WO2017211803A1

WO2017211803A1 - Co-expression of heterologous polypeptides to increase yield

Info

Publication number: WO2017211803A1
Application number: PCT/EP2017/063681
Authority: WO
Inventors: Kaihei Kojima; Hiromi AKEBOSHI
Original assignee: Novozymes A/S
Priority date: 2016-06-07
Filing date: 2017-06-06
Publication date: 2017-12-14

Abstract

The present invention relates to a method of producing a poorly expressed first heterologous polypeptide in a host cell by co-expressing it with a second heterologous polypeptide that is well- expressed in said host cell, whereby the yield or productivity of the first heterologous polypeptide is improved.

Description

TITLE: CO-EXPRESSION OF HETEROLOGOUS POLYPEPTIDES TO INCREASE YIELD

Reference to sequence listing

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Improving the yield or productivity of heterologous polypeptides is important in the highly competitive business of commercial polypeptide production. It is well-known that, for example, enzymes are produced with different yields or productivities in a host organism and the gap between the best-expressed and worst-expressed enzymes can easily be more than 100-fold. Consequently, the development of methods for improving the yield or productivity of especially poorly expressed polypeptides is desirable.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to methods for producing a first heterologous polypeptide, said methods comprising the steps of:

a) expressing the first heterologous polypeptide and a second heterologous polypeptide in a host cell, where the second heterologous polypeptide is expressed with a higher yield or productivity than the first heterologous polypeptide when each polypeptide is expressed without the other in said host cell under comparable conditions, and wherein their co- expression improves the yield/productivity of the first heterologous polypeptide compared to when it is expressed without the second heterologous polypeptide in said host cell under comparable conditions; and, optionally,

b) recovering the first heterologous polypeptide.

In a second aspect, the invention also relates to host cells expressing a first heterologous polynucleotide encoding a first heterologous polypeptide and a second heterologous polynucleotide encoding a second heterologous polypeptide, where the second heterologous polypeptide is expressed with a higher yield or productivity than the first heterologous polypeptide when each heterologous polypeptide is expressed without the other in said host cell under comparable conditions, and wherein their co-expression improves the yield/productivity of the first heterologous polypeptide compared to when it is expressed without the second heterologous polypeptide in said host cell under comparable conditions.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the plasmid map for plasmid pKKa164.

DEFINITIONS

cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g. , recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).

Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.

Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

DETAILED DESCRIPTION OF THE INVENTION

Methods of Production

The present invention relates to methods for producing a first heterologous polypeptide, said methods comprising the steps of:

b) recovering the first heterologous polypeptide.

The host cells are cultivated in a nutrient medium suitable for production of the first heterologous polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the present invention expressing the polypeptide is used as a source of the polypeptide.

Heterologous Polypeptides

In a preferred embodiment of the invention, the first heterologous polypeptide is an enzyme and the second heterologous polypeptide is an enzyme. Preferably, either of the two heterologous enzymes is a hydrolase, isomerase, ligase, lyase, oxidoreductase, and/or a transferase; preferably either of the two heterologous enzymes is an alpha-galactosidase, alpha- glucosidase, aminopeptidase, amylase, arabinofuranosidase, beta-galactosidase, beta- glucanase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and/or a xylanase. Preferably, the first heterologous polypeptide is an arabinofuranosidase and the second heterologous polypeptide is a glucoamylase.

In another embodiment, the heterologous polypeptides may be variants of wildtype polypeptides comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et ai, 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos ef a/., 1992, Science 255: 306-312; Smith et ai, 1992, J. Mol. Biol. 224: 899- 904; Wlodaver ei a/., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide. Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; er et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie ef al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48. Sources of Polypeptides

A polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a bacterial polypeptide. For example, the polypeptide may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having [enzyme] activity, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.

In another aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide.

In another aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide.

The polypeptide may be a fungal polypeptide. For example, the polypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature {e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra). Polynucleotides

The present invention also relates to heterologous polynucleotides encoding the heterologous polypeptides of the invention, as described herein. The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.

