deletion construct were excised from the plasmids and co-transformed into the CI strain DNL146, which has 12 deletions of the protease genes, as described previously by Visser, V.J., et al. {ibid.).

Transformed colonies growing on pyr4 selective medium plates were streaked again on the same selective medium. Identification of correct transformants was performed by PCR. Mycelium from streak cultures of transformants was dissolved in ₂₀ mM NaOH and incubated at 100°C to lyse the cells. 1-2 μl of this solution was used as a template for PCR using the Phire Plant PCR™ kit (Thermo Fisher). The oligonucleotide primers used in this PCR are shown in Table 2. Integration of the deletion construct into the kex2 locus was confirmed by two PCR reactions. Integration at the 5' end of the gene was confirmed by a reaction with the primers shown in SEQ ID NOs: 33 and 34. The 1187 bp fragment was amplified, indicating successful integration into the kex2 locus. Integration at the 3' end of kex2 was confirmed with the primers shown in SEQ ID NOs: 35 and 36. A 1849 bp fragment was amplified, indicating successful integration into the kex2 locus. Loss of the kex2 gene was confirmed by reaction with the primers shown in SEQ ID NOs: 37 and 38. A 510 bp fragment was not amplified, indicating deletion of the kex2 gene.

Transformants positive for both integration PCR reactions and for loss of the kex2 ORF were further analyzed by quantitative PCR with the primers shown in SEQ ID NOs: 39 and 40 and with the primers shown in SEQ ID NOs: 41 and 42 to demonstrate complete deletion of the kex2 gene from the transformants tested. One transformant clone C1, positive for integration of the deletion cassette into the kex2 locus and negative for the kex2 gene by quantitative PCR, was stored at -80°C and assigned the strain number DNL152.

Example 3: Combination of deletions of the C1 alp7 gene and the C1 kex2 gene

The creation of strain C1, in which both the alp7 and kex2 genes were deleted, was accomplished by deleting the kex2 gene from strain DNL150, in which the alp7 gene and 12 other protease genes had previously been deleted. Before deleting the kex2 gene, the pyr4 marker was deleted in strain DNL150 in order to use the same deletion cassettes for the kex2 gene deletion that were described above for the generation of strain DNL152.

Removal of the pyr4 selectable marker using the deletion cassette described above for the production of DNL150 relies on two features: a) a functional pyr4 gene converts 5-fluoroorotic acid (5-FOA) to 5-fluorouracil, a toxic metabolite, so clones that have lost the functional pyr4 gene are able to grow in the presence of 5-FOA; and b) under selection pressure from 5-FOA, a direct repeat sequence in the deletion construct allows clones to remove the pyr4 selectable marker through a homologous recombination event between the 5' flanking region and the direct repeat. Successful recombination removes the entire pyr4 marker, allowing the correct clones to grow in the presence of 5-FOA.

Removal of the pyr4 marker from DNL150 was performed according to the following protocol: a small portion of fresh mycelium from a plate was suspended in a solution of 0.9% NaCl, 0.025% Tween-20. Dilutions of the suspension were prepared. Different amounts of mycelial suspension were applied to plates containing 5-fluoroorotic acid (5-FOA) (components of the medium of 5-FOA plates: 7 mM KCl, 11 mM KH ₂ PO ₄ , 0.1% glucose, 10 mM uracil, 10 mM uridine, 2 mM MgSO ₄ , 10 mM proline, trace element solution (100x: 174 mM EDTA, 76 mM ZnSO ₄ ⋅7H ₂ O, 178 mM H ₃ BO ₃ , 25 mM MnSO ₄ ^⋅ H ₂ O, 18 mM FeSO ₄ ⋅7H ₂ O, 7.1 mM CoCl ₂ ⋅6H ₂ O, 6.4 mM _CuSO4⋅5H2O , 6.2 mM Na2MoO4⋅2H2O), 4 mM 5-fluoroorotic acid, _{2 O} g/l granular agar, pH 6.0). The plates were incubated at +35°C until colonies appeared. Colonies growing on the _{plates with} 5-FOA _medium were streaked again on the same selective medium. Since growth on the selective 5-FOA medium is weak and streaks do not expand into distinct bands, mycelium from weak bands was streaked onto a non-selective medium (medium components: 7 mM KCl, 55 mM KH ₂ PO ₄ , 1.0% glucose, 670 mM sucrose, 0.6% yeast extract, 35 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 10 mM uracil, 10 mM uridine, trace element solution (100x: 174 mM EDTA, 76 mM ZnSO ₄ ⋅7H ₂ O, 178 mM H ₃ BO ₃ , 25 mM MnSO ₄ ⋅H ₂ O, 18 mM FeSO _{4 ⋅7H2O} , 7.1 mM CoCl2 _⋅6H2O , 6.4 mM CuSO4 _⋅5H2O , 6.2 mM _{Na2MoO4 -2H2O} ), 16 g/L granular agar, _pH 6.5 ₎ to ensure good growth. Streaks growing efficiently on non-selective medium plates _were streaked onto pyr4 selective medium plates without uracil and uridine for phenotype testing. When testing the phenotype, clones in which pyr4 deletion was successful could not grow on the medium without the addition of uracil and uridine ( _medium components: 7 mM KCl, 11 mM _KH2PO4 , 1.0% glucose, ₆₇₀ mM sucrose, 35 mM ( _NH4 ) _2SO4 , 2 mM _MgSO4 , trace element solution (1000x: 174 mM _{EDTA, 76 mM ZnSO4 ⋅7H2O, 178 mM H3BO3 , 25 mM MnSO4 ⋅H2O} ^, _{18 mM FeSO4 ⋅7H2O} , _7.1 mM _{CoCl2 ⋅6H2O} , 6.4 mM CuSO ₄ ⋅5H ₂ O, 6.2 mM Na ₂ MoO ₄ ⋅2H ₂ O), 15 g/L granular agar, pH 6.5). Clones not growing on phenotype testing plates were analyzed for pyr4 deletion by quantitative PCR with the primers given as SEQ ID NOs: 43 and 44. The oligonucleotide primers used in the qPCR reactions are listed in Table 3.

One pyr4-deleted clone of DNL150, which failed to grow in phenotype testing and was negative for the pyr4 gene by qPCR, was stored at -80°C and given the strain number DNL151.

The kex2 protease was deleted from the CI strain DNL151 using the same deletion cassette and transformation method described above for DNL152. Identification of the correct integration and deletion of kex2 by PCR reaction was performed as described above for DNL152. One transformant clone C1, positive for integration of the deletion cassette into the kex2 locus and negative in the quantitative PCR test for the presence of the kex2 gene, was stored at -80°C and assigned the strain number DNL155 (ΔalplΔalp2Δpep4ΔprtlΔsrplΔalp3ΔpeplΔmtp2Δpep5Δmtp4Δpep6Δalp4Δalp7Δkex2)

Example 4: Expression of SARS-CoV-2 RBD in protease-deficient C1 strains

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein was expressed in protease-deficient C1 strains. The first construct contained the sequence encoding the endogenous CBH1 signal sequence of C1, residues 333-527 of the SARS-CoV-2 spike protein, a Gly-Ser linker, and a C-tag, flanked by recombinant sequences to the C1 expression vector and Mssl restriction enzyme sites. The fragment was synthesized by GenScript (USA) and presented as SEQ ID NO: 45 (amino acid sequence of the RBD-C-tag, including the signal sequence and the Gly/Ser linker between the RBD and the C-tag). The codon usage of the gene was optimized for expression in Thermothelomyces heterothallica. The synthesized fragment was amplified by PCR from the GenScript plasmid and cloned using the Gibson assembly method (NEBuilder® HiFi DNA Assembly Cloning kit, New England Biolabs) into the Pad site of the C1 expression vector pMYT1055 under the endogenous C1 bgl8 promoter and C1 chil terminator. The correctness of the construct sequence was confirmed by sequencing the fragment inserted into the plasmid. The plasmid with the correct sequence was assigned the plasmid number pMYT1142 (SEQ ID NO: 46). The second construct contained, in addition to the same sequences as in pMYT1142, a Gly-Ser linker and a Spytag between the RBD domain and the C-terminal Gly-Ser linker and a C-tag. This sequence is given as SEQ ID NO: 47 (amino acid sequence of RBD-Spytag-C-tag, including signal sequence and Gly/Ser linker between Spytag and C-tag). The second construct was inserted into the expression vector pMYT1055 in the same manner as pMYT1142, and the plasmid with the correct sequence was assigned the plasmid number pMYT1143 (SEQ ID NO: 48).

The expression vector pMYT1142 and the dummy partner vector pMYT1140, which is required for completion of the hygromycin resistance marker gene and integration into the bgl8 locus, were cleaved with MssI and cotransformed into the DNL155 strain, from which fourteen protease genes had been deleted. Transformation was performed by the protoplast/PEG method (Visser, VJ, et al., ibid.), and transformants were selected for the nia1+ phenotype and hygromycin resistance. Transformants were streaked onto selective medium plates and inoculated from streaks into liquid cultures in 24-well plates. The medium components were: (in g/l) glucose 5, yeast extract 1, (NH ₄ ) ₂ SO ₄ 4.6, MgSO ₄ ⋅7H ₂ O 0.49, KH ₂ PO ₄ 7.48, and (in mg/l) EDTA 45, ZnSO ₄ ⋅7H ₂ O 19.8, MnSO ₄ ⋅4H ₂ O 3.87, СоСl ₂ ⋅6Н ₂ O 1.44, CuSO ₄ ⋅5H ₂ O 1.44, Na ₂ MoO ₄ ⋅2H ₂ O 1.35, FeSO ₄ ⋅7H ₂ O 4.5, Н ₃ BO ₄ 9.9, D-biotin 0.004, 50 U/ml penicillin and 0.05 mg streptomycin. The 24-well plates were incubated at 35°C with shaking at 800 rpm for four days. Culture supernatants were collected and analyzed by Western blotting using standard methods with Capture Select Biotin Anti-C-tag conjugate primary detection agent (ThermoFisher) and IRDye 800CW Stretavidin secondary detection agent (Li-Cor). Western analysis (Fig. 1) showed that a strong signal of the expected magnitude was detected for a number of RBD-C-tag and RBD-Spytag-C-tag transformants, indicating that both of these proteins are produced in C1.

Transformants producing the RBD-C tag protein were purified by single colony plating, and the purified clone was checked by PCR for correct integration of the expression cassette and by quantitative PCR for clone purity. One verified transformant producing the RBD-C tag was stored at -80°C and assigned the strain number M4169.

The expression vector pMYT1143 carrying the RBD-Spytag-C-tag variant was co-transformed with the mock partner vector pMYT1140, and transformants were analyzed from 24-well plate cultures (Fig. 1) and purified by single colony plating in the same manner as described above for pMYT1142. After PCR verification, one clone of the C1 trans form producing the RBD-Spytag-C-tag was stored at -80°C and assigned the strain number M4173.

Plasmids pMYT1142 and pMYT1143 were also transformed into other protease-deficient C1 strains besides DNL155 to compare production levels in the different protease-deleted strains. The protease gene deletions in these strains are listed in Table 4. RBD-producing plasmids pMYT1142 and pMYT1143 were transformed into four other protease-deficient strains: 1) strain DNL145, in which 12 proteases were deleted, 2) DNL150, in which 13 proteases were deleted, 3) DNL159, which is a parallel clone of DNL155, and 4) DNL157, in which 14 proteases were deleted but the kex2 gene remained intact. Transformation, analysis of transformants, purification from single colonies and PCR analysis were carried out in the same manner as described above for the production of strains M4169 and M4173. Several production strains were confirmed to produce both the RBD-C-tag and the RBD-Spytag-C-tag in all four protease-deficient strains. Three parallel transformants from these new production strains in DNL145, DNL150, DNL157 and DNL159, together with M4169 and M4173 and two other parallel clones of both these strains were cultured in liquid medium in 24-well plates at 35°C with shaking at 800 rpm for four days. Culture supernatants were collected and analyzed on Coomassie-stained SDS gels by methods known in the art. The highest production of RBD proteins was observed in strains DNL155 and DNL159, in which kex2 was deleted (Fig. 2).

The RBD-C-tag protein-producing strain CI M4169 was grown in a 2-liter bioreactor in a fed-batch process in a medium with yeast extract as an organic nitrogen source and glucose as a carbon source. Cultivation was carried out at 38°C for five days. At the end of the cultivation, the mycelium was removed by centrifugation at 4000 g for ₂ O minutes, phenylmethylsulfonyl fluoride was added at a concentration of 1-2 mM to inhibit protease activity in the resulting liquid culture supernatant, and the supernatant was stored at -80 °C. For purification of RBD by C-labeled affinity chromatography, 100 ml of the liquid culture was thawed on ice, and after thawing, the sample was clarified by centrifugation 3x ₂ O min at 20,000 g at +4 °C, followed by filtration through a 0.45 μM filter. 90 mL of the clear supernatant _was diluted _with 1×PBS (phosphate-buffered saline) (12 _{mM Na2HPO4⋅2H2O} , 3 mM _{NaH2PO4⋅H2O} , 150 mM NaCl, _pH 7.3) to a final volume of 200 mL. C-tag affinity purification was performed using a 10 mL column packed with CaptureSelect C-tag XL resin (Thermo Fisher) attached to an AKTA Start Protein Purification System (Cytiva) and operated at a flow rate of 2.5 mL/min. The column was first equilibrated with 5 column volumes (CV) of 1×PBS before sample loading. After loading the sample, the column was washed with 15 column volumes of 1×PBS and then eluted with a one-step gradient of 5 column volumes of ₂₀ mM Tris-HCl, 2 M MgCl2, 1 mM EDTA, pH 7.5, in 3 ml fractions. The amount of eluted RBD was quantified by integrating the UV curve of the elution peak using the Unicorn 1.0 application program included in the AKTA Start system. An extinction coefficient of 1.498 was used to calculate the amount of RBD-C-tags and 1.450 to calculate the amount of RBD-Spytag-C-tags. After elution, the column was regenerated with 5 column volumes of 0.1 M glycine, pH 2.3, and washed with 1×PBS until the pH reached 7.3. The elution fractions containing protein were pooled for a dialysis step to exchange the elution buffer with 1×PBS. The pooled fractions were placed in a 12 mL dialysis cassette and the dialysis cassette was dialyzed in 1.5 L of 1×PBS for 1 h at +4°C with magnetic stirring. 1×PBS was replaced with fresh buffer after 1 h and dialysis was continued for 2 h under the same conditions. Finally, 1×PBS was replaced with fresh buffer and dialysis was continued overnight. The concentration of dialyzed RBD was determined using a Nanodrop spectrophotometer by measuring the optical density at 280 nm and using extinction coefficients of 1.498 for the RBD-C-tag and 1.450 for the RBD-Spytag-C-tag. Aliquots of the RBD preparations were stored at -80°C. Affinity purification of the RBD-C tag from M4169 fermentation is shown as an example in Fig. 3A-3B. Polyclonal rabbit antiserum to SARS-CoV-2 spike RBD antibody (SinoBiologicals) and goat anti-rabbit IRDye 680RD (Li-Cor) were used for Western blot detection.

Example 5: Protein expression and stability in different strains of Thermothelomyces heterothallica C1

Different strains of Thermothelomyces heterothallica C1 are shown in Fig. 4.

To test the stability of proteins and antibodies, addition experiments were used. Target proteins were added and incubated in the culture supernatant of fungal strains. Samples were taken at different time points and analyzed by Western blotting. As shown in Fig. 5 and 6, deletion of ALP7 had a positive effect on antibody stability.

Fig. 7 shows experiments with the addition of fibrinogen. Increased stability was found in the KEX2-deficient strain.

Fig. 8 shows experiments with addition of Fc-FGF21. Increased stability was found in strains deficient in KEX2 and SRP10.

Figure 9 shows the spiking experiment and expression of mAb in protease-deficient strains. Increased stability and protein amounts were found in the ALP7-deficient strain 13x compared to the protease-deficient strains 12x and 13x SRP10. When the same mAb was expressed in the ALP7-deleted strain 13x, significantly more intact mAb was produced.

Figure 10 shows mAb expression in 13x protease deletion strains with deletion of kex2 or alp7. The 27 kDa degradation fragment (marked with arrow) was not produced in the KEX2 deletion strain compared to the parental 12x strain. In addition, the 37 kDa degradation fragments were not produced in the ALP7-deficient 13x strain compared to the parental 12x protease-deficient strains.

Example 6: Expression of RVFV in 14x protease-deficient strains

The vaccine antigen protein from Rift Valley fever virus (RVFV) was expressed as a fusion protein with the Spycatcher domain from the same expression vector in the 13x protease deletion strain DNL150 and the 14x protease deletion strain DNL155, which has a kex2 deletion.

Strains transformed with the RVFV antigen expression vector were grown in 24-well plates, and antigen production was analyzed by Western blotting with an antibody against RVFV antigen.

As shown in Fig. 11, transformants of the 14x protease-deficient strain DNL155 showed high expression of RVFV. The expression level was much higher than that of the 13x protease-deficient strain (DNL150).

Example 7: Expression and functionality of RBD-Spytag in 14x protease-deficient strains

The structural arrangement of the receptor-binding domain of the SARS-CoV-2 spike protein fused to Spytag in the 14x protease-deficient Thermothelomyces heterothallica C1 strain is shown in Figs. 12A–12B. The protein was conjugated to the SpyCatcher virus-like particle (VLP) vaccine with recombinant hepatitis B surface antigen (HBsAg) to study the potential of using the resulting protein as a vaccine. Two lots of CI RBD-Spytag (#2 and #4) were tested. The stability of the proteins and conjugates was examined by SDS-PAGE gel and then analyzed by Western blotting using mouse anti-HBsAg antibody (1st antibody) and goat anti-mouse IgG-ALP (2nd antibody). As shown in Fig. 12A-12B, RBD-Spytag were efficiently conjugated to SpyCatcher HBsAg VLP. Importantly, RBD proteins with or without conjugated SpyCatcher were able to generate dimers/trimers. Dimerization and trimerization of the recombinant RBD mimics the natural structure of coronavirus RBS and is expected to enable the design of an effective vaccine.

The binding of RBD-Spytag to human ACE-2 protein was then examined using the CR3022 antibody. As shown in Fig. 13A-13F, the CR3022 antibody was able to bind to the RBD presented on the HPV particle. In addition, an indirect ELISA was used to show that the conjugated RBD binds hACE-2 and not the HPV particle. Taken together, the results indicate that the resulting Spytag-fused RBD is correctly assembled, presented on the HPV particle, and thus has potential for use as a vaccine.

Example 8: Production of SARS-CoV-2 receptor-binding domain fusion proteins with Fc in C1

Two potential SARS-CoV-2 coronavirus vaccine proteins were produced in C1, in which the receptor binding domain (RBD) of the SARS-CoV-2 S2 spike protein was fused at either the N-terminus or the C-terminus to the Fc domain of an IgG1 antibody. The 40 bp DNA encoding fragments overlap with the C1 bgl8 promoter, the CI CBH1 signal sequence, the coding region of the RBD-Fc or Fc-RBD amino acid sequence (shown as SEQ ID NOs: 49 and 51; these sequences include the signal sequence and the linker between the RBD and Fc), a stop codon, and an overlap with the C1 bgl8 or chil terminator. The protein-coding regions of the DNA fragments are shown as SEQ ID NOs: 50 and 52. The DNA fragments overlapping the chil terminator were cloned into the 5' region of the expression construct (plasmid pMYT1055), and the fragments overlapping the bgl8 terminator were cloned into the 3' region of the expression construct (plasmid pMYT1056). Cloning was performed by the Gibson assembly method using the NEBuilder™ HiFi DNA Assembly Kit (New England Biolabs) according to the manufacturer's instructions. The resulting expression plasmids were designated pMYT1302 (RBD-Fc 5' region), pMYT1303 (RBD-Fc 3' region), pMYT1304 (Fc-RBD 5' region), and pMYT1305 (Fc-RBD 3' region).

To construct C1 strains producing the RBD-Fc construct, the expression plasmids pMYT1302 and pMYT1303 were transformed together into three different CI strains: DNL155 (ΔalplΔalp2Δpep4ΔprtlΔsrplΔalp3ΔpeplΔmtp2-Δpep5Δmtp4Δpep6Δalp4Δalp7Δkex2), DNL157 (ΔalplΔalp2Δpep4ΔprtlΔsrplΔalp3-ΔpeplAmtp2Δpep5Δmtp4Δpep6Δalp4Δalp7Δsrpl0) and a glycoengineered strain containing 10 protease deletions, M3599 (Δа1р1Δа1р2Δрер4ΔрrtΔsrtΔаrр3Δрер1Δmtр2Δаlр6Δsrр7). Following transformation, the 5' and 3' regions of the expression construct are integrated into the bgl8 locus, and the overlapping fragments of the hygromycin resistance gene in the two regions are recombined with each other to form a final expression construct with two expression cassettes at the bgl8 locus. Transformations were performed as described by Visser, V.J., et al. {ibid.). Transformants were selected for hygromycin resistance and screened using 24-well plate cultures and Western blotting for RBD-Fc protein production. Western analysis was performed using a standard method with a 1:10,000 dilution of goat anti-human IgG polyclonal F(c) conjugate-IRDye700DX (Licor). Signal detection was performed using a Licor Odyssey fluorometer. The results show that only a small fraction of the RBD-Fc produced in the M3599 strain with deletions of 10 proteases is full-length (calculated molecular weight 49.4 kDa). In strains M155 and Ml57, most of the RBD-Fc is not cleaved by proteases and is produced as intact product (Fig. 14A). These strains have deletions of the alp7 protease (DNL157) or both alp7 and kex2 (DNL155). The level of production in DNL155 is significantly higher than in DNL157. In conclusion, it should be noted that deletions of alp7 and kex2 have a beneficial effect on RBD-Fc production.

To generate a strain expressing the Fc-RBD fusion protein, plasmids pMYT1304 and pMYT1305 were transformed together into the DNL155 strain as described above for constructing the RBD-Fc producer strain. Transformants were analyzed for the presence of Fc-RBD by Western blotting from 24-well plate cultures as described above (Fig. 14B). Several transformants were found producing good levels of Fc-RBD protein. The product was largely intact.

Example 9: Vaccination of mice with SARS-CoV-2 RBD antigen

The resulting SARS-CoV-2 spike protein from Example 4 was tested for use as a vaccine. The SARS-CoV-2 RBD antigen was administered to K18 hACE2 transgenic mice. Two groups of transgenic mice were vaccinated with 20 μg of RBD formulated with alhydrogel. Primary vaccination was performed on day 1 (“prime”) and on day 21 (“booster”). On day 42, mice were challenged with 2,000 PFU of SARS-CoV-2. Serum studies showed that the antigen produced high titers of neutralizing antibodies. Two days after SARS challenge, all control mice died, while 13 of 14 vaccinated mice survived with little or no weight loss.

Example 10: Expression of recombinant antigen αMHCII-Cal07 in protease-deficient O strains

Recombinant antigen αMHCII-Саl07, consisting of the targeting domain of MHCII and the HA antigen of the influenza A/California/07/2009 strain (subtype H1N1), was expressed in protease-deficient C1 strains. The expression construct contained a sequence encoding the endogenous C1 signal sequence CBH1, an MHCII-specific targeting element, a 20-amino acid linker, residues 18-541 of the HA protein obtained from the influenza A/California/07/2009 strain, and a C-tag flanked by recombination sequences with the C1 expression vector and recognition sites for Mssl restriction enzymes. The fragment was synthesized by GenScript (USA). Gene codon usage was optimized for expression in Thermothelomyces heterothallicus. The synthesized fragment was released from the GenScript plasmid by digestion with the restriction enzyme Mssl and cloned using the Gibson assembly method (NEBuilder® HiFi DNA Assembly Cloning kit, New England Biolabs) into the Pad site of the C1 expression vector pMYT1055 under the endogenous C1 bgl8 promoter and the C1 chil terminator. The correctness of the construct sequence was confirmed by sequencing the fragment inserted into the plasmid. The plasmid with the correct sequence was assigned the plasmid number pMYT1242.

In the second case, the synthesized fragment was amplified by PCR from the GenScript plasmid and cloned by the Gibson assembly method into the Pad site of the C1 expression vectors pMYT0987 under the synthetic AnSES promoter and the endogenous C1 chil terminator. The correctness of the construct sequence was confirmed by sequencing the fragment inserted into the plasmid. The plasmid with the correct sequence was assigned the plasmid number pMYT1243.

