WO2025226596A1

WO2025226596A1 - Methods for producing secreted polypeptides

Info

Publication number: WO2025226596A1
Application number: PCT/US2025/025607
Authority: WO
Inventors: Jonathan PHAM; Kirk EHMSEN; Sandeep SHARMA KHATIWADA; Michael Lynge NIELSEN
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2024-04-24
Filing date: 2025-04-21
Publication date: 2025-10-30
Anticipated expiration: 2026-10-24

Abstract

The present invention relates to nucleic acid constructs comprising a first polynucleotide encoding a signal peptide, and a second polynucleotide encoding a polypeptide of interest; expression vectors and host cells comprising said nucleic acid constructs; methods for producing a polypeptide of interest; and fusion proteins comprising the polypeptide of interest and a signal peptide.

Description

METHODS FOR PRODUCING SECRETED POLYPEPTIDES

Reference to a Sequence Listing

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

Background of the Invention

Field of the Invention

Description of the Related Art

Product development in industrial biotechnology includes a continuous challenge to increase recombinant protein yields at large scale to reduce costs. Two major approaches have been used for this purpose in the last decades. The first one 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 increments in yield. High-throughput screening enables large numbers of mutants to be screened in search forthe desired phenotype, i.e., higher recombinant protein yields. The second approach includes numerous strategies ranging from the use of stronger promoters and multi-copy 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 a given protein may in turn trigger several bottlenecks in the cellular machinery for secretion of the enzyme of interest into the medium, emphasizing the need for further optimization strategies.

Signal peptides (SPs) are short amino acid sequences present in the amino terminus of many newly synthesized polypeptides that target these into or across cellular membranes, thereby aiding maturation and secretion. The amino acid sequence of the SP influences secretion efficiency and thereby the yield of the polypeptide manufacturing process. Bioinformatic tools such as SignalP and SignalP5 can predict SPs from amino acid sequences, but most cannot distinguish between various types of SPs (Armenteros et al., Nat. Biotechnol. 37: 420-423, 2019). Moreover, a large degree of redundancy in the amino acid sequence of SPs makes it difficult to predict the efficiency of any given SP for production of recombinant proteins at industrial scale. There are no tools to predict the efficiency with which a given SP directs the secretion of a target protein of interest (POI) (Owji et al., Eur. J. Cell Biol. 2018, 97, 422-441). In fact, finding an efficient SP to secrete a POI is currently still based on trial and error. It is established that the SP-POI match plays a crucial role in determining secretion efficiency (Peng, C. et al., Front. Bioeng. Biotechnol. 2019, 7, 139) whereas the underlying fundamental parameters remain unknown. Hence, SP selection is an important step for manufacturing of recombinant proteins, but the optimal combination of signal peptide and mature protein is very context dependent and not easy to predict.

Although there are expression systems available, there is a need for increasing yields during recombinant production of proteins. Taking recombinant lactoferrin as an example, there is a major challenge, i.e., lactoferrin having several disulfide linkages which makes correct expression, folding and secretion difficult. Thus, in order to satisfy the growing demand for recombinant lactoferrin it is necessary to provide recombinant expression systems with increased lactoferrin yields.

Summary of the Invention

The present invention is based on the surprising and inventive finding that expression of difficult- to-express proteins (e.g., lactoferrin) with novel signal peptides provides increased yield when expressed in fungal host cells.

Using the signal peptides of the invention an improved yield of the lactoferrin product was observed, e.g., using the SP sequences of SEQ ID NO: 3, 6, 9, and 1 1 (see Tables 1 - 3).

Notably, the increased expression was achieved using several, different fermentation protocols at different scales, including microtiter plates (MTP), micro-bioreactors, and glass tank fermentation.

In a first aspect, the present invention relates to nucleic acid constructs comprising: a first polynucleotide encoding a signal peptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to any of the amino acid sequences of SEQ ID NOs: 3, 6, 9, or 11 ; and a second polynucleotide encoding a polypeptide of interest; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion, and wherein the first polynucleotide and the second polynucleotide are heterologous to another.

In a second aspect, the invention relates to expression vectors comprising a nucleic acid construct according to the first aspect.

In a third aspect, the invention relates to fungal host cells comprising in its genome: a) A nucleic acid construct according to the first aspect, and/or b) An expression vector according to the second aspect.

In a fourth aspect, the invention relates to methods of producing a polypeptide of interest, the method comprising: a) cultivating a host cell according to the third aspect under conditions conducive for production of the polypeptide of interest; and optionally b) recovering the polypeptide of interest. In a fifth aspect, the invention relates to fusion polypeptides, comprising: a signal peptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 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 to any one of SEQ ID NOs: 3, 6, 9, or 11 , and a lactoferrin polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 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 to SEQ ID NO: 13.