Modification of a polynucleotide encoding a heterologous polypeptide of the present invention may be necessary. The heterologous polypeptides may differ in some engineered way from the polypeptide isolated from their native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variants may be constructed on the basis of the polynucleotide presented as the mature polypeptide coding sequence, a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107. Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a heterologous polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The heterologous polynucleotides may be manipulated in a variety of ways to provide for expression of the polypeptides. Manipulation of the heterolgous polynucleotides prior to their insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes lor Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase I I, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II , Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I , Trichoderma reesei xylanase II , Trichoderma reesei xylanase I II, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Patent No. 6,01 1 ,147.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

Preferred terminators for filamentous fungal host cells are obtained from the genes for

Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase I I, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II , Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I , Trichoderma reesei xylanase II , Trichoderma reesei xylanase I II, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor.

The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor. Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a heterologous polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl- aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991 , Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook ef al., 1989, supra).

Host Cells

The second aspect of the invention relates to host cells expressing a first heterologous polynucleotide encoding a first heterologous polypeptide and a second heterologous polynucleotide encoding a second heterologous polypeptide, where the second heterologous polypeptide is expressed with a higher yield or productivity than the first heterologous polypeptide when each heterologous polypeptide is expressed without the other in said host cell under comparable conditions, and wherein their co-expression improves the yield/productivity of the first heterologous polypeptide compared to when it is expressed without the second heterologous polypeptide in said host cell under comparable conditions.

The heterologous polypeptides are encoded by heterolgous polynucleotides operably linked to one or more control sequences that direct the production of the polypeptides. A construct or vector comprising a heterologous polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

The host cell may be a fungal cell. "Fungi" as used herein includes the phyla Ascomycota,

Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et ai, 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.

The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton ef al. , 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen ef al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier ef al., 1989, Gene 78: 147-156, and WO 96/00787. EXAMPLES

Materials and Methods

General molecular biological methods

General methods of PCR, cloning, ligation nucleotides etc. are well-known to a person skilled in the art and may for example be found in in "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.); "Current protocols in Molecular Biology", John Wiley and Sons, (1995); Harwood, C. R., and Cutting, S. M. (eds.); "DNA Cloning: A Practical Approach, Volumes I and II", D.N. Glover ed. (1985); ligonucleotide Synthesis", M.J. Gait ed. (1984); "Nucleic Acid Hybridization", B.D. Hames & S.J. Higgins eds (1985); "A Practical Guide To Molecular Cloning", B. Perbal, (1984).

Enzymes

Enzymes for DNA manipulations (e.g. restriction endonucleases, ligases etc.) are obtainable from New England Biolabs, Inc. and were used according to the manufacturer's instructions.

Media and reagents

Chemicals used for buffers and substrates were commercial products of analytical grade. Cove: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10mM Acetamide, 30 g/L noble agar.

Cove top agar: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10mM Acetamide, 10 g/L low melt agarose

Cove-2: 30 g/L Sucrose, 20 ml/L COVE salt solution, 10mM Acetamide, 30 g/L noble agar.

Cove-N(tf) plates are composed of 342.3 g of sucrose, 20 ml of Cove salt solution, 3g of NaN03, and 30 g of noble agar, water to 1 L.

Cove-N plates are composed of 30 g of sucrose, 20 ml of Cove salt solution, 3g of NaN03, and 30 g of noble agar, water to 1 L.

COVE salt solution is composed of 26 g KCI, 26 g MgS0₄-7H₂0, 76 g KH₂P0₄ and 50ml Cove trace metals, water to 1 L.

Trace metal solution for COVE is composed of 0.04 g NaB O₇-10H₂O, 0.4 g of

CuS0 -5H₂0, 1.2 g of FeS0₄-7H₂0, 1 .0 g of MnS0 -H20, 0.8 g of Neutral amylase II Mo0₂-2H₂0, and 10.0 g of ZnS0₄-7H20, water to 1 L.

Cove-N top agarose is composed of 342.3 g of Sucrose, 20 ml of COVE salt solution, 3g of NaN03, and 10 g of low melt agarose, water to 1 L.

