Tandem protein expression

Abstract

The present invention provides means and methods for increasing the yield of polypeptide expression by increasing the amount of polypeptide that can be transported to the endoplasmic reticulum (ER) per translocation event.

Description

[0001] References to sequence listings

[0002] This application contains a sequence listing in computer readable form. This computer readable form is incorporated herein by reference. Technical Field

[0003] The present invention provides means and methods for increasing the yield of polypeptide expression by increasing the amount of polypeptide that can be transported to the endoplasmic reticulum (ER) per translocation event. Background Art

[0004] Product development in industrial biotechnology includes the ongoing challenge of massively increasing enzyme production to reduce costs. Over the past few decades, two main approaches have been used for this purpose. The first is based on classical mutagenesis and screening. Here, the specific genetic modification is not predefined, and the main requirement is a screening assay that is sensitive to detect relatively discrete yield increments. High-throughput screening enables the screening of large numbers of mutants to search for the desired phenotype, i.e., higher enzyme production. The second approach includes numerous strategies that range from the use of stronger promoters and multicopy strains to ensure high expression of the gene of interest to the use of codon-optimized gene sequences to aid translation. However, high-level production of proteins may trigger several bottlenecks in the cellular machinery for secretion of the enzyme of interest into the culture medium.

[0005] For secretases whose amino acid sequences contain a signal peptide (SP), the SP is cleaved by a signal peptidase after translation and the mature protein is translocated into the endoplasmic reticulum (ER; Voss et al., 2013; Aviram and Schuldiner 2017). To secrete proteins through the ER, the signal recognition particle (SRP) recognizes the SP in a highly conserved manner. The SRP associates with the ribosome and recognizes secretory proteins that are being translated and have a hydrophobic motif through a hydrophobic cleft and binds to the SRP receptor (SR) present in the eukaryotic ER membrane (Aviram and Schuldiner 2017). Therefore, the limiting step of protein secretion may be the translocation of nascent polypeptides from the cytosol to the ER. Since this process is very energy-intensive, there is a capacity upper limit to the number of translocations per cell per time unit.

[0006] Means and methods to overcome the translocation ceiling would constitute entirely new approaches to yield optimization and would therefore be highly desirable in industrial biotechnology. Summary of the invention

[0007] The present invention provides means and methods for increasing polypeptide expression yields by increasing the number of polypeptides that can be translocated to the ER while still using one signal peptide per translocation-dependent event, thereby increasing the number of secretable polypeptide units per translocation event.

[0008] In a first aspect, the present invention relates to a nucleic acid construct comprising a heterologous promoter operably linked to:

[0009] a) a polynucleotide encoding a signal peptide; and

[0010] b) at least two polynucleotides encoding one or more polypeptides of interest;

[0011] wherein the at least two polynucleotides encoding one or more polypeptides of interest are each separated by a linker polynucleotide encoding a linker polypeptide comprising a proteolytic cleavage site; and

[0012] Wherein the signal peptide, the one or more polypeptides of interest and the one or more linker polypeptides comprising a proteolytic cleavage site are encoded in-frame as a single polypeptide.

[0013] In a second aspect, the present invention relates to an expression vector comprising the nucleic acid construct according to the first aspect.

[0014] In a third aspect, the present invention relates to a fungal host cell comprising the nucleic acid construct according to the first aspect and / or the expression vector according to the second aspect.

[0015] In a fourth aspect, the present invention relates to a method for producing one or more polypeptides of interest, the method comprising:

[0016] a) providing a fungal host cell according to the third aspect;

[0017] b) culturing the host cell under conditions conducive to the expression of the one or more polypeptides of interest; and optionally

[0018] c) recovering the one or more polypeptides of interest. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The lipVR expression cassette is schematically shown. Briefly, one or more "units" of the lipVR coding sequence (using different codon-optimized versions of the gene, lipVR, lipVR*, etc.) were cloned to make gene constructions in which lipVR was transcribed in single lines, in tandem, or more. SP: native signal peptide from the lipVR gene or the cutinase prepro coding sequence; CP: cutinase pro coding sequence; KexB: KexB cleavage site ("KR"). In all cases, a single transcript contained all "lipVR units".

[0020] Figure 2 A map of plasmid pAT652 (single gene construct) used for expression and secretion of LipVR lipase in Aspergillus oryzae is shown.

[0021] Figure 3A map of plasmid pAT1509 (tandem gene construct) used for expression and secretion of LipVR lipase in Aspergillus oryzae is shown.

[0022] Figure 4 (Left) Shown is the production of LipVR as demonstrated by SDS PAGE for comparison of single-line production of LipVR versus tandem production of LipVR by Aspergillus oryzae strains AT969 (lane 1: 18 copies of single expression plasmid pAT652) and AT1684 (lane 2: 9 copies of pAT1509 (lipVR tandem)) at the end of fermentation (day 7). Figure 4 (Right) Shown are the lipolytic activities (expressed in arbitrary units, AU) of strains AT969 and AT1684 at the end of fermentation.

[0023] Figure 5 The extent of correct processing to produce the complete LipVR molecule (produced during fed-batch fermentation of Aspergillus oryzae strain AT1684 containing the lipVR tandem cassette) is shown. Samples (supernatants) were taken at the end of the fermentation at 30°C or 34°C and at pH 6.5 or 7.4 as indicated and analyzed by mass spectrometry. The level of tandem processing is shown as a percentage of the full-length LipVR molecule (black boxes) and the aggregate percentage of different but not fully processed LipVR molecules (grey boxes).

[0024] definition

[0025] 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 pre-mRNA, which is processed through a series of steps (including RNA splicing) and then appears as a mature mRNA.

[0026] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are usually determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0027] Control sequences: The term "control sequences" means nucleic acid sequences necessary for the 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 exogenous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or exogenous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, and transcription and translation termination signals. For the purpose of introducing specific restriction sites that facilitate connection of control sequences to the coding region of a polynucleotide encoding a polypeptide, these control sequences may be provided with multiple linkers.

[0028] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0029] 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.

[0030] Heterologous promoter: The term "heterologous promoter" means a promoter that is foreign (ie, from a different gene) to the polynucleotide to which it is operably linked.

[0031] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. 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.

[0032] Isolated: The term "isolated" means material that is in a form or environment that does not occur in nature.

[0033] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 289 of SEQ ID NO:5 (corresponding to amino acids 1 to 289 of SEQ ID NO:6). Amino acids -20 to -1 of SEQ ID NO:5 are a signal peptide. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus a host cell expressing a polynucleotide can produce different mature polypeptides (e.g., having different C-terminal and / or N-terminal amino acids) when compared to another host cell expressing the same polynucleotide.

[0034] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having lipolytic activity as determined in WO 2018 / 150021. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 1056 of SEQ ID NO: 3 or its cDNA sequence (corresponding to nucleotides 61 to 927 of SEQ ID NO: 4), and nucleotides 1 to 60 of SEQ ID NO: 3 (corresponding to nucleotides 1 to 60 of SEQ ID NO: 4) encode a signal peptide.

[0035] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to contain a nucleic acid segment in a manner not originally found in nature, or is synthetic, and that contains one or more control sequences.

[0036] 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 the expression of the coding sequence.

[0037] Secreted: The term "secreted" means that one or more polypeptides of interest are processed and released into the extracellular environment (e.g., growth medium). In one embodiment, one or more polypeptides of interest are processed and released via a conventional secretory pathway comprising the endoplasmic reticulum, the Golgi apparatus, and secretory vesicles. In another embodiment, one or more polypeptides of interest are processed and released via an unconventional secretory pathway (Rabouille, Trends in Cell Biology [Cell Biology Trends] 2017, Vol. 27, pp. 230-240; Kim et al., Journal of Cell Science [Cell Science Magazine] 2018, Vol. 131, jcs213686).

[0038] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0039] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453) implemented in the Needleman program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Genetics Trend] 16:276-277) (preferred 5.0.0 version or updated version). The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output (obtained using non-simplified option (nobrief option)) of the "longest identity" marked by Needleman is used as the identity percentage and is calculated as follows:

[0040] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0041] For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferably 5.0.0 version or updated version). The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output (obtained using the non-simplified option) of the "longest identity" marked by Needle is used as the identity percentage and is calculated as follows:

[0042] (number of identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in alignment)

[0043] Variant: The term "variant" means a polypeptide comprising an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions compared to the corresponding native polypeptide. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. DETAILED DESCRIPTION

[0044] The present invention provides means and methods for increasing polypeptide expression yields by increasing the number of polypeptides that can be transported to the ER while still using only one SP per translocation event, thereby increasing the number of secretable polypeptide units per translocation event.

[0045] The present invention is based on the design of nucleic acid constructs comprising a heterologous promoter operably linked to a polynucleotide encoding a signal peptide and at least two polynucleotides encoding one or more polypeptides of interest. Each polynucleotide encoding a polypeptide of interest is separated by a linker polynucleotide encoding a linker polypeptide comprising a proteolytic cleavage site ( Figure 1 ). The signal peptide, one or more polypeptides of interest, and one or more linker polypeptides are encoded in-frame to allow translation into a single polypeptide. The term "in-frame encoding" refers to the mature mRNA obtained after RNA splicing and removal of any introns present in the pre-mRNA.

[0046] The nucleic acid construct of the present invention can be transformed into a suitable host cell, and fermentation of such host cells under suitable conditions will allow transcription of the polynucleotide, followed by ribosomal translation of the resulting mRNA. The resulting polypeptide (which contains a signal peptide and one or more target polypeptides each separated by a linker polypeptide) is then targeted into the secretory pathway as a single unit in a translocation event. After proteolytic processing by a protease that recognizes the proteolytic cleavage site contained by the linker polypeptide, the one or more target polypeptides are released and secreted into the extracellular medium.