SEQUENCE OVERVIEW

SEQ ID NO:1 is the TREMBL_U9V5V1_synthetic signal peptide coding sequence

SEQ ID NO:2 is the TREMBL_U9V5V1_plect_int signal peptide coding sequence

SEQ ID NO:3 is the TREMBLJJ9V5V1 signal peptide sequence

SEQ ID NO:4 is the TREMBL_A0A015K2K4_synthetic signal peptide coding sequence

SEQ ID NO:5 is the TREMBL_A0A015K2K4_plect_int signal peptide coding sequence

SEQ ID NO:6 is the TREMBL_A0A015K2K4 signal peptide sequence

SEQ ID NO:7 is the TREMBL_A0A015IUP6_synthetic signal peptide coding sequence

SEQ ID NO:8 is the TREMBL_A0A015IUP6_plect_int signal peptide coding sequence

SEQ ID NO:9 is the TREMBL_A0A015IUP6 signal peptide sequence

SEQ ID NO:10 is the AoPlectasinPrePro_SP signal peptide coding sequence

SEQ ID NO:11 is the AoPlectasinPrePro_SP signal peptide sequence

SEQ ID NO: 12 is the pectjntron coding sequence

SEQ ID NO: 13 is the bovine lactoferrin amino acid sequence

SEQ ID NO: 14 is the bovine lactoferrin DNA sequence

Definitions

In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Lactoferrin: The term “lactoferrin”, “LF”, or “lactoferrin polypeptide” means a polypeptide comprising iron-binding capabilities, including human lactoferrin and bovine lactoferrin. LF is categorized under EC 3.4.21 . LF comprises at least three different isoforms: (i) LF-alpha which is the iron-binding form, and (ii) LF-beta and (iii) LF-gamma. Both, LF-beta and LF-gamma are associated with ribonuclease activity (E.C. 3.1.21.1) and with reduced iron-binding activity, or with no iron-binding activity. Also, the term lactoferrin comprises hololactoferrin (iron-rich forms) and apolactoferrin (iron-free forms). LF quantification can be carried out as described with the aptamer-based assay described in the section “Examples”.

A non-limiting example of a lactoferrin polypeptide is the bovine lactoferring polypeptide of SEQ ID NOs: 13. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

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

Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

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

Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

Extension: The term “extension” means an addition of one or more amino acids to the amino and/or carboxyl terminus of a polypeptide, wherein the “extended” is a lactoferrin polypeptide which comprises iron-binding capabilities. Persons skilled in the art will know that a polypeptide having a given amino acid sequence and enzymatic activity may be produced with one or a few additional amino acids at the N- and/or C-terminus, and that such a polypeptide can have essentially the same enzyme activity. Such extended polypeptides are intended to be encompassed by the present invention.

Fragment: The term “fragment” as used in the context of a polypeptide means a polypeptide having one or more amino acids absent from its amino and/or carboxyl terminus, wherein the fragment is a lactoferrin fragment which comprises iron-binding capabilities. The fragment may be produced naturally during expression and/or purification of the polypeptide, or may be the result of expression of a modified nucleotide sequence expressing the fragment or of targeted removal of amino acids from the amino and/or carboxy terminus.

Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and/or the C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras etal., 1991 , Biotechnology 9: 378-381 ; Eaton etal., 1986, Biochemistry 25: 505-512; Collins- Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest e.g., an amylase) has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following translation and any post-translational modifications such as N-terminal processing (e.g. removal of signal peptide), C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (/.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g. having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide. Mature polypeptides of the invention may therefore have slight differences at the N- and/or C-terminal due to such differentiated expression by the host cell. A mature polypeptide having one or more amino acids absent from the N- and/or C-terminal may be considered to be a “fragment” of the full-length polypeptide.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature lactoferrin polypeptide.

Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

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

Obtained polypeptide/peptide/polynucleotide: The term “obtained” or “derived” when used in reference to a polynucleotide sequence, lactoferrin sequence, polypeptide sequence, variant sequence or signal peptide sequence, means that the molecule originally has been isolated from the given source and that the molecule can either be utilized in its native sequence or that the molecule is modified by methods known to the skilled person.

Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.

Parent: The term “parent” means a polypeptide functioning as a signal peptide, or a lactoferrin polypeptide, to which an alteration is made to produce variants of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof. Lactoferrin quantification: For the purpose of the present invention, lactoferrin is quantified using the assays described in the Examples.

Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

Subsequence: The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having lactoferrin activity.

Variant: The term “variant” means a polypeptide having lactoferrin activity, comprising a man-made mutation, i.e., a substitution, insertion (including extension), and/or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally-occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

Detailed Description of the Invention

The present invention is based on the surprising and inventive finding that expression of difficult- to-express proteins (e.g., lactoferrin) with the disclosed novel signal peptides provides increased yield in fungal host cells.

Using of the novel signal peptides of the invention, an improved yield of lactoferrin is achieved.

Polynucleotides

The present invention also relates to polynucleotides encoding a polypeptide of the present invention, as described herein.

The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof. The polynucleotide may be cloned from a strain of Rhizophagus, e.g., Rhizophagus irregularis, or a related organism and thus, for example, may be a polynucleotide sequence encoding a variant of the polypeptide of the invention.