Amyloglycosidase trace metal solution is composed of 6.8 g ZnCI₂-7H₂0, 2.5 g

CuS0₄-5H₂0, 0.24 g NiCI₂-6H₂0, 13.9 g FeS0 -7H₂0, 13.5 g MnS0₄-H₂0 and 3 g citric acid, water to 1 L. YPG is composed of 4 g of yeast extract, 1 g of KH2PO4, 0.5 g of MgS04-7H₂0 and 15 g of Glucose (pH 6.0) , water to 1 L.

STC buffer is composed of 0.8 M of sorbitol, 25 mM of Tris (pH 8), and 25 mM of CaCI₂, water to 1 L.

STPC buffer is composed of 40 % PEG4000 in STC buffer.

MLC is composed of 40 g Glucose, 50 g Soybean powder, 4 g/ Citric acid (pH 5.0), water to 1 L.

MSS is composed of 70 g Sucrose, 100 g Soybean powder (pH 6.0), water to 1 L.

MU-1 is composed 260 g of Maltodextrin, 3 g of MgS0 -7H₂0, 5 g of KH2PO4, 6 g of K2SO4, amyloglycosidase trace metal solution 0.5 ml and urea 2 g (pH 4.5), water to 1 L.

MU-1 glu is composed of 260 g of glucose, 3 g of MgSCv7H₂0, 5 g of KH₂P0₄, 6 g of K₂S0₄, amyloglycosidase trace metal solution 0.5 ml and urea 2 g (pH 4.5), water to 1 L; for expression of arabinofuranosidase, amount of urea added was 1.5 g.

Purchased material {E.coli, plasmid and kits)

E.coli DH5-alpha (Toyobo) is used for plasmid construction and amplification. The commercial plasmids/ vectors TOPO cloning kit (Invitrogen) and pBluescript II SK- (Stratagene #212206) are used for cloning of PCR fragments. Amplified plasmids are recovered with Qiagen^® Plasmid Kit (Qiagen). Ligation is done with DNA ligation kit (Roche). Polymerase Chain Reaction (PCR) is carried out with KOD plus (TOYOBO). QIAquick™ Gel Extraction Kit (Qiagen) is used for the purification of PCR fragments and extraction of DNA fragment from agarose gel. Integration of Arabinofuranosidase gene into plasmid is carried out with In-Fusion HD Cloning Kit (Clon tech).

Strains

Aspergillus niger strain NN059280 (C2218) is described in WO 2012/160093.

Aspergillus niger strain C3085 is described in "materials and methods" of WO 2015/144936. Plasmids

Construction of phahi025

The 1.0 kb region of a BG025 was amplified from the plasmid NC000148 by PCR with primer pairs:

HA098: agtcttgatcggatccaccatgaagctcggctctct (SEQ ID NO:1 ) and

HA099: tatcgtacgcaccacgtgtcaaagatacggagtctatcgtacgcaccacgtgtcaaagatacggagtc (SEQ ID NO:2) The obtained 1.0 kb DNA fragment was ligated by In-Fusion® HD Cloning Kit (Clontech Laboratories, Inc) into the pHUda1260 (described in WO 2015/144936) digested with BamHI and Pmll. Five μΙ of the ligation mixture were transformed into DH5-alpha chemically competent E. coli cells. Transformants were spread onto LB plus ampicillin plates and incubated at 37°C overnight. Plasmid DNA was purified from several transformants using a QIA mini-prep kit. The plasmid DNA was screened for proper ligation by use of proper restriction enzymes followed by 0.8% agarose gel electrophoresis using TAE buffer. One plasmid was designated as phahi025.

Construction of phahi026

The 1 .7 kb region of araf was amplified from the plasmid NC000990 by PCR with primer pairs HA100: agtcttgatcggatccaccatgctaggcttgaaggtcttg (SEQ ID NO:3) and

HA101 : tatgcgttatcgtacgcaccacgtgctagatcatgttcatc (SEQ ID NO:4).