[0047] As shown in the examples disclosed herein, the use of the nucleic acid constructs of the present invention in the construction of fungal host cells results in increased production of the polypeptide of interest and uniform processing of individual copies of the polypeptide of interest. The latter is particularly important for functional polypeptides such as enzymes, since the integrity of the primary structure is important for the folding and correct formation of the tertiary structure, which is important for activity.

[0048] Thus, in a first aspect, the present invention relates to a nucleic acid construct comprising a heterologous promoter operably linked to:

[0049] a) a polynucleotide encoding a signal peptide; and

[0050] b) at least two polynucleotides encoding one or more polypeptides of interest;

[0051] wherein the at least two polynucleotides encoding one or more polypeptides of interest are each separated by a linker polynucleotide encoding a linker polypeptide comprising a proteolytic cleavage site; and

[0052] Wherein the signal peptide, the one or more polypeptides of interest and the one or more linker polypeptides comprising a proteolytic cleavage site are encoded in-frame as a single polypeptide.

[0053] Preferably, the nucleic acid construct of the invention comprises at least two, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more polynucleotides encoding one or more, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more polypeptides of interest.

[0054] The signal peptide may be any signal peptide suitable for targeting one or more polypeptides of interest to the secretory pathway of a host cell, preferably a fungal host cell. Preferably, the signal peptide is a heterologous signal peptide.

[0055] In a preferred embodiment, the signal peptide has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2. Preferably, the signal peptide comprises or consists of SEQ ID NO: 2.

[0056] In a preferred embodiment, the polynucleotide encoding the signal peptide has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1; preferably, the polynucleotide encoding the signal peptide comprises SEQ ID NO: 1 or consists of it.

[0057] The one or more polypeptides of interest may be any polypeptide. Preferably, the polypeptide of interest comprises an enzyme; preferably, the enzyme is selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase or a transferase; more preferably, an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, an α-glucosidase, a β-glucosidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, a nuclease, an oxidase, a pectinolytic enzyme, a peroxidase, a phosphodiesterase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, a xylanase, and a β-xylosidase; most preferably, the enzyme is a lipase.

[0058] In a preferred embodiment, the one or more target polypeptides have at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:5; preferably, the one or more target polypeptides comprise or consist of the mature polypeptide of SEQ ID NO:5.

[0059] In a preferred embodiment, the at least two polynucleotides encoding one or more polypeptides of interest independently have at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 4; preferably, the at least two polynucleotides encoding one or more polypeptides of interest comprise or consist of the mature polypeptide coding sequence of SEQ ID NO: 4; also preferably, the at least two polynucleotides encoding one or more polypeptides are different, i.e., have no identity.

[0060] In another preferred embodiment, the nucleic acid construct of the present invention comprises at least two polynucleotides encoding at least two polypeptides of interest having the same amino acid sequence, and the at least two polypeptides of interest are secreted as separate polypeptides having the same amino acid sequence. Preferably, the at least two polypeptides of interest have at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO: 5; preferably, the at least two polypeptides of interest comprise or consist of the mature polypeptide of SEQ ID NO: 5. Preferably, the at least two polynucleotides encoding at least two polypeptides of interest independently have at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 4; preferably, the at least two polynucleotides encoding at least two polypeptides of interest comprise or consist of the mature polypeptide coding sequence of SEQ ID NO: 4; also preferably, the at least two polynucleotides encoding at least two polypeptides are different, i.e., have no identity.

[0061] The joint polynucleotide should be selected so that the joint polypeptide of coding has enough length and flexibility to allow the suitable protease of recognizing proteolytic cleavage site to approach the proteolytic cleavage site. Preferably, the joint polypeptide comprises at least 10 amino acids, for example at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 95 amino acids, at least 100 amino acids or more amino acids.

[0062] The linker polypeptide comprises a proteolytic cleavage site recognized by a suitable protease, preferably a KexB protease. Preferably, the proteolytic cleavage site is a binary amino acid motif, such as a Lys-Arg or Arg-Arg motif. Preferably, the proteolytic cleavage site is a KexB cleavage site.

[0063] The one or more polypeptides of interest are expressed as part of a single polypeptide, but should be released intracellularly before secreting the individual polypeptides of interest into the extracellular medium. Therefore, preferably, the proteolytic cleavage site encoded by the linker polypeptide is recognized by intracellular proteases. Preferably, proteolytic cleavage occurs intracellularly, i.e., inside a suitable host cell comprising the nucleic acid construct of the present invention. Alternatively, proteolytic cleavage occurs extracellularly and during and / or after culture of a suitable host cell comprising the nucleic acid construct of the present invention.

[0064] Nucleic acid construct

[0065] The present invention relates to a nucleic acid construct comprising polynucleotides encoding a signal peptide, one or more polypeptides of interest and one or more linker polypeptides, which are operably linked to one or more control sequences that direct the expression of these polynucleotides in a suitable host cell under conditions compatible with the control sequences.

[0066] Polynucleotides can be manipulated in a variety of ways to provide for their expression. Manipulation of the polynucleotide may be desirable or necessary, depending on the nucleic acid construct, expression vector, and / or host cell into which the polynucleotide is introduced. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0067] The control sequence may be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter comprises a transcriptional control sequence that mediates the expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including variants, truncated and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0068] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in a filamentous fungal host cell are promoters obtained from the genes for 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 (WO 00 / 56900), and Aspergillus oryzae acetamidase. miehei) lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, 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 III, Trichoderma reesei β-xylosidase and Trichoderma reesei translation elongation factor , and NA2-tpi promoter (a modified promoter from an Aspergillus neutral α-amylase gene in which the untranslated leader sequence has been replaced with an untranslated leader sequence from an Aspergillus triose phosphate isomerase gene; non-limiting examples include a modified promoter from an Aspergillus niger neutral α-amylase gene in which the untranslated leader sequence has been replaced with an untranslated leader sequence from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and variants, truncations, and hybrid promoters thereof. Other promoters are described in U.S. Pat. No. 6,011,147.

[0069] In yeast hosts, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0070] The promoter selected should be a heterologous promoter in which the polynucleotide or polynucleotides to which it is operably linked are foreign (ie, from a different gene).

[0071] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator may be operably linked to the 3'-end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

[0072] 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 β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, 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 III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor.

[0073] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al. (1992, supra).

[0074] The control sequence may also be a leader sequence, i.e., a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell may be used.

[0075] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0076] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0077] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0078] 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.

[0079] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0080] The control sequence can also be a signal peptide coding region that encodes a signal peptide connected to the N-terminus of the polypeptide and directs the polypeptide to enter the secretory pathway of the cell. The 5' end of the coding sequence of the polynucleotide itself can contain a signal peptide coding sequence that is naturally connected to the coding sequence segment of the coded polypeptide in the translation reading frame. Alternatively, the 5' end of the coding sequence can contain a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide to enter the secretory pathway of the host cell can be used.

[0081] Effective signal peptide coding sequences for filamentous fungal host cells are those 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.

[0082] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (1992, supra).

[0083] In a preferred embodiment, the signal peptide has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2; preferably, the signal peptide comprises or consists of SEQ ID NO: 2.

[0084] In a preferred embodiment, the signal peptide comprises or consists of SEQ ID NO:2.

[0085] The control sequence can also be a propeptide coding sequence encoding a propeptide at the N-terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptides are generally inactive and can be converted into active polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the following genes: Myceliophthora thermophila laccase (WO95 / 33836), Rhizomucor miehei aspartic proteinase and Saccharomyces cerevisiae α-factor.

[0086] Where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located immediately adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located immediately adjacent to the N-terminus of the propeptide sequence.

[0087] It may also be desirable to add regulatory sequences that regulate the expression of polypeptides associated with host cell growth. Examples of regulatory sequences are those that cause gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.

[0088] In yeast, the ADH2 system or the GAL1 system can be used.

[0089] In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, and the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter may be used.

[0090] Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate and the metallothionein gene that is amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide will be operably linked to the regulatory sequence.

[0091] Polynucleotide

[0092] The present invention also relates to polynucleotides encoding signal peptides, polypeptides encoding one or more polypeptides of interest, and one or more linker polynucleotides encoding one or more linker polypeptides. In embodiments, these polynucleotides have been isolated.

[0093] Techniques for isolating or cloning polynucleotides are known in the art and include separation from genomic DNA or cDNA or a combination thereof. Cloning of polynucleotides from genomic DNA can be achieved, for example, by detecting cloned DNA fragments with common structural features using well-known polymerase chain reaction (PCR) or antibody screening of expression libraries. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application [PCR: Methods and Application Guide], Academic Press [Academic Press], New York. Other nucleic acid amplification programs such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) can be used. These polynucleotides can be cloned from Aspergillus strains or related organisms, and therefore, for example, can be allele variants or species variants of the polypeptide coding region of the polynucleotide.

[0094] Modification of the polynucleotide encoding the signal peptide, one or more polypeptides of interest, or the linker polypeptide may be necessary to synthesize polypeptides substantially similar to these polypeptides.The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide.

[0095] Expression vector

[0096] In a second aspect, the present invention further relates to a recombinant expression vector comprising a polynucleotide of the present invention, a promoter and a transcription and translation termination signal. A plurality of nucleotides and control sequences can be linked together to produce a recombinant expression vector, which can include one or more convenient restriction sites to allow the insertion or replacement of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting a polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. When producing an expression vector, the encoding sequence is so located in the vector that the encoding sequence is operably connected to the suitable control sequence for expression.