In one embodiment the polynucleotide encoding the signal peptide of the present invention is isolated from an Rhizophagus cell, such as an Rhizophagus irregularis cell.

The polynucleotide may be cloned from a strain of Aspergillus, e.g., Aspergillus oryzae, or a related organism and thus, for example, may be a polynucleotide sequence encoding a variant of the polypeptide of the invention.

In one embodiment the polynucleotide encoding the signal peptide of the present invention is isolated from an Aspergillus cell, such as an Aspergillus oryzae cell.

The polynucleotide may also be mutated by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2 95-107.

In an aspect, the polynucleotide is isolated.

In another aspect, the polynucleotide is purified. Nucleic Acid Constructs

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

In a first aspect, the invention relates to nucleic acid constructs comprising: a first polynucleotide encoding a signal peptide comprising or consisting of an amino acid sequence having a sequence identity of at least 80% to any of the amino acid sequences of SEQ ID NOs: 3, 6, 9, or 11 ; and a second polynucleotide encoding a polypeptide of interest; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion, and wherein the first polynucleotide and the second polynucleotide are heterologous to another.

In one embodiment, the second polynucleotide is located downstream from the first polynucleotide.

In one embodiment, the signal peptide is a naturally occurring signal peptide, or a functional fragment or functional variant of a naturally occurring signal peptide.

In one embodiment, the signal peptide is from a filamentous fungal cell.

In one embodiment, the first polynucleotide comprises a third polynucleotide.

In one embodiment, the third polynucleotide is a non-coding intron.

In one embodiment, the third polynucleotide has a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO:12.

In one embodiment, the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, the second polynucleotide, and optionally the third polynucleotide, are operably linked.

In one embodiment, the promoter is an amylase promoter, preferably the heterologous promoter is an Aspergillus neutral amylase II promoter, e.g. the Aspergillus niger neutral amylase II promoter.

In one embodiment, the promoter is an Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi).

In one embodiment, the promoter is operably linked to an mRNA stabilizer region; preferably the mRNA stabilizer region is the cry I II A mRNA stabilizer region.

In one embodiment, the signal peptide is a naturally occurring signal peptide, or a functional fragment or functional variant of a naturally occurring signal peptide. In one embodiment, the signal peptide is obtained from a polypeptide expressed by a Rhizophagus host cell, such as an Rhizophagus irregularis.

In one embodiment, the signal peptide is obtained from a polypeptide expressed by a Aspergillus host cell, such as an Aspergillus oryzae.

In one embodiment, the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO:1 ; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 1.

In one embodiment, the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 2; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 2.

In one embodiment, the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 4; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 4.

In one embodiment, the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 5; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 5.

In one embodiment, the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 7; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 7.

In one embodiment, the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 8; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 8.

In one embodiment, the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 10; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 10.

In one embodiment, the signal peptide is obtained from a Rhizophagus cell.

In one embodiment, the signal peptide is obtained from a Rhizophagus irregularis cell.

In one embodiment, the signal peptide is obtained from a Aspergillus cell.

In one embodiment, the signal peptide is obtained from a Aspergillus oryzae cell.

In one embodiment, the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO: 3.

In one embodiment, the signal peptide comprises, consists essentially of, or consists of SEQ ID NO: 3.

In one embodiment, the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO: 6.

In one embodiment, the signal peptide comprises, consists essentially of, or consists of SEQ ID NO: 6.

In one embodiment, the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO: 9.

In one embodiment, the signal peptide comprises, consists essentially of, or consists of SEQ ID NO: 9.

In one embodiment, the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO: 1 1 .

In one embodiment, the signal peptide comprises, consists essentially of, or consists of SEQ ID NO: 11 .

In one embodiment, the signal peptide consists of the amino acid sequence of any one of SEQ ID NOs: 1 , 3, 6, 9, or 11 with or without its C-terminal alanine, or a peptide fragment thereof that retains the ability to direct the polypeptide into or across a cell membrane.

In one embodiment, the N- and/or C-terminal end of the signal peptide has been extended by addition of one or more amino acids. In one embodiment, the signal peptide is a fragment of the signal peptides of any of any of the foregoing embodiments.

In one embodiment, the second polynucleotide encoding the polypeptide of interest has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, 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%, to the mature polypeptide coding sequence of SEQ ID NO:14; most preferably the second polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:14.

In one embodiment, the polypeptide of interest is a lactoferrin, e.g., a bovine lactoferrin, or a human lactoferrin.

In one embodiment, the one or more polypeptide of interest is a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

In one embodiment, the polypeptide of interest is a bovine lactoferrin.

In one embodiment, the N- and/or C-terminal end of the polypeptide of interest has been extended by addition of one or more amino acids.

In one embodiment, the polypeptide of interest is a fragment of the polypeptide of interest of any of the foregoing embodiments.