The expression vector pMYT1242 and the mock partner vector pMYT1140, which is required for completion of the hygromycin resistance marker gene and integration into the bgl8 locus, were digested with Mssl and cotransformed into strain DNL155, from which fourteen protease genes were deleted, and into strain M3599, from which ten protease genes were deleted. The proteases deleted in the above strains are listed in Table 5. Transformation was performed by the protoplast/PEG method according to Visser, VJ, et al. (ibid.), and transformants were selected for the nial+ phenotype and hygromycin resistance. Transformants were streaked onto plates containing selective medium and inoculated from the streaks into liquid cultures in 24-well plates. The medium components were: (in g/l) glucose 5, yeast extract 1, (NH ₄ ) ₂ SO ₄ 4.6, MgSO ₄ ⋅7H ₂ O 0.49, KH ₂ PO ₄ 7.48, and (in mg/l) EDTA 45, ZnSO ₄ ⋅7H ₂ O 19.8, MnSO ₄ ⋅4H ₂ O 3.87, СоСl ₂ ⋅6Н ₂ O 1.44, CuSO ₄ ⋅5H ₂ O 1.44, Na ₂ MoO ₄ ⋅2H ₂ O 1.35, FeSO ₄ ⋅7H ₂ O 4.5, Н ₃ BO ₄ 9.9, D-biotin 0.004, 50 U/ml penicillin and 0.05 mg streptomycin. The 24-well plates were incubated at 35°C with shaking at 800 rpm for four days. The culture supernatants were collected and analyzed by Western blotting using standard methods with Capture Select Biotin Anti-C-tag conjugate (ThermoFisher) as the primary detection agent and IRDye 680RD Streptavidin (Li-Cor) as the secondary detection agent. Western analysis (Fig. 15) showed a strong signal of the expected size (87 kDa) for a number of αMHCII-Cal07 transformants derived from strain DNL155, confirming protein production in C1. However, none of the transformants derived from M3599 showed products of the expected size. Additional proteases present in M3599-derived transformants compared to DNL155-derived transformants caused proteolytic degradation of the product.

Transformants producing αMHCII-Cal07 protein were purified by single colony plating, and the purified clone was checked by PCR for correct integration of the expression cassette and by quantitative PCR for clone purity. One confirmed αMHCII-Cal07-producing transformant was stored as a stock solution in glycerol at -80°C and assigned the strain number M4540.

C1 DNL155 strains were further co-transformed with the Mssl-digested pMYT1243 expression vector carrying the αMHCII-Cal07 construct under the control of the synthetic AnSES promoter, together with the mock partner vector pMYT1141, and transformants were assayed in 24-well plate cultures (Fig. 15) and purified by single colony plating in the same manner as described above for pMYT1242. After PCR confirmation, one C1 transformant clone producing αMHCII-Cal07 was stored at -80°C and assigned the strain number M4543.

In addition, strain C1 M4621 with fourteen deletions of protease genes and deletion of the alg3 gene encoding dolichol-β-Man-dependent alpha-(1-3)mannosyltransferase was cotransformed with the Mss I-digested pMYT1243 expression vector and the mock pMYT1 141 vector, similar to the description given above for DNL155. Deletion of the alg3 gene causes a change in the structure of N-glycans associated with glycoproteins, which leads to a shift towards smaller N-glycan species with fewer mannose residues. Transformants obtained after such transformation were cultured in liquid medium in 24-well plates at 35°C with shaking at 800 rpm for four days. Culture supernatants were collected and analyzed by Western blotting by standard methods using the primary detection agents Capture Select Biotin Anti-C-tag conjugate (ThermoFisher) and mouse monoclonal antibody 29E3 raised against the HA antigen of influenza A/California/07/2009 strain (Manicassamy et al., 2010; PLoS Pathog 6(1): el000745. doi: 10.1371/journal.ppat. 1000745), and the secondary agents IRDye 680RD Streptavidin (Li-Cor) and IRDye 800CW goat anti-mouse IgG secondary antibody (Li-Cor). Western analysis revealed the presence of a signal of the expected size (87 kDa) for a number of transformants, confirming protein production in transformants derived from M4621 (data not shown).

The C1 M4540 strain producing the recombinant αMHCII-Cal07 protein was cultured in a 0.25 L bioreactor using a fed-batch process in a medium containing yeast extract as an organic nitrogen source and glucose as a carbon source. Cultivation was carried out at 38°C for seven days. After completion of cultivation, the fermentation broth was stored at -80°C. For purification of αMHCII-Cal07 by C-labeled affinity chromatography, 50 mL of the liquid culture was thawed on ice, and after thawing, the sample was purified by centrifugation for 3×20 min at 20,000 × g at +4°C, followed by filtration through a 0.45 μm filter. 33 ml of the clear supernatant was diluted with 1×PBS/0.5 M NaCl (12 mM Na ₂ HPO ₄ ⋅2H ₂ O, 3 mM NaH ₂ PO ₄ ⋅H ₂ O, 650 mM NaCl, pH 7.3) to a final volume of 100 ml.

C-tag affinity purification was performed using a 1 mL CaptureSelect C-tag XL column (Thermo Fisher) attached to the AKTA Start Protein Purification System (Cytiva) running at a flow rate of 1 mL/min. The column was first equilibrated with 5 column volumes (CV) of 1×PBS/0.5 M NaCl before sample loading. After sample loading, the column was washed with 15 CVs of 1×PBS/0.5 M NaCl and then eluted with a one-step gradient of 10 CVs of 20 mM Tris-HCl, 2 M MgCl, 1 mM EDTA, pH 7.5, in 1 mL fractions. The amount of eluted αMHCII-Cal07 was quantified by integration of the UV peak elution curve using the Unicorn 1.0 software included in the AKTA Start system. An extinction coefficient of 1.7 was used to calculate the amount of αMHCII-Cal07. After elution, the column was regenerated with 5 column volumes of 0.1 M glycine, pH 2.3, and washed with 1×PBS until pH 7.3 was achieved. The elution fractions containing protein were pooled for a dialysis step to replace the elution buffer with 1×PBS buffer. The pooled fractions were placed in a 12 mL dialysis cassette, and the dialysis cassette was dialyzed in 1.5 L of 1×PBS for 1 h at +4°C with magnetic stirring. 1×PBS was replaced with fresh buffer after 1 h, and dialysis was continued for 2 h under the same conditions. Finally, 1×PBS was replaced and dialysis was continued overnight. The concentration of dialyzed αMHCII-Cal07 was determined using a Nanodrop spectrophotometer by measuring the optical density at 280 nm and using an extinction coefficient of 1.7. Aliquots of the RBD preparations were stored at -80°C. Affinity purification of αMHCII-Cal07 from the M4540 fermentation supernatant is shown as an example in Figs. 16A-16C. For Western blot detection, the primary agents used were CaptureSelect Biotin Anti-C-tag conjugate (ThermoFisher) and mouse monoclonal antibody 29E3 raised against influenza HA antigen, and the secondary agents were IRDye 680RD Streptavidin (Li-Cor) and IRDye 800CW goat anti-mouse IgG secondary antibody (Li-Cor).

Example 11: Expression of SARS-CoV-2 RBD variants in the 14-fold protease-deficient O strain

Three variants of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were expressed in the protease-deficient C1 DNL155 strain. The three variants were: 1) RBD_B.1.1.7-UK with the N501Y mutation, 2) RBD_B.1.351-SA with the K417N, E484K, and N501Y mutations, and 3) RBD_1.1.28.1(P.1)-BR with the K417T, E484K, and N501Y mutations. A fragment of each variant was synthesized by GenScript (USA), and the optimized Wuhan RBD sequence (in pMYT1142 of Example 4) was used as a backbone in which the mutated amino acids were replaced with the codon most frequently occurring in C1. The design of the synthesized fragment was similar to the Wuhan C-tag RBD (used in pMYT1142 of Example 4), except that the Gly/Ser linker between the variant RBD and the C-tag was three amino acids long, whereas in the Wuhan RBD-C -tag the linker was five amino acids long. The variant RBDs were expressed as two copies of the gene in C1, and to express a double copy at the same genomic locus, two plasmid constructs (5' region and 3' region) containing both one copy of the gene were constructed for each variant. In C1 cells, recombination between the selectable marker fragments in the plasmids with the 5' and 3' regions renders the marker gene functional and allows transformants to grow under selection conditions. For plasmids with 5' regions, the synthesized fragments were PCR amplified from GenScript plasmids and cloned using the Gibson assembly method (NEBuilder® HiFi DNA Assembly kit, New England Biolabs) into the Pad site of the CI expression vector pMYT1055 under the endogenous C1 bgl8 promoter and C1 chil terminator. The correctness of the construct sequences was confirmed by sequencing the fragments inserted into the plasmids. Plasmids with the correct sequences were assigned the plasmid numbers pMYT1572 for RBD B.1.1.7-UK, pMYT1574 for RBDB.1.351-SA, and pMYT1576 for RBD1.1.28.1(P. 1)-BR, respectively. For plasmids with 3' regions, the synthesized fragments in GenScript plasmids were cut with the restriction enzyme Mssl and cloned using the Gibson assembly method (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs) into the Pad site of the CI expression vector pMYT1056 under the endogenous CI bgl8 promoter and CI bgl8 terminator. Plasmids with the correct sequences were assigned the plasmid numbers pMYT1573 for RBD_B.1.1.7-UK, pMYT1575 for RBD_B.1.351-SA, and pMYT1577 for RBD_1.1.28.1 (P. 1) -BR, respectively.

For expression, a double copy of the plasmid with both the 5' and 3' region was digested with Mssl, and plasmids carrying the same gene variant were co-transformed into strain DNL155, from which fourteen protease genes were deleted. DNL155 was chosen as the host strain because the production of the Wuhan RBD was tested in several C1 protease deletion strains (Example 4), and the highest production was observed in strains DNL155 and DNL159, which are both 14-fold protease deletion strains with kex2 deletion. Transformation and screening of transformants by culturing in 24-well plates were performed as for Wuhan RBD (Example 4), except that the culture supernatants were analyzed by Western blotting with two primary detection agents simultaneously: SARS-CoV-2 (2019-nCoV) spike protein RBD antibody, rabbit polyclonal antiserum (SinoBiologicals, Cat. No. 40592-T62), and Capture Select Biotin Anti-C-tag conjugate (ThermoFisher). The secondary detection agents were IRDye 680RD goat anti-rabbit antibody (Li-Cor) and IRDye 800CW Stretavidin (Li-Cor). Figure 17 shows an example of Western blot results with at least one positive transformant for each RBD variant. Strong signals of the expected size were detected with both primary antibodies, and the levels of production of variant RBD-C-tag proteins appeared to be equal to those observed for the Wuhan RBD-C-tag-producing control strain M4169.

The amino acid sequence of RBD_B.1.1.7-UK is shown in SEQ ID NO: 53 and the DNA sequence is shown in SEQ ID NO: 54. The sequence includes a signal sequence, a Gly/Ser linker and a C-tag.

The amino acid sequence of RBD_B.1.351-SA is shown in SEQ ID NO: 55 and the DNA sequence is shown in SEQ ID NO: 56. The sequence includes a signal sequence, a Gly/Ser linker and a C-tag.

The amino acid sequence of RBD_1.1.28.1(P.1)-BR is shown in SEQ ID NO: 57 and the DNA sequence is shown in SEQ ID NO: 58. The sequence includes a signal sequence, a Gly/Ser linker and a C-tag.

The foregoing description of specific embodiments will so fully disclose the general nature of the invention that others, using current knowledge, can easily modify and/or adapt such specific embodiments to various applications without undue experimentation and without departing from the general concept, and therefore such adaptations and modifications should be considered as being within the spirit and range of equivalents of the disclosed embodiments. It should be understood that the language or terminology used herein is intended to be descriptive and not limiting. The means, materials, and steps for performing the various functions disclosed may take many alternative forms without departing from the scope of the invention.

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SEQUENCE LIST

<110> DYADIC INTERNATIONAL (USA), INC.

<120> MODIFIED MYCELIAL FUNGI FOR THE PRODUCTION OF EXOGENOUS

BELKOV

<130> DYD/005 PCT

<150> US 62/024550

<151> 2020-05-14

<160> 58

<170> PatentIn version 3.5

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Gly Val Ala Lys Lys Thr Lys Leu Tyr Ala Val Lys Val Leu Gly Ser

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Asn Phe Thr Leu Gly Pro Tyr Asp Tyr Val Leu Glu Val Gln Gly Ser