[0097] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0098] The vector can be an autonomously replicating vector, i.e. a vector existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicates with one or more chromosomes into which it has been integrated when it is introduced into the host cell. In addition, a single vector or plasmid or two or more vectors or plasmids can be used, which contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0099] The vector preferably contains one or more selectable markers that permit easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0100] Suitable selectable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0101] Suitable selectable markers for filamentous fungal host cells include, but are not limited to, adeA (phosphoribosylamidoimidazole-succinylcarboxamide synthase), adeB (phosphoribosylamidoimidazole 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), and their equivalents. Preferred for use in Aspergillus cells are the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and the Streptomyces hygroscopicus bar gene. Preferred for use in Trichoderma cells are the adeA, adeB, amdS, hph, and pyrG genes.

[0102] The selectable marker may be a dual selectable marker system as described in WO 2010 / 039889. In one aspect, the dual selectable marker is the hph-tk dual selectable marker system.

[0103] The vector preferably contains one or more elements that allow the vector to be integrated into the genome of the host cell or the vector to replicate autonomously in the cell independently of the genome. For integration into the host cell genome, the vector can rely on the polynucleotide sequence of the encoding polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector can contain other polynucleotides at one or more precise positions for guiding integration into one or more chromosomes in the host cell genome by homologous recombination. In order to increase the possibility of integration at the precise position, the integration element 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, and these nucleic acids have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integration element can be any sequence homologous to the target sequence in the host cell genome. In addition, the integration element can be a non-coding or coded polynucleotide. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0104] For autonomous replication, the vector may further comprise an origin of replication, which enables autonomous replication of the vector in the host cell in question. The origin of replication may be any plasmid replicon mediating autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0105] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0106] Examples of origins of replication useful in filamentous fungal cells are AMA1 and ANS1 (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 a plasmid or vector containing the gene can be accomplished according to the method disclosed in WO 00 / 24883.

[0107] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the production of the polypeptide. The increased 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 together with the polynucleotide, wherein cells containing an amplified copy of the selectable marker gene and thus additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0108] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

[0109] Host cells

[0110] In a third aspect, the present invention also relates to a recombinant host cell comprising a nucleic acid construct of the present invention or an expression vector. The construct of the present invention or a vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The term "host cell" encompasses any parent cell progeny that is not identical to the parent cell due to mutations that occur during replication. The selection of the host cell will depend to a large extent on the polynucleotides encoding one or more polypeptides of interest and their source.

[0111] The host cell may be any cell useful for recombinant production of a polypeptide of interest, such as a eukaryotic cell, preferably a fungal cell.

[0112] As used herein, "fungi" include Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all mitotic spore fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0113] The fungal host cell can be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of the present invention, yeast should be defined as described in Biology and Activities of Yeast (Skinner, Passmore and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0114] The yeast host cell can be a Candida, Hansenula, Kluyveri, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Saccharomyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or a Yarrowia lipolytica cell.

[0115] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subphylum Eumycota and Oomycota (as defined by Hawksworth et al., 1995 (supra)). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is carried out by hyphal extension, and carbon catabolism is obligate aerobic. In contrast, vegetative growth of yeasts (such as Saccharomyces cerevisiae) is carried out by budding of unicellular thallus, and carbon catabolism can be fermentative.

[0116] The filamentous fungal host cell can be a Acremonium, Aspergillus, Pullulan, Nicotiana, Ceroplasts, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Ustilago, Fusarium, Humicola, Pyrrospora, Mucor, Myceliophthora, Neotrichia, Neurospora, Paecilomyces, Penicillium, Pseudoceratium, Radius, Chytrid, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolylcorrhiza, Trametes, or Trichoderma cell.

[0117] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus odoratus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, black thorn tobacco pipe fungus, dry wax fungus, Carnegie wax fungus, pale yellow wax fungus, Pannohita wax fungus, annular wax fungus, red wax fungus, insect wax fungus, narrow side golden spore, horny golden spore, Lukenovinsi golden spore, fecal golden spore, rent spore, queen Ledum golden spore, tropical golden spore, brown thin golden spore, gray capped cap mushroom, hairy leather cap mushroom, rod spore shaped Fusarium, cereal Fusarium, library Fusarium wei, Fusarium maculatum, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium albizia, Fusarium oxysporum, Fusarium multi-branched, Fusarium pink, Fusarium elderberry, Fusarium color, Fusarium pseudo-branched spore, Fusarium sulfur color, Fusarium round, Fusarium pseudo-tricholoma, Fusarium venezuelae, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Pleurotus eryngii, Thielavia terrestris, Trametes longifolia, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longifolia, Trichoderma reesei, or Trichoderma viride cells.

[0118] Fungal cells can be transformed in a manner known per se by methods involving protoplast formation, protoplast transformation, and cell wall regeneration. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 81: 1470-1474 and Christensen et al., 1988, Bio / Technology [Biology / Technology] 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene [Gene] 78: 147-156 and WO 96 / 00787. Yeast can be transformed using the procedures described by Becker and Guarente, in Abelson, JN and Simon, MI, eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0119] Generation method

[0120] In a fourth aspect, the present invention also relates to a method for producing one or more polypeptides of interest, the method comprising:

[0121] a) providing a fungal host cell of the present invention;

[0122] b) culturing the host cell under conditions conducive to the production of the one or more polypeptides of interest; and optionally

[0123] c) recovering the one or more polypeptides of interest.

[0124] In one embodiment, the host cell is an Aspergillus cell. In a preferred embodiment, the host cell is an Aspergillus oryzae cell.

[0125] These fungal host cells are cultivated in a nutrient medium suitable for producing the target polypeptide using methods known in the art. For example, cells can be cultured by shaking flasks or in small or large-scale fermentation (including continuous, batch, fed-batch or solid-state fermentation) in a laboratory or industrial fermentor, which is cultured in a suitable medium and under conditions that allow expression and / or separation of the target polypeptide. Using procedures as known in the art, cultivation occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable medium can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalog of the American Type Culture Collection). If the target polypeptide is secreted into the nutrient medium, the target polypeptide can be directly recovered from the medium. If the target polypeptide is not secreted, it can be recovered from the cell lysate.

[0126] The one or more target polypeptides can be detected using methods known in the art that are specific for the polypeptide. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of the target polypeptide.

[0127] The one or more polypeptides of interest may be recovered using methods known in the art. For example, the one or more polypeptides of interest may be recovered from the nutrient medium by conventional methods, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. In one aspect, the fermentation broth containing the one or more polypeptides of interest is recovered.

[0128] The one or more polypeptides of interest may be purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing electrophoresis), differential solubilization (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, eds., VCH Publishers, New York, 1989), in order to obtain a substantially pure polypeptide.

[0129] In an alternative aspect, the one or more polypeptides of interest are not recovered, but rather a host cell of the invention expressing the one or more polypeptides of interest is used as a source of the one or more polypeptides.

[0130] Preferred Embodiments

[0131] 1) A nucleic acid construct comprising a heterologous promoter operably linked to:

[0132] a) a polynucleotide encoding a signal peptide; and

[0133] b) at least two polynucleotides encoding one or more polypeptides of interest;

[0134] wherein the at least two polynucleotides encoding one or more polypeptides of interest are each separated by a linker polynucleotide encoding a linker polypeptide comprising a proteolytic cleavage site; and

[0135] Wherein the signal peptide, the one or more polypeptides of interest and the one or more linker polypeptides comprising a proteolytic cleavage site are encoded in-frame as a single polypeptide.

[0136] 2) The nucleic acid construct according to claim 1, wherein the at least two polynucleotides encode at least two polypeptides of interest having the same amino acid sequence, and wherein the at least two polypeptides of interest are secreted as separate polypeptides having the same amino acid sequence.

[0137] 3) The nucleic acid construct according to any one of the preceding embodiments, wherein the signal peptide has at least 80% sequence identity with SEQ ID NO: 2; preferably, the signal peptide comprises or consists of SEQ ID NO: 2.

[0138] 4) The nucleic acid construct according to any one of the preceding embodiments, wherein the polynucleotide encoding the signal peptide has at least 80% sequence identity with SEQ ID NO: 1; preferably, the polynucleotide encoding the signal peptide comprises or consists of SEQ ID NO: 1.

[0139] 5) The nucleic acid construct of any one of the preceding embodiments, wherein the one or the polypeptide of interest comprises an enzyme; preferably, the enzyme is selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase or a transferase; more preferably, an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, an α-glucosidase, a β-glucosidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, a nuclease, an oxidase, a pectinolytic enzyme, a peroxidase, a phosphodiesterase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, a xylanase, and a β-xylosidase; most preferably, the polypeptide of interest is a lipase.

[0140] 6) The nucleic acid construct according to any one of the preceding embodiments, wherein the one or more target polypeptides have at least 80% sequence identity with the mature polypeptide of SEQ ID NO:5; preferably, the one or more target polypeptides comprise or consist of the mature polypeptide of SEQ ID NO:5.

[0141] 7) A nucleic acid construct according to any one of the preceding embodiments, wherein the at least two polynucleotides encoding one or more target polypeptides independently have at least 80% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:4; preferably, the at least two polynucleotides encoding one or more target polypeptides comprise or consist of the mature polypeptide coding sequence of SEQ ID NO:4.

[0142] 8) The nucleic acid construct according to any one of the preceding embodiments, wherein the linker polypeptide comprises at least 10 amino acids.

[0143] 9) The nucleic acid construct according to any one of the preceding embodiments, wherein the proteolytic cleavage site is a binary amino acid motif; preferably, the proteolytic cleavage site is a Lys-Arg motif or an Arg-Arg motif.

[0144] 10) The nucleic acid construct according to any one of the preceding embodiments, wherein the proteolytic cleavage site is a KexB cleavage site.