It is expected that the invention will be just as effective when employing a signal peptide that is highly similar to the signal peptide disclosed in SEQ ID NOs: 3, 6, 9, or 11 , encoded by any one of SEQ ID NOs: 1 , 2, 4, 5, 7, 8, or 10, respectively. One or more non-essential amino acids may, for example, be altered. Non-essential amino acids in a signal peptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for signal peptide activity to identify amino acid residues that are critical to the activity of the molecule and residues that are non-essential. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The identity of essential and non-essential amino acids can also be inferred from an alignment with one or more related signal peptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g. Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner e a/., 1988, DNA 7: 127).

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

In one aspect, the signal peptide is a variant (/.e., functional variant) or fragment (/.e., functional fragment) of the signal peptide of any one of SEQ ID NOs: 3, 6, 9, or 11. In one aspect, the number of alterations in the signal peptide variant of the present invention is 1-10, e.g., 1-5, such as 1 , 2, 3, 4, or 5 alterations. Alterations includes substitutions, insertions, and/or deletions at one or more (e.g., several) positions compared to the parent. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

In a preferred embodiment, the signal peptide is a variant of the mature polypeptide of any one of SEQ ID NOs: 3, 6, 9, or 11 comprising 1-10 alterations, e.g., 1-5, such as 1 , 2, 3, 4, or 5 alterations, compared to SEQ ID NOs: 3, 6, 9, or 11 respectively.

The first and second polynucleotide are operably linked in translational fusion. In the context of the present invention, the term “operably linked in translation fusion” means that the signal peptide encoded by the first polynucleotide and the polypeptide encoded by the second polynucleotide are encoded in frame and translated together as a single polypeptide. Preferably, following translation, the signal peptide is removed to provide the mature polypeptide of interest. Alternatively, the signal peptide is not removed, or only removed partly to provide the mature polypeptide of interest.

The first and second polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a nucleic acid construct or expression vector may be desirable or necessary depending on the construct or vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

Besides a signal peptide, the nucleic acid constructs of the invention may be operably linked to one or more further control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

Promoters

The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. In one embodiment, the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, and the second polynucleotide are operably linked. The promoter is orientated upstream of the first polynucleotide.

In an embodiment, the promoter is a heterologous promoter. Preferably, the promoter is a tandem promoter. More preferably, the promoter is a P3 promoter or a P3-based promoter.

Further suitable promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma'. Biology and Applications”, and by Schmoll and Dattenbock, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

For expression in a yeast host, examples of useful promoters are described by Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae), and by Schmoll and Dattenbock, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

In one embodiment, the promoter is a promoter, such as a P3 promoter, operably linked to an mRNA stabilizer region. Preferably, the mRNA stabilizer region is the cry I II A mRNA stabilizer region. mRNA Stabilizers

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

Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471). Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce// 5(11): 1838-1846.

Terminators

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

Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma'. Biology and Applications”, and by Schmoll and Dattenbock, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology. Preferred terminators for yeast host cells may be 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, Yeast 8: 423-488.

Propeptides

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

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. Additionally or alternatively, when both signal peptide and propeptide sequences are present, the polypeptide may comprise only a part of the signal peptide sequence and/or only a part of the propeptide sequence. Alternatively, the final or isolated polypeptide may comprise a mixture of mature polypeptides and polypeptides which comprise, either partly or in full length, a propeptide sequence and/or a signal peptide sequence.

Regulatory Sequences

It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In fungal systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.

Leader Sequences

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

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

Suitable leaders for yeast host cells may be 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).

Transcription Factors

The control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. The transcription factor may function alone and/or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor. The transcription factor may regulate the expression of a protein of interest either directly, i.e. by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, i.e. by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., Subcell Biochem 2011 ; 52:7-23, as well in Balleza et al., FEMS Microbiol Rev 2009, 33(1 ): 133-151 .

Polyadenylation Sequences

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

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

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

Expression Vectors

In a second aspect, the present invention also relates to recombinant expression vectors comprising a nucleic acid construct according to the first aspect. The expression vectors comprise a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

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

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

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

For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non-homologous recombination, such as non- homologous end-joining (NHEJ).

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

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

Host Cells

In a third aspect, the invention relates to fungal host cells comprising in its genome: a) a nucleic acid construct according to the first aspect; and/or b) an expression vector according to the second aspect.

A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The polypeptide encoded by the introduced polynucleotide can be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may comprise a single copy, or at least two copies, e.g. three, four, five or more copies of the polynucleotide of the present invention.

In one embodiment, the host cell comprises two or more copies of the nucleic acid construct and/or the expression vector.

The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Christensen et al., 1988, Bio/TechnologyB'. 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.

The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). For purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell (Komagataella phaffii).

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

The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

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

In one embodiment the host cell is an Aspergillus cell.

In another embodiment, the host cell is an Aspergillus niger cell.

In one embodiment, the host cell is an Aspergillus oryzae cell.

In an aspect, the host cell is isolated.

In one embodiment, the host cell comprises at least two copies of the nucleic acid construct and/or the expression vector, such as two copies, three copies, four copies or more than four copies.