340 345 350

Cys Ile Ser Thr Phe Met Gly Met Asp Phe Pro Ala Pro Thr Gly Pro

355 360 365

Leu Ala Ile Leu Gly Asp Ala Phe Leu Arg Arg Tyr Tyr Ser Ile Tyr

370 375 380

Asp Leu Gly Ala Asp Thr Val Gly Leu Ala Glu Ala Lys

385 390 395

<210> 3

<211> 534

<212> PRT

<213> Thermothelomyces thermophilus

<400> 3

Met Arg Gly Leu Val Ala Phe Ser Leu Ala Ala Cys Val Ser Ala Ala

1 5 10 15

Pro Ser Phe Lys Thr Glu Thr Ile Asn Gly Glu His Ala Pro Ile Leu

20 25 30

Ser Ser Ser Asn Ala Glu Val Val Pro Asn Ser Tyr Ile Ile Lys Phe

35 40 45

Lys Lys His Val Asp Glu Ser Ser Ala Ser Ala His His Ala Trp Ile

50 55 60

Gln Asp Ile His Thr Ser Arg Glu Lys Val Arg Gln Asp Leu Lys Lys

65 70 75 80

Arg Gly Gln Val Pro Leu Leu Asp Asp Val Phe His Gly Leu Lys His

85 90 95

Thr Tyr Lys Ile Gly Gln Glu Phe Leu Gly Tyr Ser Gly His Phe Asp

100 105 110

Asp Glu Thr Ile Glu Gln Val Arg Arg His Pro Asp Val Glu Tyr Ile

115 120 125

Glu Arg Asp Ser Ile Val His Thr Met Arg Val Thr Glu Glu Thr Cys

130 135 140

Asp Gly Glu Leu Glu Lys Ala Ala Pro Trp Gly Leu Ala Arg Ile Ser

145 150 155 160

His Arg Asp Thr Leu Gly Phe Ser Thr Phe Asn Lys Tyr Leu Tyr Ala

165 170 175

Ala Glu Gly Gly Glu Gly Val Asp Ala Tyr Val Ile Asp Thr Gly Thr

180 185 190

Asn Ile Glu His Val Asp Phe Glu Gly Arg Ala Lys Trp Gly Lys Thr

195 200 205

Ile Pro Ala Gly Asp Ala Asp Val Asp Gly Asn Gly His Gly Thr His

210 215 220

Cys Ser Gly Thr Ile Ala Gly Lys Lys Tyr Gly Val Ala Lys Lys Ala

225 230 235 240

Asn Val Tyr Ala Val Lys Val Leu Arg Ser Asn Gly Ser Gly Thr Met

245 250 255

Ala Asp Val Val Ala Gly Val Glu Trp Ala Ala Lys Ser His Leu Glu

260 265 270

Gln Val Gln Ala Ala Lys Asp Gly Lys Arg Lys Gly Phe Lys Gly Ser

275 280 285

Val Ala Asn Met Ser Leu Gly Gly Gly Lys Thr Arg Ala Leu Asp Asp

290 295 300

Thr Val Asn Ala Ala Val Ser Val Gly Ile His Phe Ala Val Ala Ala

305 310 315 320

Gly Asn Asp Asn Ala Asp Ala Cys Asn Tyr Ser Pro Ala Ala Ala Glu

325 330 335

Lys Ala Val Thr Val Gly Ala Ser Ala Ile Asp Asp Ser Arg Ala Tyr

340 345 350

Phe Ser Asn Tyr Gly Lys Cys Thr Asp Ile Phe Ala Pro Gly Leu Ser

355 360 365

Ile Leu Ser Thr Trp Ile Gly Ser Lys Tyr Ala Thr Asn Thr Ile Ser

370 375 380

Gly Thr Ser Met Ala Ser Pro His Ile Ala Gly Leu Leu Ala Tyr Tyr

385 390 395 400

Leu Ser Leu Gln Pro Ala Thr Asp Ser Glu Tyr Ser Val Ala Pro Ile

405 410 415

Thr Pro Glu Lys Met Lys Ser Asn Leu Leu Lys Ile Ala Thr Gln Asp

420 425 430

Ala Leu Thr Asp Ile Pro Asp Glu Thr Pro Asn Leu Leu Ala Trp Asn

435 440 445

Gly Gly Gly Cys Asn Asn Tyr Thr Ala Ile Val Glu Ala Gly Gly Tyr

450 455 460

Lys Ala Lys Lys Lys Thr Thr Thr Asp Lys Val Asp Ile Gly Ala Ser

465 470 475 480

Val Ser Glu Leu Glu Lys Leu Ile Glu His Asp Phe Glu Val Ile Ser

485 490 495

Gly Lys Val Val Lys Gly Val Ser Ser Phe Ala Asp Lys Ala Glu Lys

500 505 510

Phe Ser Glu Lys Ile His Glu Leu Val Asp Glu Glu Leu Lys Glu Phe

515 520 525

Leu Glu Asp Ile Ala Ala

530

<210> 4

<211> 307

<212> PRT

<213> Thermothelomyces thermophilus

<400> 4

Met Lys Pro Thr Val Leu Phe Thr Leu Leu Ala Ser Gly Ala Tyr Ala

1 5 10 15

Ala Ala Thr Pro Ala Ile Pro Gly Tyr Ser Pro Arg Thr Arg Gly Met

20 25 30

Asn Pro His His His Ala Pro Leu Arg Leu Leu His Thr Phe Thr Pro

35 40 45

Ile Ser Thr Ser Gly Lys Ser Phe Arg Leu Leu Ala Ser Ser Thr Glu

50 55 60

Ser Thr Lys Gly Gly Ala Ile Leu Gly Leu Pro Asp Asn Asp Leu Ser

65 70 75 80

Thr Val Arg Thr Thr Ile Arg Ile Pro Ala Ala Lys Met Pro Thr Ala

85 90 95

Gly Pro Thr Ala Asn Asn Thr Val Gly Glu Tyr Ala Ala Ser Phe Trp

100 105 110

Val Gly Ile Asp Ser Ala Thr Asp Ala Cys Gly Ala Gly Gly Ser Leu

115 120 125

Arg Ala Gly Val Asp Ile Phe Trp Asp Gly Thr Leu Gly Gly Gln Gln

130 135 140

Thr Pro Phe Ala Trp Tyr Gln Gly Pro Gly Gln Ala Asp Val Val Gly

145 150 155 160

Phe Gly Gly Gly Phe Pro Val Gly Glu Gly Asp Leu Val Arg Leu Thr

165 170 175

Leu Glu Ala Gly Pro Ala Gly Gly Glu Glu Ile Ala Val Val Ala Glu

180 185 190

Asn Phe Gly Arg Asn Val Thr Arg Ala Asp Glu Gly Ala Val Pro Val

195 200 205

Arg Lys Val Arg Lys Val Leu Pro Ala Glu Ala Gly Gly Gln Lys Leu

210 215 220

Cys Arg Gly Glu Ala Ala Trp Met Val Glu Asp Phe Pro Leu Gln Gly

225 230 235 240

Arg Pro Glu Phe Pro Thr Ala Leu Ala Asn Phe Thr Ser Val Thr Phe

245 250 255

Asn Thr Gly Ile Thr Leu Asp Asp Gly Thr Glu Lys Asp Leu Thr Gly

260 265 270

Ala Glu Val Leu Asp Ile Gln Leu Glu Ala Gln Gly Gly Arg Leu Thr

275 280 285

Ser Cys Glu Val Val Asp Asp Arg Asn Val Lys Cys Ala Arg Val Val

290 295 300

Gly Asp Asn

305

<210> 5

<211> 554

<212> PRT

<213> Thermothelomyces thermophilus

<400> 5

Met Arg Ile Ala Ala Ser Thr Val Leu Leu Gly Ala Ala Ser Ala Ala

1 5 10 15

Ser Phe Gln Gln Gln Ala Gln His Val Leu Ser Asp Gly Phe Gly Lys

20 25 30

Ala Gln Glu Ala Met Lys Pro Leu Ser Asp Ala Leu Ala Asp Ala Ala

35 40 45

Gly Arg Pro Ile Glu Asn Phe Glu Glu Ala Phe Ser Gly Met Thr Ala

50 55 60

Glu Ala Lys Ala Leu Trp Glu Glu Ile Lys Leu Leu Val Pro Asp Ser

65 70 75 80

Ala Phe Lys Asn Pro Ser Trp Phe Ser Lys Pro Lys Pro His Arg Arg

85 90 95

Arg Asp Asp Trp Asp His Val Val Lys Gly Ala Asp Val Gln Lys Ile

100 105 110

Trp Val Gln Asp Ala Asn Gly Glu Ser His Arg Gln Val Gly Gly Arg

115 120 125

Ile Glu Asp Tyr Asn Leu Arg Val Lys Thr Val Asp Pro Ser Lys Leu

130 135 140

Gly Val Asp Ser Val Lys Gln Phe Ser Gly Tyr Leu Asp Asp Glu Ala

145 150 155 160

Asn Asp Lys His Leu Phe Tyr Trp Phe Phe Glu Ser Arg Asn Asp Pro

165 170 175

Lys Asn Asp Pro Val Val Leu Trp Leu Asn Gly Gly Pro Gly Cys Ser

180 185 190

Ser Leu Thr Gly Leu Phe Leu Glu Leu Gly Pro Ser Ser Ile Asp Lys

195 200 205

Asn Leu Lys Val Val Asn Asn Glu Phe Ser Trp Asn Asn Asn Ala Ser

210 215 220

Val Ile Phe Leu Asp Gln Pro Val Asn Val Gly Tyr Ser Tyr Ser Gly

225 230 235 240

Ser Ser Val Ser Asn Thr Ile Ala Ala Gly Lys Asp Val Tyr Ala Leu

245 250 255

Leu Thr Leu Phe Phe His Gln Phe Pro Glu Tyr Ala Lys Gln Asp Phe

260 265 270

His Ile Ala Gly Glu Ser Tyr Ala Gly His Tyr Ile Pro Val Phe Ala

275 280 285

Ser Glu Ile Leu Ser His Lys Asn Arg Asn Ile Asn Leu Lys Ser Ile

290 295 300

Leu Ile Gly Asn Gly Leu Thr Asp Gly Leu Thr Gln Tyr Glu Tyr Tyr

305 310 315 320

Arg Pro Met Ala Cys Gly Glu Gly Gly Tyr Pro Ala Val Leu Ser Glu

325 330 335

Ser Glu Cys Arg Ser Met Asp Asn Ala Leu Pro Arg Cys Gln Ser Leu

340 345 350

Ile Arg Asn Cys Tyr Asp Ser Gly Ser Val Trp Ser Cys Val Pro Ala

355 360 365

Ser Ile Tyr Cys Asn Asn Ala Leu Ile Gly Pro Tyr Gln Arg Thr Gly

370 375 380

Gln Asn Val Tyr Asp Ile Arg Gly Lys Cys Glu Asp Ser Ser Asn Leu

385 390 395 400

Cys Tyr Ser Ala Leu Gly Tyr Ile Ser Asp Tyr Leu Asn Gln Gln Ser

405 410 415

Val Met Asp Ala Leu Gly Val Glu Val Ser Ser Tyr Glu Ser Cys Asn

420 425 430

Phe Asp Ile Asn Arg Asn Phe Leu Phe Gln Gly Asp Trp Met Gln Pro

435 440 445

Phe His Arg Leu Val Pro Asn Ile Leu Lys Glu Ile Pro Val Leu Ile

450 455 460

Tyr Ala Gly Asp Ala Asp Tyr Ile Cys Asn Trp Leu Gly Asn Arg Ala

465 470 475 480

Trp Thr Glu Lys Leu Glu Trp Pro Gly Gln Lys Ala Phe Asn Gln Ala

485 490 495

Lys Val His Asp Leu Lys Leu Ala Gly Ala Asp Glu Glu Tyr Gly Lys

500 505 510

Val Lys Ala Ser Gly Asn Phe Thr Phe Met Gln Ile Tyr Gln Ala Gly

515 520 525

His Met Val Pro Met Asp Gln Pro Glu Asn Ser Leu Asp Phe Leu Asn

530 535 540

Arg Trp Leu Ser Gly Glu Trp Phe Ala Lys

545 550

<210> 6

<211> 897

<212> PRT

<213> Thermothelomyces thermophilus

<400> 6

Met Val Arg Leu Asp Trp Ala Ala Val Leu Leu Ala Ala Thr Ala Val

1 5 10 15

Ala Lys Ala Val Thr Pro His Thr Pro Ser Phe Val Pro Gly Ala Tyr

20 25 30

Ile Val Glu Tyr Glu Glu Asp Gln Asp Ser His Ala Phe Val Asn Lys

35 40 45

Leu Gly Gly Lys Ala Ser Leu Arg Lys Asp Leu Arg Phe Lys Leu Phe

50 55 60

Lys Gly Ala Ser Ile Gln Phe Lys Asp Thr Glu Thr Ala Asp Gln Met

65 70 75 80

Val Ala Lys Val Ala Glu Met Pro Lys Val Lys Ala Val Tyr Pro Val

85 90 95

Arg Arg Tyr Pro Val Pro Asn His Val Val His Ser Thr Gly Asn Val

100 105 110

Ala Asp Glu Val Leu Val Lys Arg Gln Ala Ala Gly Asn Asp Thr Phe

115 120 125

Ser Thr His Leu Met Thr Gln Val Asn Lys Phe Arg Asp Ala Gly Ile

130 135 140

Thr Gly Lys Gly Ile Lys Ile Ala Val Ile Asp Thr Gly Ile Asp Tyr

145 150 155 160

Leu His Glu Ala Leu Gly Gly Cys Phe Gly Pro Asp Cys Leu Val Ser

165 170 175

Tyr Gly Thr Asp Leu Val Gly Asp Asp Phe Asn Gly Ser Asn Thr Pro

180 185 190

Lys Pro Asp Pro Asp Pro Ile Asp Asn Cys Gln Gly His Gly Thr His

195 200 205

Val Ala Gly Ile Ile Ala Ala Gln Thr Asn Asn Pro Phe Gly Ile Ile

210 215 220

Gly Ala Ala Thr Asp Val Thr Leu Gly Ala Tyr Arg Val Phe Gly Cys

225 230 235 240

Asn Gly Asp Thr Pro Asn Asp Val Leu Ile Ala Ala Tyr Asn Met Ala

245 250 255

Tyr Glu Ala Gly Ser Asp Ile Ile Thr Ala Ser Ile Gly Gly Pro Ser

260 265 270

Gly Trp Ser Glu Asp Pro Trp Ala Ala Val Val Thr Arg Ile Val Glu

275 280 285

Asn Gly Val Pro Cys Val Val Ser Ala Gly Asn Asp Gly Asp Ala Gly

290 295 300

Ile Phe Tyr Ala Ser Thr Ala Ala Asn Gly Lys Lys Val Thr Ala Ile

305 310 315 320

Ala Ser Val Asp Asn Ile Val Thr Pro Ala Leu Leu Ser Asn Ala Ser

325 330 335

Tyr Thr Leu Asn Gly Thr Asp Asp Phe Phe Gly Phe Thr Ala Gly Asp

340 345 350

Pro Gly Ser Trp Asp Asp Val Asn Leu Pro Leu Trp Ala Val Ser Phe

355 360 365

Asp Thr Thr Asp Pro Ala Asn Gly Cys Asn Pro Tyr Pro Asp Ser Thr

370 375 380

Pro Asp Leu Ser Gly Tyr Ile Val Leu Ile Arg Arg Gly Thr Cys Thr

385 390 395 400

Phe Val Glu Lys Ala Ser Tyr Ala Ala Ala Lys Gly Ala Lys Tyr Val

405 410 415

Met Phe Tyr Asn Asn Val Gln Gln Gly Thr Val Thr Val Ser Ala Ala

420 425 430

Glu Ala Lys Gly Ile Glu Gly Val Ala Met Val Thr Ala Gln Gln Gly

435 440 445

Glu Ala Trp Val Arg Ala Leu Glu Ala Gly Ser Glu Val Val Leu His

450 455 460

Met Lys Asp Pro Leu Lys Ala Gly Lys Phe Leu Thr Thr Thr Pro Asn

465 470 475 480

Thr Ala Thr Gly Gly Phe Met Ser Asp Tyr Thr Ser Trp Gly Pro Thr

485 490 495

Trp Glu Val Glu Val Lys Pro Gln Phe Gly Thr Pro Gly Gly Ser Ile

500 505 510

Leu Ser Thr Tyr Pro Arg Ala Leu Gly Ser Tyr Ala Val Leu Ser Gly

515 520 525

Thr Ser Met Ala Cys Pro Leu Ala Ala Ala Ile Tyr Ala Leu Leu Ile

530 535 540

Asn Thr Arg Gly Thr Lys Asp Pro Lys Thr Leu Glu Asn Leu Ile Ser

545 550 555 560

Ser Thr Ala Arg Pro Asn Leu Phe Arg Leu Asn Gly Glu Ser Leu Pro

565 570 575

Leu Leu Ala Pro Val Pro Gln Gln Gly Gly Gly Ile Val Gln Ala Trp

580 585 590

Asp Ala Ala Gln Ala Thr Thr Leu Leu Ser Val Ser Ser Leu Ser Phe

595 600 605

Asn Asp Thr Asp His Phe Lys Pro Val Gln Thr Phe Thr Ile Thr Asn

610 615 620

Thr Gly Lys Lys Ala Val Thr Tyr Ser Leu Ser Asn Val Gly Ala Ala

625 630 635 640

Thr Ala Tyr Thr Phe Ala Asp Ala Lys Ser Ile Glu Pro Ala Pro Phe

645 650 655

Pro Asn Glu Leu Thr Ala Asp Phe Ala Ser Leu Thr Phe Val Pro Lys

660 665 670

Arg Leu Thr Ile Pro Ala Gly Lys Arg Gln Thr Val Thr Val Ile Ala

675 680 685

Lys Pro Ser Glu Gly Val Asp Ala Lys Arg Leu Pro Val Tyr Ser Gly

690 695 700

Tyr Ile Ala Ile Asn Gly Ser Asp Ser Ser Ala Leu Ser Leu Pro Tyr

705 710 715 720

Leu Gly Val Val Gly Ser Leu His Ser Ala Val Val Leu Asp Ser Asn

725 730 735

Gly Ala Arg Ile Ser Leu Ala Ser Asp Asp Thr Asn Lys Pro Leu Pro

740 745 750

Ala Asn Thr Ser Phe Val Leu Pro Pro Ala Gly Phe Pro Asn Asp Thr

755 760 765

Ser Tyr Ala Asn Ser Thr Asp Leu Pro Lys Leu Val Val Asp Leu Ala

770 775 780

Met Gly Ser Ala Leu Leu Arg Ala Asp Val Val Pro Leu Ser Gly Gly

785 790 795 800

Ala Ala Thr Ala Thr Ala Arg Leu Thr Arg Thr Val Phe Gly Thr Arg

805 810 815

Thr Ile Gly Gln Pro Tyr Gly Leu Pro Ala Arg Tyr Asn Pro Arg Gly

820 825 830

Thr Phe Glu Tyr Ala Trp Asp Gly Arg Leu Asp Asp Gly Ser Tyr Ala

835 840 845

Pro Ala Gly Arg Tyr Arg Phe Ala Val Lys Ala Leu Arg Ile Phe Gly

850 855 860

Asp Ala Lys Arg Ala Arg Glu Tyr Asp Ala Ala Glu Thr Val Glu Phe

865 870 875 880

Asn Ile Glu Tyr Leu Pro Gly Pro Ser Ala Lys Phe Arg Arg Arg Leu

885 890 895

Phe

<210> 7

<211> 566

<212> PRT

<213> Thermothelomyces thermophilus

<400> 7

Met Lys Pro Ser Ser Ala Ile Leu Leu Ala Leu Ala Pro Gly Ser Ser

1 5 10 15

Ser Lys Asn Val Val Glu Phe Ser Val Ser Arg Gly Leu Pro Gly Asn

20 25 30

Arg Thr Pro Leu Ser Phe Pro Pro Leu Thr Arg Arg Glu Thr Tyr Ser

35 40 45

Glu Arg Leu Ile Asn Asn Ile Ala Gly Gly Gly Tyr Tyr Val Gln Val

50 55 60

Gln Val Gly Thr Pro Pro Gln Asn Leu Thr Met Leu Leu Asp Thr Gly

65 70 75 80

Ser Ser Asp Ala Trp Val Leu Ser His Glu Ala Asp Leu Cys Ile Ser

85 90 95

Pro Ala Leu Gln Asp Phe Tyr Gly Met Pro Cys Thr Asp Thr Tyr Asp

100 105 110

Pro Ser Lys Ser Ser Ser Lys Lys Met Val Glu Glu Gly Gly Phe Lys

115 120 125

Ile Thr Tyr Leu Asp Gly Gly Thr Ala Ser Gly Asp Tyr Ile Thr Asp

130 135 140

His Phe Thr Ile Gly Gly Val Thr Val Gln Ser Leu Gln Met Ala Cys

145 150 155 160

Val Thr Lys Ala Val Arg Gly Thr Gly Ile Leu Gly Leu Gly Phe Ser

165 170 175

Ile Ser Glu Arg Ala Ser Thr Lys Tyr Pro Asn Ile Ile Asp Glu Met

180 185 190

Tyr Ser Gln Gly Leu Ile Lys Ser Lys Ala Phe Ser Leu Tyr Leu Asn

195 200 205

Asp Arg Arg Ala Asp Ser Gly Thr Leu Leu Phe Gly Gly Ile Asp Thr

210 215 220

Asp Lys Phe Ile Gly Pro Leu Gly Val Leu Pro Leu His Lys Pro Pro

225 230 235 240

Gly Asp Arg Asp Tyr Ser Ser Phe Glu Val Asn Phe Thr Ser Val Ser

245 250 255

Leu Thr Tyr Thr Asn Gly Ser Arg His Thr Ile Pro Thr Ala Ile Leu

260 265 270

Asn His Pro Ala Pro Ala Val Leu Asp Ser Gly Thr Thr Leu Ser Tyr

275 280 285

Leu Pro Asp Glu Leu Ala Asp Pro Ile Asn Thr Ala Leu Asp Thr Phe

290 295 300

Tyr Asp Asp Arg Leu Gln Met Thr Leu Ile Asp Cys Ser His Pro Leu

305 310 315 320

Leu Arg Thr Asp Pro Asp Phe His Leu Ala Phe Thr Phe Thr Pro Thr

325 330 335

Thr Ser Ile Thr Val Pro Leu Gly Asp Leu Val Leu Asp Ile Leu Pro

340 345 350

Pro Thr Tyr Pro Gln Ser Asn Ser Asn Asn Asn Asn Glu Val Glu Asp

355 360 365

Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Lys Val Pro Pro

370 375 380

Ala Thr Glu Arg Arg Trp Cys Val Phe Gly Ile Gln Ser Thr Thr Arg

385 390 395 400

Phe Ala Ala Ser Ser Gly Gln Ser Glu Ala Asn Phe Thr Leu Leu Gly

405 410 415

Asp Thr Phe Leu Arg Ser Ala Tyr Val Val Tyr Asp Leu Ser His Tyr

420 425 430

Gln Ile Gly Leu Ala Gln Ala Asn Leu Asn Ser Ser Ser Ser Ser Thr

435 440 445

Asn Thr Asn Thr Ile Val Glu Leu Thr Ala Asp Asn His Asp Asp Gly

450 455 460

Ala Ser Glu Arg Gly Glu Gly Ala Gly Ala Gly Ala Asp Ala Gly Thr

465 470 475 480

Arg Thr Val Ile Ala Gly Gly Leu Pro Ser Gly Leu Met Gly Val Glu

485 490 495

Ala Gln Gln Thr Thr Phe Thr Pro Thr Ala Thr Ala Asn Gly His Pro

500 505 510

Gly Tyr Gly Gly Gly Pro Gly Gly Ser Thr Arg Pro Gly Ser Glu Arg

515 520 525

Asn Ala Ala Ala Gly Gly Phe Thr Ala Val Arg Thr Gly Leu Leu Gly

530 535 540

Glu Leu Val Gly Val Ala Ala Val Thr Ala Leu Phe Ile Leu Leu Gly

545 550 555 560

Gly Ala Leu Ile Ala Val

565

<210> 8

<211> 874

<212> PRT

<213> Thermothelomyces thermophilus

<400> 8

Met Ala Gly Gly Val Asn Val Gln Ala Arg Glu Leu Leu Pro Thr Asn

1 5 10 15

Val Ile Pro Arg His Tyr Asn Ile Thr Leu Glu Pro Asp Phe Lys Lys

20 25 30

Leu Thr Phe Asp Gly Thr Val Val Ile Asp Leu Asp Val Val Glu Asp

35 40 45

Ser Lys Ser Ile Ser Leu His Thr Leu Glu Leu Asp Ile His Asp Ala

50 55 60

Lys Ile Thr Ser Gly Gly Gln Thr Val Ser Ser Ser Pro Thr Val Ser

65 70 75 80

Tyr Asn Glu Asp Thr Gln Val Ser Thr Phe Glu Phe Gly Asn Ala Val

85 90 95

Thr Lys Gly Ser Lys Ala Gln Leu Glu Ile Lys Phe Thr Gly Gln Leu

100 105 110

Asn Asp Lys Met Ala Gly Phe Tyr Arg Ser Thr Tyr Lys Asn Pro Asp

115 120 125

Gly Ser Glu Gly Ile Met Ala Val Thr Gln Met Glu Pro Thr Asp Ala

130 135 140

Arg Arg Ser Phe Pro Cys Phe Asp Glu Pro Ser Leu Lys Ala Glu Phe

145 150 155 160

Thr Val Thr Leu Val Ala Asp Lys Lys Leu Thr Cys Leu Ser Asn Met

165 170 175

Asp Val Ala Tyr Glu Lys Glu Val Lys Ser Glu Gln Thr Gly Gly Ile

180 185 190

Lys Lys Ala Val Thr Phe Asn Lys Ser Pro Leu Met Ser Thr Tyr Leu

195 200 205

Val Ala Phe Val Val Gly Glu Leu Asn Tyr Ile Glu Thr Asn Glu Phe

210 215 220

Arg Val Pro Val Arg Val Tyr Ala Pro Pro Gly Gln Asp Ile Glu His

225 230 235 240

Gly Arg Phe Ser Leu Asn Leu Ala Ala Lys Thr Leu Ala Phe Tyr Glu

245 250 255

Lys Val Phe Gly Ile Glu Phe Pro Leu Pro Lys Met Asp Gln Ile Ala

260 265 270

Ile Pro Asp Phe Ala Gln Gly Ala Met Glu Asn Trp Gly Leu Val Thr

275 280 285

Tyr Arg Val Val Asp Leu Leu Leu Asp Glu Lys Ala Ser Gly Ala Ala

290 295 300

Thr Lys Glu Arg Val Ala Glu Val Val Gln His Glu Leu Ala His Gln

305 310 315 320

Trp Phe Gly Asn Leu Val Thr Met Asp Trp Trp Asp Gly Leu Trp Leu

325 330 335

Asn Glu Gly Phe Ala Thr Trp Ala Ser Trp Tyr Ser Cys Asn Ile Phe

340 345 350

Tyr Pro Glu Trp Lys Val Trp Glu Ser Tyr Val Val Asp Asn Leu Gln

355 360 365

Arg Ala Leu Ser Leu Asp Ser Leu Arg Ser Ser His Pro Ile Glu Val

370 375 380

Pro Val Lys Arg Ala Asp Glu Ile Asn Gln Ile Phe Asp Ala Ile Ser

385 390 395 400

Tyr Ser Lys Gly Ser Cys Val Leu Arg Met Ile Ser Thr Tyr Leu Gly

405 410 415

Glu Glu Thr Phe Leu Glu Gly Val Arg Arg Tyr Leu Lys Lys His Ala

420 425 430

Tyr Gly Asn Thr Gln Thr Gly Asp Leu Trp Ala Ser Leu Ala Glu Ala

435 440 445

Ser Gly Lys Lys Val Glu Glu Val Met Gln Val Trp Thr Lys Asn Ile

450 455 460

Gly Phe Pro Val Val Thr Val Thr Glu Lys Asp Asp Lys Thr Ile His

465 470 475 480

Leu Lys Gln Asn Arg Phe Leu Arg Thr Gly Asp Thr Lys Pro Glu Glu

485 490 495

Asp Gln Val Ile Tyr Pro Val Phe Leu Gly Leu Arg Thr Lys Asp Gly

500 505 510

Ile Asp Glu Ser Gln Thr Leu Thr Lys Arg Glu Asp Thr Phe Thr Val

515 520 525

Pro Ser Thr Asp Phe Phe Lys Leu Asn Ala Asn His Thr Gly Leu Tyr

530 535 540

Arg Thr Ala Tyr Ser Pro Glu Arg Leu Lys Lys Leu Gly Asp Ala Ala

545 550 555 560

Lys Glu Gly Leu Leu Ser Val Glu Asp Arg Ala Gly Met Ile Ala Asp

565 570 575

Ala Gly Ala Leu Ala Thr Ser Gly Tyr Gln Arg Thr Ser Gly Val Leu

580 585 590

Ser Leu Leu Lys Gly Phe Asn Ser Glu Pro Glu Phe Val Val Trp Asn

595 600 605

Glu Ile Ile Ala Arg Val Ser Ser Val Gln Ser Ala Trp Ile Phe Glu

610 615 620

Asp Gln Ala Asp Arg Asp Ala Leu Asp Ala Phe Leu Arg Asp Leu Ala

625 630 635 640

Ser Pro Lys Ala His Glu Leu Gly Trp Gln Phe Ser Glu Lys Asp Gly

645 650 655

His Ile Leu Gln Gln Phe Lys Ala Met Met Phe Gly Thr Ala Gly Leu

660 665 670

Ser Gly Asp Glu Thr Ile Ile Lys Ala Ala Lys Asp Met Phe Lys Lys

675 680 685

Phe Met Ala Gly Asp Arg Thr Ala Ile His Pro Asn Ile Arg Gly Ser

690 695 700

Val Phe Ser Met Ala Leu Lys Tyr Gly Gly Thr Glu Glu Tyr Asp Ala

705 710 715 720

Val Ile Asn Phe Tyr Arg Thr Ser Thr Asn Ser Asp Glu Arg Asn Thr

725 730 735

Ala Leu Arg Cys Leu Gly Arg Ala Lys Ser Pro Glu Leu Ile Lys Arg

740 745 750

Thr Leu Asp Leu Leu Phe Ser Gly Glu Val Lys Asp Gln Asp Ile Tyr

755 760 765

Met Pro Ala Ser Gly Leu Arg Ser His Pro Glu Gly Ile Glu Ala Leu

770 775 780

Phe Thr Trp Met Thr Glu Asn Trp Asn Glu Leu Ile Lys Lys Leu Pro

785 790 795 800

Pro Ala Leu Ser Met Leu Gly Thr Met Val Thr Ile Phe Thr Ser Ser

805 810 815

Phe Thr Lys Lys Glu Gln Leu Glu Arg Val Glu Lys Phe Phe Glu Gly

820 825 830

Lys Asn Thr Asn Gly Phe Asp Gln Ser Leu Ala Gln Ser Leu Asp Ala

835 840 845

Ile Arg Ser Lys Ile Ser Trp Ile Glu Arg Asp Arg Ala Asp Val Thr

850 855 860

Ala Trp Leu Lys Glu Asn Gly Tyr Arg Ser

865 870

<210> 9

<211> 454

<212> PRT

<213> Thermothelomyces thermophilus

<400> 9