[0145] 11) An expression vector comprising a nucleic acid construct containing a heterologous promoter operably linked to:

[0146] a) a polynucleotide encoding a signal peptide; and

[0147] b) at least two polynucleotides encoding one or more polypeptides of interest;

[0148] wherein the at least two polynucleotides encoding one or more polypeptides of interest are each separated by a linker polynucleotide encoding a linker polypeptide comprising a proteolytic cleavage site; and

[0149] Wherein the signal peptide, the one or more polypeptides of interest and the one or more linker polypeptides comprising a proteolytic cleavage site are encoded in-frame as a single polypeptide.

[0150] 12) The expression vector according to claim 11, wherein the at least two polynucleotides encode at least two target polypeptides having the same amino acid sequence, and wherein the at least two target polypeptides are secreted as separate polypeptides having the same amino acid sequence.

[0151] 13) The expression vector according to any one of Examples 11-12, wherein the signal peptide has at least 80% sequence identity with SEQ ID NO: 2; preferably, the signal peptide comprises or consists of SEQ ID NO: 2.

[0152] 14) An expression vector according to any one of Examples 11-3, wherein the polynucleotide encoding the signal peptide has at least 80% sequence identity with SEQ ID NO: 1; preferably, the polynucleotide encoding the signal peptide comprises SEQ ID NO: 1 or consists of it.

[0153] 15) An expression vector according to any one of embodiments 11-14, wherein the one or more polypeptides of interest comprise an enzyme; preferably, the enzyme is selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase or a transferase; more preferably, an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, an α-glucosidase, a β-glucosidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, a nuclease, an oxidase, a pectinolytic enzyme, a peroxidase, a phosphodiesterase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, a xylanase, and a β-xylosidase; most preferably, the one or more polypeptides of interest are lipases.

[0154] 16) An expression vector according to any one of embodiments 11-15, wherein the one or more target polypeptides have at least 80% sequence identity with the mature polypeptide of SEQ ID NO:5; preferably, the one or more target polypeptides comprise or consist of the mature polypeptide of SEQ ID NO:5.

[0155] 17) An expression vector according to any one of Examples 11-16, wherein the at least two polynucleotides encoding one or more target polypeptides independently have at least 80% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:4; preferably, the at least two polynucleotides encoding one or more target polypeptides comprise or consist of the mature polypeptide coding sequence of SEQ ID NO:4.

[0156] 18) An expression vector according to any one of Examples 11-17, wherein the linker polypeptide comprises at least 10 amino acids.

[0157] 19) An expression vector according to any one of Examples 11-18, wherein the proteolytic cleavage site is a binary amino acid motif; preferably, the proteolytic cleavage site is a Lys-Arg motif or an Arg-Arg motif.

[0158] 20) An expression vector according to any one of Examples 11-19, wherein the proteolytic cleavage site is a KexB cleavage site.

[0159] 21) A fungal host cell comprising in its genome:

[0160] I) A nucleic acid construct containing a heterologous promoter operably linked to:

[0161] a) a polynucleotide encoding a signal peptide; and

[0162] b) at least two polynucleotides encoding one or more polypeptides of interest;

[0163] wherein the at least two polynucleotides encoding one or more polypeptides of interest are each separated by a linker polynucleotide encoding a linker polypeptide comprising a proteolytic cleavage site; and

[0164] wherein the signal peptide, the one or more polypeptides of interest and the one or more linker polypeptides comprising a proteolytic cleavage site are encoded in-frame as a single polypeptide;

[0165] and / or

[0166] II) an expression vector containing the nucleic acid construct.

[0167] 22) The fungal host cell according to claim 21, wherein the at least two polynucleotides encode at least two polypeptides of interest having the same amino acid sequence, and wherein the at least two polypeptides of interest are secreted as separate polypeptides having the same amino acid sequence.

[0168] 23) The fungal host cell according to any one of embodiments 21-22, wherein the fungal host cell is a yeast host cell; preferably, the yeast host cell is selected from the group consisting of: Candida, Hansenula, Kluyveri, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia cells; more preferably, the yeast host cell is selected from the group consisting of: Kluyveri lactis, Karlsaccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasus, Kluyveri, Saccharomyces norbensis, Saccharomyces oviformis and Yarrowia lipolytica cells.

[0169] 24) The fungal host cell according to any one of embodiments 21-22, wherein the fungal host cell is a filamentous fungal host cell; preferably, the filamentous fungal host cell is selected from the group consisting of: Acremonium, Aspergillus, Acidis, Nicotiana, Ceroplastes, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Ustilagoraceae, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neobes, Neurospora, Paecilomyces, Penicillium, More preferably, the filamentous fungal host cell is selected from the group consisting of: Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, black-spined tobacco pipe fungus, dry wax fungus, Carnegie wax fungus, pale yellow wax fungus, Pannohita wax fungus, annular wax fungus, red wax fungus, insect wax fungus, narrow Chrysosporium edge, Chrysosporium hornophilum, Chrysosporium Lukenowens, Chrysosporium feces, Chrysosporium rent, Chrysosporium queen Ledum, Chrysosporium tropical, Chrysosporium brown thin, Coprinus cinerea, Leather cover mushroom, Fusarium rod-shaped, Fusarium cereal, Fusarium kuwei, Fusarium broadsword, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium albizia, Fusarium oxysporum, Fusarium multi-branched, Fusarium pink, Fusarium elderberry, Fusarium color, Fusarium pseudo-branched, Fusarium sulfur color, Fusarium round, Fusarium pseudo-sporum, Fusarium veneer Fusarium, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Pseudomonas chrysosporium, Pleurotus eryngii, Thielavia terrestris, Trametes longiflorus, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride cells; even more preferably, the filamentous fungal host cell is selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Fusarium venenatum and Trichoderma reesei; most preferably, the filamentous fungal host cell is an Aspergillus oryzae cell.

[0170] 25) The fungal host cell according to any one of embodiments 21-24, wherein the signal peptide has at least 80% sequence identity with SEQ ID NO: 2; preferably, the signal peptide comprises or consists of SEQ ID NO: 2.

[0171] 26) The fungal host cell according to any one of embodiments 21-25, wherein the polynucleotide encoding the signal peptide has at least 80% sequence identity with SEQ ID NO: 1; preferably, the polynucleotide encoding the signal peptide comprises or consists of SEQ ID NO: 1.

[0172] 27) The fungal host cell according to any one of embodiments 21-26, wherein the one or more target polypeptides comprise an enzyme; preferably, the enzyme is selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase or a transferase; more preferably, an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, an α-glucosidase, a β-glucosidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, a nuclease, an oxidase, a pectinolytic enzyme, a peroxidase, a phosphodiesterase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, a xylanase, and a β-xylosidase; most preferably, the target polypeptide is a lipase.

[0173] 28) The fungal host cell according to any one of embodiments 21-27, wherein the one or more target polypeptides have at least 80% sequence identity with the mature polypeptide of SEQ ID NO:5; preferably, the one or more target polypeptides comprise or consist of the mature polypeptide of SEQ ID NO:5.

[0174] 29) A fungal host cell according to any one of embodiments 21-28, wherein the at least two polynucleotides encoding one or more target polypeptides independently have at least 80% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:4; preferably, the at least two polynucleotides encoding one or more target polypeptides comprise or consist of the mature polypeptide coding sequence of SEQ ID NO:4.

[0175] 30) The fungal host cell according to any one of embodiments 21-29, wherein the linker polypeptide comprises at least 10 amino acids.

[0176] 31) The fungal host cell according to any one of embodiments 21-30, wherein the proteolytic cleavage site is a binary amino acid motif; preferably, the proteolytic cleavage site is a Lys-Arg motif or an Arg-Arg motif.

[0177] 32) The fungal host cell of any one of embodiments 21-31, wherein the proteolytic cleavage site is a KexB cleavage site.

[0178] 33) A fungal host cell according to any one of embodiments 21-32, wherein the proteolytic cleavage occurs intracellularly, or wherein the proteolytic cleavage occurs extracellularly and during and / or after culturing the fungal host cell.

[0179] 34) A method for producing one or more polypeptides of interest, the method comprising:

[0180] A) providing a fungal host cell comprising:

[0181] I) A nucleic acid construct containing a heterologous promoter operably linked to:

[0182] a) a polynucleotide encoding a signal peptide; and

[0183] b) at least two polynucleotides encoding one or more polypeptides of interest;

[0184] wherein the at least two polynucleotides encoding one or more polypeptides of interest are each separated by a linker polynucleotide encoding a linker polypeptide comprising a proteolytic cleavage site; and

[0185] wherein the signal peptide, the one or more polypeptides of interest and the one or more linker polypeptides comprising a proteolytic cleavage site are encoded in-frame as a single polypeptide;

[0186] and / or

[0187] II) an expression vector containing the nucleic acid construct.

[0188] B) culturing the host cell under conditions conducive to the expression of the one or more polypeptides of interest; and optionally

[0189] C) recovering the one or more polypeptides of interest.

[0190] 35) The method according to claim 34, wherein the at least two polynucleotides encode at least two target polypeptides having the same amino acid sequence, and wherein the at least two target polypeptides are secreted as separate polypeptides having the same amino acid sequence.

[0191] 36) The method according to any one of embodiments 34-35, wherein the one or more polypeptides of interest comprise an enzyme; preferably, the enzyme is selected from the group consisting of: a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase or a transferase; more preferably, an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, an α-glucosidase, a β-glucosidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, a nuclease, an oxidase, a pectinolytic enzyme, a peroxidase, a phosphodiesterase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, a xylanase, and a β-xylosidase; most preferably, the one or more polypeptides of interest are lipases.