In another aspect, the host cell is purified.

Methods of Production

In a fourth aspect, the present invention also relates methods of producing a polypeptide of interest, the method comprising: a) cultivating a host cell according to the third aspect under conditions conducive for production of the polypeptide of interest; and optionally b) recovering the polypeptide of interest.

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

The polypeptide may be detected using methods known in the art that are specific for the polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an assay determining the relative or specific activity of the polypeptide.

The polypeptide may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the polypeptide is recovered. In another aspect, a cell- free fermentation broth comprising the polypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and/or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

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

Fusion Polypeptide

In a fifth aspect, the invention relates to a fusion polypeptide, comprising a) a signal peptide comprising or consisting of an amino acid sequence having at least 60% sequence identity to any one of SEQ ID NOs: 3, 6, 9, or 11 , and b) a lactoferrin polypeptide comprising or consisting of an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 13.

In one embodiment, the polypeptide of interest comprises a lactoferrin polypeptide comprising or consisting of an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 13.

In one embodiment, the signal peptide is located upstream of the polypeptide of interest.

In one embodiment, the signal peptide is located at the N-terminal end of the polypeptide of interest.

In one embodiment, the lactoferrin polypeptide is selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 13;

(b) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 14;

(c) a polypeptide derived from SEQ ID NO: 13, having 1 -30 alterations, e.g., substitutions, deletions and/or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(d) a polypeptide derived from the polypeptide of (a), (b), or (c) wherein the N- and/or C- terminal end has been extended by addition of one or more amino acids; (e) a fragment of the polypeptide of (a), (b), (c), or (d), and

(f) the fragment of (e) wherein the polypeptide has 1 - 10 deletions at the N-terminal end.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide of interest, e.g., a fusion protein according to the fifth aspect and/or a cell according to the third aspect. The fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the nucleic acid constructs of the present invention which are used to produce the polypeptide having phytase activity), cell debris, biomass, fermentation media and/or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.

The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.

In some embodiments, the fermentation broth formulation or the cell composition comprises a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris. In some embodiments, the killed cells and/or cell debris are removed from a cell- killed whole broth to provide a composition that is free of these components.

The fermentation broth formulation or cell composition may further comprise a preservative and/or anti-microbial (e.g. , bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or cell composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.

The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Examples

Example 1 : Signal peptide library for expression of bovine lactoferrin

Signal peptide constructs for the expression of bovine lactoferrin (bLF) (SEQ ID NO: 13) were prepared in a single pooled transformation approach. 285 different signal peptides were ordered as a single pool and PCR amplified. The signal peptides were designed both with and without the presence of the intron shown in SEQ ID NO: 12. For expression cassette, three fragments were prepared separately and transformed into Aspergillus oryzae via the direct overlay recombination system using homologous recombination. The first fragment contained part of the niaD locus. The second fragment consisted of the pooled signal peptides with the ends homologous to the first and the third fragment. The third fragment consisted of the bovine lactoferrin gene and part of the niiA locus. In the transformation (Aspergillus oryzae host CQLS1300), the bLF expression cassette along with the amplified pool of signal peptides were integrated using a CRISPR system. We used the promoter of the Aspergillus niger neutral amylase II fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi). The transformants growing on selection plates were pooled and spores were FACS sorted into individual wells of 32 96-welled plates to obtain around 2816 individual transformants through single spores. The strains generated with this method comprise a single copy of the expression cassette in the genome, confirmed by amplicon sequencing.

Example 2: Signal peptides showing expression of bovine lactoferrin

Using MTP, all 2816 strains were screened for production of bovine lactoferrin. The strains were cultivated in expression medium for 4 days. The supernatant from the samples was filtered and bovine lactoferrin was quantified with aptamer-based assay that gives millipolarization values corresponding to expression of bovine lactoferrin. As shown in Table 1 , we obtained 6 strains that showed high milipolarization values which correlated with bovine lactoferrin expression. Notably, using the native lactoferrin signal peptide no lactoferrin could be quantified (data not shown).

As also shown in Table 1 , lactoferrin expression is not directly dependent on the absence or presence of the intron with SEQ ID NO: 12. Table 1. Milipolarization values for signal peptides showing expression of bovine lactoferrin in Aspergillus oryzae. Example 3: Expression of bovine lactoferrin confirmed in glass tank fermentation

The 1-copy strains showing expression of bovine lactoferrin in MTP assay were selected for fermentations in glass tanks under current optimized conditions. The expression of bovine lactoferrin with the selected signal peptides was compared with signal peptide of SEQ ID NO: 9. A further signal peptide (SEQ ID NO: 11) encoded by SEQ ID NO: 10 was added to the experiment, cloned according to Example 1 and comprising the intron of SEQ ID NO: 12. Expression of bovine lactoferrin was quantified using Labchip. As shown in Table 2, all four tested selected signal peptide (SP) sequences resulted in bLF expression. Out of the four tested SP sequences, the SP with SEQ ID NO: 3 (without intron sequence) resulted in the highest bLF expression. SP sequences SEQ ID NO: 6 (without intron) and SEQ ID NO: 11 (with intron) achieved similarly high bLF expression yields.