Met Lys Phe Ala Ala Leu Ala Leu Ala Ala Ser Leu Val Ala Ala Ala

1 5 10 15

Pro Arg Val Val Lys Val Asp Pro Ser Asp Ile Lys Pro Arg Arg Leu

20 25 30

Gly Gly Thr Lys Phe Lys Leu Gly Gln Ile His Asn Asp Leu Phe Arg

35 40 45

Gln His Gly Arg Gly Pro Arg Ala Leu Ala Lys Ala Tyr Glu Lys Tyr

50 55 60

Asn Ile Glu Leu Pro Pro Asn Leu Leu Glu Val Val Gln Arg Ile Leu

65 70 75 80

Lys Asp Leu Gly Ile Glu Pro His Ser Lys Lys Ile Pro Gly Ser Lys

85 90 95

Ser Ser Tyr Gly Asn Gly Ala Pro Tyr Thr Asn Glu Thr Asp Asp Ser

100 105 110

Gly Glu Val Ser Ala Ile Pro Gln Leu Phe Asp Val Glu Tyr Leu Ala

115 120 125

Pro Val Gln Ile Gly Thr Pro Pro Gln Thr Leu Met Leu Asn Phe Asp

130 135 140

Thr Gly Ser Ser Asp Leu Trp Val Phe Ser Ser Glu Thr Pro Ser Arg

145 150 155 160

Gln Gln Asn Gly Gln Lys Ile Tyr Lys Ile Glu Glu Ser Ser Thr Ala

165 170 175

Arg Arg Leu Ser Asn His Thr Trp Ser Ile Gln Tyr Gly Asp Gly Ser

180 185 190

Arg Ser Ala Gly Asn Val Tyr Leu Asp Thr Val Ser Val Gly Gly Val

195 200 205

Asn Val Phe Asn Gln Ala Val Glu Ser Ala Thr Phe Val Ser Ser Ser

210 215 220

Phe Val Thr Asp Ala Ala Ser Ser Gly Leu Leu Gly Leu Gly Phe Asp

225 230 235 240

Ser Ile Asn Thr Val Lys Pro Thr Lys Gln Lys Thr Phe Ile Ser Asn

245 250 255

Ala Leu Glu Ser Leu Glu Met Gly Leu Phe Thr Ala Asn Leu Lys Lys

260 265 270

Ala Glu Pro Gly Asn Tyr Asn Phe Gly Phe Ile Asp Glu Thr Glu Phe

275 280 285

Val Gly Pro Leu Ser Phe Ile Asp Val Asp Ser Thr Asp Gly Phe Trp

290 295 300

Gln Phe Asp Ala Thr Gly Tyr Ser Ile Gln Leu Pro Glu Pro Ser Gly

305 310 315 320

Asn Ile Thr Gly Thr Pro Phe Arg Ala Val Ala His Thr Ala Ile Ala

325 330 335

Asp Thr Gly Thr Thr Leu Leu Leu Leu Pro Pro Gly Ile Ala Gln Ala

340 345 350

Tyr Tyr Trp Gln Val Gln Gly Ala Arg Gln Ala Pro Glu Val Gly Gly

355 360 365

Trp Val Met Pro Cys Asn Ala Ser Met Pro Asp Leu Thr Leu His Ile

370 375 380

Gly Thr Tyr Lys Ala Val Ile Pro Gly Glu Leu Ile Pro Tyr Ala Pro

385 390 395 400

Val Asp Thr Asp Asp Met Asp Thr Ala Thr Val Cys Tyr Gly Gly Ile

405 410 415

Gln Ser Ala Ser Gly Met Pro Phe Ala Ile Tyr Gly Asp Ile Phe Phe

420 425 430

Lys Ala Gln Phe Thr Val Phe Asp Val Glu Asn Leu Lys Leu Gly Phe

435 440 445

Ala Pro Lys Pro Glu Leu

450

<210> 10

<211> 428

<212> PRT

<213> Thermothelomyces thermophilus

<400> 10

Met Arg Val Ser Phe Gln Ser Leu Leu Leu Leu Gly Ala Leu Ser Ala

1 5 10 15

Gln Ala Ser Ala Tyr Ala Ser Leu Glu Tyr Gln Gln Gln Thr Phe Pro

20 25 30

Glu Asp Asn Ala Pro Pro Tyr Arg Val Pro Leu Leu Thr Leu His Arg

35 40 45

Ala Leu Val Asn Val Ser Ser Ile Ser Asp Ser Glu Gly Glu Val Gly

50 55 60

Leu Leu Leu Lys Arg Leu Leu Lys Asp Leu Asn Tyr Thr Val Glu Leu

65 70 75 80

Gln Pro Val Pro Pro Ser Glu Ala Gly Gln Gly Pro Asp Asp Arg Pro

85 90 95

Thr Arg Tyr Asn Val Leu Ala Trp Pro Gly Arg Asn Ala Ser Arg Ala

100 105 110

Leu Asp Lys Arg Thr Ile Ile Thr Ser His Ile Asp Val Val Pro Pro

115 120 125

Tyr Ile Pro Tyr Ala Ile Asp Asn Glu Thr Val Pro Pro Ser Glu Val

130 135 140

Val Asp Phe Ala Ala Leu Pro Pro Thr Thr Leu Ile Ser Gly Arg Gly

145 150 155 160

Ser Val Asp Ala Lys Ala Ser Val Ala Ala Gln Ile Thr Ala Thr Asn

165 170 175

Ala Leu Leu Ser Glu Gly Ala Ile Ser Pro Asp Ser Val Val Leu Leu

180 185 190

Tyr Val Val Gly Glu Glu Asn Ser Gly Ser Gly Met Lys His Phe Ser

195 200 205

Asp Ser Leu Ser Asn Ser Ser Ala Tyr Pro Val Arg Pro Gln Phe Arg

210 215 220

Ala Ala Ile Phe Gly Glu Pro Thr Glu Asn Lys Leu Ala Cys Gly His

225 230 235 240

Lys Gly Val Thr Gly Gly Thr Val Ser Ala Val Gly Lys Ala Gly His

245 250 255

Ser Gly Tyr Pro Trp Leu Gly Lys Ser Ala Ile His Val Leu Ile Arg

260 265 270

Ala Leu Asp Arg Leu Leu Glu Glu Asp Leu Gly Ser Ser Glu Arg Tyr

275 280 285

Gly Asn Thr Thr Val Asn Val Gly Leu Ile Glu Gly Gly Val Ala Ala

290 295 300

Asn Val Ile Ala Pro Ala Ala Ser Ala Arg Val Ser Ala Arg Val Ala

305 310 315 320

Val Gly Asn Gln Thr Thr Gly Gly Gln Ile Val Ala Glu Arg Ile Lys

325 330 335

Lys Leu Ile Lys Asp Val Asp Ser Glu Ala Leu Gln Val Asn Ile Thr

340 345 350

Ser Gly Val Gly Pro Val Glu Cys Glu Cys Glu Val Asp Gly Phe Glu

355 360 365

Thr Val Val Ala Asn Tyr Gly Thr Asp Ile Pro Asn Leu Lys Gly Asn

370 375 380

His Val Lys Tyr Leu Tyr Gly Pro Gly Ser Ile Leu Val Ala His Gly

385 390 395 400

Asp Asn Glu Gly Leu Gln Ile Lys Asp Leu Glu Asp Ser Val Glu Gly

405 410 415

Tyr Lys Arg Leu Ile Lys His Ala Val Gly Ser Ser

420 425

<210> 11

<211> 444

<212> PRT

<213> Thermothelomyces thermophilus

<400> 11

Met Glu Ile Glu Ile Gly Thr Pro Pro Gln Lys Val Met Leu Ile Val

1 5 10 15

Asp Thr Gly Ser Pro Asn Thr Trp Val Asn Pro Gln Cys Glu Thr Ser

20 25 30

Asn Thr Pro Ser Asp Cys Ala Lys Tyr Pro Gln Phe Asp Tyr Thr Glu

35 40 45

Ser Ser Ser Ile Asn Ile Thr Asp Tyr Val Asp Val Leu Arg Tyr Gly

50 55 60

Ser Gly Ser Ala Thr Val Gln Tyr Val Tyr Glu Thr Val Ser Ile Gly

65 70 75 80

Ser Ala Thr Leu Lys Asp Gln Ile Ile Gly Ile Ala Leu Glu Ser Glu

85 90 95

Asp Ile Pro Leu Gly Ile Leu Gly Leu Ser Pro Pro Val Arg Gly Val

100 105 110

Asn Gln Tyr Pro Tyr Ile Leu Asp Thr Met Val Asp Gln Gly Leu Ile

115 120 125

Lys Ser Arg Ala Phe Ser Leu Asp Leu Arg Gly Val Asp Asn Pro Thr

130 135 140

Gly Ala Val Ile Phe Gly Gly Ile Asp Thr Gly Lys Tyr Ile Gly Thr

145 150 155 160

Leu Ala Lys Leu Pro Ile Ile Ala Pro Ser Ser Ala Pro Gly Gly Ala

165 170 175

Asp Arg Tyr Tyr Ile Thr Met Thr Gly Val Gly Leu Thr Leu Pro Asp

180 185 190

Gly Thr Met Val Arg Ser Glu Glu Leu Asp Val Pro Val Phe Leu Asp

195 200 205

Ser Gly Ser Thr Leu Ser Arg Leu Pro Thr Val Ile His Gln Ala Leu

210 215 220

Ala Ala Ser Phe Thr Glu Ala Met Leu Asp Gln Glu Ser Gly Leu Phe

225 230 235 240

Ile Leu Pro Cys Glu Tyr Thr Asp Met Ala Gly Ser Ile Asp Phe Tyr

245 250 255

Phe Ala Gly Lys Thr Ile Arg Val Pro Leu Arg Glu Phe Ile Trp Arg

260 265 270

Ser Gly Asp Tyr Cys Ile Leu Gly Val Ala Pro Glu Asp Asp Glu Pro

275 280 285

Ile Leu Gly Asp Thr Phe Leu Arg Ala Ala Tyr Val Val Tyr Asp Gln

290 295 300

Asp Asn Arg Asn Val His Leu Ala Gln Ala Ala Asp Cys Gly Thr Asn

305 310 315 320

Leu Val Ala Ile Gly Ser Gly Glu Asp Ala Val Pro Ser Ser Thr Gly

325 330 335

Arg Cys Thr Glu Leu Pro Thr Pro Thr Gly Asp Pro Thr Arg Thr Arg

340 345 350

Ala Gly Ser Ser Asn Leu Asp Met Thr Ala Thr Arg Pro Pro Ala Asn

355 360 365

Thr Phe Thr Gly Arg Leu Pro Thr Gly Ile Ala Gly Gly Pro Gly Pro

370 375 380

Ala Arg Asp Gly Ser Thr Thr Thr Val Thr Gly Gly Gly Leu Gln Pro

385 390 395 400

Met Leu Pro Thr Gly Ser Pro Lys Gly Ser Glu Gly Thr Glu Gln Asn

405 410 415

Ala Ala Gly Arg Gly Val Asp Ser Gly Leu Gly Ala Ala Val Ala Ala

420 425 430

Val Leu Gly Val Val Ser Leu Leu Val Leu Met Leu

435 440

<210> 12

<211> 621

<212> PRT

<213> Thermothelomyces thermophilus

<400> 12

Met Leu Arg Asn Ile Phe Leu Thr Ala Ala Leu Ala Ala Phe Gly Gln

1 5 10 15

Cys Gly Ser Thr Val Phe Glu Ser Val Pro Ala Lys Pro Arg Gly Trp

20 25 30

Thr Arg Leu Gly Asp Ala Ser Ala Asp Gln Pro Leu Arg Leu Arg Ile

35 40 45

Ala Leu Gln Gln Pro Asn Glu Asp Leu Phe Glu Arg Thr Leu Tyr Glu

50 55 60

Val Ser Asp Pro Ser His Ala Arg Tyr Gly Gln His Leu Ser Arg Asp

65 70 75 80

Glu Leu Ser Ala Leu Leu Ala Pro Arg Ala Glu Ser Thr Ala Ala Val

85 90 95

Leu Asn Trp Leu Arg Asp Ala Gly Ile Pro Ser Asp Lys Ile Glu Glu

100 105 110

Asp Gly Glu Trp Ile Asn Leu Arg Val Thr Val Arg Glu Ala Ser Glu

115 120 125

Leu Leu Asp Ala Asp Phe Gly Val Trp Ala Tyr Glu Gly Thr Asn Val

130 135 140

Lys Arg Val Arg Ala Leu Gln Tyr Ser Val Pro Glu Glu Ile Ala Pro

145 150 155 160

His Ile Arg Met Val Ala Pro Val Val Arg Phe Gly Gln Ile Arg Pro

165 170 175

Glu Arg Ser Gln Val Phe Glu Val Val Glu Thr Ala Pro Ser Gln Val

180 185 190

Lys Val Ala Ala Ala Ile Pro Pro Gln Asp Leu Asp Val Lys Ala Cys

195 200 205

Asn Thr Ser Ile Thr Pro Glu Cys Leu Arg Ala Leu Tyr Lys Val Gly

210 215 220

Ser Tyr Gln Ala Glu Pro Ser Lys Lys Ser Leu Phe Gly Val Ala Gly

225 230 235 240

Tyr Leu Glu Gln Trp Ala Lys Tyr Asp Gln Leu Glu Leu Phe Ala Ser

245 250 255

Thr Tyr Ala Pro Tyr Ala Ala Asp Ala Asn Phe Thr Ser Val Gly Val

260 265 270

Asn Gly Gly Glu Asn Asn Gln Gly Pro Ser Asp Gln Gly Asp Ile Glu

275 280 285

Ala Asn Leu Asp Ile Gln Tyr Ala Val Ala Leu Ser Tyr Lys Thr Pro

290 295 300

Ile Thr Tyr Tyr Ile Thr Gly Gly Arg Gly Pro Leu Val Pro Asp Leu

305 310 315 320

Asp Gln Pro Asp Pro Asn Asp Val Ser Asn Glu Pro Tyr Leu Glu Phe

325 330 335

Phe Ser Tyr Leu Leu Lys Leu Pro Asp Ser Glu Leu Pro Gln Thr Leu

340 345 350

Thr Thr Ser Tyr Gly Glu Asp Glu Gln Ser Val Pro Arg Pro Tyr Ala

355 360 365

Glu Lys Val Cys Gln Met Ile Gly Gln Leu Gly Ala Arg Gly Val Ser

370 375 380

Val Ile Phe Ser Ser Gly Asp Thr Gly Val Gly Ser Ala Cys Gln Thr

385 390 395 400

Asn Asp Gly Lys Asn Thr Thr Arg Phe Leu Pro Ile Phe Pro Gly Ala

405 410 415

Cys Pro Tyr Val Thr Ser Ile Gly Ala Thr Arg Tyr Val Glu Pro Glu

420 425 430

Gln Ala Ala Ala Phe Ser Ser Gly Gly Phe Ser Asp Ile Phe Lys Arg

435 440 445

Pro Ala Tyr Gln Glu Ala Ala Val Ser Thr Tyr Leu His Lys His Leu

450 455 460

Gly Ser Arg Trp Lys Gly Leu Tyr Asn Pro Gln Gly Arg Gly Phe Pro

465 470 475 480

Asp Val Ser Ala Gln Gly Val Ala Tyr His Val Phe Ser Gln Asp Lys

485 490 495

Asp Ile Lys Val Ser Gly Thr Ser Ala Ser Ala Pro Leu Phe Ala Ala

500 505 510

Leu Val Ser Leu Leu Asn Asn Ala Arg Leu Ala Gln Gly Arg Pro Pro

515 520 525

Leu Gly Phe Leu Asn Pro Trp Leu Tyr Ser Glu Lys Val Gln Lys Ala

530 535 540

Gly Ala Leu Thr Asp Ile Val His Gly Gly Ser Ser Gly Cys Thr Gly

545 550 555 560

Lys Asp Met Tyr Ser Gly Leu Pro Thr Pro Tyr Val Pro Tyr Ala Ser

565 570 575

Trp Asn Ala Thr Pro Gly Trp Asp Pro Val Thr Gly Leu Gly Thr Pro

580 585 590

Val Phe Asp Lys Leu Leu Glu Leu Ser Ser Pro Gly Lys Lys Leu Pro

595 600 605

His Ile Gly Gly Gly His Gly His Gly Ala Gly Gly His

610 615 620

<210> 13

<211> 420

<212> PRT

<213> Thermothelomyces thermophilus

<400> 13

Met Ala Gly Arg Leu Leu Leu Cys Leu Thr Ala Ala Leu Ser Ala Leu

1 5 10 15

Gly Val Ser Ala Ala Pro Ala Pro Asp Ala Ser Gly Arg Pro Phe Ile

20 25 30

Gly Val Pro Val Ser Asn Pro Gly Ile Ala Asn Ala Ile Pro Asn Arg

35 40 45

Tyr Ile Val Val Tyr Asn Asn Thr Phe Asn Asp Glu Asp Ile Asp Leu

50 55 60

His Gln Ser Asn Val Ile Lys Thr Ile Ala Lys Arg Asn Ile Ala Lys

65 70 75 80

Arg Ser Leu Thr Gly Lys Leu Leu Ser Thr Thr Val Asn Thr Tyr Lys

85 90 95

Ile Asn Asn Trp Arg Ala Met Ala Leu Glu Ala Asp Asp Ala Thr Ile

100 105 110

Asn Glu Ile Phe Ala Ala Lys Glu Val Ser Tyr Ile Glu Gln Asp Ala

115 120 125

Val Ile Ser Leu Asn Val Arg Gln Met Gln Ser Gln Ala Thr Thr Gly

130 135 140

Leu Ala Arg Ile Ser His Ala Gln Pro Gly Ala Arg Thr Tyr Ile Phe

145 150 155 160

Asp Ser Ser Ala Gly Glu Gly Ile Thr Ala Tyr Val Val Asp Thr Gly

165 170 175

Ile Arg Val Thr His Glu Glu Phe Glu Gly Arg Ala Thr Phe Ala Ala

180 185 190

Asn Phe Ile Asp Asp Val Asp Thr Asp Glu Gln Gly His Gly Ser His

195 200 205

Val Ala Gly Thr Ile Gly Gly Lys Thr Phe Gly Val Ala Lys Lys Val

210 215 220

Asn Leu Val Ala Val Lys Val Leu Gly Ala Asp Gly Ser Gly Ser Asn

225 230 235 240

Ser Gly Val Ile Ala Gly Met Gln Phe Val Ala Ser Asn Ala Thr Ala

245 250 255

Met Gly Leu Lys Gly Arg Ala Val Met Asn Met Ser Leu Gly Gly Pro

260 265 270

Ala Ser Arg Ala Val Asn Ser Ala Ile Asn Gln Val Glu Ala Ala Gly

275 280 285

Val Val Pro Val Val Ala Ala Gly Asn Glu Ser Gln Asp Thr Ala Asn

290 295 300

Thr Ser Pro Gly Ser Ala Glu Ala Ala Ile Thr Val Gly Ala Ile Asp

305 310 315 320

Gln Thr Asn Asp Arg Met Ala Ser Phe Ser Asn Phe Gly Glu Leu Val

325 330 335

Asp Ile Phe Ala Pro Gly Val Asn Val Gln Ser Val Gly Ile Arg Ser

340 345 350

Asp Thr Ser Thr Asn Thr Leu Ser Gly Thr Ser Met Ala Ser Pro His

355 360 365

Val Ala Gly Leu Ala Ala Tyr Ile Met Ser Leu Glu Asn Ile Thr Gly

370 375 380

Val Gln Ala Val Ser Asp Arg Leu Lys Glu Leu Ala Gln Ala Thr Gly

385 390 395 400

Ala Arg Ala Arg Gly Val Pro Arg Gly Thr Thr Thr Leu Ile Ala Asn

405 410 415

Asn Gly Phe Ala

420

<210> 14

<211> 892

<212> PRT

<213> Thermothelomyces thermophilus

<400> 14

Met Lys Ile Trp Ser Gly Ala Ala Leu Leu Gly Leu Ala Ala Leu Ala

1 5 10 15

Thr Ala Ser His Ile Leu Pro Arg Asp Trp Glu Ala Asn Asp Tyr Tyr

20 25 30

Val Leu His Leu Asp Ala Asp Thr Ser Pro Gln Glu Val Ala Arg Ser

35 40 45

Leu Gly Leu Ser His Glu Gly Pro Leu Gly Glu Leu Arg Asp His His

50 55 60

Val Phe Val Ala Lys Arg Ala Glu His Asp Val Val Lys Arg Glu Leu

65 70 75 80

Ala Arg Arg Arg Lys Lys Arg Ser Leu Gly Leu Gly Gly Arg Asp Val

85 90 95

Leu Asp Gly Val Leu Phe Ser Gln Lys Gln Arg Leu Arg Lys Pro Trp

100 105 110

Glu Lys Arg Val Val Pro Arg Leu Phe Gly Pro Leu Pro Arg Arg Ser

115 120 125

Val Asp Glu Pro Val Glu Ser Leu Val Gln Arg Gln Thr Glu Val Ala

130 135 140

Arg Lys Leu Asp Ile Lys Asp Pro Ile Phe His Glu Gln Trp His Leu

145 150 155 160

Phe Asn Thr Val Gln Ala Gly His Asp Val Asn Val Thr Asp Val Trp

165 170 175

Leu Gln Gly Val Thr Gly Lys Asn Ala Thr Val Ala Ile Val Asp Asp

180 185 190

Gly Leu Asp Met Tyr Ser Asp Asp Leu Arg Asp Asn Tyr Tyr Ala Leu

195 200 205

Gly Ser Tyr Asp Phe Asn Asp Lys Ala Asp Glu Pro Arg Pro Arg Leu

210 215 220

Ala Asn Asp Asn His Gly Thr Arg Cys Ala Gly Glu Val Ala Ala Gly

225 230 235 240

Arg Asn Asn Ala Cys Gly Val Gly Val Ala Tyr Asp Ser Asn Ile Ala

245 250 255

Gly Leu Arg Ile Leu Ser Lys Leu Ile Ser Asp Ala Asp Glu Ala Val

260 265 270

Ala Leu Asn Tyr Asp Phe Gln His Asn Gln Ile Tyr Ser Cys Ser Trp

275 280 285

Gly Pro Pro Asp Asp Gly Lys Ser Met Asp Ala Pro Gly Ile Leu Ile

290 295 300

Arg Arg Ala Met Leu Asn Ala Val Gln Asn Gly Arg Gly Gly Leu Gly

305 310 315 320

Ser Ile Tyr Val Phe Ala Ser Gly Asn Gly Ala His Asn Glu Asp Asn

325 330 335

Cys Asn Phe Asp Gly Tyr Thr Asn Ser Ile Tyr Ser Ile Thr Val Gly

340 345 350

Ala Leu Asp Arg Lys Gly Gln His Pro Tyr Tyr Ser Glu Ser Cys Ser

355 360 365

Ala Gly Leu Val Val Thr Tyr Ser Ser Gly Ser Gly Asp Ala Ile His

370 375 380

Thr Thr Asp Val Gly Gln Asn Thr Cys Thr Ser Ser His Gly Gly Thr

385 390 395 400

Ser Ala Ala Ala Pro Leu Ala Ala Gly Ile Phe Ala Leu Val Leu Gln

405 410 415

Val Arg Pro Asp Leu Ser Trp Arg Asp Met Gln Tyr Leu Ala Met Asp

420 425 430

Thr Ala Val Pro Val Asn Val Asp Thr Gly Asp Tyr Gln Asp Thr Thr

435 440 445

Ile Gly Lys Lys Phe Ser His Thr Tyr Gly Tyr Gly Lys Leu Asp Ser

450 455 460

Tyr Ala Ile Val Glu Ala Ala Lys Lys Trp Lys Lys Val Lys Pro Gln

465 470 475 480

Ala Trp Phe Tyr Ser Pro Trp Ile His Val Asn Gln Pro Ile Pro Gln

485 490 495

Gly Asp Lys Gly Val Val Val Glu Phe Glu Val Thr Lys Glu Met Leu

500 505 510

Glu Glu Ala Asn Leu Asp Arg Leu Glu His Val Thr Val Thr Met Asn

515 520 525

Val Glu His Gly Arg Arg Gly Asp Leu Ser Val Asp Leu Ile Ser Pro

530 535 540

Asn Lys Ile Val Ser His Leu Ser Val Thr Arg Lys Asn Asp Asp Ser

545 550 555 560

Asp Lys Gly Tyr Asn Asp Trp Thr Phe Met Ser Val Ala His Trp Gly

565 570 575

Glu Ser Gly Val Gly Thr Trp Thr Ile Val Val Lys Asp Thr Glu Ile

580 585 590

Asn Gln Tyr Thr Gly Lys Phe Ile Asp Trp His Leu Lys Leu Trp Gly

595 600 605

Glu Thr Arg Asp Ala Ser Lys Ala Gln Leu Leu Pro Met Pro Thr Glu

610 615 620

Glu Asp Asp Asp Asp His Asp Val Ile Ala Thr Thr Thr Ala Thr Ala

625 630 635 640

Ala Thr Thr Thr Val Ser Lys Pro Glu Ala Thr Gly Ser Val Pro Ala

645 650 655

Asp Ala Thr Asp Gln Pro Asn Arg Pro Val Asn Ser Lys Pro Thr Asp

660 665 670

Thr Ser Pro Ala Glu Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser

675 680 685

Ala Glu Thr Asp Lys Thr Asn Thr Trp Leu Pro Ser Phe Leu Pro Thr

690 695 700

Phe Gly Val Ser Ala Ala Thr Gln Ala Trp Ile Tyr Gly Ser Leu Val

705 710 715 720

Leu Ile Val Leu Phe Cys Ala Gly Leu Gly Ile Tyr Leu Tyr Leu Ala

725 730 735

Arg Arg Lys Arg Leu Arg Asn Lys Thr Arg Thr Asp Tyr Glu Phe Glu

740 745 750

Leu Leu Asp Asp Asp Asp Asp Asp Asp Glu Glu Ala Ala Ala Leu Thr

755 760 765

Arg Gly Gly Gly Gly Gly Glu Lys Gly Val Val Gly Gly Gly Gly Gly

770 775 780

Gly Gly Gly Lys Arg Gly Arg Arg Thr Arg Gly Gly Glu Leu Tyr Asp

785 790 795 800

Ala Phe Ala Gly Glu Ser Asp Glu Asp Ser Asp Asp Asn Asp Phe Ala

805 810 815

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr Arg Asp Arg Ser Asp

820 825 830

Ser Arg Ser Arg Ser Arg Ser Asp Gly Ser Gly Ser Pro Ile Gly Ile

835 840 845

Ser Glu Lys Leu Pro Gly Arg Arg Asp Ser Leu Ser Gly Glu Glu Glu

850 855 860

His His Val Val Gly Asp Asp Asp Asp Asp Asp Glu Asp Gly Thr Gly

865 870 875 880

Asn Asp Gln Ala Arg Pro Leu Gln Gly Gly Ala Arg

885 890

<210> 15

<211> 387

<212> PRT

<213> Thermothelomyces thermophilus

<400> 15

Met Gln Leu Leu Ser Leu Ala Ala Leu Leu Pro Leu Ala Leu Ala Ala

1 5 10 15

Pro Val Ile Lys Pro Gln Gly Leu Gln Leu Ile Pro Gly Asp Tyr Ile

20 25 30

Val Lys Leu Lys Asp Gly Ala Ser Glu Ser Thr Leu Gln Asp Thr Ile

35 40 45

Arg His Leu Gln Ala Gly Glu Ala Lys His Val Tyr Arg Ala Arg Arg

50 55 60

Phe Lys Gly Phe Ala Ala Lys Leu Ser Pro Gln Val Val Asp Thr Leu

65 70 75 80

Ser Lys Leu Pro Glu Val Glu Tyr Ile Glu Gln Asp Ala Val Val Thr

85 90 95

Ile Gln Ala Leu Val Thr Gln Glu Asp Val Pro Trp Gly Leu Ala Arg

100 105 110

Ile Ser His His Glu Leu Gly Pro Thr Ser Tyr Val Tyr Asp Asp Ser

115 120 125

Ala Gly Glu Gly Thr Cys Ala Tyr Val Ile Asp Thr Gly Ile Tyr Val

130 135 140

Ala His Ser Gln Phe Glu Gly Arg Ala Thr Trp Leu Ala Asn Phe Ile

145 150 155 160

Asp Ser Ser Asp Ser Asp Gly Ala Gly His Gly Thr His Val Ser Gly

165 170 175

Thr Ile Gly Gly Val Thr Tyr Gly Val Ala Lys Lys Thr Lys Leu Phe

180 185 190

Ala Val Lys Val Leu Asn Ala Ser Gly Ser Gly Thr Val Ser Ser Val

195 200 205

Leu Ala Gly Leu Glu Phe Val Ala Ser Asp Ala Pro Ala Arg Val Ala

210 215 220

Ser Gly Glu Cys Ala Asn Gly Ala Val Ala Asn Leu Ser Leu Gly Gly

225 230 235 240

Gly Arg Ser Thr Ala Ile Asn Ala Ala Ala Ala Ala Ala Val Asp Ala

245 250 255

Gly Val Phe Val Ala Val Ala Ala Gly Asn Ser Asn Thr Asp Ala Gln

260 265 270

Ser Thr Ser Pro Ala Ser Glu Pro Ser Val Cys Thr Val Gly Ala Thr

275 280 285

Asp Asp Ser Asp Ala Arg Ala Tyr Phe Ser Asn Tyr Gly Ser Val Val

290 295 300

Asp Val Phe Ala Pro Gly Val Asp Val Leu Ser Ser Trp Ile Gly Gly

305 310 315 320

Val Asp Ala Thr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His

325 330 335

Ile Ala Gly Leu Gly Ala Tyr Leu Leu Ala Leu Leu Gly Pro Arg Ser

340 345 350

Pro Glu Glu Leu Cys Glu Tyr Ile Lys Gln Thr Ala Thr Ile Gly Thr

355 360 365

Ile Thr Ser Leu Pro Ser Gly Thr Ile Asn Ala Ile Ala Tyr Asn Gly

370 375 380

Ala Thr Ala

385

<210> 16

<211> 1468

<212> PRT

<213> Thermothelomyces thermophilus

<400> 16

Met Val Ala Ser Ser Trp Phe Thr Ala Pro Leu Val Ala Val Ala Leu

1 5 10 15

Leu Leu Ser Leu Asp Gly Ala Val Ala Lys Lys Pro Thr Phe Arg Pro

20 25 30

Pro Ser Leu Pro Thr Tyr Asp Asp Asp Ala Ala Cys Pro Glu Arg Cys

35 40 45

Ser Val Ser Gly Pro Ser Thr Gly Asn Trp Ser Val Tyr Pro Asn Phe

50 55 60

Glu Pro Ile Arg Lys Cys Thr Gln Thr Met Phe Tyr Asp Phe Ser Leu

65 70 75 80

Tyr Asp Ser Val Asp Asp Pro Thr Val Asn His Arg Ile His Ala Cys

85 90 95

Ser Ser Phe Gly Pro Asp Phe Ser Ile Ile Pro Gly Ser Ile Thr Lys

100 105 110

Thr Ala Tyr Ala Ser Pro Ala Pro Ala Lys Ile Arg Phe Glu Leu Gly

115 120 125

Trp Trp Asn Arg Gly Tyr Gly Leu Ala Ala Pro Gly Leu Arg Ser Leu

130 135 140