The obtained 1.7 kb DNA fragment was ligated by In-Fusion® HD Cloning Kit (Clontech Laboratories, Inc) into the pHUda1260 digested with BamHI and Pmll. Five μΙ of the ligation mixture were transformed into DH5-alpha chemically competent E. coli cells. Transformants were spread onto LB plus ampicillin plates and incubated at 37°C overnight. Plasmid DNA was purified from several transformants using a QIA mini-prep kit. The plasmid DNA was screened for proper ligation by use of proper restriction enzymes followed by 0.8% agarose gel electrophoresis using TAE buffer. One plasmid was designated as phahi026.

Construction of phahi033

Plasmid phuda1719 and phahi026 were digested with Nhe\ and Pml\, and the 8,324 bp and 2,458 bp fragments, respectively, were purified by 0.8% agarose gel electrophoresis using TAE buffer, excised from the gel and extracted using a QIAQUICK^® Gel Extraction Kit. The 8,324 bp fragment was ligated to the 2,458 bp fragment containing A. niger na2 promoter and H. insolens GH43 arabinofuranosidase gene {araf} in a reaction composed of 2 μΙ of the 9,241 bp fragment, 6 μΙ of the 3, 1 14 bp fragment, 2 μΙ of 5X ligase Buffer, 10 μΙ of 2X Ligase Buffer and 1 μΙ of Ligase (Roche Rapid DNA Ligation Kit). The ligation reaction was incubated at room temperature for 10 minutes. Five μΙ of the ligation mixture were transformed into DH5-alpha chemically competent E. coli cells. Transformants were spread onto LB plus ampicillin plates and incubated at 37°C overnight. Plasmid DNA was purified from several transformants using a QIA mini-prep kit. The plasmid DNA was screened for proper ligation by use of proper restriction enzymes followed by 0.8% agarose gel electrophoresis using TAE buffer. One plasmid was designated as phahi033.

Construction of pKKa35

The plasmid pKKa35 with pyrG marker gene for integration of Gloeophyllum trabeum

AMG expression cassette into A. niger strain by using FLP integration manner was constructed by changing from asaA signal, synthetic AM782 and TMV3'UTR-AMG terminator to CDS of Gloeophyllum trabeum AMG gene in pRika147 (described in example 9 in WO2012160093).

Gloeophyllum trabeum AMG gene was isolated as cDNA from mRNA transcribed from total RAN extracted from an MBin1 18 strain expressing Gloeophyllum trabeum AMG. The 1.72 kb region of Gloeophyllum trabeum AMG gene was amplified from reversetranscribed cDNA by PCR with primer pairs:

KA 35-1 : cccggatccatgtaccgcttccttgtctg (SEQ ID NO:5) and

KA35-2: gggcacgtgttaacgccaagtgtcattc (SEQ ID NO:6).

The obtained fragment was digested by BamHI and Pmll, and ligated into the Aspergillus expression cassette pRika147 digested with BamHI and Pmll to create pKKa35.

Construction of pKKa150

The plasmid pKKa150 with pyrG marker gene for integration of arabinofuranosidase expression cassette into A niger strain by FLP integration manner was constructed by changing from Gloeophyllum sepiarium AMG gene to arabinofuranosidase gene in pHUda1732 harboring 5, and 3, flanking region of pepA locus. pHUda1732 was used for pepA gene deletion to generate pepA deleted mutants (described in WO 2015/025055). pHUda1732 was digested with Xbal for removal of 6.91 kbp fragment, and 3.31 kbp fragment containing arabinofuranosidase gene driven by NA2 promoter and AMG terminator obtained by digestion of phahi026 with Nhel and Spel was integrated into it.

The obtained intermediate plasmid harboring arabinofuranosidase was digested with Notl and Xbal, and the 2.14 kbp fragment of pyrG gene from pHUda 1732 digested with Notl and Xbal after amplification by PCR with primer pairs:

KA 150-1 : aaagcggccgcctagcaaagtattttcctag (SEQ ID NO:7) and

KA150-2: attatcatgacccagcccag (SEQ ID NO:8)

The resulting fragment was used to create pKKa150.