[0192] 37) The method according to any one of embodiments 34-36, wherein the fungal host cell is a yeast host cell; preferably, the yeast host cell is selected from the group consisting of: Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia cells; more preferably, the yeast host cell is selected from the group consisting of: Kluyveromyces lactis, Karlsaccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasus, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis and Yarrowia lipolytica cells.

[0193] 38) The method according to any one of embodiments 34-36, wherein the fungal host cell is a filamentous fungal host cell; preferably, the filamentous fungal host cell is selected from the group consisting of: Acremonium, Aspergillus, Brevicaulis, Nicotiana, Ceroplastes, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Ustilagoraceae, Fusarium, Humicola, Pyrrospora, Mucor, Myceliophthora, Neobes, Neurospora, Paecilomyces, Penicillium, and Flat-leaved More preferably, the filamentous fungal host cell is selected from the group consisting of: Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Black-spined tobacco pipe fungus, Dry wax fungus, Carnegie wax fungus, Light yellow wax fungus, Pannohita wax fungus, Annular wax fungus, Red wax fungus, Insect wax fungus, Narrow edge Chrysosporium, keratinophilic chrysosporium, Lukenowens chrysosporium, fecal chrysosporium, rent spore, queen's Ledum chrysosporium, tropical chrysosporium, brown thin chrysosporium, gray capped cap mushroom, hairy leather cap mushroom, rod-shaped fusarium, cereal fusarium, Kuwei fusarium, broadsword fusarium, gramineous fusarium, gramineous fusarium, heterosporic fusarium, albizzia fusarium, sharp spore fusarium, multi-branched fusarium, pink fusarium, elderberry fusarium, skin color fusarium, pseudo-branched fusarium, sulfur-colored fusarium, round fusarium, pseudo-silk spore fusarium, veneer fusarium Preferably, the filamentous fungal host cell is selected from the group consisting of: Aspergillus niger, Aspergillus oryzae, Fusarium venenatum, and Trichoderma reesei; and most preferably, the filamentous fungal host cell is an Aspergillus oryzae cell.

[0194] 39) The method according to any one of embodiments 34-38, wherein the signal peptide has at least 80% sequence identity with SEQ ID NO:2; preferably, the signal peptide comprises or consists of SEQ ID NO:2.

[0195] 40) The method according to any one of embodiments 34-39, wherein the polynucleotide encoding the signal peptide has at least 80% sequence identity with SEQ ID NO: 1; preferably, the polynucleotide encoding the signal peptide comprises or consists of SEQ ID NO: 1.

[0196] 41) The method according to any one of embodiments 34-40, wherein the one or more polypeptides of interest comprise an enzyme; preferably, the enzyme is selected from the group consisting of: a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase or a transferase; more preferably, an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, an α-glucosidase, a β-glucosidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, a nuclease, an oxidase, a pectinolytic enzyme, a peroxidase, a phosphodiesterase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, a xylanase, and a β-xylosidase; most preferably, the one or more polypeptides of interest are lipases.

[0197] 42) The method according to any one of embodiments 34-41, wherein the one or more target polypeptides have at least 80% sequence identity with the mature polypeptide of SEQ IDNO:5; preferably, the one or more target polypeptides comprise or consist of the mature polypeptide of SEQ IDNO:5.

[0198] 43) The method according to any one of embodiments 34-42, wherein the at least two polynucleotides encoding one or more target polypeptides independently have at least 80% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:4; preferably, the at least two polynucleotides encoding one or more target polypeptides comprise or consist of the mature polypeptide coding sequence of SEQ ID NO:4.

[0199] 44) A method according to any one of embodiments 34-43, wherein the linker polypeptide comprises at least 10 amino acids.

[0200] 45) The method according to any one of embodiments 34-44, wherein the proteolytic cleavage site is a binary amino acid motif; preferably, the proteolytic cleavage site is a Lys-Arg motif or an Arg-Arg motif.

[0201] 46) A method according to any one of embodiments 34-45, wherein the proteolytic cleavage site is a KexB cleavage site.

[0202] 47) A method according to any one of embodiments 34-46, wherein the proteolytic cleavage occurs intracellularly, or wherein the proteolytic cleavage occurs extracellularly and during and / or after culturing of the fungal host cell.

[0203] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0204] Examples

[0205] Materials and Methods

[0206] General methods of PCR, cloning, ligating nucleotides, etc. are well known to the person skilled in the art and can be found, for example, in 'Molecular Cloning: A Laboratory Manual', Sambrook et al. (1988), Cold Spring Harbor Lab., Cold Spring Harbor, NY; Ausubel, FM et al. (eds.); 'Current Protocols in Molecular Biology', John Wiley and Sons (1995); Harwood, CR and Cutting, SM (eds.); 'DNA Cloning: A Practical Approach, Volumes I and II', DN Glover, ed. (1985); 'Oligonucleotide Synthesis', MJ Gait, ed. (1984); 'Nucleic Acid Hybridization', BD Hames and SJ Higgins, eds. (1985); 'A Practical Guide To Molecular Cloning [A Practical Guide to Molecular Cloning]', B. Perbal (1984).

[0207] Chemicals used as buffers and substrates were commercial products of at least reagent grade.

[0208] Aspergillus transformation

[0209] Aspergillus transformation was performed as described in US 9,487,767. Transformants that had repaired the target niaD gene and contained the pyrG gene were selected for the ability to grow on a basal plate containing nitrate as a nitrogen source and thiamine (Cove D.J., 1966. Biochem. Biophys. Acta [Biological Chemistry and Biophysics Journal] 113: 51-56). After growing for 5-7 days at 30°C, stable transformants appeared to grow vigorously and sporulate the colonies. Transformants were purified by conidia production.

[0210] Strain culture

[0211] Use method well known in the art to cultivate transformed cells in a nutrient medium suitable for producing lipase protein. For example, shake flask culture (wherein inoculating 10mL YPD medium (2g / L yeast extract, 2g / L peptone and 2% glucose) with spores from transformant and incubating 4 days at 30 ℃) can be carried out in a suitable medium and under the condition that allows lipase protein to be expressed and recovered) and in a laboratory or industrial fermenter, small-scale or large-scale fermentation (including, for example, batch or fed-batch fermentation) is carried out to cultivate cells. Using procedures as known in the art, cultivation occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable medium can be obtained from commercial suppliers or can be prepared according to disclosed composition. Lipase protein is secreted into the nutrient medium, and can be directly recovered from this medium.

[0212] In-Fusion Cloning

[0213] In-Fusion cloning was performed using the In-Fusion Cloning Kit and Manual provided by Clontech Laboratories, Inc.

[0214] Determination of copy number by ddPCR

[0215] BioRad QX200 was used according to the manufacturer. TM Droplet Generator and QX200 TM Droplet reader, using BioradQuantaSoft TM Version 1.7.4.0917, using oliC as a single copy gene, and determining copy number by ddPCR.

[0216] SDS-PAGE

[0217] Using Criterion TM Protein production was visualized by SDS-PAGE analysis with XT precast gels, 10% Bis-Tris (from BIO-RAD), and gels were run as recommended by the manufacturer and stained with Coomassie blue.

[0218] Plasmids

[0219] pAT652 is described in Example 1.

[0220] pAT1509 is described in Example 1.

[0221] strain

[0222] The Aspergillus oryzae host strain was derived from BECh2 described in WO 2000 / 39322.

[0223] Example 1. Increasing lipase production in Aspergillus oryzae using an expression cassette containing two lipase molecules in one mRNA (tandem construct)

[0224] The aim of this experiment was to construct a plasmid for expressing the lipase lipVR originally isolated from Polypodium vulgare (WO 2018 / 150021, mature polypeptide of SEQ ID NO: 5) as a tandem construct in an Aspergillus oryzae strain, i.e., containing two lipase molecules ( Figure 1 The rationale for this experiment is that if removal of the signal peptide and translocation of the newly synthesized polypeptide to the endoplasmic reticulum (ER) is the rate-limiting step for protein secretion in fungi, then coupling a signal peptide to two or more polypeptides of interest should help increase secretion.

[0225] 1A. Construction of pAT652 and pAT1509 for expression of lipVR as single lines or in tandem

[0226] The aim of this experiment was to construct a plasmid for expressing lipVR in an Aspergillus oryzae strain (WO 2018 / 150021).

[0227] By using In-Fusion The lipVR gene (SEQ ID NO: 3) amplified using primers lipVR-A (SEQ ID NO: 7) and lipVR-B (SEQ ID NO: 8) was cloned into a NotI+AsiSI digested plasmid derived from pCOls1175 (WO 2013 / 178674) to construct plasmid pAT652 ( Figure 2 ).

[0228] Plasmid pAT1509 contains the following elements downstream of the Pna2 promoter sequence: 1) the native lipVR gene lacking a stop codon (wherein the native signal peptide sequence is replaced by the signal peptide sequence of the cutinase pre-pro-sequence (CPP), followed by a KexB cleavage sequence (encoding the amino acids Lys-Arg), a new DNA sequence encoding the cutinase Pro sequence without the pro-region, and finally a second codon-optimized lipVR gene (optimized for Aspergillus oryzae and lacking introns) ( Figure 3The lipVR tandem expression cassette (SEQ ID NO: 9) was constructed into a NotI+AsiSI digested plasmid derived from pCOls1175 (WO 2013 / 178674) by splicing of overlap extension amplification using primers lipVR-C (SEQ ID NO: 10) and lipVR-D (SEQ ID NO: 11) (Higuchi et al., 1988; Horton et al., 2013).