Val Lys Gln Leu Arg Ala Tyr Ile Asp His Gly His Gly Asp Gly Ala

145 150 155 160

Ala Asp Arg Pro Phe Ile Ile Tyr Gly Gln Ser Gly Gln Ala Thr Ile

165 170 175

Gly Leu Tyr Ile Gly Gln Gly Leu Leu Ser Gln Gly Leu Ser Lys Ser

180 185 190

Ala Leu Lys Ile Leu Gln Asp Asn Leu Ala Asn Ser Asp Val Ser Ala

195 200 205

Pro Ser Leu Ala Ile Gln Leu Cys Gly Gln Gly Tyr Gly Ser Ser His

210 215 220

Ile Phe Gly Ala Met Val Thr Ser Asn Gly Thr Phe Ala Pro Ile Gln

225 230 235 240

Glu Ala Ile Arg Thr Trp Ala Asn Ala Thr Cys Leu Ser Phe Ala Gly

245 250 255

Ser Lys Glu Phe Pro Gly Glu Val Met Phe Thr Thr Pro Leu Leu Leu

260 265 270

Ala Asn Gly Thr Ala Asn Ser Thr Val Arg Ala Arg Ser Leu Arg Pro

275 280 285

Tyr Ala Ala Glu Cys Arg Thr Val Gln Val Glu Ala Gly Asp Ser Cys

290 295 300

Gly Thr Leu Ala Lys Lys Cys Gly Ile Ser Gly Ala Asp Phe Thr Asn

305 310 315 320

Tyr Asn Pro Gly Ala Ser Phe Cys Ser Thr Leu Lys Pro Lys Gln His

325 330 335

Val Cys Cys Ser Ser Gly Thr Leu Pro Asp Phe Arg Pro Val Thr Asn

340 345 350

Pro Asp Gly Ser Cys Tyr Ser Tyr Lys Val Lys Ser Asn Asp Asn Cys

355 360 365

Ala Asp Leu Ala Ala Glu Tyr Gly Leu Thr Val Asp Glu Ile Glu Ser

370 375 380

Phe Asn Lys Asn Thr Trp Gly Trp Gly Gly Cys Lys Val Leu Phe Leu

385 390 395 400

Asp Thr Ile Met Cys Leu Ser Lys Gly Ala Pro Pro Phe Pro Ala Pro

405 410 415

Ile Ser Asn Ala Ile Cys Gly Pro Gln Lys Leu Gly Thr Ile Pro Pro

420 425 430

Thr Asp Gly Ser Asn Ile Ala Asp Leu Asn Pro Cys Pro Ile Asn Ala

435 440 445

Cys Cys Asn Ile Trp Gly Gln Cys Gly Ile Ser Lys Asp Phe Cys Ile

450 455 460

Asp Thr Asn Thr Gly Pro Pro Gly Thr Ala Ala Pro Gly Thr Tyr Gly

465 470 475 480

Cys Ile Ser Asn Cys Gly Leu Asp Ile Val Lys Gly Lys Gly Thr Gly

485 490 495

Ser Ile Lys Ile Ala Tyr Phe Glu Gly Phe Gly Leu Glu Arg Glu Cys

500 505 510

Leu Phe Arg Asp Ala Ser Gln Ile Asp Arg Ser Lys Tyr Thr His Val

515 520 525

His Phe Ala Phe Gly Thr Leu Thr Pro Thr Tyr Glu Val Asn Val Gly

530 535 540

Asp Ile Leu Ser Ser Tyr Gln Phe Thr Gln Phe Lys Leu Ile Ser Gly

545 550 555 560

Pro Lys Lys Ile Leu Ser Phe Gly Gly Trp Asp Phe Ser Thr Ser Lys

565 570 575

Ala Thr Tyr Ser Ile Phe Arg Asn Gly Val Lys Ala Glu Asn Arg Leu

580 585 590

Thr Met Ala Lys Ser Ile Ala Asn Phe Ile Lys Glu His Asp Leu Asp

595 600 605

Gly Val Asp Ile Asp Trp Glu Tyr Pro Gly Ala Pro Asp Ile Pro Asp

610 615 620

Ile Pro Ala Gly Glu Glu Asp Glu Gly Thr Asn Tyr Leu Ala Phe Leu

625 630 635 640

Val Val Leu Lys Asn Leu Leu Pro Gly Lys Ser Ile Ser Ile Ala Ala

645 650 655

Pro Ser Ser Tyr Trp Tyr Leu Lys Gln Phe Pro Ile Lys Ala Ile Ser

660 665 670

Arg Ile Val Asp Tyr Ile Val Phe Met Ser Tyr Asp Ile His Gly Gln

675 680 685

Trp Asp Ala His Asn Met Trp Ser Gln Asp Gly Cys Val Thr Gly Asn

690 695 700

Cys Leu Arg Ser His Val Asn Leu Thr Glu Thr Arg Leu Ala Leu Val

705 710 715 720

Met Ile Thr Lys Ala Gly Val Pro Gly Glu Lys Val Ile Val Gly Val

725 730 735

Thr Ser Tyr Gly Arg Ser Phe Asp Met Ala Gln Pro Gly Cys Trp Ser

740 745 750

Pro Asp Cys Gln Phe Thr Gly Asp Arg Leu Asn Ser Asn Ala Lys Pro

755 760 765

Gly Arg Cys Thr Gly Thr Ala Gly Tyr Ile Ser Asn Ala Glu Ile Asp

770 775 780

Glu Ile Leu Ala Gly Gly Gly Ser Ser Gly Gly Ser Ser Gln Ala Arg

785 790 795 800

Ala Gly Arg Val Val Ala Ser Phe Val Asp Thr Ser Ser Asn Thr Asp

805 810 815

Val Leu Val Tyr Asp Asn Asn Gln Trp Val Gly Tyr Met Ser Glu Lys

820 825 830

Thr Lys Lys Thr Arg Thr Thr Leu Tyr Thr Gly Trp Gly Leu Gly Gly

835 840 845

Thr Thr Asp Trp Ala Ser Asp Leu Gln Gln Tyr His Asp Val Pro Gly

850 855 860

Pro Ala Lys Asp Trp Thr Glu Phe Lys Gln Leu Ile Arg Ala Gly Glu

865 870 875 880

Asp Pro Lys Ser Asp His Ser Arg Glu Gly Asp Trp Thr Lys Phe Asp

885 890 895

Cys Thr Asn Pro Tyr Leu Val Asp Lys Thr Phe Tyr Thr Pro Thr Gln

900 905 910

Arg Trp Lys Asn Leu Asp Thr Asp Ala Ala Trp Arg Asp Val Val Arg

915 920 925

Ile Trp Lys Glu Thr Asp Lys Pro Arg Asn Ile Met Phe Thr Ala Ser

930 935 940

Val Ser Thr Thr Leu Tyr Ile Ser Ala Asp Val Asp Cys Arg Asn Leu

945 950 955 960

Glu Asp Cys Asn Thr Thr Glu Glu Cys Ser Ala Gly Leu Asn Gly Pro

965 970 975

Tyr Ser Gly Pro Ala Ala Gln Phe Ile Trp Asn Ser Met Val Lys Ile

980 985 990

His Ala Met Tyr His Asn Tyr Val Leu Met Leu Glu Arg Ala Thr Ser

995 1000 1005

Leu Val Ser Met Ala Leu Asp Asp Met Gln Lys Thr Phe Ala Pro

1010 1015 1020

Val Pro Val Glu Glu Asp Lys Ala Trp Leu Tyr Leu Leu Ile Asp

1025 1030 1035

Leu Ile Thr Leu Gly Thr Leu Thr Val Ala Gly Pro Leu Tyr Asn

1040 1045 1050

Arg Gln Leu Gly Met Tyr Val Tyr Phe Ser Asp Lys Ser Val Asp

1055 1060 1065

Asp Ile Lys Asp Thr Thr Met Thr Leu Ile Gly Gln Ser Thr Thr

1070 1075 1080

Ile Ala Lys Asp Val Leu Ser Thr Lys Gln Glu Ala Trp Thr Glu

1085 1090 1095

Asn Leu Gln Ala Ser Phe Asn Asn Met Leu Ser Arg Val Ile Glu

1100 1105 1110

Gly Trp Gln Asn Ala Thr Ser Leu Ala Val Asn Lys Ile Phe Ser

1115 1120 1125

Gly Ser Glu Thr Ser Leu Asn Ile Leu Trp Asp Val Met Ser Asp

1130 1135 1140

Gly Lys Leu Ile Glu Gly Met Pro Pro Pro Gly Ser Gly Pro Pro

1145 1150 1155

Pro Asp Pro Gly Asn Ile His Asn Glu Leu Gln Ala Asn Val Lys

1160 1165 1170

Lys Ser Ile Tyr Ala Phe Ala Ile Pro Asn Leu Trp Arg Val Ser

1175 1180 1185

Gln Thr Phe Ala Phe Ile Leu Asp Ser Gly Phe Gly Cys Asp Val

1190 1195 1200

Glu Lys Pro Leu Gln Asp Tyr Leu Glu Asp Glu Thr Met Glu Ala

1205 1210 1215

Thr Gly Ala Cys Val Asp Gly Lys Arg Tyr Tyr Leu Val Ala Pro

1220 1225 1230

Ile Gly Glu Ser Arg Thr Cys Asp Trp Val Asn Gly Met Trp Asp

1235 1240 1245

Cys Thr Leu Ser Asn Lys Phe Ser Ala Pro Pro Gly Leu Asp Arg

1250 1255 1260

Leu Gly Ala Asp Phe Gly Tyr Leu Thr Lys Glu Asp Phe Ile Lys

1265 1270 1275

Gly Ser Ile Arg Thr Trp Leu Lys Asn Gly Lys Arg Asn Ala Gly

1280 1285 1290

Gly Gly Met Pro Asp Val Thr Asp Ile Asp Thr Ile Asn Ser Leu

1295 1300 1305

Ile Asp Leu Asp Phe Thr Thr Pro Gly Phe Ile His Leu Pro Val

1310 1315 1320

Cys Ser Pro Glu Arg Ala Tyr Gln Thr Trp Asp Thr Ser Ser Ser

1325 1330 1335

Gly Tyr Gly Ala Asn Tyr Pro Cys Asp Pro Pro Pro Gly Ile Asn

1340 1345 1350

Asn Cys Gly Asp Ser Thr Phe Glu Asp Gln Thr Ser Ala Ala Ser

1355 1360 1365

Pro Lys Val Glu Asp Cys Leu Gln Ile Ile Lys Asn Ile Gln Asp

1370 1375 1380

Asp Gly Lys Thr Glu Trp Thr Ile Gln Val Leu Gly Lys Asn Gln

1385 1390 1395

Arg Glu Ile Ala Lys Phe Gly Glu Cys Arg Phe Gly Val Glu Ala

1400 1405 1410

Thr Glu Gln Thr Gly Asn Ala Asp Phe Lys Val Gly Gly Gln Asp

1415 1420 1425

Val Ile Asp Ile Ile Asn Asp Ala Val Glu Lys Phe Gly Gly Ser

1430 1435 1440

Gly Arg Val Gly Ala Lys Gly Asp Met Ser Cys Asn Gly Asn Ile

1445 1450 1455

Lys Gly Gln Ala Val Lys Trp Gly Ile Tyr

1460 1465

<210> 17

<211> 561

<212> PRT

<213> Thermothelomyces thermophilus

<400> 17

Met Leu Arg Gly Thr Ile Ala Val Gly Val Ala Cys Leu Ala Gln Leu

1 5 10 15

Val Ala Gly Leu Asp Gly Pro Leu Phe Arg Thr Ser Leu Thr Leu Arg

20 25 30

Asp Phe Arg Glu Gln Leu Glu Arg Arg Gln Ala Arg Asp Gly Ala Ala

35 40 45

Leu Glu Ala Arg Ser Ser Asp Leu Gln Asp Leu Tyr Pro Ala His Thr

50 55 60

Leu Gln Val Pro Val Asp His Phe His Asn Asp Ser Leu Tyr Glu Pro

65 70 75 80

His Ser Ser Glu Thr Phe Pro Leu Arg Tyr Trp Phe Asp Ala Ser His

85 90 95

Tyr Lys Lys Gly Gly Pro Ile Ile Val Leu Gln Ser Gly Glu Thr Asp

100 105 110

Gly Val Gly Arg Leu Pro Phe Leu Gln Lys Gly Ile Val Ala Gln Leu

115 120 125

Ala Arg Ala Thr Asn Gly Leu Gly Val Ile Leu Glu His Arg Tyr Tyr

130 135 140

Gly Glu Ser Ile Pro Thr Pro Asp Phe Ser Thr Glu Lys Leu Arg Phe

145 150 155 160

Leu Thr Thr Asp Gln Ala Leu Ala Asp Met Ala Tyr Phe Ala Arg His

165 170 175

Val Val Phe Lys Gly Leu Glu His Leu Asp Leu Thr Ser Ala Lys Asn

180 185 190

Pro Tyr Ile Ala Tyr Gly Gly Ser Tyr Ala Gly Ala Phe Val Ala Phe

195 200 205

Leu Arg Lys Leu Tyr Pro Asp Val Tyr Trp Gly Ala Ile Ser Ser Ser

210 215 220

Gly Val Pro Glu Ala Ile Tyr Asp Tyr Trp Gln Tyr Tyr Glu Ala Ala

225 230 235 240

Arg Ile Tyr Ala Pro His Asp Cys Val Val Ala Thr Gln Lys Leu Thr

245 250 255

His Ile Val Asp Asn Ile Leu Leu Asp Lys Ala Asp Thr Asp Tyr Val

260 265 270

Arg Arg Leu Lys Thr Gly Phe Gly Leu Gly Gly Val Thr Arg Asn Asp

275 280 285

Asp Phe Ala Asn Ala Ile Ser Trp Gly Ile Gly Gly Leu Gln Gly Leu

290 295 300

Asn Trp Asp Pro Ala Leu Asn Asp Thr Gly Phe Gly Glu Tyr Cys Asn

305 310 315 320

Asn Leu Thr Ala Thr Lys Pro Leu Tyr Pro Thr Ser Pro Ala Leu Glu

325 330 335

Gln Glu Ala Arg Glu Leu Val Lys Ala Gly Gly Tyr Gly Lys Glu Ala

340 345 350

Asp Thr Leu Thr Thr Gln Leu Leu Asn Tyr Met Gly Tyr Val Asn Ala

355 360 365

Thr Thr Val Gln Thr Cys His Lys Asp Ser Gln Asp Glu Cys Phe Thr

370 375 380

Asn Tyr Asn Ser Thr Phe Tyr Gln Gln Asp Asp Lys Thr Gln Asp Trp

385 390 395 400

Arg Leu Trp Pro Tyr Gln Tyr Cys Phe Glu Trp Gly Tyr Leu Gln Thr

405 410 415

Gly Ser Gly Val Pro Ala Asn Gln Leu Pro Leu Ile Ser Arg Leu Ile

420 425 430

Asp Leu Asn Phe Thr Ser Val Val Cys Arg Glu Ala Phe Asn Ile Thr

435 440 445

Thr Pro Ser Gln Val Glu Arg Ile Asn Lys Leu Gly Gly Val Asn Ile

450 455 460

Ser Tyr Pro Arg Leu Ala Phe Val Asp Gly Glu Arg Asp Pro Trp Arg

465 470 475 480

Tyr Ala Ser Pro His Arg Ile Gly Leu Pro Glu Arg Lys Asn Thr Ile

485 490 495

Ser Glu Pro Phe Ile Leu Ile Lys Asp Gly Val His His Trp Asp Glu

500 505 510

Asn Gly Leu Phe Pro Asn Glu Thr Arg Pro Gly Leu Pro Pro Lys Pro

515 520 525

Val Ala Asp Ala Gln Arg Ala Glu Val Lys Phe Val Lys Ala Trp Leu

530 535 540

Lys Glu Trp Lys Glu Lys Glu Lys Cys Arg Gly Arg Lys Phe Cys Trp

545 550 555 560

Pro

<210> 18

<211> 640

<212> PRT

<213> Thermothelomyces thermophilus

<400> 18

Met Thr Met Lys Gly Ser Thr Leu Leu Ala Leu Ala Leu Gly Phe Gly

1 5 10 15

Ala His Ala Gln Phe Pro Pro Lys Arg Glu Gly Ile Thr Val Ile Glu

20 25 30

Ser Lys Phe Tyr Lys Asn Val Ser Ile Ser Phe Lys Glu Pro Gly Ile

35 40 45

Cys Glu Thr Thr Pro Gly Val Lys Ser Tyr Ser Gly Tyr Val His Leu

50 55 60

Pro Pro Asn Leu Ile Glu Gly Ala Asp Gln Asp Tyr Pro Ile Asn Thr

65 70 75 80

Phe Phe Trp Phe Phe Glu Ala Arg Lys Asp Pro Ala Asn Ala Pro Leu

85 90 95

Ala Ile Trp Leu Asn Gly Gly Pro Gly Gly Ser Ser Met Met Gly Leu

100 105 110

Leu Glu Glu Asn Gly Pro Cys Phe Val Gly Pro Asp Ser Lys Thr Thr

115 120 125

Tyr Leu Asn Arg Trp Ser Trp Asn Asn Glu Ala Asn Met Leu Tyr Ile

130 135 140

Asp Gln Pro Val Gln Thr Gly Phe Ser Tyr Asp Val Leu Thr Asn Val

145 150 155 160

Thr Val Gln Leu Asp Val Asp Asp Pro Ser Glu Pro Ile Ile Thr Pro

165 170 175

Thr Asn Phe Thr Asp Gly His Ile Pro Arg Thr Asn Asn Thr Phe Arg

180 185 190

Ile Gly Thr Val Gly Ser Gln Lys Ala Ser Gln Val Thr Asn Ser Thr

195 200 205

Glu Leu Ser Ala His Ala Met Trp His Phe Leu Gln Thr Trp Leu Phe

210 215 220

Glu Phe Pro His Tyr Arg Ser Asp Asp Gly Arg Ile Ser Leu Trp Ala

225 230 235 240

Glu Ser Tyr Gly Gly Thr Tyr Gly Pro Ala Phe Phe Arg Phe Phe Gln

245 250 255

Gln Gln Asn Glu Arg Ile Ala Asp Gly Gln Leu Glu Gly Arg Tyr Leu

260 265 270

His Leu Asp Thr Leu Gly Ile Ile Asn Gly Ala Val Asp Trp Pro Ile

275 280 285

Leu Ala Glu Ser Leu Ile Asp Tyr Pro Tyr Asn Asn Ser Tyr Gly Ile

290 295 300

Gln Phe Tyr Asn Asp Thr Phe His Ala Ala Leu Lys His Asn Trp Thr

305 310 315 320

Arg Pro Ser Gly Trp Arg Glu Gln Met Gln Ala Cys Thr Glu Ser Leu

325 330 335

Ala Ser Ser Ser Ser Ser Ser Pro Pro Ala Ala Gly Cys Glu Ala

340 345 350

Val Arg Ser Val Leu Asp Asp Val Leu Ala Ala Ala Phe Pro Arg Gln

355 360 365

Ser Gly Arg Ala Pro Phe Asp Leu Ala His Pro Arg Ala Asp Pro Phe

370 375 380

Pro Pro Pro His Pro His Gly Phe Leu Ala Arg Ala Asp Val Gln Ala

385 390 395 400

Ala Leu Gly Val Pro Val Asn His Thr Ala Val Ser Leu Pro Val Asn

405 410 415

Arg Ala Phe Asp Ala Thr Phe Asp Pro Leu Arg Gly Gly Gln Leu Asp

420 425 430

Ala Leu Ala Gly Leu Leu Asp Arg Arg Ala Gly Gly Gly Val Lys Val

435 440 445

His Leu Val Tyr Gly Asp Arg Asp Pro Ser Cys Asn Trp Ala Gly Gly

450 455 460

Glu Lys Val Ser Leu Ala Val Pro Trp Ser Arg Arg Asp Val Phe Ala

465 470 475 480

Ala Ala Gly Tyr Ala Pro Leu Val Val Val Ser Gly Lys Gly Gly Gly

485 490 495

Asp Gly Gly Asn Thr Gly Gly Gly Asn Thr Gly Gly Gly Glu Glu Glu

500 505 510

Val Val Val Val Arg Gly Leu Thr Arg Gln Val Gly Arg Phe Ser Phe

515 520 525

Thr Arg Val Phe Gln Ala Gly His Glu Val Pro Ser Tyr Gln Pro Gln

530 535 540

Ala Gly Tyr Glu Ile Phe Arg Arg Ala Met Ala Gly Leu Asp Leu Pro

545 550 555 560

Thr Gly Arg Val Arg Ala Gly Asp Asp Phe Val Thr Ala Gly Leu Arg

565 570 575

Asp Ala Trp Ala Val Lys Asn Ala Ala Pro Asp Met Val Glu Pro Arg

580 585 590

Cys Tyr Val Leu Lys Pro Glu Ser Cys Glu Pro Glu Val Trp Lys Thr

595 600 605

Val Val Asp Gly Thr Ala Ile Val Lys Asp Trp Phe Val Val Gly Ser

610 615 620

Thr Gly Gly Glu Gly Arg Gly Val Glu Gly Gly Ile Asp Gly Asp Glu

625 630 635 640

<210> 19

<211> 571

<212> PRT

<213> Thermothelomyces thermophilus

<400> 19

Met Leu Trp Thr Thr Leu Leu Ser Ala Leu Leu Leu Thr Gly Thr Ala

1 5 10 15

Glu Ala Ala Gly Arg Ser Ile Ala His Ala Gly Lys Arg His Val Glu

20 25 30

His Ala Ala Lys Arg Ala Lys Pro Ile Met Pro Ala Gly Pro Tyr His

35 40 45

Pro Val Ile Glu Arg Glu Glu Lys Ala Pro Lys Phe Leu Thr Pro Lys

50 55 60

Thr Glu Lys Phe Ala Val Asp Gly Lys Gly Ile Pro Asp Val Asp Phe

65 70 75 80

Asp Val Gly Glu Ser Tyr Ala Gly Leu Leu Pro Leu Ser Ser Asp Pro

85 90 95

Asn Asp Asp Lys Asn Leu Phe Phe Trp Phe Phe Pro Ser Thr Asn Pro

100 105 110

Ala Ala Glu Lys Glu Ile Leu Ile Trp Leu Asn Gly Gly Pro Gly Cys

115 120 125

Ser Ser Phe Glu Gly Leu Leu Gln Glu Asn Gly Pro Phe Leu Trp Gln

130 135 140

Tyr Gly Thr Tyr Lys Pro Val Gln Asn Pro Trp Ser Trp His Thr Leu

145 150 155 160

Thr Asn Ile Val Tyr Val Glu Gln Pro Val Gly Thr Gly Phe Thr Thr

165 170 175

Gly Thr Pro Thr Ile Thr Asn Glu Glu Glu Leu Ala Ala Glu Phe Met

180 185 190

Gly Phe Trp Lys Asn Phe Val Asp Thr Phe Gly Leu His Gly Tyr Lys

195 200 205

Val Tyr Ile Ala Gly Glu Ser Tyr Ala Gly Tyr Tyr Cys Pro Tyr Ile

210 215 220

Ala Ala Ala Phe Leu Asp Glu Glu Asp Lys Thr Tyr Tyr Asp Met Ser

225 230 235 240

Gly Met Thr Ile Tyr Asn Pro Ser Leu Ala Pro Asp Glu Ile Gln Glu

245 250 255

Pro Ile Pro Val Val Ala Phe Thr Glu Tyr Trp Ser Gly Leu Phe Pro

260 265 270

Phe Asn Asp Thr Phe Arg Ala Asp Ile Lys Arg Arg Glu Lys Glu Cys

275 280 285

Gly Tyr Ala Asp Phe Leu Ala Glu Tyr Leu Val Tyr Pro Pro Lys Gly

290 295 300

Pro Leu Pro Ser Arg Leu Pro Gly Thr His Arg Asp Gly Thr Thr Arg

305 310 315 320

Glu Glu Cys Trp Asn Ile Tyr Trp Asp Ile Phe Asp Ala Ile Ser Val

325 330 335

Leu Asn Pro Cys Phe Asp Ile Tyr Gln Val Ala Thr Thr Cys Pro Leu

340 345 350

Leu Trp Asp Val Leu Gly Phe Pro Gly Ser Met Pro Tyr Leu Pro Glu

355 360 365

Gly Thr Lys Val Tyr Phe Asp Arg Glu Asp Val Lys Arg Ala Ile His

370 375 380

Ala Pro Val Asn Ala Thr Trp Glu Glu Cys Ser Ser Arg Asp Val Phe

385 390 395 400

Val Asn Gly Thr Asp His Ser Val Pro Ser Thr Val Arg Ala Leu Pro

405 410 415

Arg Val Ile Asp Gly Thr Lys Asn Val Ile Ile Gly His Ser Ala Leu

420 425 430

Asp Met Ile Leu Leu Ala Asn Gly Thr Leu Leu Ala Leu Gln Asn Met

435 440 445

Thr Trp Gly Gly Lys Arg Gly Phe Gln Ser Arg Pro Asp Gln Pro Phe

450 455 460

Tyr Val Pro Leu Asn Asn Ile Thr Thr Leu Ser Thr Leu Ala Ala Ala

465 470 475 480

Gly Val Phe Gly Ser Leu Val Ser Glu Arg Gly Leu Thr Tyr Val Gly

485 490 495

Val Asp Leu Ala Gly His Met Val Pro Gln Tyr Ala Pro Ser Ala Ala

500 505 510

Tyr Arg His Val Glu Tyr Met Leu Gly Arg Val Asp Cys Met Asn Cys

515 520 525

Thr Lys Pro Phe Thr Thr Asp Pro Phe Thr Pro Gln Ser Lys Gly Lys

530 535 540

Leu Gly Lys Gly Thr Ala Pro Gln Gly Trp Ser Asn Ala Ser Gly His

545 550 555 560

Gly Lys Gly Asn Gly Pro Arg Arg Ile Arg Ala

565 570

<210> 20

<211> 8017

<212> DNA

<213> Thermothelomyces thermophilus

<400> 20

agggtaggtg ggatgggcgg ggtgtagggt aggtcggtgt agggtaggtc ggctgggcgg 60

ggtgtagggt aggtcggttg ggcggggtgt agggtaggtc ggttgggatg ggtgtagggt 120

aggtcggccg ggtgtagggt aggtcggctg ggcggggtgt agggtaggtc ggtgtagggt 180

aggtgggatg gggcgctatg tgcggccgcg agctcgcgag cccattttta gcgaaggcca 240

tacaaacgag ttttgcggaa cccgggattc cacccccgaa gccgccggcg cgtgcgcccc 300

gctgcgcatc ggtcggtggg tatatgagaa gggggcgggc aagccggaag ccagaggcaa 360

ctgctactgt tagctgccgc tggcctccgc ggcccagggc gcggcacggc tgcgttgaag 420

tctcccagtc tcccacccgt tggctgcgcg gatccgcccg tcttggtggt tgcgagctcg 480

cgagcccatt tttagcgaag gccatacaaa cgagttttgc ggggcccggg attccacccc 540

ggaacccgcc ggcgcgtgcg ccccgctgcg catcggtcgg tgggtatgtg agggaggaag 600

aagaaaaaaa aaaaaagctc ctgcgggggg gctgtcgggc acgcctactt tcgggcgacc 660

cggcacctct ccgcggcagc cttcgcaggc cgctgttggt cccatttcat acgtcgccgc 720

cttcgcgtgg tgccctacgg tctgccgggg taccgacgat tgcggcgagc accgcctcag 780

caccgctgct gccaccggcg cgacctcgcc cggggggtgcg cgcggcatct gggaagactc 840

tgcaggcgta agggaatacc ccatgtgcgc cgaggggtgg gctatgtggg tgcttggcgg 900

ttcgccagac ctttctaaag ccaccggggg tacctaccgg ttggggacgc ctacagggct 960

gaacctcccg gtcgggcctc ctcttggggc gcttaggcgg cgacttcggg gcgcgatcgc 1020

tccccgctct cgcccgccga cggcgctctg gggaattcag gaggggaaag cagatgtgac 1080

ccgcggctcg accggcgcat tgccggacga gctgcgcggc cacgcgggcc cccgcgcccg 1140

ccgacccagt aacttagtga actcttccgc cctgaaacac gggcggttgg ccctaaccgg 1200

ctcacgatag ttacctggtt gattctgcca gtagtcatat gcttgtctca aagattaagc 1260

catgcatgtc taagtataag caattataca gcgaaactgc gaatggctca ttaaatcagt 1320

tatcgtttat ttgatagtac cttactacat ggataaccgt ggtaattcta gagctaatac 1380

atgctaaaaa tcccgacttc ggaagggatg tatttattag attaaaaacc aatgccctcc 1440

ggggctctct ggtgattcat gataacttct cgaatcgcac ggccttgcgc cggcgatggt 1500

tcattcaaat ttctgcccta tcaactttcg acggctgggt cttggccagc cgtggtgaca 1560

acgggtaacg gagggttagg gctcgacccc ggagaaggag cctgagaaac ggctactaca 1620

tccaaggaag gcagcaggcg cgcaaattac ccaatcccga cacggggagg tagtgacaat 1680

aaatactgat acagggctct tttggggtctt gtaattggaa tgagtacaat ttaaatccct 1740

taacgaggaa caattggagg gcaagtctgg tgccagcagc cgcggtaatt ccagctccaa 1800

tagcgtatat taaagttgtt gaggttaaaa agctcgtagt tgaaccttgg gcctagccgg 1860

ccggtccgcc tcaccgcgtg cactggctcg gctgggtctt tccttctgga gaaccgcatg 1920

cccttcactg ggtgtgccgg ggaaccagga cttttactct gaacaaatta gatcgcttaa 1980

agaaggccta tgctcgaata cattagcatg gaataataga ataggacgtg tggttctatt 2040

ttgttggttt ctaggaccgc cgtaatgatt aatagggaca gtcggggggca tcagtattca 2100

attgtcagag gtgaaattct tggatttatt gaagactaac tactgcgaaa gcatttgcca 2160

aggatgtttt cattaatcag gaacgaaagt taggggatcg aagacgatca gataccgtcg 2220

tagtcttaac cataaactat gccgattagg gatcggacgg cgttattttt tgacccgttc 2280

ggcaccttac gataaatcaa aatgtttggg ctcctggggg agtatggtcg caaggctgaa 2340

acttaaagaa attgacggaa gggcaccacc aggggtggag cctgcggctt aatttgactc 2400

aacacgggga aactcaccag gtccagacac gatgaggatt gacagattga gagctctttc 2460

ttgatttcgt gggtggtggt gcatggccgt tcttagttgg tggagtgatt tgtctgctta 2520

attgcgataa cgaacgagac cttaacctgc taaatagccc gtattgcttt ggcagtacgc 2580

cggcttctta gagggactat cggctcaagc cgatggaagt ttgaggcaat aacaggtctg 2640

tgatgccctt agatgttctg ggccgcacgc gcgctacact gacagagcca gcgagtactc 2700

ccttggccgg aaggcccggg taatcttgtt aaactctgtc gtgctgggga tagagcattg 2760

caattattgc tcttcaacga ggaatcccta gtaagcgcaa gtcatcagct tgcgttgatt 2820

acgtccctgc cctttgtaca caccgcccgt cgctactacc gattgaatgg ctcagtgagg 2880