Construction of pKKa164 pKKa164 for integration of mock expression cassettes with hph marker gene into a host strain by using FLP integration manner was constructed. The plasmid map for pKKa164 is shown in Figure 1. Construction of pKKa199

pKKa199 for integration of mock expression cassettes with amdS marker gene by using FLP integration manner was constructed by removal JTP196 gene driven by pNA2 promoter and Tamg terminator from pHUda1260 (described in example 7 in 12691 -WO-PCT). To create pKKa199p, HUda1260 was digested for removal of 3.72 kpb JTP196 fragment with Nhel and then integrated by 0.98 kbp fragment containing Ptefl obtained by digestion of pHUda1260 with Nhel and Spel.

Genome DNA extraction and purification

The genomic DNA was extracted from frozen mycelial samples by using DNA extraction kit, FastDNA Spin Kit for Soil (MP Biomedicals. LLC Cat: 1 1-6560-200). We used the kit in accordance with the standard protocol attached to the kit. Fast Prep-24 (MP Biomedicals. LLC) was used as beads beater.

Transformation of Aspergillus niger strains

Transformation of Aspergillus species can be achieved using the general methods for yeast transformation. The preferred procedure for the invention is described below.

Aspergillus niger host strain was inoculated to 100 ml of YPG medium supplemented with 10 mM uridine and incubated for 16 hrs at 32°C at 80 rpm. Pellets were collected and washed with 0.6 M KCI, and resuspended 20 ml 0.6 M KCI containing a commercial β-glucanase product (GLUCANEX™, Novozymes A/S, Bagsvaerd, Denmark) at a final concentration of 20 mg per ml. The suspension was incubated at 32 °C at 80 rpm until protoplasts were formed, and then washed twice with STC buffer. The protoplasts were counted with a hematometer and resuspended and adjusted in an 8:2:0.1 solution of STC:STPC:DMSO to a final concentration of 2.5x10⁷ protoplasts/ml. Approximately 4μ9 of plasmid DNA was added to 100 μΙ of the protoplast suspension, mixed gently, and incubated on ice for 30 minutes. One ml of SPTC was added and the protoplast suspension was incubated for 20 minutes at 37°C. After the addition of 10 ml of 50°C Cove or Cove-N top agarose, the reaction was poured onto Cove or Cove-N (tf) agar plates and the plates were incubated at 32°C for 5days. Table 1. PCR amplification KOD plus (TOYOBO)

3-step cycle:

1 . Pre-denaturation: 94 °C, 2 min.

2. Denaturation: 94 °C, 15 sec.

3. Annealing: Tm-[5-10] °C^*, 30 sec.

4. Extension: 68 °C, 1 min./ kb

35 cycles for 2-4 Spore PCR

This is a method to amplify DNA fragment by PCR with A. niger spores as template by using Phire® Plant Direct PCR Kit (New England Biolabs). We followed the description with this kit. Total RNA extraction and reverse-transcription for cDNA

The total RNA was extracted from frozen samples of - . niger mycelia by TRIzol (Termo

Fisher Scientific: cat. No. 15596026) method. 200 ng total RNA was reverse-transcribed with oligo-(dT)i8 primer to generate template cDNA for following qPCR reaction. Transcriptor High

Fidelity cDNA Synthesis Kit was purchased from Roche Applied Science (cat. no. 05081955001 ), containing Transcriptor High Fidelity Reverse Transcriptase, Transcriptor High Fidelity Reverse

Transcriptase Reaction Buffer, Protector RNase inhibitor, Deoxynucleotide Mix, Anchored-oligo (dT) is Primer, DTT and PCR-grade Water. We followed Two steps RT-PCR procedure in the protocol.

Table 2. Step 1

10 min at 65 °C, then chilled on ice

To the tube containing the template-primer mix, add the remaining components of the RT mix in the order listed below.