[0229] 1B. Construction and analysis of A. oryzae strains expressing lipVR as a single line or in tandem in multiple copies

[0230] Plasmids pAT652 (single) and pAT1509 (tandem) were used individually to transform an Aspergillus oryzae host strain, and transformants were selected for the ability to grow on sucrose medium supplemented with sodium nitrate (as a nitrogen source) and thiamine (to downregulate expression of the pyrG marker to increase copy number preference) but lacking uridine (to select for pyrG complementation).

[0231] Selected transformants were cultured in 10 mL YPD at 30 °C for 4 days and the supernatants were analyzed for lipase production by SDS-PAGE. The identified lipase-producing transformants were genetically characterized for the copy number of the introduced expression construct and selected for fermentation. Strain AT969 contained 18 copies of the single-line lipVR cassette inserted into the genome (or 1 SP / LipVR unit), while strain AT1684 contained 9 copies of the tandem lipVR cassette, so 18 LipVR "units" were made of 9 tandem molecules with a single SP (or 1 SP / 2 LipVR units). Samples collected from the fermentation on day 7 were analyzed for lipase production by SDS-PAGE and lipolytic activity assay ( Figure 4 As shown, the secretion of LipVR in the tandem strain AT1684 was significantly increased compared to the single-line strain AT969 (75% increase in lipolytic activity, Figure 4 ).

[0232] These results strongly suggest that the use of a tandem lipVR expression cassette can significantly increase the yield of secreted and functionally active enzyme under strong expression conditions at relevant high copy numbers in a production strain background.

[0233] Example 2. Increased enzyme production and conditions for correct processing of enzyme units

[0234] The aim of this experiment was to investigate whether Aspergillus oryzae strains (like AT1684) constructed using multiple copies of a tandem expression cassette and a suitable promoter (e.g. the Pna2 promoter) (WO 2012 / 160093) could be used not only to increase enzyme production (see Example 1), but also to ensure the correct processing of a given polypeptide of interest, thereby enabling the method to be used in industrial enzyme production, where it is important that the produced enzyme molecules are functional and identical to ensure uniform activity.

[0235] LipVR is a 30 kDa protein that runs at an apparent molecular weight of 40 kDa. Fermentations of AT1684 were performed at two different temperatures (30°C and 34°C) and two different pH values ​​(6.5 and 7.4). MS analysis of the culture supernatants was performed to investigate the composition of LipVR-derived molecules. Figure 5 As shown in , correct processing of LipVR was high in all conditions tested after 7 days of culture. Notably, complete processing of LipVR was obtained at pH 6.5, regardless of the growth temperature, providing evidence that these growth conditions are sufficient to process the tandem protein without, for example, overexpressing KexB. The exact conditions (temperature, pH, etc.) required for complete processing of KexB will depend on the fungal host cell chosen. The skilled person will be able to optimize the conditions for any given fungal host cell of choice using methods known in the art.

[0236] in conclusion

[0237] When comparing A. oryzae strains containing the same number of copies of the same polypeptide of interest, configured as one signal peptide - one polypeptide of interest (single line) or one signal peptide - two polypeptides of interest (tandem), a two-fold increase in polypeptide yield was observed in the tandem configuration. In addition, the use of the nucleic acid constructs of the present invention resulted in correct and uniform processing of each copy of the polypeptide of interest under different growth conditions. These results provide clear evidence that significant increases in the yield of industrial functional enzymes can be obtained using genetic constructions comprising one SP and more than one CDS of the enzyme of interest, which are separated by protease cleavage recognition sites. In addition, we demonstrated that correct and complete processing of the polypeptide can lead to the production of a single functional LipVR molecule.

[0238] References:

[0239] Aviram N,Schuldiner M(2017). Targeting and translocation of proteins to the endoplasmic reticulum at a glance. J Cell Sci 130:4079-4085.

[0240] Higuchi R, Krummel B, Saiki R (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16(15):7351-7367.

[0241] Horton RM,Cai Z,Ho SN,and Pease LR(2013).Gene Splicing by OverlapExtension:Tailor-Made Genes Using the Polymerase Chain Reaction.BioTechniques54(3):129-133.