Table 2. Relative bLF expression in glass tanks using different signal peptides.

Example 4: Increased bLF yield in micro-bioreactors

A. oryzae multicopy strains were generated starting from a parental strain comprising the IreA mutation described in WO 2018/015443 A1 . The expression cassettes were integrated into the strain genome using the method described in WO 2013/178674.

These strains express bLF either with the SP of SEQ ID NO:9 (encoded by SEQ ID NO:7) or the SP of SEQ ID NO:11 (encoded by SEQ ID NO:10), either comprising 2 copies of the expression cassette, or 3 copies (see Table 3). Strains were cultivated in micro-bioreactors each with a cultivation volume of 250 ml. After 7 days of cultivation bLF yield was measured by LabChip® (Revvity) with results shown in Table 3. The bLF yields of Table 3 were normalized to the bLF yield of strains comprising the SP with SEQ ID NO: 11 . Also, the bLF yield was normalized to the copy number of expression vector copies for each strain. As shown in Table 3, for multicopy strains the SP of SEQ ID NO: 9 shows 69-75% increased bLF yield compared to the yield of strains with the SP of SEQ ID NO: 11 .

Table 3. Relative bLF expression in micro-bioreactors using different signal peptides.

Materials and Methods

General PCR was performed using standard methods of molecular biology as described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology", John Wiley and Sons, 1995; Harwood, C. R., and Cut-ting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990.

Medium

COVE trace metals solution was composed of 0.04 g of NaB4O7»10H2O, 0.4 g of CuSO4»5H2O, 1.2 g of FeSO4«7H2O, 0.7 g of MnSO4«H2O, 0.8 g of Na2MoO2«2H20, 10 g of ZnSO4«7H2O, and deionized water to 1 liter.

50X COVE salts solution was composed of 26 g of KCI, 26 g of MgSO4»7H2O, 76 g of KH2PO4, 50 ml of COVE trace metals solution, and deionized water to 1 liter.

COVE-Sucrose+ 10 mM Sodium nitrate agar was composed of 342.3 g of sucrose, 20 ml of 50X COVE salts solution, 0.85 g of NaNO3, 20 g of Noble Agar, and deionized water to 1 liter.

STC buffer was composed of 0.8 M sorbitol, 25 mM Tris pH 8, and 25 mM CaCI2.

TAE buffer was composed of 4.84 g of Tris Base, 1 .14 ml of Glacial acetic acid, 2 ml of 0.5 M EDTA pH 8.0, and deionized water to 1 liter.

YP broth with 2% Glucose is composed of 10 g of Bacto Yeast Extract, 20 g of Bacto Peptone, 20 g Glucose and deionized water to 1 liter.

Aspergillus oryzae seed medium was composed of 18 g of yeast extract, 21 g of glycerol, 1 ml of pluronic acid, 0.6 g of Urea and deionized water to 1 liter.

MDU2-C media is composed of 45 g of Maltose Monohydrate, 1 g of MgSO4.7H2O, 1 g of NaCI, 2 g of K2SO4, 2 g of KH2PO4, 2 g of Urea, 0.5 ml of AMG trace elements and deionized water to 1 liter.

Transformation of Aspergillus oryzae

Transformation of Aspergillus species can be achieved using the general methods for yeast transformation. Aspergillus oryzae host strain was inoculated to 100 ml of YP + 2% Glucose medium medium supplemented with 10 mM urea and incubated for 16-24 h at 30°C at 160 rpm. Tissue was harvested by filtration and washed with 0.6 M MgSO4, and resuspended 10 ml protoplasting solution containing a commercial beta-glucanase product (GLUCANEX™, Novozymes A/S, Bagsvaerd, Denmark) at a final concentration of 20 mg per ml and chitinase (Sigma). The suspension was incubated at 34°C at 80 rpm until protoplasts were formed, and then washed twice with STC buffer. The protoplasts were counted with a hematometer and resuspended to a final concentration of 2 x10⁷ protoplasts/ml. Approximately 2 micrograms of DNA fragments and MAD7 plasmid were added to 100 pl of the protoplast suspension, mixed gently. Approximately 250 ul of poly-ethylene solution (PEG) was added to the mixture and mixed gently by swirling. The protoplast, DNA and PEG mixture were incubated at 37 C for 30 minutes. After incubation, 1 ml of STC was added to the transformation mixture and were spread onto Cove + sucrose + NaNO3 plates and incubated at 30 C for 7-10 days for colonies to appear.

Sorting transformed Aspergillus spores

After the colonies formed on selection plates, the spores were collected by flooding plates with 0.01 % Tween-20 and scraping plates lightly with a spreader. All the spores from different plates were collected and pooled into single tube. A Sony FACS sorter was used to sort individual spores into 96-welled plates containing Cove + sucrose + NaNO3.