ctttcggact ggcccagaga ggtcggcaac gaccactcag ggccggaaag ttatccaaac 2940

tcggtcattt agaggaagta aaagtcgtaa caaggtctcc gttggtgaac cagcggaggg 3000

atcattacag agctgcaaaa ctccctaaac catcgtgaac gctacctaga ccgttgcttc 3060

ggcgggcggc gccctcgcgc gccccccctg gggcccgcac cgcgggcgcc cgccggaggt 3120

acaccaaact cttgatatgt tatggccact ctgagtctcc tgtactgaat aagtcaaaac 3180

tttcaacaac ggatctcttg gttctggcat cgatgaagaa cgcagcgaaa tgcgataagt 3240

aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcacattg cgcccgccag 3300

catcctggcg ggcatgcctg ttcgagcgtc atttcaaccc atcaagccca cggcttgtgt 3360

tggggacctg cggctgcccg caggccctga aaaccagtgg cgggctcgct agtcacaccg 3420

ggcgtagtag catacgacct cgctcagggc gtgctgcggg ttccagccgt aaaacgacct 3480

tcacaaccca aggttgacct cggatcaggt aggaggaccc gctgaactta agcatatcaa 3540

taagcggagg aaaagaaacc aacagggatt gccctagtaa cggcgagtga agcggcaaca 3600

gctcaaattt gaaatctggc ttcggcccga gttgtaattt gcagaggaag ctttaggcgc 3660

ggcaccttct gagtcccctg gaacggggcg ccatagaggg tgagagcccc gtatagttgg 3720

atgcctagcc tgtgtaaagc tccttcgacg agtcgagtag tttgggaatg ctgctcaaaa 3780

tgggaggtaa atttcttcta aagctaaata ccggccagag accgatagcg cacaagtaga 3840

gtgatcgaaa gatgaaaagc actttgaaaa gagggttaaa tagcacgtga aattgttgaa 3900

agggaagcgc ttgtgaccag acttgcgccg ggctgatcat ccggtgttct caccggtgca 3960

ctctgcccgg ctcaggccag catcggttct cgcgggggga taaaggcccg gggaatgtag 4020

ctcctccggg agtgttatag ccccgggtgt aataccctcg cggggaccga ggttcgcgca 4080

tctgcaagga tgctggcgta atggtcatca gcgacccgtc ttgaaacacg gaccaaggag 4140

tcaaggtttt gcgcgagtgt ttgggtgtaa aacccgcacg cgtaatgaaa gtgaacgtag 4200

gtgagagctt cggcgcatca tcgaccgatc ctgatgtttt cggatggatt tgagtaggag 4260

cgttaagcct tggacccgaa agatggtgaa ctatgcttgg atagggtgaa gccagaggaa 4320

actctggtgg aggctcgcag cggttctgac gtgcaaatcg atcgtcaaat ctgagcatgg 4380

gggcgaaaga ctaatcgaac catctagtag ctggttaccg ccgaagtttc cctcaggata 4440

gcagtgttgt cttcagtttt atgaggtaaa gcgaatgatt agggactcgg gggcgctttt 4500

tagccttcat ccattctcaa actttaaata tgtaagaagc ccttgttact tagttgaacg 4560

tgggccttcg aatgtatcaa cactagtggg ccatttttgg taagcagaac tggcgatgcg 4620

ggatgaaccg aacgcggggt taaggtgccg gagtggacgc tcatcagaca ccacaaaagg 4680

cgttagtaca tcttgacagc aggacggtgg ccatggaagt cggaatccgc taaggactgt 4740

gtaacaactc acctgccgaa tgtactagcc ctgaaaatgg atggcgctca agcgtcccac 4800

ccataccccg ccctcagggt agaaacgacg ccctgaggag taggcggccg tggaggtcag 4860

tgacgaagcc tagggcgtga gcccgggtcg aacggcctct agtgcagatc ttggtggtag 4920

tagcaaatac ttcaatgaga acttgaagga ccgaagtggg gaaaggttcc atgtgaacag 4980

cggttggaca tgggttagtc gatcctaagc catagggaag ttccgtttca aaggggcact 5040

cgtgccccgt gtggcgaaag ggaagccggt taacattccg gcacctggat gtgggttttg 5100

cgcggtaacg caactgaacg cggagacgac ggcggggggcc ccgggcagag ttctcttttc 5160

ttcttaacgg tctatcaccc tggaaacagt ttgtctggag atagggttta acggccggaa 5220

gagcccgaca cttctgtcgg gtccggtgcg ctctcgacgt cccttgaaaa tccgcggggag 5280

ggaataattc tcacgccagg tcgtactcat aaccgcagca ggtccccaag gtgaacagcc 5340

tctggttgat agaacaatgt agataaggga agtcggcaaa atagatccgt aacttcggga 5400

taaggattgg ctctaagggt tgggcacgtt gggctttggg cggacgccct gggagcaggt 5460

cgcctctagc cgggcaaccg gcggggggct tccagcatcc gggtgcagat gcccttagca 5520

ggcttcggcc gtccggcgcg cggttaacaa ccaacttaga actggtacgg acagggggaa 5580

tctgactgtc taattaaaac atagcattgc gatggccaga aagtggtgtt gacgcaatgt 5640

gatttctgcc cagtgctctg aatgtcaaag tgaagaaatt caaccaagcg cgggtaaacg 5700

gcgggagtaa ctatgactct cttaaggtag ccaaatgcct cgtcatctaa ttagtgacgc 5760

gcatgaatgg attaacgaga ttcccactgt ccctatctac tatctagcga aaccacagcc 5820

aagggaacgg gcttggcaga atcagcgggg aaagaagacc ctgttgagct tgactctagt 5880

ttgacattgt gaaaagacat aggaggtgta gaataggtgg gagcttcggc gccggtgaaa 5940

taccactact cctattgttt ttttacttat tcaatgaagc ggggctggat tttcgtccaa 6000

cttctggttt taaggtcctt cgcggggccga cccgggttga agacattgtc aggtggggag 6060

tttggctggg gcggcacatc tgttaaacca taacgcaggt gtcctaaggg gggctcatgg 6120

agaacagaaa tctccagtag aacaaaaggg taaaagtccc cttgattttg attttcagtg 6180

tgaatacaaa ccatgaaagt gtggcctatc gatcctttag tccctcgaaa tttgaggcta 6240

gaggtgccag aaaagttacc acagggataa ctggcttgtg gcggccaagc gttcatagcg 6300

acgtcgcttt ttgatccttc gatgtcggct cttcctatca taccgaagca gaattcggta 6360

agcgttggat tgttcaccca ctaataggga acgtgagctg ggtttagacc gtcgtgagac 6420

aggttagttt taccctactg atgaactcat cgcaatggta attcagctta gtacgagagg 6480

aaccgctgat tcagataatt ggtttttgcg gttgtccgac cgggcagtgc cgcgaagcta 6540

ccatctgctg gataatggct gaacgcctct aagtcagaat ccatgccaga acgcgatgat 6600

actacccgca cgttgtagac gtataagaat aggctccggc ctcgtatcct agcaggcgat 6660

tcctccgccg gcctcgaagt tggccggcgg taattcgcgt attgcaattt cgacacgcgc 6720

gggatcaaat cctttgcaga cgacttagat gtgcgaaagg gtcctgtaag cagtagagta 6780

gccttgttgt tacgatctgc tgagggtaag ccctccttcg cctagatttc ccagcgagag 6840

cccgccggcg gaacagccgg gcgagcctta cgggggaagc cttaagggga ttgagaagtg 6900

gtgccgtgcg ttcgcgcgcc cctaggtcct ttagccggcc gcaggtgtag ggtaggtcgg 6960

ttgggaggat ggggtgtagg gtaggtcggt gtagggtagg ttggttggga ggatggggtg 7020

tagggtaggt cggccggggtg tagggtaggt cggtgtaggg taggtgggat ggggcgctat 7080

atgcggccgc gagctcgcga gcctattttt agtgaaggct atataaataa gctttacgtt 7140

accgggcctt gctaccctcg agtggcgtgg gccgtgctgc ctactgggca ttgctcgccg 7200

ggctgtataa gggaggggtc ggggtcgcgg tctagggtag gtcgggtggg atggggtgta 7260

gggtaggaga agcgctctag tcgtgtgtct ttttctctag gtctattatt agtactggct 7320

gtagggcgac gtgccctgcc ttgttataat attatattgt atgtttaggc ctaactagc 7380

ttgtaatcta tttgtatctg gcttattagg tacggcttcc tttgtatata actagagagg 7440

ctctggtatg cttcttagta tagcggtata ggattcataa tcatagtaat gataatcata 7500

atagtaataa taataataat agtaatgata ataataataa tctatttata tcttatttaa 7560

aatgcttgta cggctgcctg ctcttaagga gtagctagat atgagatggt agggtagcta 7620

gctaacctag gctagacgtt ctcgtccctt agctatataa gtgctatata ttatagttag 7680

ttatctaacc taccttctta cttgagcaga agaggtaggg ttctagtata gctagtaggg 7740

cttctaggcc taagggcctg ttattcgagt tattataggt tagtatttaa tatagttata 7800

gggataggcc tcgattacgg gtataggata ggtaggatag gtatagggta ggtcggttag 7860

gaggataggg tgtaaggtag gtcggccggg tatagggtag gtagtaggtt aggcggggtg 7920

tagggtaggt cggtgtaggg taggtgggat gggcggggtg tagggtaggt cggttgggag 7980

gatggggtgt agggtaggtc ggtgtaggt aggtcgg 8017

<210> 21

<211> 8334

<212> DNA

<213> Artificial sequence

<400> 21

gtttaaacga aaggatctct cgcccaggtg gacaacccgc ataatggagg cgccgtggtg 60

gattttgcca tgcaggagcc aaccgccagc ggccatgaac ccgcccagca cgatcactct 120

accaagtaac gttaaagagg cgatccctca tgcgccggag taactaacgg agtaccttct 180

cattgatctt actgacttgt tagtccgcgc tgacggccaa cagttcaacc agcccggtga 240

tcagcagctc gagggcttgc actggccatc cgccgggcgc ttggctgagc gagcatccgc 300

gggtcagcca cagccgccgg tctgatgtgg ccgtggcatg acctcagctt gtttatgggt 360

tattagatct ggcttagatc cggcttattt agatatctat ctagtcgagg caggggggttc 420

gagagaccgt ttggtttggt tcatccgacg tttgcctctc tgagctcggt gagtgagagc 480

ttcggttgcg acggccaagc atcgcggggc cgcactcgca ggctgcttcg caccacggaa 540

ctcatccgag ctacggagtc cctccgtacg ctactcgagt acgtccgtcc tctgtaggca 600

tctcgagccg tcgccagcat ttgagaccaa tggaaggggg tttcctcccg tgcaactttg 660

gttgaaacct tcaaagcgtc ccccgactgg ggcaaccgtt tgacttggat ccacacttca 720

ggggtagaga ggctcctctc gacaaccccc tgtatttctg taacccccct tgaacgccgt 780

acatggcacc acaacaccaa acaagccatg ctgcttggaa tcttcgctcc gaaaagagcc 840

ccccccgggc cctccaactt catgcgctcc ctcctcagtt aaatagcggc cgctctgctc 900

catggagtat tgctctcttc attaggctgt tgtgatttgc ttgcttgttt tcctttcctt 960

ttccgcacac ccattccctc ttttcactgg gtcgaggctc tctccaccga taaccttcga 1020

tccgcggcgc gccgttggcg gtaacagcag gtgccgttcg ctgctgctcg gtccttttcc 1080

cgaaagtacc ctgccttcct ggcctcgacc tccgcctgcg gggctctggg cactgtcaaa 1140

ctttcgcagt tttgcacccg cagcgattcg agcgggaccc gcttcgccaa cgcggatcag 1200

gagaggtaaa agtctcccct gtcctttctg ccggagcatc ctccgtccgt cgttcgctgg 1260

ttgttgtgtg tgtgtgtgtg tacagtactg taggtacata gtgctcaccc cagggcattt 1320

ccccaatccg cgattgcgca taccataggc gtcatctgaa tctgcgttac gtccggataa 1380

cactacgcag tacgaaggag tcggtgctac tacgcccgca agttcaagtc tacggtgtgg 1440

ctaaggtgct caagggcata gttacacgca ccccgggcga gcttctcgca cctcgaccct 1500

ttgccggggc gtcactggtc cgacaactca taatccacca tcgcttgggc aggcctgcat 1560

cccccgtggg tcgcatccaa gccgccggta aataccggcc gacacacaca cacacacacg 1620

caccctgtct agcgcgacgc tacaaaagag accggcaggc actccccgcg accgcttgtt 1680

ctggccatcc cgtctctcgc ctctgcagtt atcgcctgcc tcatctctgc gtcctgataa 1740

cttttgtcac ctctgatccc cccccgacga gacggtctcg ccgaccagga cccgagatgg 1800

cggaccgcca tcctactctg tcccagtcct tcgccgagcg ggcgaagacg gctagccacc 1860

cgctcacccg ctacctcttt cggctcatgg acctcaaggc ctcgaacctg tgcctgagcg 1920

ccgacgtgtc caccgcgcgc gagcttctga cgctggccga ccgggtcggc ccctcgatcg 1980

tcgtgctcaa gacgcactac gacctgatct cgggctggga ctacaacccg caaaccggca 2040

ccggcgcgaa gctggccgcc ctggcgagga agcatggctt cctcatcttt gaggaccgca 2100

agtttgtcga cattggtaag acggtgcaga tgcagtacac ggctggcact gcgcgcataa 2160

tagagtgggc gcacatcacc aacgccaaca tcgacgccgg caaggacatg gtgcgcgcca 2220

tggccgaggc ggccgccaag tggaaggaac gcatcaacta cgaggtcaag acctccgtca 2280

cggtgggcac gcccgtctcg gaccagttcg acgatgcgga agagcaagcg cagtggccgc 2340

agcaccagca gcaccagcac cagcaccagc accagcaaca gcgagatgaa aaaggtgggc 2400

cccgcaggct cggcactcgg gagggagcagc accaacagga caacggagac ggtgacggcc 2460

ggaaagggag cattgtctcg atcactacgg tgacgcagtc atttgagccc gctcactccc 2520

cacgcctgtc caagagcaac gagctgggcg acgacgccgt cttccccggc atcgaggagg 2580

cccccgtcga ccgcggcctg cttctgctcg cccagatgtc gtccaagggc tgcctcatga 2640

ccaaggagta cacccaggcc tgcgtcgagg ccgcgcgcga gcataaggat tttgtcatgg 2700

gcttcgtctc acaggagtcg ctcaactcgg ccccggacga cactttcatc cacatgaccc 2760

ccggatgcaa gcttccgccg ccaggcgagg acgaagagag cggccagatc gagtttaaac 2820

ggcgcgccgc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga 2880

gccggaagca taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt 2940

gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 3000

atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 3060

actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 3120

gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 3180

cagcaaaagg cccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 3240

ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 3300

ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 3360

ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 3420

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 3480

cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 3540

aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 3600

gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 3660

agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 3720

ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 3780

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3840

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3900

aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3960

tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 4020

atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 4080

cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 4140

gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 4200

gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 4260

tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 4320

tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 4380

tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 4440

aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 4500

atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 4560

tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 4620

catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 4680

aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 4740

tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 4800

gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 4860

tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 4920

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaacga 4980

agcatctgtg cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac 5040

aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca 5100

acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt 5160

caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt 5220

taccaacaaa gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat 5280

ttttctaaca aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc 5340

tcttgataac tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct 5400

attttctctt ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa 5460

gctgcgggtg cattttttca agataaaggc atccccgatt atattctata ccgatgtgga 5520

ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat 5580

tatgaacggt ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc 5640

gtattgtttt cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta 5700

atactagaga taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga 5760

aaggtggatg ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt 5820

ttgagcaatg tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg 5880

cgtttttggt tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga 5940

agttcctata ctttctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa 6000

aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc 6060

acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt 6120

ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac 6180

ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt 6240

tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat 6300

ttcctttgat attggatcat actaagaaac cattattatc atgacattaa cctataaaaa 6360

taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 6420

acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 6480

agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 6540

atcagagcag attgtactga gagtgcacca taccacagct tttcaattca attcatcatt 6600

ttttttttat tctttttttt gatttcggtt tctttgaaat ttttttgatt cggtaatctc 6660

cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata tacgcatatg 6720

tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca gaacaaaaac 6780

ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg ctgctactca 6840

tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa caaacttgtg 6900

tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag cattaggtcc 6960

caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca tggagggcac 7020

agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg aagacagaaa 7080

atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat acagaatagc 7140

agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg ttagcggttt 7200

gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt tagcagaatt 7260

gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg acattgcgaa 7320

gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg gaagagatga 7380

aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg gagacgcatt 7440

gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca ttattattgt 7500

tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac gttacagaaa 7560

agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa ctgtattata 7620

agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat atcagttatt 7680

accctatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat 7740

tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 7800

taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 7860

gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 7920

caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 7980

aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 8040

atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 8100

aggagcgggc gctaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 8160

cgccgcgctt aatgcgccgc tacagggcgc gtcgcgccat tcgccattca ggctgcgcaa 8220

ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg 8280

atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacg 8334

<210> 22

<211> 8273

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 22

ggcgcgccgt ttaaacgaac ctgtgcctga gcgccgacgt gtccaccgcg cgcgagcttc 60

tgacgctggc cgaccgggtc ggcccctcga tcgtcgtgct caagacgcac tacgacctga 120

tctcgggctg ggactacaac ccgcaaaccg gcaccggcgc gaagctggcc gccctggcga 180

ggaagcatgg cttcctcatc tttgaggacc gcaagtttgt cgacattggt aagacggtgc 240

agatgcagta cacggctggc actgcgcgca taatagagtg ggcgcacatc accaacgcca 300

acatcgacgc cggcaaggac atggtgcgcg ccatggccga ggcggccgcc aagtggaagg 360

aacgcatcaa ctacgaggtc aagacctccg tcacggtggg cacgcccgtc tcggaccagt 420

tcgacgatgc ggaagagcaa gcgcagtggc cgcagcacca gcagcaccag caccagcacc 480

agcaccagca acagcgagat gaaaaaggtg ggccccgcag gctcggcact cgggaggagc 540

agcaccaaca ggacaacgga gacggtgacg gccggaaagg gagcattgtc tcgatcacta 600

cggtgacgca gtcatttgag cccgctcact ccccacgcct gtccaagagc aacgagctgg 660

gcgacgacgc cgtcttcccc ggcatcgagg aggcccccgt cgaccgcggc ctgcttctgc 720

tcgcccagat gtcgtccaag ggctgcctca tgaccaagga gtacacccag gcctgcgtcg 780

aggccgcgcg cgagcataag gattttgtca tgggcttcgt ctcacaggag tcgctcaact 840

cggccccgga cgacactttc atccacatga cccccggatg caagcttccg ccgccaggcg 900

aggacgaaga gagcggccag atcgagggcg acggcctcgg ccagcagtac aactcgccca 960

gcaagttgat caacatttgc ggcaccgaca ttgtcatcgt agggcgtggc atcaccgccg 1020

ccggcgaccc gccctccgag gctgagaggt acaggagaaa agcctggaag gcctatctgg 1080

cgcgtctggc gtgatttggg gggaggggga gaggagatgg gggacgggag gggtcgcctt 1140

ggtcagtctt gtgcgtgtcc tgcagcggat tcgtcaccgg ggcagcaccc aaaagaggga 1200

gaaaaagggg aaaaaaaata aataaataaa aagggttaag ttgttgaaaa aagtgttgtg 1260

agctctctgg caaggcgcgc cttcgcacca cggaactcat ccgagctacg gagtccctcc 1320

gtacgctact cgagtacgtc cgtcctctgt aggcatctcg agccgtcgcc agcatttgag 1380

accaatggaa gggggtttcc tcccgtgcaa ctttggttga aaccttcaaa gcgtcccccg 1440

actggggcaa ccgtttgact tggatccaca cttcaggggt agagaggctc ctctcgacaa 1500

ccccctgtat ttctgtaacc ccccttgaac gccgtacatg gcaccacaac accaaacaag 1560

ccatgctgct tggaatcttc gctccgaaaa gagccccccc cgggccctcc aacttcatgc 1620

gctccctcct cagttaaata gcggccgctc tgctccatgg agtattgctc tcttcattag 1680

gctgttgtga tttgcttgct tgttttcctt tccttttccg cacacccatt ccctcttttc 1740

actgggtcga ggctctctcc accgataacc ttcgatccgc aggcgcgccc cccatgtttc 1800

ccaaagggtc ctgtttgttt gttttttctt cctcttcttt aagggctcgg gtgacagctg 1860

gagagccgag caagcagcac ataaaatggc ttgcgaattc aggatacatt gattatgtca 1920

tggacccaag gaaacaccct cttcctgcgc cgtccactgc accaacttct cctcaaacac 1980

gatcaaccac gtaggaacag cagacgaaaa cgtcacctgg ccgcgattga catacccaaa 2040

gtgagacgtg gaggacgg gggttgcgtg cttcggccgg atgaggtttt cagaccgact 2100

acggtactgg atcttagcag cacaaagatc acctacccag agtaagtagt tggacaagcg 2160

ctttcactcg gaactcagag ccaacggaaa tcggatgagg catcaagatt tttcgagggg 2220

caactactcc gtcggacaac cgagtcctgt gtgcaagcgc cgcatttcgt cattgtagat 2280

gttggagaca tgtttgcagt ccgccctaag caggccattc cgtcggagaa gagggaaaga 2340

cccggccgag ggcgtgcccg ggttccaggc tacttgccac gagggtttca tatcagcaca 2400

tcttcggcac aaccacagcc attgagcccg attgccccga gaggggaaag ggcggctgaa 2460

ttgcaatttg acatccgtgc ttgttgttac ccttgtttag caaagccagt gggggtcatc 2520

aataccacct ccaaggcgcg catatcacgg caacacctgg cccgataaaa cagaagccaa 2580

acacgtgtgt attatgttgg tattagatgt tcgcttctcc caaccggagc tgatgcccgc 2640

gccagatccc gcgcccaaca gttcactcgt aatcgttgta tacatgaccg cctgtatcga 2700

agggtatgtg tcattagcaa ggtatataga aacgtgaacc gaaaatgctc atctcgccgg 2760