[0242] Voss M, B, Fluhrer R(2013).Mechanism,specificity andphysiology of signal peptide peptidase(SPP)and SPP-like proteases.BiochBiophys Acta 1828:2828-2839. Sequence Listing <110> Novozymes A / S <120> Tandem protein expression <130> 14646-WO-PCT <160> 11 <170> PatentIn Version 3.5 <210> 1 <211> 105 <212> DNA <213> Artificial sequence <220> <223> DNA sequence of CPP used as signal peptide <400> 1 atgaagttct tcaccaccat cctcagcacc gccagccttg ttgctgctct ccccgccgct 60 gttgactcga accatacccc ggccgctcct gaacttgttg cccgg 105 <210> 2 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of CPP used as signal peptide <400> 2 Met Lys Phe Phe Thr Thr Ile Leu Ser Thr Ala Ser Leu Val Ala Ala 1 5 10 15 Leu Pro Ala Ala Val Asp Ser Asn His Thr Pro Ala Ala Pro Glu Leu 20 25 30 Val Ala Arg 35 <210> 3 <211> 1059 <212> DNA <213> Polyceras rubrum <400> 3 atgaagtccg cttcgatctt actcagggta gctgccctcc tcctccctgc tgtatctgca 60 ctgccacttg aaagaagagg tatggacgaa ctatcctagc gatcagtgtg tctattttgc 120 ctaacctagc aaagctatat ccgcggatct cctggcaacc ttcagcctct tcgagcagtt 180 cgcagccgca gcatattgtc cggataacaa cgacagtccc gacaccaagc ttacttgctc 240 tgtcggaaac tgcccgcttg tcgaagctga cacgaccagc acggtcactg aattcgaaaa 300 gtacatctta cacgaccccg ttcacctaca gacaaagtcc cagctaacgt ccacctctat 360 ctctgtccct ttagctcgct cgaaaccgac gtcactggct acgtcgcgac tgacagcaca 420 cgagagctca tcgttgtggc attccgcggg agttcctcga tccggaactg gatcgccgac 480 atcgactttc ccttcaccga caccgacctc tgcgatggct gccaggcagc ctcgggcttc 540 tggacgtcct ggacggaggc acggacaggg gtgctggcgg cggtggcgag cgctgccgcg 600 gccaacccgt cctataccgt tgccgtgacg ggccacagcc tcggcggggc cgtggccgcg 660 ctggccgctg gcgccctccg gaacgcgggc tacacggtcg cgctatacag cttcggagcg 720 cctcgcgtgg gtgacgagac cctcagcgag tacatcactg cgcaggcggg tggaaactac 780 cgcatcacgc acctcaacga cccagtgccg aagctgcccc cgctgctcct ggggtatcgc 840 cacatcagcc cggaatacta catcagcagc gggaacaacg tgaccgtgac ggcggatgac 900 gtggaggagt acaccggcac gatcaacctg agtgggaaca cgggcgatct gacgttcgac 960 acggatgcgc acagttggta cttcaacgag atcggggcat gcgatgatgg tgaggctttg 1020 gagtggaaga agcggggggt agaagttcag tgggtttaa 1059 <210> 4 <211> 930 <212> DNA <213> Artificial sequence <220> <223> cDNA sequence of LipVR gene <220> <221> CDS <222> (1)..(927) <220> <221> Signal peptide <222> (1)..(60) <220> <221> Mature peptide <222> (61)..(927) <400> 4 atg aag tcc gct tcg atc tta ctc agg gta gct gcc ctc ctc ctc cct 48 Met Lys Ser Ala Ser Ile Leu Leu Arg Val Ala Ala Leu Leu Leu Pro -20 -15 -10 -5 gct gta tct gca ctg cca ctt gaa aga aga gct ata tcc gcg gat ctc 96 Ala Val Ser Ala Leu Pro Leu Glu Arg Arg Ala Ile Ser Ala Asp Leu -1 1 5 10 ctg gca acc ttc agc ctc ttc gag cag ttc gca gcc gca gca tat tgt 144 Leu Ala Thr Phe Ser Leu Phe Glu Gln Phe Ala Ala Ala Ala Tyr Cys 15 20 25 ccg gat aac aac gac agt ccc gac acc aag ctt act tgc tct gtc gga 192 Pro Asp Asn Asn Asp Ser Pro Asp Thr Lys Leu Thr Cys Ser Val Gly 30 35 40 aac tgc ccg ctt gtc gaa gct gac acg acc agc acg gtc act gaa ttc 240 Asn Cys Pro Leu Val Glu Ala Asp Thr Thr Ser Thr Val Thr Glu Phe 45 50 55 60 gaa aac tcg ctc gaa acc gac gtc act ggc tac gtc gcg act gac agc 288 Glu Asn Ser Leu Glu Thr Asp Val Thr Gly Tyr Val Ala Thr Asp Ser 65 70 75 aca cga gag ctc atc gtt gtg gca ttc cgc ggg agt tcc tcg atc cgg 336 Thr Arg Glu Leu Ile Val Val Ala Phe Arg Gly Ser Ser Ser Ile Arg 80 85 90 aac tgg atc gcc gac atc gac ttt ccc ttc acc gac acc gac ctc tgc 384 Asn Trp Ile Ala Asp Ile Asp Phe Pro Phe Thr Asp Thr Asp Leu Cys 95 100 105 gat ggc tgc cag gca gcc tcg ggc ttc tgg acg tcc tgg acg gag gca 432 Asp Gly Cys Gln Ala Ala Ser Gly Phe Trp Thr Ser Trp Thr Glu Ala 110 115 120 cgg aca ggg gtg ctg gcg gcg gtg gcg agc gct gcc gcg gcc aac ccg 480 Arg Thr Gly Val Leu Ala Ala Val Ala Ser Ala Ala Ala Ala Asn Pro 125 130 135 140 tcc tat acc gtt gcc gtg acg ggc cac agc ctc ggc ggg gcc gtg gcc 528 Ser Tyr Thr Val Ala Val Thr Gly His Ser Leu Gly Gly Ala Val Ala 145 150 155 gcg ctg gcc gct ggc gcc ctc cgg aac gcg ggc tac acg gtc gcg cta 576 Ala Leu Ala Ala Gly Ala Leu Arg Asn Ala Gly Tyr Thr Val Ala Leu 160 165 170 tac agc ttc gga gcg cct cgc gtg ggt gac gag acc ctc agc gag tac 624 Tyr Ser Phe Gly Ala Pro Arg Val Gly Asp Glu Thr Leu Ser Glu Tyr 175 180 185 atc act gcg cag gcg ggt gga aac tac cgc atc acg cac ctc aac gac 672 Ile Thr Ala Gln Ala Gly Gly Asn Tyr Arg Ile Thr His Leu Asn Asp 190 195 200 cca gtg ccg aag ctg ccc ccg ctg ctc ctg ggg tat cgc cac atc agc 720 Pro Val Pro Lys Leu Pro Pro Leu Leu Leu Gly Tyr Arg His Ile Ser 205 210 215 220 ccg gaa tac tac atc agc agc ggg aac aac gtg acc gtg acg gcg gat 768 Pro Glu Tyr Tyr Ile Ser Ser Gly Asn Asn Val Thr Val Thr Ala Asp 225 230 235 gac gtg gag gag tac acc ggc acg atc aac ctg agt ggg aac acg ggc 816 Asp Val Glu Glu Tyr Thr Gly Thr Ile Asn Leu Ser Gly Asn Thr Gly 240 245 250 gat ctg acg ttc gac acg gat gcg cac agt tgg tac ttc aac gag atc 864 Asp Leu Thr Phe Asp Thr Asp Ala His Ser Trp Tyr Phe Asn Glu Ile 255 260 265 ggg gca tgc gat gat ggt gag gct ttg gag tgg aag aag cgg ggg gta 912 Gly Ala Cys Asp Asp Gly Glu Ala Leu Glu Trp Lys Lys Arg Gly Val 270 275 280 gaa gtt cag tgg gtt taa 930 Glu Val Gln Trp Val 285 <210> 5 <211> 309 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 5 Met Lys Ser Ala Ser Ile Leu Leu Arg Val Ala Ala Leu Leu Leu Pro -20 -15 -10 -5 Ala Val Ser Ala Leu Pro Leu Glu Arg Arg Ala Ile Ser Ala Asp Leu -1 1 5 10 Leu Ala Thr Phe Ser Leu Phe Glu Gln Phe Ala Ala Ala Ala Tyr Cys 15 20 25 Pro Asp Asn Asn Asp Ser Pro Asp Thr Lys Leu Thr Cys Ser Val Gly 30 35 40 Asn Cys Pro Leu Val Glu Ala Asp Thr Thr Ser Thr Val Thr Glu Phe 45 50 55 60 Glu Asn Ser Leu Glu Thr Asp Val Thr Gly Tyr Val Ala Thr Asp Ser 65 70 75 Thr Arg Glu Leu Ile Val Val Ala Phe Arg Gly Ser Ser Ser Ile Arg 80 85 90 Asn Trp Ile Ala Asp Ile Asp Phe Pro Phe Thr Asp Thr Asp Leu Cys 95 100 105 Asp Gly Cys Gln Ala Ala Ser Gly Phe Trp Thr Ser Trp Thr Glu Ala 110 115 120 Arg Thr Gly Val Leu Ala Ala Val Ala Ser Ala Ala Ala Ala Asn Pro 125 130 135 140 Ser Tyr Thr Val Ala Val Thr Gly His Ser Leu Gly Gly Ala Val Ala 145 150 155 Ala Leu Ala Ala Gly Ala Leu Arg Asn Ala Gly Tyr Thr Val Ala Leu 160 165 170 Tyr Ser Phe Gly Ala Pro Arg Val Gly Asp Glu Thr Leu Ser Glu Tyr 175 180 185 Ile Thr Ala Gln Ala Gly Gly Asn Tyr Arg Ile Thr His Leu Asn Asp 190 195 200 Pro Val Pro Lys Leu Pro Pro Leu Leu Leu Gly Tyr Arg His Ile Ser 205 210 215 220 Pro Glu Tyr Tyr Ile Ser Ser Gly Asn Asn Val Thr Val Thr Ala Asp 225 230 235 Asp Val Glu Glu Tyr Thr Gly Thr Ile Asn Leu Ser Gly Asn Thr Gly 240 245 250 Asp Leu Thr Phe Asp Thr Asp Ala His Ser Trp Tyr Phe Asn Glu Ile 255 260 265 Gly Ala Cys Asp Asp Gly Glu Ala Leu Glu Trp Lys Lys Arg Gly Val 270 275 280 Glu Val Gln Trp Val 285 <210> 6 <211> 289 <212> PRT <213> Rhodotorula glutinis <400> 6 Leu Pro Leu Glu Arg Arg Ala Ile Ser Ala Asp Leu Leu Ala Thr Phe 1 5 10 15 Ser Leu Phe Glu Gln Phe Ala Ala Ala Ala Tyr Cys Pro Asp Asn Asn 20 25 30 Asp Ser Pro Asp Thr Lys Leu Thr Cys Ser Val Gly Asn Cys Pro Leu 35 40 45 Val Glu Ala Asp Thr Thr Ser Thr Val Thr Glu Phe Glu Asn Ser Leu 50 55 60 Glu Thr Asp Val Thr Gly Tyr Val Ala Thr Asp Ser Thr Arg Glu Leu 65 70 75 80 Ile Val Val Ala Phe Arg Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala 85 90 95 Asp Ile Asp Phe Pro Phe Thr Asp Thr Asp Leu Cys Asp Gly Cys Gln 100 105 110 Ala Ala Ser Gly Phe Trp Thr Ser Trp Thr Glu Ala Arg Thr Gly Val 115 120 125 Leu Ala Ala Val Ala Ser Ala Ala Ala Ala Asn Pro Ser Tyr Thr Val 130 135 140 Ala Val Thr Gly His Ser Leu Gly Gly Ala Val Ala Ala Leu Ala Ala 145 150 155 160 Gly Ala Leu Arg Asn Ala Gly Tyr Thr Val Ala Leu Tyr Ser Phe Gly 165 170 175 Ala Pro Arg Val Gly Asp Glu Thr Leu Ser Glu Tyr Ile Thr Ala Gln 180 185 190 Ala Gly Gly Asn Tyr Arg Ile Thr His Leu Asn Asp Pro Val Pro Lys 195 200 205 Leu Pro Pro Leu Leu Leu Gly Tyr Arg His Ile Ser Pro Glu Tyr Tyr 210 215 220 Ile Ser Ser Gly Asn Asn Val Thr Val Thr Ala Asp Asp Val Glu Glu 225 230 235 240 Tyr Thr Gly Thr Ile Asn Leu Ser Gly Asn Thr Gly Asp Leu Thr Phe 245 250 255 Asp Thr Asp Ala His Ser Trp Tyr Phe Asn Glu Ile Gly Ala Cys Asp 260 265 270 Asp Gly Glu Ala Leu Glu Trp Lys Lys Arg Gly Val Glu Val Gln Trp 275 280 285 Val <210> 7 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Primer lipVR-A <400> 7 tacacaactg ggggccacca tgaagtccgc ttcgatctta c 41 <210> 8 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primer lipVR-B <400> 8 gtgtcagtca ccgcgttaaa cccactgaac ttctaccc 38 <210> 9 <211> 2028 <212> DNA <213> Artificial sequence <220> <223> lipVR-concatenated DNA sequences <400> 9 atgaagttct tcaccaccat cctcagcacc gccagccttg ttgctgctct ccccgccgct 60 gttgactcga accatacccc ggccgctcct gaacttgttg cccggctgcc acttgaaaga 120 agaggtatgg acgaactatc ctagcgatca gtgtgtctat tttgcctaac ctagcaaagc 180 tatatccgcg gatctcctgg caaccttcag cctcttcgag cagttcgcag ccgcagcata 240 ttgtccggat aacaacgaca gtcccgacac caagcttact tgctctgtcg gaaactgccc 300 gcttgtcgaa gctgacacga ccagcacggt cactgaattc gaaaagtaca tcttacacga 360 ccccgttcac ctacagacaa agtcccagct aacgtccacc tctatctctg tccctttagc 420 tcgctcgaaa ccgacgtcac tggctacgtc gcgactgaca gcacacgaga gctcatcgtt 480 gtggcattcc gcgggagttc ctcgatccgg aactggatcg ccgacatcga ctttcccttc 540 accgacaccg acctctgcga tggctgccag gcagcctcgg gcttctggac gtcctggacg 600 gaggcacgga caggggtgct ggcggcggtg gcgagcgctg ccgcggccaa cccgtcctat 660 accgttgccg tgacgggcca cagcctcggc ggggccgtgg ccgcgctggc cgctggcgcc 720 ctccggaacg cgggctacac ggtcgcgcta tacagcttcg gagcgcctcg cgtgggtgac 780 gagaccctca gcgagtacat cactgcgcag gcgggtggaa actaccgcat cacgcacctc 840 aacgacccag tgccgaagct gcccccgctg ctcctggggt atcgccacat cagcccggaa 900 tactacatca gcagcggga caacgtgacc gtgacggcgg atgacgtgga ggagtacacc ggcacgatca acctgagtgg gaacacgggc gatctgacgt tcgacacgga tgcgcacagt tggtacttca acgagatcgg ggcatgcgat gatggtgagg ctttggagtg gaagaagcgg 1080 ggggtagaag ttcagtgggt taaacgagcc gctgttgact cgaaccatac cccggccgct cctgaacttg ttgcccggtt gcctttgga aggagggcga tttccgcaga cctcctcgcg accttctcct tgttcgaaca gttcgcagca gcagcctact gtcccgacaa caacgactcc cctgatacta agttgacatg ttcggtcggc aactgtcctt tggtcgaagc cgacacaaca tcgaccgtga cagagttcga gaactccctc gagacagacg tcacgggtta tgtggcaacc gattccacgc gtgagctcat cgtcgtcgcc ttcaggggat cctcgtccat ccggaactgg 1440 attgccgaca tcgatttccc cttcaccgat accgatctct gtgatggttg tcaggcagca tcgggcttct ggacctcgtg gacagaagcc cgaacaggtg tgctcgcagc cgtggcgtcc gcagcagcag cgaacccctc ctacacggtg gcagtgacag gccattcgtt gggtggtgcg gtcgcagccc tcgcagcagg tgccttgagg aacgcaggct acacagtggc gttgtactcc 1680 ttcggtgcac ctcgggtcgg cgacgagaca ttgtcggagt atatcactgc gcaggcaggt 1740 ggcaactaca ggattactca cctcaacgac ccggtcccta agctccctcc gttgctcttg 1800 ggctatcgac acatttcgcc tgagtattac atctcgtccg gcaacaacgt cacggtgaca 1860 gccgatgatg tggaggagta cactggaaca atcaacttgt cgggtaacac aggcgatttg 1920 accttcgaca cagacgccca ctcgtggtat ttcaacgaga ttggtgcgtg tgatgatggc 1980 gaggcattgg aatggaagaa acgcggagtc gaagtgcagt gggtctaa 2028 <210> 10 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer lipVR-C <400> 10 tacacaactg ggggccacca tgaagttctt caccaccatc c 41 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer lipVR-D <400> 11 gtgtcagtca ccgcgttaga cccactgcac ttcgac 36