Table 3. Step 2

30 min at 50 °C, then 5 min at 85 °C to stop reaction

Quantitative PCR assay for copy number detection

Quantitative PCR (qPCR) was performed using the Light Cycler 480 System (Roche

Applied Science) in 96-well plate format with 20 μΙ working volume. 1/100 dilutions of reverse- transcribed cDNA samples were used in qPCR reactions. A negative control was always rum with the samples. In the negative control, cDNA template was replaced with PCR grade water. All samples were run in triplicate applied science (samples for standard curve was run once for each gene). LightCycler 480 Probes Master buffer (2 x concentrate) was purchased from Roche (cat. no. 04707494001 ). Probes were designed by Universal ProbeLibrary Assay Design Center on Roche website (www.roche-diagnostics.jp).

Cultivation of transformants in shake flasks for beta-glucanase and arabinofuranosidase production

Spores of the selected transformants were inoculated in 100 ml of MSS media and cultivated at 30 C for 2 days. 10 ml of MSS was inoculated to 150 ml of MU-1 glu medium and cultivated at 30 C for 7 days. The supernatant was obtained by centrifugation.

Arabinofuranosidase activity

Arabinofuranosidase hydrolyzes arabinoxylane and releases reducing carbohydrate. This reaction is stopped by an alkaline reagent including PAH BAH and Bi³⁺, which complexes with reducing sugar, producing color detected at 405 nm. The produced color is proportional to the arabinofuranosidase activity. Enzymatic reaction and absorption measurement proceed automatically in the Konelab analyzer.

Bi³⁺ concentration 4.5 mM

NaOH concentration 146 mM pH Alkaline

Temperature 50°C

Time 20 minutes

Absorbance wavelength 405 nm

Beta-glucanase activity

Beta-glucanase hydrolyzes beta-glucan and releases reducing carbohydrate. This reaction is stopped by an alkaline reagent including PAHBAH and Bi3+, which complexes with reducing sugar, producing color detected at 405 nm. The produced color is proportional to the beta- glucanase activity. Enzymatic reaction and absorption measurement proceed automatically in the Konelab analyzer.

Table 6.

Bi³⁺ concentration 4.5 mM

NaOH concentration 146 mM pH Alkaline

Temperature 50°C

Time 20 minutes

Absorbance wavelength 405 nm

Glucoamylase activity

Glucoamylase activity is measured by Relative AmyloGlucosidase activity (RAG). Glucoamylase hydrolyzes p-Nitrophenyl-a-D-glucopyranoside (pNPG) and releases p- Nitrophenol. This reaction is stopped by pH shift, producing color detected at 400 nm. The produced color is proportional to the glucoamylase activity.

Table 8.

Example 1

Arabinofuranosidase production level is increased by expression of Trametes cingulata AMG Transformation of 4C4-4 with pKKa150

4C4-4 is an A. niger strain of pyrG minus strain generated from C2218 described in WO 2015/025055. 4C4-4 was isolated as a pyrG gene deleted mutant from C2218 after selection on the Cove-N plate containing 1 g / L 5-fluoro-orotic acid (FOA) and 2 mM uridine. Modifications of 4C4-4 were made at pepA gene locus by homologues recombination with the expression cassette for arabinofuranosidase, pKKa150. For the selection of 4C4-4-ReAraf-7 from the transformants integrated by pKKa150 arabinofuranosidase expression cassette, they were incubated on Cove-N plate containing 1 g / L 5-Fluoro-2-deoxyuridine (FdU), an agent which kills cells expressing the herpes simplex virus (HSV) thymidine kinase gene (tk1) harboring in pKKa150. Strains which grew well on Cove-N plates supplemented with FdU were purified and subjected to PCR analysis to confirm whether the FRT sites in 4C4-4 was introduced correctly or not. Resulting strain 4C4-4-ReAraf-7 was expressing 4 copies of Trametes cingulata AMG genes and 1 copy of arabinofuranosidase gene.

Transformation of 4C4-4-ReAraf-7 with pKKa164

The method of transformation with the strains which has FLP integration sites is described in WO 2012/160093. Modifications of 4C4-4-ReAraf-7 were made at four loci {amyA, amyB, asaA and payA) that will be targeted for integration by FLP integration manner. At these loci, insertion of hph selective marker of pKKa164 was performed by kicking out the Trametes cingulata AMG genes at these loci. Randomly selected transformants were inoculated onto Cove plates with 200 ug / L hygromycin. Strains which could grow on Cove plates with 200 ug / L hygromycin were purified.