PCR amplifications

Polymerase Chain Reaction (PCR) was carried out with Phusion Hot Start II DNA polymerase (Thermo Fisher). The PCR reaction mixtures is shown in table 4 below and the corresponding PCR cycle is shown in in table 5.

Table 4. Phusion Hot Start II PCR reaction mix:

Table 5. Phusion Hot Start II PCR program MTP fermentation

Spores of the selected transformants were inoculated in 0.15 mL of Ao seed medium, in 96 well MTP and cultivated at 30°C for 2 days. 1 ml of MDU2-C medium was added to each well after 2 days and cultivated at 30° C for 4 days at 350 RPM.

Lab tank fermentation

Fermentation was done as fed-batch fermentation (H. Pedersen 2000, Appl Microbiol Biotechnol, 53: 272-277). Selected strains were pre-cultured in Ao seed medium then grown mycelia were transferred to the tanks for further cultivation of protein production. Cultivation was done at pH 4 to 7, 30 to 34°C, for 6~8 days with the feeding of maltodextrin without over-dosing. Culture supernatant after centrifugation was used for yield evaluation.

Quantification of bovine lactoferrin using aptamers

Quantification of bovine lactoferrin in the strains was performed with aptamers from a previously published method by Zhu C. et al., (Taianta, 2019;205:120088. doi: 10.1016/j.talanta.2019.06.088. Epub 2019 Jun 27. PMID: 31450439). Supernatants from the MTP assay were taken for quantification of bovine lactoferrin. Bovine lactoferrin was quantified based on the milipolarization values of the aptamers.

Quantification of bovine lactoferrin using LabChip® (digital SDS-PAGE)

• Supernatants were prepared, keeping the samples frozen in between time points; storage in a standard microplate.

• Samples were thawed and then mixed with wide bore pipet itps, then diluted 10X with 1X PBS

• Samples were filtered using a 0.45 um PES milli-pore 96w filter plate (1000 x g for 10 min, ambient temperature)

• LabChip sample plate was set up, including blanks (1X PBS) and a standard dilution series

• Samples were prepped via the high sensitivity assay recommendation from Revvity.

• Samples were run using the Protein Express 200 high sensitivity method and the 384w 4 mm sip height parameters on a LabChip® GXII Touch (Revvity).

• Data was analyzed using a smear analysis. The standard curve was fit with a 4PL in JMP and the reverse prediction formula used to determine titers in the supernatants.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. LIST OF EMBODIMENTS

The invention is further defined by the following numbered embodiments:

[1] A nucleic acid construct comprising: a first polynucleotide encoding a signal peptide comprising or consisting of an amino acid sequence having a sequence identity of at least 80% to any of the amino acid sequences of SEQ ID NOs: 3, 6, 9, or 11 ; and a second polynucleotide encoding a polypeptide of interest; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion, and wherein the first polynucleotide and the second polynucleotide are heterologous to another.

[2] The nucleic acid construct according to embodiment 1 , wherein the second polynucleotide is located downstream from the first polynucleotide.

[3] The nucleic acid construct according to any of embodiment 1 or 2, wherein the signal peptide is a naturally occurring signal peptide, or a functional fragment or functional variant of a naturally occurring signal peptide.

[4] The nucleic acid construct according to any of embodiments 1 to 3, wherein the signal peptide is from a filamentous fungal cell.

[4a] The nucleic acid construct according to any of embodiments 1 to 4, wherein the first polynucleotide comprises a third polynucleotide.

[4b] The nucleic acid construct according to any previous embodiment, wherein the third polynucleotide is a non-coding intron.

[4c] The nucleic acid construct according to any previous embodiment, wherein the third polynucleotide has a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO:12.

[5] The nucleic acid construct according to any previous embodiment, wherein the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, the second polynucleotide, and optionally the third polynucleotide, are operably linked.

[6] The nucleic acid construct according to any previous embodiment, wherein the promoter is an amylase promoter, preferably the heterologous promoter is an Aspergillus neutral amylase II promoter, e.g. the Aspergillus niger neutral amylase II promoter.

[6a] The nucleic acid construct according to any previous embodiment, wherein the promoter is an Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi). [7] The nucleic acid construct according to any previous embodiment, wherein the promoter is operably linked to an mRNA stabilizer region; preferably the mRNA stabilizer region is the crylllA mRNA stabilizer region.

[8] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is a naturally occurring signal peptide, or a functional fragment or functional variant of a naturally occurring signal peptide.

[9] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a polypeptide expressed by a Rhizophagus host cell, such as an Rhizophagus irregularis.

[9a] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a polypeptide expressed by a Aspergillus host cell, such as an Aspergillus oryzae.

[10] The nucleic acid construct according to any of the preceding embodiments, wherein the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO:1 ; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 1 .

[11] The nucleic acid construct according to any of the preceding embodiments, wherein the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 2; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 2.

[12] The nucleic acid construct according to any of the preceding embodiments, wherein the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 4; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 4.