Claims

1. A nucleic acid construct comprising a heterologous promoter operably linked to: a) a polynucleotide encoding a signal peptide; and b) at least two polynucleotides encoding one or more polypeptides of interest; wherein the at least two polynucleotides encoding one or more polypeptides of interest are each separated by a linker polynucleotide encoding a linker polypeptide comprising a proteolytic cleavage site; wherein the signal peptide, the one or more polypeptides of interest and the one or more linker polypeptides comprising a proteolytic cleavage site are encoded in-frame as a single polypeptide; and The at least two polynucleotides encode at least two polypeptides of interest consisting of the same amino acid sequence, and the at least two polypeptides of interest are secreted as separate polypeptides consisting of the same amino acid sequence. 2 . The nucleic acid construct according to claim 1 , wherein the signal peptide has at least 80% sequence identity with SEQ ID NO:

2. 3 .

3. The nucleic acid construct according to claim 2, wherein the signal peptide comprises or consists of SEQ ID NO:

2. 4 . The nucleic acid construct according to claim 1 , wherein the polynucleotide encoding the signal peptide has at least 80% sequence identity with SEQ ID NO:

1. 5 . The nucleic acid construct according to claim 4 , wherein the polynucleotide encoding the signal peptide comprises or consists of SEQ ID NO:

1.

6. The nucleic acid construct according to any one of the preceding claims, wherein the one or more polypeptides of interest comprise an enzyme.

7. The nucleic acid construct according to claim 6, wherein the enzyme is selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase or a transferase.

8. The nucleic acid construct according to claim 7, wherein the enzyme is selected from the group consisting of carbohydrases, chitinases, cutinases, esterases, invertases, laccases, nucleases, oxidases, pectinolytic enzymes, phytases, and proteolytic enzymes.

9. The nucleic acid construct according to claim 6, wherein the one or more polypeptides of interest are lipases.

10. The nucleic acid construct according to any one of the preceding claims, wherein the one or more polypeptides of interest have at least 80% sequence identity with the mature polypeptide of SEQ ID NO:

5.

11. The nucleic acid construct according to claim 10, wherein the one or more polypeptides of interest comprise or consist of the mature polypeptide of SEQ ID NO:

5.

12. The nucleic acid construct according to any one of the preceding claims, wherein the at least two polynucleotides encoding one or more polypeptides of interest independently have at least 80% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:

4.

13. The nucleic acid construct according to claim 12, wherein the at least two polynucleotides encoding one or more polypeptides of interest comprise or consist of the mature polypeptide coding sequence of SEQ ID NO:

4.

14. The nucleic acid construct according to any one of the preceding claims, wherein the linker polypeptide comprises at least 10 amino acids.

15. The nucleic acid construct according to any one of the preceding claims, wherein the proteolytic cleavage site is a dibasic amino acid motif.

16. The nucleic acid construct according to claim 15, wherein the proteolytic cleavage site is a Lys-Arg motif or an Arg-Arg motif.

17. The nucleic acid construct according to any one of the preceding claims, wherein the proteolytic cleavage site is a KexB cleavage site.

18. The nucleic acid construct according to claim 7, wherein the enzyme is selected from the group consisting of aminopeptidase, amylase, carboxypeptidase, cellulase, cyclodextrin glycosyltransferase, deoxyribonuclease, α-galactosidase, β-galactosidase, lipase, mannosidase, mutanase, peroxidase, phosphodiesterase, polyphenol oxidase, ribonuclease, transglutaminase, xylanase, and β-xylosidase.

19. The nucleic acid construct according to claim 7, wherein the enzyme is selected from the group consisting of catalase, cellobiohydrolase, alpha-glucosidase, beta-glucosidase, endoglucanase, and glucoamylase.

20. An expression vector comprising the nucleic acid construct according to any one of claims 1-19.

21. A fungal host cell comprising the nucleic acid construct according to any one of claims 1 to 19 and / or the expression vector according to claim 20.

22. The fungal host cell of claim 21, which is a yeast host cell.

23. The fungal host cell of claim 22, wherein the yeast host cell is selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia cells.

24. The fungal host cell of claim 22, wherein the yeast host cell is selected from the group consisting of Saccharomyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia lipolytica cells.

25. The fungal host cell of claim 21, which is a filamentous fungal host cell.

26. The fungal host cell of claim 25, wherein the filamentous fungal host cell is selected from the group consisting of Acremonium, Aspergillus, Pullulan, Nicotiana, Ceroplasts, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Ustilagoraceae, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neotrichia, Neurospora, Paecilomyces, Penicillium, Pseudoceratium, Radius, Chytrid, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolylcorrhiza, Trametes, and Trichoderma cells.

27. The fungal host cell of claim 25, wherein the filamentous fungal host cell is selected from the group consisting of: Aspergillus awamori, Aspergillus odoratus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus niger, Pseudomonas aeruginosa, Pseudomonas carnegiei, Pseudomonas flavoceae, Pseudomonas pannoxita, Pseudomonas annularis, Pseudomonas erythrorhizium, Pseudomonas wormii, Chrysosporium stenospermum, Chrysosporium keratinum, Chrysosporium lukenowensi, Chrysosporium feces-like, Chrysosporium rent, Chrysosporium queen, Chrysosporium tropicalis, Chrysosporium brown thin, Coprinus cinereus, and Chrysosporium pubescens. The invention relates to the invention to a kind of bacteria, rod-shaped fusarium, cereal fusarium, kuwei fusarium, broad-sword fusarium, graminearum fusarium, gram red fusarium, heterosporic fusarium, albizzia fusarium, oxysporic fusarium, multi-branched fusarium, pink fusarium, elder fusarium, skin-colored fusarium, pseudo-branched fusarium, sulfur-colored fusarium, round fusarium, pseudo-sporic fusarium, embellished fusarium, Humicola insolens, Humicola lanuginosa, Mucor miehei, thermophilic myceliophthora, Neurospora crassa, Penicillium purpurogenum, Pleurotus eryngii, terrestrial thaliana, long-domain hairy Trametes, color-changing Trametes, Trichoderma harzianum, Trichoderma koningii, long-branched Trichoderma, Trichoderma reesei, and Trichoderma viride cells.

28. The fungal host cell of claim 25, wherein the filamentous fungal host cell is selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Fusarium venenatum, and Trichoderma reesei.

29. The fungal host cell of claim 25, wherein the filamentous fungal host cell is an Aspergillus oryzae cell.

30. A method for producing one or more polypeptides of interest, the method comprising: a) providing a fungal host cell according to any one of claims 21 to 29; b) culturing the host cell under conditions conducive to the expression of the one or more polypeptides of interest; and optionally c) recovering the one or more polypeptides of interest.

31. The method of claim 30, wherein the one or more polypeptides of interest comprise an enzyme.

32. The method of claim 31, wherein the enzyme is selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase or a transferase.

33. The method of claim 31, wherein the enzyme is selected from the group consisting of carbohydrase, chitinase, cutinase, esterase, invertase, laccase, nuclease, oxidase, pectinolytic enzyme, phytase, and proteolytic enzyme.

34. The method of claim 31, wherein the one or more polypeptides of interest are lipases.

35. The method according to claim 31, wherein the enzyme is selected from the group consisting of aminopeptidase, amylase, carboxypeptidase, cellulase, cyclodextrin glycosyltransferase, deoxyribonuclease, α-galactosidase, β-galactosidase, lipase, mannosidase, mutanase, peroxidase, phosphodiesterase, polyphenol oxidase, ribonuclease, transglutaminase, xylanase, and β-xylosidase.

36. The method according to claim 31, wherein the enzyme is selected from the group consisting of catalase, cellobiohydrolase, alpha-glucosidase, beta-glucosidase, endoglucanase, and glucoamylase.

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