Determination of Tc-AMG gene copy number after transformation

After transformation of 4C4-4-ReAraf-7 with pKKa164, number of remaining Trametes cingulata AMG gene copies at amyA, amyB, asaA or payA loci were decided by amplification of DNA fragments amplified by spore PCR (described in Material and methods) with specific primers (HA057, HA059, HA196 and HA095) and NA2 promoter specific primer. Primer sequence and lengths of expected fragments after spore PCR, and the PCR condition were described below. Table 9.

Table 10.

Table 11.

Table 12.

Table 13.

Assays for glucoamylase and arabinofuranosidase activity of the transformants

The transformants which have 1 -copy arabinofuranosidase with 0-4 copies of Trametes cingulata AMG genes were cultivated by shake flasks. The collected culture broths of the transformants at day 7 were used for measurement of glucoamylase activity (RAG) and arabinofuranosidase activities as shown in the table below.

Table 14.

copy# (Tc/Araf) 0/1 1/1 2/1 3/1 4/1

Average RAG

(stdev) 0.65(±0.6) 10.66(±2.9) 41.15(±0.3) 66.69(±10.3) 96.92(±18.48)

Average AFU

(stdev) -0.14(±0.03) -0.01 (±0.12) 0.6(±0.03) 0.74(±0.08) 0.65(±0.07) Example 2

Arabinofuranosidase production level is increased by expression with Beta-glucanase

The strains expressing following proteins with specified copy numbers were generated Reference 1 : Araf 1 copy

Reference 2: BG025 3 copy

Sample 1 : Araf 1 copy + BG025 3 copy

Sample 2: Araf 2 copy + BG025 2 copy

Transformation of C3085 with phahi025 and phahi033

Chromosomal insertion into A. niger of both arabinofuranosidase (phahi033) with pyrG and beta-glucanase (phahi025) with amdS selective markers were performed as described in WO 2012/160093. The reference strains were performed by chromosomal insertion into A niger of either arabinofuranosidase (phahi026) and beta-glucanase (phahi025) both with amdS selective marker as described in WO 2012/160093. Strains which grew well were purified and subjected to southern blotting analysis to confirm whether the arabinofuranosidase gene in phahi033 or phahi026 and beta-glucanase gene in phahi025 were introduced at amyA, amyB, asaA or payA loci correctly or not. The following sets of primers to make non-radioactive probe were used to analyze the selected transformants.

Table 15. For arabinofuranosidase coding region:

Genomic DNA extracted from the selected transformants was digested by Spel and Pmll, and then probed with arabinofuranosidase coding region. By the right gene introduction event, hybridized signals at the size of 5.9 kb (amyA), 2.7 kb (asaA), 3.9 kb (amyB) and 4.8 kb (payA) by Spel and Pmll digestion was observed probed described above. Table 16. For beta-glucanase coding region:

Genomic DNA extracted from the selected transformants was digested by Spel and Pmll, and then probed with beta-glucanase coding region. By the right gene introduction event, hybridized signals at the size of 5.3 kb (amyA), 2.0 kb (asaA), 3.2 kb (amyB) and 4.1 kb (payA) by Spel and Pmll digestion was observed probed described above.

Assays for arabinofuranosidase and beta-glucanase activity of the transformants

The collected culture broths from the transformants below at day 7 in shake flasks were used for measurement of Arabinofuranosidase activity and beta-glucanase activity assays for their activities.

Table 17. Relative activities achieved

Example 3

Gloeophyllum trabeum AMG production level is increased by expression with beta- glucanase

Transformation of 4C4-4 with pKKa35 and phahi025

In order to generate strains expressing Gloeophyllum trabeum AMG and beta-glucanase, expression cassettes pKKa35 and phahi025 were integrated by FLP integration manner into four integration sites of 4C4-4 (amyA, amyB, asaA and payA) by kicking out of Trametes cingulata AMG genes in the presence of 10 μg ml 5-fluorocytosine (5FC). As a control experiment, pKKa35 and pKKa199, which is a construct for integration of amdS marker gene but not for beta- glucanase gene, were integrated into 4C4-4 by FLP integration manner.