tttaaacgct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacataggag 2820

ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gaggtaactc acattaattg 2880

cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 2940

tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 3000

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 3060

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 3120

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 3180

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 3240

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 3300

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 3360

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 3420

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 3480

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 3540

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 3600

gaagggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 3660

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 3720

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 3780

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 3840

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 3900

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3960

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 4020

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 4080

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 4140

caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 4200

cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 4260

cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 4320

cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 4380

agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 4440

tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 4500

agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 4560

atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 4620

ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 4680

cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 4740

caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 4800

attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 4860

agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaacgaa 4920

gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca 4980

aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct attttaccaa 5040

cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc 5100

aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt 5160

accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt 5220

tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct 5280

cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta 5340

ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag 5400

ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat 5460

tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt 5520

atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg 5580

tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa 5640

tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa 5700

aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt 5760

tgagcaatgt ttgtggaagc ggtattcgca atattttagt agctcgttac agtccggtgc 5820

gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa 5880

gttcctatac tttctagaga ataggaactt cggaatagga acttcaaagc gtttccgaaa 5940

acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt tcacgtcgca 6000

cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat agtgcgtgtt 6060

tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc 6120

tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc actacccttt 6180

agctgttcta tatgctgcca ctcctcaatt ggattagtct catccttcaa tgctatcatt 6240

tcctttgata ttggatcata ctaagaaacc attattatca tgacattaac ctataaaaat 6300

aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 6360

cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 6420

gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 6480

tcagagcaga ttgtactgag agtgcaccat accacagctt ttcaattcaa ttcatcattt 6540

tttttttatt cttttttttg atttcggttt ctttgaaatt tttttgattc ggtaatctcc 6600

gaacagaagg aagaacgaag gaaggagcac agacttagat tggtatatat acgcatatgt 6660

agtgttgaag aaacatgaaa ttgcccagta ttcttaaccc aactgcacag aacaaaaacc 6720

tgcaggaaac gaagataaat catgtcgaaa gctacatata aggaacgtgc tgctactcat 6780

cctagtcctg ttgctgccaa gctatttaat atcatgcacg aaaagcaaac aaacttgtgt 6840

gcttcattgg atgttcgtac caccaaggaa ttactggagt tagttgaagc attaggtccc 6900

aaaatttgtt tactaaaaac acatgtggat atcttgactg atttttccat ggagggcaca 6960

gttaagccgc taaaggcatt atccgccaag tacaattttt tactcttcga agacagaaaa 7020

tttgctgaca ttggtaatac agtcaaattg cagtactctg cgggtgtata cagaatagca 7080

gaatgggcag acattacgaa tgcacacggt gtggtgggcc caggtattgt tagcggtttg 7140

aagcaggcgg cagaagaagt aacaaaggaa cctagaggcc ttttgatgtt agcagaattg 7200

tcatgcaagg gctccctatc tactggagaa tatactaagg gtactgttga cattgcgaag 7260

agcgacaaag attttgttat cggctttatt gctcaaagag acatgggtgg aagagatgaa 7320

ggttacgatt ggttgattat gacacccggt gtgggtttag atgacaaggg agacgcattg 7380

ggtcaacagt atagaaccgt ggatgatgtg gtctctacag gatctgacat tattattgtt 7440

ggaagaggac tatttgcaaa gggaagggat gctaaggtag agggtgaacg ttacagaaaa 7500

gcaggctggg aagcatattt gagaagatgc ggccagcaaa actaaaaaac tgtattataa 7560

gtaaatgcat gtatactaaa ctcacaaatt agagcttcaa tttaattata tcagttatta 7620

ccctatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt 7680

gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 7740

aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 7800

ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 7860

aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 7920

agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 7980

tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 8040

ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 8100

gccgcgctta atgcgccgct acagggcgcg tcgcgccatt cgccattcag gctgcgcaac 8160

tgttgggaag ggcgatcggt gcggggcctct tcgctattac gccagctggc gaaaggggga 8220

tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acg 8273

<210> 23

<211> 8375

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 23

gtttaaacgt ttaaacggtg gacacaaaca gttgccaatt agatctcctg aacttaataa 60

agcgccattc tgttgaaaga cgagatcgcc gagcgtacca agtcttgttc gggctctgaa 120

caaaactgga accaccactc caaaacggta gttgatgcga atacctatat cctccagatc 180

gagtctgcag tgcagcgtgg atatgggtat cggcacttgc gtgcacagta gtgtagctcc 240

gtagtacgga gtgctgcagg atatcaactt ctaagccaca gccgttccga gcggaacatt 300

tcttcattgg ctccctcgcg catggggcag gtcgtgcagt gttcgtggct atttgcggat 360

catacagaca ccttttgggg tccgcttcgg gaaactgccg cccccctaca ctacggagta 420

gtgcgacgcc gcattcccgt tcggcgcttg gcagccaatt ctaccactcg gggccgtctg 480

caaggccacc tctgcatttt caagtcgaat agtcaatagt cacaaaaact ggtcaaactg 540

gccaaactgg tcaacccggt tgccaccttt tggaaagagc accgcttctt tttcgttcct 600

cggcctgagc cgtcgaatgc gaacgtcaaa aggcgaacta gaaattctga aacatagtac 660

ggattactcc gtacccggtt gttttgcacc gggattttgc ttcaatcgcc accgagttcc 720

acccactttc gccaaggtac ggattacagt aatccgtaca tacctacgga cgtactccgt 780

cgtgtatcta ggtgttcccc cttggcacgc tttccacctg cgacaacgcg gcctcagatc 840

ccgacctcga accccccccc cccccccccc aaacaacaac ccagctcttc ggctgtgcgc 900

ccgccaactc gacaaacaac aacatccaac aagtgcgaat ttgaattcga ctcgacagcc 960

catcgattcg tctctcttca tgcgcatcaa tccgatccgg aaccgccgac tttaacaaca 1020

cccgtgccgg gctcgaccac ggggctcccg tagtccgcca aatacaggcg cgccgttggc 1080

ggtaacagca ggtgccgttc gctgctgctc ggtccttttc ccgaaagtac cctgccttcc 1140

tggcctcgac ctccgcctgc ggggctctgg gcactgtcaa actttcgcag ttttgcaccc 1200

gcagcgattc gagcgggacc cgcttcgcca acgcggatca ggagaggtaa aagtctcccc 1260

tgtcctttct gccggagcat cctccgtccg tcgttcgctg gttgttgtgt gtgtgtgtgt 1320

gtacagtact gtaggtacat agtgctcacc ccagggcatt tccccaatcc gcgattgcgc 1380

ataccatagg cgtcatctga atctgcgtta cgtccggata acactacgca gtacgaagga 1440

gtcggtgcta ctacgcccgc aagttcaagt ctacggtgtg gctaaggtgc tcaagggcat 1500

agttacacgc accccggggcg agcttctcgc acctcgaccc tttgccgggg cgtcactggt 1560

ccgacaactc ataatccacc atcgcttggg caggcctgca tcccccgtgg gtcgcatcca 1620

agccgccggt aaataccggc cgacacacac acacacac gcaccctgtc tagcgcgacg 1680

ctacaaaaga gaccggcagg cactccccgc gaccgcttgt tctggccatc ccgtctctcg 1740

cctctgcagt tatcgcctgc ctcatctctg cgtcctgata acttttgtca cctctgatcc 1800

ccccccgacg agacggtctc gccgaccagg acccgagatg gcggaccgcc atcctactct 1860

gtcccagtcc ttcgccgagc gggcgaagac ggctagccac ccgctcaccc gctacctctt 1920

tcggctcatg gacctcaagg cctcgaacct gtgcctgagc gccgacgtgt ccaccgcgcg 1980

cgagcttctg acgctggccg accgggtcgg cccctcgatc gtcgtgctca agacgcacta 2040

cgacctgatc tcgggctggg actacaaccc gcaaaccggc accggcgcga agctggccgc 2100

cctggcgagg aagcatggct tcctcatctt tgaggaccgc aagtttgtcg acattggtaa 2160

gacggtgcag atgcagtaca cggctggcac tgcgcgcata atagagtggg cgcacatcac 2220

caacgccaac atcgacgccg gcaaggacat ggtgcgcgcc atggccgagg cggccgccaa 2280

gtggaaggaa cgcatcaact acgaggtcaa gacctccgtc acggtgggca cgcccgtctc 2340

ggaccagttc gacgatgcgg aagagcaagc gcagtggccg cagcaccagc agcaccagca 2400

ccagcaccag caccagcaac agcgagatga aaaaggtggg ccccgcaggc tcggcactcg 2460

ggagaggagcag caccaacagg acaacggaga cggtgacggc cggaaaggga gcattgtctc 2520

gatcactacg gtgacgcagt catttgagcc cgctcactcc ccacgcctgt ccaagagcaa 2580

cgagctgggc gacgacgccg tcttccccgg catcgaggag gcccccgtcg accgcggcct 2640

gcttctgctc gccgatgt cgtccaaggg ctgcctcatg accaaggagt acacccaggc 2700

ctgcgtcgag gccgcgcgcg agcataagga ttttgtcatg ggcttcgtct cacaggagtc 2760

gctcaactcg gccccggacg acactttcat ccacatgacc cccggatgca agcttccgcc 2820

gccaggcgag gacgaagaga gcggccagat cgagtttaaa cggcgcgccg ctgtttcctg 2880

tgtgaaattg ttatccgctc acaattccac acaacatagg agccggaagc ataaagtgta 2940

aagcctgggg tgcctaatga gtgaggtaac tcacattaat tgcgttgcgc tcactgcccg 3000

ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 3060

gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 3120

tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 3180

aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 3240

gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 3300

aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 3360

ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 3420

tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 3480

tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 3540

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 3600

tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 3660

ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 3720

tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 3780

aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 3840

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 3900

aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 3960

ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 4020

acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 4080

ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 4140

gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 4200

taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 4260

tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 4320

gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 4380

cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 4440

aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 4500

cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 4560

tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 4620

gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 4680

tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 4740

gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 4800

ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 4860

cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 4920

agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 4980

gggttccgcg cacatttccc cgaaaagtgc cacctgaacg aagcatctgt gcttcatttt 5040

gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt 5100

tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca 5160

tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct 5220

gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa agaatctata 5280

cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac aaagcatctt 5340

agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa ctttttgcac 5400

tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct tccataaaaa 5460

aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt gcattttttc 5520

aagataaagg catccccgat tatattctat accgatgtgg attgcgcata ctttgtgaac 5580

agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg tttcttctat 5640

tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt tcgattcact 5700

ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag ataaacataa 5760

aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat gggtaggtta 5820

tatagggata tagcacagag atatatagca aagagatact tttgagcaat gtttgtggaa 5880

gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa 5940

gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat actttctaga 6000

gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat 6060

gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc tgcgtgttgc 6120

ctgtatatat atatacatga gaagaacggc atagtgcgtg tttatgctta aatgcgtact 6180

tatatgcgtc tatttatgta ggatgaaagg tagtctagta cctcctgtga tattatccca 6240

ttccatgcgg ggtatcgtat gcttccttca gcactaccct ttagctgttc tatatgctgc 6300

cactcctcaa ttggattagt ctcatccttc aatgctatca tttcctttga tattggatca 6360

tactaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 6420

tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 6480

acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 6540

gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg 6600

agagtgcacc ataccacagc ttttcaattc aattcatcat ttttttttta ttcttttttt 6660

tgatttcggt ttctttgaaa tttttttgat tcggtaatct ccgaacagaa ggaagaacga 6720

aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga agaaacatga 6780

aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa acgaagataa 6840

atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc 6900

aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt ggatgttcgt 6960

accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg tttactaaaa 7020

acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc gctaaaggca 7080

ttatccgcca agtacaattt tttactcttc gaagacagaa aatttgctga cattggtaat 7140

acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc agacattacg 7200

aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc ggcagaagaa 7260

gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa gggctcccta 7320

tctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa agattttgtt 7380

atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga ttggttgatt 7440

atgacacccg gtgtgggttt agatgacaag ggagacgcat tgggtcaaca gtatagaacc 7500

gtggatgatg tggtctctac aggatctgac attattattg ttggaagagg actatttgca 7560

aagggaaggg atgctaaggt agaggtgaa cgttacagaa aagcaggctg ggaagcatat 7620

ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc atgtatacta 7680

aactcacaaa ttagagcttc aatttaatta tatcagttat taccctatgc ggtgtgaaat 7740

accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt taatattttg 7800

ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 7860

ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 7920

tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 7980

tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 8040

tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 8100

aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 8160

ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 8220

ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg 8280

gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta 8340

agttgggtaa cgccagggtt ttcccagtca cgacg 8375

<210> 24

<211> 8196

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 24

ggcgcgccgt ttaaacgaac ctgtgcctga gcgccgacgt gtccaccgcg cgcgagcttc 60

tgacgctggc cgaccgggtc ggcccctcga tcgtcgtgct caagacgcac tacgacctga 120

tctcgggctg ggactacaac ccgcaaaccg gcaccggcgc gaagctggcc gccctggcga 180

ggaagcatgg cttcctcatc tttgaggacc gcaagtttgt cgacattggt aagacggtgc 240

agatgcagta cacggctggc actgcgcgca taatagagtg ggcgcacatc accaacgcca 300

acatcgacgc cggcaaggac atggtgcgcg ccatggccga ggcggccgcc aagtggaagg 360

aacgcatcaa ctacgaggtc aagacctccg tcacggtggg cacgcccgtc tcggaccagt 420

tcgacgatgc ggaagagcaa gcgcagtggc cgcagcacca gcagcaccag caccagcacc 480

agcaccagca acagcgagat gaaaaaggtg ggccccgcag gctcggcact cgggaggagc 540

agcaccaaca ggacaacgga gacggtgacg gccggaaagg gagcattgtc tcgatcacta 600

cggtgacgca gtcatttgag cccgctcact ccccacgcct gtccaagagc aacgagctgg 660

gcgacgacgc cgtcttcccc ggcatcgagg aggcccccgt cgaccgcggc ctgcttctgc 720

tcgcccagat gtcgtccaag ggctgcctca tgaccaagga gtacacccag gcctgcgtcg 780

aggccgcgcg cgagcataag gattttgtca tgggcttcgt ctcacaggag tcgctcaact 840

cggccccgga cgacactttc atccacatga cccccggatg caagcttccg ccgccaggcg 900

aggacgaaga gagcggccag atcgagggcg acggcctcgg ccagcagtac aactcgccca 960

gcaagttgat caacatttgc ggcaccgaca ttgtcatcgt agggcgtggc atcaccgccg 1020

ccggcgaccc gccctccgag gctgagaggt acaggagaaa agcctggaag gcctatctgg 1080

cgcgtctggc gtgatttggg gggaggggga gaggagatgg gggacgggag gggtcgcctt 1140

ggtcagtctt gtgcgtgtcc tgcagcggat tcgtcaccgg ggcagcaccc aaaagaggga 1200

gaaaaagggg aaaaaaaata aataaataaa aagggttaag ttgttgaaaa aagtgttgtg 1260

agctctctgg caaggcgcgc cccttttgga aagagcaccg cttctttttc gttcctcggc 1320

ctgagccgtc gaatgcgaac gtcaaaaggc gaactagaaa ttctgaaaca tagtacggat 1380

tactccgtac ccggttgttt tgcaccggga ttttgcttca atcgccaccg agttccaccc 1440

actttcgcca aggtacggat tacagtaatc cgtacatacc tacggacgta ctccgtcgtg 1500

tatctaggtg ttcccccttg gcacgctttc cacctgcgac aacgcggcct cagatcccga 1560

cctcgaaccc cccccccccc ccccccaaac aacaacccag ctcttcggct gtgcgcccgc 1620

caactcgaca aacaacaaca tccaacaagt gcgaatttga attcgactcg acagccatc 1680

gattcgtctc tcttcatgcg catcaatccg atccggaacc gccgacttta acaacacccg 1740

tgccgggctc gaccacgggg ctcccgtagt ccgccaaata catcgggtct gggatgtctt 1800

ttttttattt tattttttta tttttgtcgc ggtgtgagtg tgtttgtcgg gtccggttcg 1860

gcagttcatg atcattcctc tataaataag gtatggatcg tatatattat atattacata 1920

cagttgaagc cttagcacag tatgaatctc catataaatc tcttttttct tttcttttct 1980

tctttttttt ttttttttgc accccaccca cgtgctttcc ttatattcat catgcccttc 2040

atggctaggt gagttgatac caggactacg agatgtatat atatctcttg aacgattctc 2100

ctagagtttg tttagacgtg cactgtcctc tgataataat aaatcagctg ctgcattcat 2160

ccacgtgcga aaccagcttt gttaggttcg aatgtagacc gttttggtat ttcaaacggc 2220

agccattgcc tccgccttta gaatctgtcc aagctattgt tcagcaacta atgtcaaaaa 2280

aaaaaaaaaa aaaacgccta agcccccaac gtccggatag ataagaatac agcagggtga 2340

cgggttgggg ggacggggag gttgtcttcc gctgagcatg ccaccacatc acatgaatgc 2400

tttttcttcg ctgcctggac ctgaaccacc cccggagggg ctttcctccc cccgcttgac 2460

tactgcgctg acctccagac ctcggacgga tcctcaatgg cggctaacca ggggtaagtt 2520

cccatcaggc taccaccacc accagaaggg ccggaactcg cgctccccgc gtccgaaact 2580

tcgccgtctc tctcggtctc ggcctcggtc tcggtctcgg cagaagcacc gtggccgccc 2640

ccaatcacca tccacccgtc cctcgtctcg cgaggatcgg ccgtttaaac gctgtttcct 2700

gtgtgaaatt gttatccgct cacaattcca cacaacatag gagccggaag cataaagtgt 2760

aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa ttgcgttgcg ctcactgccc 2820

gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 2880

agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 2940

gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 3000

gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 3060

cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 3120

aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 3180

tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 3240

ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 3300

ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 3360

cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 3420

ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 3480

gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 3540

atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 3600

aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 3660

aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 3720

gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 3780

cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 3840

gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 3900

tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 3960

ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 4020

ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 4080

atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 4140

cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 4200

tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 4260

aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 4320

tcactcatgg ttatggcagc actngcataat tctcttactg tcatgccatc cgtaagatgc 4380

ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 4440

agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 4500

gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 4560

agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 4620

accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 4680

gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 4740

cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 4800

ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt 4860

tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat 4920

ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc 4980

atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc 5040

tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat 5100

acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct 5160

tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca 5220

ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa 5280

aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt 5340

caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa 5400

cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta 5460

ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac 5520

tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata 5580

aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt 5640

atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga 5700

agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa 5760

agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag 5820

agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa 5880

tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg 5940

cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac 6000

ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc 6060

attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg 6120

ccactcctca attggattag tctcatcctt caatgctatc atttcctttg atattggatc 6180

atactaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 6240

ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 6300

gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 6360

agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact 6420

gagagtgcac cataccacag cttttcaatt caattcatca tttttttttt attctttttt 6480

ttgatttcgg tttctttgaa atttttttga ttcggtaatc tccgaacaga aggaagaacg 6540

aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg aagaaacatg 6600

aaattgccca gtattcttaa cccaactgca cagaacaaaa acctgcagga aacgaagata 6660

aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc ctgttgctgc 6720

caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg 6780

taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa 6840

aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc cgctaaaggc 6900

attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa 6960

tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac 7020

gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcagaaga 7080

agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct 7140

atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt 7200

tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat 7260

tatgacaccc ggtgtgggtt tagatgacaa ggggacgca ttgggtcaac agtatagaac 7320

cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag gactatttgc 7380

aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata 7440

tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact 7500

aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg cggtgtgaaa 7560

taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaaacg ttaatatttt 7620

gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 7680

cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt 7740

ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt 7800

ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag 7860

gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg 7920

aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc 7980

gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc 8040

gctacagggc gcgtcgcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 8100

ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 8160

aagttgggta acgccagggt tttcccagtc acgacg 8196

<210> 25

<211> 20

<212> DNA

<213> Primer

<400> 25

cctgcattgc aagttcccac 20

<210> 26

<211> 20

<212> DNA

<213> Primer

<400> 26

agtttgacag tgccccagagc 20

<210> 27

<211> 20

<212> DNA

<213> Primer

<400> 27

agcctggaag gcctatctgg 20

<210> 28

<211> 20

<212> DNA

<213> Primer

<400> 28

ggtcggattg gcttggtaca 20

<210> 29

<211> 20

<212> DNA

<213> Primer

<400> 29

accaccgtca acacgtacaa 20

<210> 30

<211> 20

<212> DNA

<213> Primer

<400> 30

caaaggtctt gccaccgatg 20

<210> 31

<211> 20

<212> DNA

<213> Primer

<400> 31

ttcgttgcta acactccccc 20

<210> 32

<211> 20

<212> DNA

<213> Primer

<400> 32

ctggttgatg gccgagttga 20

<210> 33

<211> 20

<212> DNA

<213> Primer

<400> 33

ggcagattat tccggaccgt 20

<210> 34

<211> 20

<212> DNA

<213> Primer

<400> 34

agtttgacag tgccccagagc 20

<210> 35

<211> 20

<212> DNA

<213> Primer

<400> 35

agcctggaag gcctatctgg 20

<210> 36

<211> 20

<212> DNA

<213> Primer

<400> 36

tcaacgtgtg ggagcagtac 20

<210> 37

<211> 20

<212> DNA

<213> Primer

<400> 37

gggctccatc tacgtcttcg 20

<210> 38

<211> 20

<212> DNA

<213> Primer

<400> 38

tggatccagg gcgagtagaa 20

<210> 39

<211> 20

<212> DNA

<213> Primer

<400> 39

tgggctcgta cgacttcaac 20

<210> 40

<211> 20

<212> DNA

<213> Primer

<400> 40

cggcgatgtt ggagtcgtat 20

<210> 41

<211> 20

<212> DNA

<213> Primer

<400> 41

cgagaccgac aagaccaaca 20

<210> 42

<211> 20

<212> DNA

<213> Primer

<400> 42

gaagagcacg atgagcacga 20

<210> 43

<211> 20

<212> DNA

<213> Primer

<400> 43

ttggtaagac ggtgcagatg 20

<210> 44

<211> 21

<212> DNA

<213> Primer

<400> 44

gtagttgatg cgttccttcc a 21

<210> 45

<211> 221

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acids

<400> 45

Met Tyr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Gly Ala Ala

1 5 10 15

Ala Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

20 25 30

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

35 40 45

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

50 55 60

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

65 70 75 80

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

85 90 95

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

100 105 110

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

115 120 125

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

130 135 140

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

145 150 155 160

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

165 170 175

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

180 185 190

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

195 200 205

Val Cys Gly Pro Gly Gly Gly Gly Ser Glu Pro Glu Ala

210 215 220

<210> 46

<211> 666

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 46

atgtacgcca agttcgcgac cctcgccgcc cttgtggctg gcgccgctgc taccaacctc 60

tgcccgttcg gcgaggtctt caacgccacc cgcttcgcct ccgtctacgc ctggaaccgc 120

aagcgcatct ccaactgcgt cgccgactac agcgtcctgt acaacagcgc ctcgttctcc 180

accttcaagt gctacggcgt cagccccacc aagctcaacg acctgtgctt caccaacgtc 240

tacgccgact ccttcgtcat ccgcggcgac gaggtccgcc agatcgcccc cggccagacc 300

ggcaagatcg ccgactacaa ctacaagctc cccgacgact tcaccggctg cgtcatcgcc 360

tggaacagca acaacctgga ctcgaaggtc ggcggcaact acaactacct ctaccgcctg 420

ttccgcaagt cgaacctcaa gccgttcgag cgcgacatct cgaccgagat ctaccaggcc 480

ggctccaccc cctgcaacgg cgtcgagggc ttcaactgct acttccccct ccagtcctac 540

ggcttccagc ccaccaacgg cgtcggctac cagccctacc gcgtcgtcgt cctctccttc 600

gagctcctgc acgcccccgc caccgtctgc ggccctggcg gcggcggcag cgagccggag 660

gcctaa 666

<210> 47

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acids

<400> 47

Met Tyr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Gly Ala Ala

1 5 10 15

Ala Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

20 25 30

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

35 40 45

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

50 55 60

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

65 70 75 80

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

85 90 95

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

100 105 110

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

115 120 125

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

130 135 140

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

145 150 155 160

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

165 170 175

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

180 185 190

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

195 200 205

Val Cys Gly Pro Gly Gly Gly Gly Ser Ala His Ile Val Met Val Asp

210 215 220

Ala Tyr Lys Pro Thr Lys Gly Gly Gly Gly Ser Glu Pro Glu Ala

225 230 235

<210> 48

<211> 720

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 48

atgtacgcca agttcgcgac cctcgccgcc cttgtggctg gcgccgctgc taccaacctc 60

tgcccgttcg gcgaggtctt caacgccacc cgcttcgcct ccgtctacgc ctggaaccgc 120

aagcgcatct ccaactgcgt cgccgactac agcgtcctgt acaacagcgc ctcgttctcc 180

accttcaagt gctacggcgt cagccccacc aagctcaacg acctgtgctt caccaacgtc 240

tacgccgact ccttcgtcat ccgcggcgac gaggtccgcc agatcgcccc cggccagacc 300

ggcaagatcg ccgactacaa ctacaagctc cccgacgact tcaccggctg cgtcatcgcc 360

tggaacagca acaacctgga ctcgaaggtc ggcggcaact acaactacct ctaccgcctg 420

ttccgcaagt ccaacctcaa gccgttcgag cgcgacatct cgaccgagat ctaccaggcc 480

ggctccaccc cctgcaacgg cgtcgagggc ttcaactgct acttccccct ccagagctac 540

ggcttccagc ccaccaacgg cgtcggctac cagccctacc gcgtcgtcgt cctctccttc 600

gagctcctgc acgccccggc caccgtctgc ggccctggcg gcggcggcag cgcccacatc 660

gtcatggtcg acgcctacaa gccgaccaag ggcggcggcg gctcggagcc cgaggcctaa 720

<210> 49

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acids

<400> 49

Met Tyr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Gly Ala Ala

1 5 10 15

Ala Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

20 25 30

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

35 40 45

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

50 55 60

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

65 70 75 80

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

85 90 95

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

100 105 110

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

115 120 125

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

130 135 140

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

145 150 155 160

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

165 170 175

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

180 185 190

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

195 200 205

Val Cys Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 50

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 50

atgtacgcca agttcgcgac cctcgccgcc cttgtggctg gcgccgctgc taccaacctc 60

tgcccgttcg gcgaggtctt caacgccacc cgcttcgcct ccgtctacgc ctggaaccgc 120

aagcgcatct ccaactgcgt cgccgactac agcgtcctgt acaacagcgc ctcgttctcc 180

accttcaagt gctacggcgt cagccccacc aagctcaacg acctgtgctt caccaacgtc 240

tacgccgact ccttcgtcat ccgcggcgac gaggtccgcc agatcgcccc cggccagacc 300

ggcaagatcg ccgactacaa ctacaagctc cccgacgact tcaccggctg cgtcatcgcc 360

tggaacagca acaacctgga ctcgaaggtc ggcggcaact acaactacct ctaccgcctg 420

ttccgcaagt cgaacctcaa gccgttcgag cgcgacatct cgaccgagat ctaccaggcc 480

ggctccaccc cctgcaacgg cgtcgagggc ttcaactgct acttccccct ccagtcctac 540

ggcttccagc ccaccaacgg cgtcggctac cagccctacc gcgtcgtcgt cctctccttc 600

gagctcctgc acgcccccgc caccgtctgc ggccctggcg gcggcggcag cggcggcggc 660

ggcagcgaca agacccacac ctgcccgccc tgccccgccc cggaggccgc tggcggcccc 720

agcgtcttcc tcttcccgcc caagccgaag gacaccctga tgatctcgcg caccccggag 780

gtcacctgcg tcgtcgtcga cgtcagccac gaggacccgg aggtcaagtt caactggtac 840

gtcgacggcg tcgaggtcca caacgccaag accaagccgc gcgaggagca gtacaactcg 900

acctaccgcg tcgtctccgt cctcaccgtc ctgcaccagg actggctcaa cggcaaggag 960

tacaagtgca aggtctcgaa caaggccctg cccgccccga tcgagaagac catctcgaag 1020

gccaagggcc agccccgcga gccccaggtc tacaccctcc cgcccagccg cgacgagctg 1080

accaagaacc aggtctcgct cacctgcctg gtcaagggct tctacccctc cgacatcgcc 1140

gtcgagtggg agagcaacgg ccagccggag aacaactaca agaccacccc gcccgtcctg 1200

gactccgacg gctccttctt cctctacagc aagctgaccg tcgacaagtc gcgctggcag 1260

cagggcaacg tcttcagctg ctcggtcatg cacgaggccc tgcacaacca ctacacccag 1320

aagtccctca gcctgtcgcc cggctaa 1347

<210> 51

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acids

<400> 51

Met Tyr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Gly Ala Ala

1 5 10 15

Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

20 25 30

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

35 40 45

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

50 55 60

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

65 70 75 80

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

85 90 95

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

100 105 110

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

115 120 125

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

130 135 140

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

145 150 155 160

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

165 170 175

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

180 185 190

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

195 200 205

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

210 215 220

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

225 230 235 240

Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asn Leu

245 250 255

Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr

260 265 270

Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val

275 280 285

Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser

290 295 300

Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser

305 310 315 320

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

325 330 335

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly

340 345 350

Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly

355 360 365

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

370 375 380

Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro

385 390 395 400

Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr

405 410 415

Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val

420 425 430

Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro

435 440 445

<210> 52

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 52

atgtacgcca agttcgcgac cctcgccgcc cttgtggctg gcgccgctgc tgacaagacc 60

cacacctgcc cgccctgccc cgccccggag gccgctggcg gccccagcgt cttcctcttc 120

ccgcccaagc cgaaggacac cctgatgatc tcgcgcaccc cggaggtcac ctgcgtcgtc 180

gtcgacgtca gccacgagga cccggaggtc aagttcaact ggtacgtcga cggcgtcgag 240

gtccacaacg ccaagaccaa gccgcgcgag gagcagtaca actcgaccta ccgcgtcgtc 300

tccgtcctca ccgtcctgca ccaggactgg ctcaacggca aggagtacaa gtgcaaggtc 360

tcgaacaagg ccctgcccgc cccgatcgag aagaccatct cgaaggccaa gggccagccc 420

cgcgagcccc aggtctacac cctcccgccc agccgcgacg agctgaccaa gaaccaggtc 480

tcgctcacct gcctggtcaa gggcttctac ccctccgaca tcgccgtcga gtggggagagc 540

aacggccagc cggagaacaa ctacaagacc accccgcccg tcctggactc cgacggctcc 600

ttcttcctct acagcaagct gaccgtcgac aagtcgcgct ggcagcaggg caacgtcttc 660

agctgctcgg tcatgcacga ggccctgcac aaccactaca cccagaagtc cctcagcctg 720

tcgcccggcg gcggcggcgg cagcggcggc ggcggcagca ccaacctctg cccgttcggc 780

gaggtcttca acgccacccg cttcgcctcc gtctacgcct ggaaccgcaa gcgcatctcc 840

aactgcgtcg ccgactacag cgtcctgtac aacagcgcct cgttctccac cttcaagtgc 900

tacggcgtca gccccaccaa gctcaacgac ctgtgcttca ccaacgtcta cgccgactcc 960

ttcgtcatcc gcggcgacga ggtccgccag atcgcccccg gccagaccgg caagatcgcc 1020

gactacaact acaagctccc cgacgacttc accggctgcg tcatcgcctg gaacagcaac 1080

aacctggact cgaaggtcgg cggcaactac aactacctct accgcctgtt ccgcaagtcg 1140

aacctcaagc cgttcgagcg cgacatctcg accgagatct accaggccgg ctccaccccc 1200

tgcaacggcg tcgagggctt caactgctac ttccccctcc agtcctacgg cttccagccc 1260

accaacggcg tcggctacca gccctaccgc gtcgtcgtcc tctccttcga gctcctgcac 1320

gcccccgcca ccgtctgcgg cccttaa 1347

<210> 53

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acids

<400> 53

Met Tyr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Gly Ala Ala

1 5 10 15

Ala Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

20 25 30

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

35 40 45

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

50 55 60

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

65 70 75 80

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

85 90 95

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

100 105 110

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

115 120 125

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

130 135 140

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

145 150 155 160

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

165 170 175

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro

180 185 190

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

195 200 205

Val Cys Gly Pro Gly Ser Gly Glu Pro Glu Ala

210 215

<210> 54

<211> 660

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 54

atgtacgcca agttcgcgac cctcgccgcc cttgtggctg gcgccgctgc taccaacctc 60

tgcccgttcg gcgaggtctt caacgccacc cgcttcgcct ccgtctacgc ctggaaccgc 120

aagcgcatct ccaactgcgt cgccgactac agcgtcctgt acaacagcgc ctcgttctcc 180

accttcaagt gctacggcgt cagccccacc aagctcaacg acctgtgctt caccaacgtc 240

tacgccgact ccttcgtcat ccgcggcgac gaggtccgcc agatcgcccc cggccagacc 300

ggcaagatcg ccgactacaa ctacaagctc cccgacgact tcaccggctg cgtcatcgcc 360

tggaacagca acaacctgga ctcgaaggtc ggcggcaact acaactacct ctaccgcctg 420

ttccgcaagt cgaacctcaa gccgttcgag cgcgacatct cgaccgagat ctaccaggcc 480

ggctccaccc cctgcaacgg cgtcgagggc ttcaactgct acttccccct ccagtcctac 540

ggcttccagc ccacctacgg cgtcggctac cagccctacc gcgtcgtcgt cctctccttc 600

gagctcctgc acgcccccgc caccgtctgc ggccctggca gcggcgagcc ggaggcctaa 660

<210> 55

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acids

<400> 55

Met Tyr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Gly Ala Ala

1 5 10 15

Ala Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

20 25 30

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

35 40 45

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

50 55 60

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

65 70 75 80

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

85 90 95

Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

100 105 110

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

115 120 125

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

130 135 140

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

145 150 155 160

Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro

165 170 175

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro

180 185 190

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

195 200 205

Val Cys Gly Pro Gly Ser Gly Glu Pro Glu Ala

210 215

<210> 56

<211> 660

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 56

atgtacgcca agttcgcgac cctcgccgcc cttgtggctg gcgccgctgc taccaacctc 60

tgcccgttcg gcgaggtctt caacgccacc cgcttcgcct ccgtctacgc ctggaaccgc 120

aagcgcatct ccaactgcgt cgccgactac agcgtcctgt acaacagcgc ctcgttctcc 180

accttcaagt gctacggcgt cagccccacc aagctcaacg acctgtgctt caccaacgtc 240

tacgccgact ccttcgtcat ccgcggcgac gaggtccgcc agatcgcccc cggccagacc 300

ggcaacatcg ccgactacaa ctacaagctc cccgacgact tcaccggctg cgtcatcgcc 360

tggaacagca acaacctgga ctcgaaggtc ggcggcaact acaactacct ctaccgcctg 420

ttccgcaagt cgaacctcaa gccgttcgag cgcgacatct cgaccgagat ctaccaggcc 480

ggctccaccc cctgcaacgg cgtcaagggc ttcaactgct acttccccct ccagtcctac 540

ggcttccagc ccacctacgg cgtcggctac cagccctacc gcgtcgtcgt cctctccttc 600

gagctcctgc acgcccccgc caccgtctgc ggccctggca gcggcgagcc ggaggcctaa 660

<210> 57

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acids

<400> 57

Met Tyr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Gly Ala Ala

1 5 10 15

Ala Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

20 25 30

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

35 40 45

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

50 55 60

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

65 70 75 80

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

85 90 95

Pro Gly Gln Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

100 105 110

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

115 120 125

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

130 135 140

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

145 150 155 160

Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro

165 170 175

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro

180 185 190

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

195 200 205

Val Cys Gly Pro Gly Ser Gly Glu Pro Glu Ala

210 215

<210> 58

<211> 660

<212> DNA

<213> Artificial sequence

<220>

<223> DNA Sequence

<400> 58

atgtacgcca agttcgcgac cctcgccgcc cttgtggctg gcgccgctgc taccaacctc 60

tgcccgttcg gcgaggtctt caacgccacc cgcttcgcct ccgtctacgc ctggaaccgc 120

aagcgcatct ccaactgcgt cgccgactac agcgtcctgt acaacagcgc ctcgttctcc 180

accttcaagt gctacggcgt cagccccacc aagctcaacg acctgtgctt caccaacgtc 240

tacgccgact ccttcgtcat ccgcggcgac gaggtccgcc agatcgcccc cggccagacc 300

ggcaccatcg ccgactacaa ctacaagctc cccgacgact tcaccggctg cgtcatcgcc 360

tggaacagca acaacctgga ctcgaaggtc ggcggcaact acaactacct ctaccgcctg 420

ttccgcaagt cgaacctcaa gccgttcgag cgcgacatct cgaccgagat ctaccaggcc 480

ggctccaccc cctgcaacgg cgtcaagggc ttcaactgct acttccccct ccagtcctac 540

ggcttccagc ccacctacgg cgtcggctac cagccctacc gcgtcgtcgt cctctccttc 600

gagctcctgc acgcccccgc caccgtctgc ggccctggca gcggcgagcc ggaggcctaa 660

<---

Claims (42)

1. A genetically modified ascomycete filamentous fungus for producing an exogenous protein of interest, wherein said genetically modified filamentous fungus comprises at least one cell having reduced expression and/or activity of KEX2 protease, as well as reduced expression and/or activity of at least 12 additional proteases, wherein at least one cell comprises an exogenous polynucleotide encoding the protein of interest. 2. A genetically modified ascomycete mycelial fungus according to claim 1, having reduced expression and/or activity of KEX2 and ALP7. 3. A genetically modified ascomycete filamentous fungus according to any one of claims 1 or 2, characterized in that KEX2 comprises an amino acid sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or 100% identity to the amino acid sequence of KEX2 of Thermothelomyces heterothallica . 4. A genetically modified ascomycete mycelial fungus according to claim 3, characterized in that KEX2 Thermothelomyces heterothallica contains the amino acids of SEQ ID NO: 14. 5. A genetically modified ascomycete filamentous fungus according to any one of claims 2 to 4, characterized in that ALP7 comprises an amino acid sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or 100% identity to the amino acid sequence of ALP7 of Thermothelomyces heterothallica . 6. A genetically modified ascomycete mycelial fungus according to claim 5, characterized in that ALP7 Thermothelomyces heterothallica contains the amino acids of SEQ ID NO: 13. 7. A genetically modified ascomycete mycelial fungus according to claim 1, having reduced expression and/or activity of a protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6 and ALP4. 8. A genetically modified ascomycete mycelial fungus according to claim 7, characterized in that said ascomycete has reduced expression and/or activity of a plurality of proteases selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4. 9. A genetically modified ascomycete filamentous fungus according to any one of claims 1-8, having reduced expression and/or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4 and KEX2. 10. A genetically modified ascomycete filamentous fungus according to any one of claims 1 to 9, having reduced expression and/or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, ALP7 and KEX2. 11. A genetically modified ascomycete filamentous fungus according to any one of claims 1-10, having reduced expression and/or activity of at least one additional protease selected from the group consisting of ALP5, ALP6, SRP3, SRP5 and SRP8. 12. A genetically modified ascomycetous mycelial fungus according to any one of claims 1-11, characterized in that said genetically modified ascomycetous mycelial fungus produces a protein of interest in an increased amount compared to the amount produced by its non-genetically modified original strain of said ascomycete, cultivated under similar conditions. 13. A genetically modified ascomycetous mycelial fungus according to any one of claims 1-12, characterized in that the protein produced by said genetically modified ascomycetous mycelial fungus has increased stability compared to said protein produced by a non-genetically modified original strain of said ascomycetous mycelial fungus, cultivated under similar conditions. 14. A genetically modified ascomycetous mycelial fungus according to any one of claims 1-13, characterized in that said ascomycetous mycelial fungus belongs to a genus belonging to Pezizomycotina . 15. A genetically modified ascomycetous mycelial fungus according to claim 14, characterized in that said ascomycetous mycelial fungus belongs to a genus selected from the group consisting of Thermothelomyces, Myceliophthora, Trichoderma, Aspergillus, Penicillium, Rasamsonia, Chrysosporium, Corynascus, Fusarium, Neurospora and Talaromyces . 16. The genetically modified ascomycetous mycelial fungus of claim 15, wherein said ascomycetous mycelial fungus is a species selected from the group consisting of Thermothelomyces heterothallica, Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma viride, Rasamsonia emersonii, Penicillium chrysogenum, Penicillium verrucosum, Sporotrichum thermophile, Corynascus fumimontanus, Corynascus thermophilus, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venenatum, Neurospora crassa and Talaromyces piniphilus . 17. A genetically modified ascomycetous filamentous fungus according to claim 16, characterized in that said ascomycetous filamentous fungus is a strain of Thermothelomyces heterothallica containing an rDNA sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identity to the nucleic acid sequence set forth in SEQ ID NO: 20. 18. A genetically modified ascomycetous mycelial fungus according to claim 17, characterized in that said ascomycetous mycelial fungus is Thermothelomyces heterothallica C1. 19. A genetically modified ascomycetous filamentous fungus according to any one of claims 1-18, characterized in that at least one exogenous polynucleotide is a DNA construct or expression vector, additionally containing at least one regulatory element functional in said ascomycetous filamentous fungus. 20. A genetically modified ascomycete mycelial fungus according to any one of claims 1-19, characterized in that the protein of interest is selected from the group consisting of an antigen, an antibody, an enzyme, a vaccine, and a structural protein. 21. A genetically modified ascomycete mycelial fungus according to claim 20, characterized in that the protein of interest is an antibody. 22. A genetically modified ascomycete mycelial fungus according to any one of claims 1-20, characterized in that the protein of interest is fused to a label. 23. A genetically modified ascomycete mycelial fungus according to claim 22, characterized in that the label is selected from the group consisting of Spytag, HA-label, chitin-binding protein (CBP), maltose-binding protein (MBP), Strep-tag, glutathione-8-transferase (GST), FLAG-label, C-label, ALFA-label, V5-label, Myc-label, Spot-label, T7-label, NE-label, and polyhistidine (poly(His))-label. 24. A genetically modified ascomycete mycelial fungus according to claim 20, characterized in that the protein of interest is a viral component. 25. A genetically modified ascomycete mycelial fungus according to claim 24, characterized in that the viral component is the receptor-binding domain (RBD) of the SARS-CoV2 spike domain or a fragment thereof. 26. A genetically modified ascomycete filamentous fungus according to any one of claims 22-25, characterized in that the protein of interest comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55 and SEQ ID NO: 57. 27. A genetically modified ascomycete mycelial fungus according to claim 24, characterized in that the viral component is an antigenic protein of the Rift Valley fever virus (RVFV). 28. A genetically modified ascomycete mycelial fungus according to claim 24, characterized in that the viral component is an influenza virus protein. 29. A genetically modified ascomycete mycelial fungus according to claim 20, characterized in that the protein of interest is fibrinogen. 30. A method for producing a genetically modified ascomycete filamentous fungus capable of producing a protein of interest, comprising transforming at least one fungal cell with at least one exogenous polynucleotide encoding the protein of interest; wherein said at least one fungal cell has reduced expression and/or protease activity of KEX2 protease and at least 12 additional proteases. 31. The method of claim 30, further comprising engineering said fungus with inhibited expression and/or protease activity of ALP7. 32. The method according to any one of claims 30, 31, characterized in that said genetically modified ascomycete mycelial fungus produces the protein of interest in an increased amount compared to the amount produced by the corresponding original non-transformed strain of the fungus, cultivated under similar conditions. 33. The method according to any one of paragraphs 30-32, characterized in that the said ascomycete mycelial fungus belongs to a genus belonging to Pezizomycotina . 34. The method according to claim 33, characterized in that said ascomycete mycelial fungus belongs to a genus selected from the group consisting of Thermothelomyces, Myceliophthora, Trichoderma, Aspergillus, Penicillium, Rasamsonia, Chrysosporium, Corynascus, Fusarium, Neurospora and Talaromyces . 35. The method according to No. 34, characterized in that the said ascomycete mycelial fungus belongs to a species selected from the group consisting of Thermothelomyces heterothallica, Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma viride, Rasamsonia emersonii, Penicillium chrysogenum, Penicillium verrucosum, Sporotrichum thermophile, Corynascus fumimontanus, Corynascus thermophilus, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venenatum, Neurospora crassa and Talaromyces piniphilus . 36. The method according to item 35, characterized in that said ascomycetous filamentous fungus is a strain of Thermothelomyces heterothallica containing an rDNA sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identity to the nucleic acid sequence presented in SEQ ID NO: 20. 37. The method according to claim 36, characterized in that said ascomycete mycelial fungus is Thermothelomyces heterothallica C1. 38. A method for producing at least one protein of interest, comprising culturing a genetically modified ascomycete filamentous fungus according to any one of claims 1-29 in a suitable medium and isolating the produced protein of interest. 39. The method according to claim 38, characterized in that the medium contains a carbon source selected from the group consisting of glucose, sucrose, xylose, arabinose, galactose, fructose, lactose, cellobiose, glycerol and any combination thereof. 40. The method according to any one of claims 30-39, characterized in that at least one protein of interest is a viral component. 41. The method according to claim 40, characterized in that the viral component relates to a coronavirus. 42. The method according to paragraph 41, characterized in that the coronavirus is SARS-CoV-2.