Selection of strains integrated by three copies of Gloeophyllum trabeum AMG genes

To select the transformants expressing three copies of Gloeophyllum trabeum AMG genes, quantitative PCR (qPCR) was performed with genomic DNA extracted in accordance with the method described in "Materials and methods". Prepared genomic DNA was diluted to 25 ng/uL for the following reactions. Tables below outline the concentrations and reaction conditions for qPCR assays to determine copy number of Gloeophyllum trabeum AMG genes.

Table 18. Primers and probes for qPCR

Table 19. Dilution series for standards

1/12 Dilution from 25

Reference gene 1/5 1/25 1/625

5 - ng/uL DNA solution

(cpyl gene) (5) (1 ) (0.04)

(0.2) (ng/uL)

Table 20. Mixture

Table 21. PCR parameters for a Light cycler 480 System PCR run

Program Name Cooling

Cycles 1

Analysis Mode None

Temperature °C Hold (hh:mm:ss) Ramp Rate (°C/s) Acquisitions mode

40 00:10:00 2.2 Nome

22. Transformants expressing three copies of Gloeophyllum trabeum AMG genes

Assays for qlucoamylase of the transformants expressinq three copies of Gloeophyllum trabeum AMG genes

The collected culture broths from 13 transformants above at day 7 in shake flasks were used for measurement of glucoamylase activity as shown in the table below. Table 23. Relative glucoamylase activity of transformants

Table 24. Abbreviations

Claims

1 . A method for producing a first heterologous polypeptide, said method comprising the steps of:

c) expressing the first heterologous polypeptide and a second heterologous polypeptide in a host cell, where the second heterologous polypeptide is expressed with a higher yield or productivity than the first heterologous polypeptide when each polypeptide is expressed without the other in said host cell under comparable conditions, and wherein their co- expression improves the yield/productivity of the first heterologous polypeptide compared to when it is expressed without the second heterologous polypeptide in said host cell under comparable conditions; and, optionally,

d) recovering the first heterologous polypeptide.

2. The method of claim 1 , wherein the first heterologous polypeptide is an enzyme and the second heterologous polypeptide is an enzyme.

3. The method of claim 2, wherein either of the two heterologous enzymes is a hydrolase, isomerase, ligase, lyase, oxidoreductase, and/or a transferase; preferably either of the two heterologous enzymes is an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, arabinofuranosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and/or a xylanase.

4. The method of any preceding claim, wherein the host cell is a fungal host cell, preferably a filamentous fungal host cell, more preferably an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliop t ora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, P anerochaete, Phlebia, Piromyces, Pleurotus, Schizop yllum, Talaromyces, Thermoascus, T ielavia, Tolypocladium, Trametes, or Trichoderma cell; and most preferably an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

5. A host cell expressing a first heterologous polynucleotide encoding a first heterologous polypeptide and a second heterologous polynucleotide encoding a second heterologous polypeptide, where the second heterologous polypeptide is expressed with a higher yield or productivity than the first heterologous polypeptide when each heterologous polypeptide is expressed without the other in said host cell under comparable conditions, and wherein their co- expression improves the yield/productivity of the first heterologous polypeptide compared to when it is expressed without the second heterologous polypeptide in said host cell under comparable conditions.

6. The host cell of claim 5, wherein the first heterologous polypeptide is an enzyme and the second heterologous polypeptide is an enzyme.

7. The host cell of claim 6, wherein either of the two heterologous enzymes is a hydrolase, isomerase, ligase, lyase, oxidoreductase, and/or a transferase; preferably either of the two heterologous enzymes is an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, arabinofuranosidase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and/or a xylanase.

8. The host cell of any preceding claim, wherein the first heterologous polypeptide is an arabinofuranosidase and the second heterologous polypeptide is a glucoamylase or a beta- glucanase.

9. The host cell of any preceding claim which is a fungal host cell, preferably a filamentous fungal host cell, more preferably an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell; and most preferably an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium ops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.