[13] The nucleic acid construct according to any of the preceding embodiments, wherein the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 5; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 5.

[14] The nucleic acid construct according to any of the preceding embodiments, wherein the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 7; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 7.

[15] The nucleic acid construct according to any of the preceding embodiments, wherein the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 8; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 8.

[16] The nucleic acid construct according to any of the preceding embodiments, wherein the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 80%, e.g. 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%, to the mature polypeptide coding sequence of SEQ ID NO: 10; most preferably the first polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 10.

[17] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a Rhizophagus cell.

[17a] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a Rhizophagus irregularis cell.

[18] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a Aspergillus cell.

[18a] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a Aspergillus oryzae cell.

[19] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO: 3.

[20] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises, consists essentially of, or consists of SEQ ID NO: 3.

[21] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO: 6.

[22] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises, consists essentially of, or consists of SEQ ID NO: 6. [23] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO: 9.

[24] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises, consists essentially of, or consists of SEQ ID NO: 9.

[25] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 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%, to SEQ ID NO: 11 .

[26] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises, consists essentially of, or consists of SEQ ID NO: 11 .

[27] The nucleic acid construct according to any preceding embodiments, wherein the signal peptide consists of the amino acid sequence of any one of SEQ ID NOs: 1 , 3, 6, 9, or 11 with or without its C- terminal alanine, or a peptide fragment thereof that retains the ability to direct the polypeptide into or across a cell membrane.

[28] The nucleic acid construct according to any of the preceding embodiments, wherein the N- and/or C- terminal end of the signal peptide has been extended by addition of one or more amino acids.

[29] The nucleic acid construct according to any of embodiments 1 to 28, wherein the signal peptide is a fragment of the signal peptides of any of embodiments 1 to 28.

[30] The nucleic acid construct according to any of the preceding embodiments, wherein the second polynucleotide encoding the polypeptide of interest has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, 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%, to the mature polypeptide coding sequence of SEQ ID NO:14; most preferably the second polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:14.

[31] The nucleic acid construct according to any of the preceding embodiments, wherein the polypeptide of interest is a lactoferrin, e.g., a bovine lactoferrin, or a human lactoferrin.

[31 a] The nucleic acid construct according to any of embodiments 1-29, wherein the one or more polypeptide of interest is a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. [32] The nucleic acid construct according to any of the preceding embodiments, wherein the polypeptide of interest is a bovine lactoferrin.

[33] The nucleic acid construct according to any of the preceding embodiments, wherein the N- and/or C- terminal end of the polypeptide of interest has been extended by addition of one or more amino acids.

[34] The nucleic acid construct according to any of embodiments 1 to 33, wherein the polypeptide of interest is a fragment of the polypeptide of interest of any of embodiments 1 to 33.

[35] An expression vector comprising a nucleic acid construct according to any one of embodiments 1 to 34.

[36] A fungal host cell comprising in its genome: a) a nucleic acid construct according to any of embodiments 1 to 34; and/or b) an expression vector according to embodiment 35.

[37] The host cell of embodiment 36, wherein the fungal host cell is a filmentous fungal host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe , Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[38] The host cell of any of embodiments 36 to 37, wherein the host cell is an Aspergillus cell.

[39] The host cell of any of embodiments 36 to 38, wherein the host cell is an Aspergillus niger or Aspergillus oryzae host cell.

[40] The host cell of any of embodiments 36 to 39, wherein the host cell comprises at least two copies of the nucleic acid construct and/or the expression vector, such as two copies, three copies, four copies or more than four copies. [41] A method of producing a polypeptide of interest, the method comprising: a) cultivating a host cell according to any of embodiments 36 to 40 under conditions conducive for production of the polypeptide of interest; and optionally b) recovering the polypeptide of interest.

[42] A fusion polypeptide, comprising a) a signal peptide comprising or consisting of an amino acid sequence having at least 60% sequence identity to any one of SEQ ID NOs: 1 , 3, 6, 9, or 11 , and b) a polypeptide of interest.

[43] The fusion polypeptide of embodiment 42, wherein the polypeptide of interest comprises a lactoferrin polypeptide comprising or consisting of an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 13.

[44] The fusion polypeptide according to any one of embodiments 42 - 43, wherein the signal peptide is located upstream of the polypeptide of interest.

[45] The fusion polypeptide according to any of embodiments 42 to 44, wherein the signal peptide is located at the N-terminal end of the polypeptide of interest.

[46] The fusion polypeptide according to any of embodiments 42 to 45, wherein the lactoferrin polypeptide is selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 13;

(c) a polypeptide derived from SEQ ID NO: 13, having 1 -30 alterations, e.g., substitutions, deletions and/or insertions at one or more positions, e.g. , 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

(d) a polypeptide derived from the polypeptide of (a), (b), or (c) wherein the N- and/or C- terminal end has been extended by addition of one or more amino acids;

(e) a fragment of the polypeptide of (a), (b), (c), or (d), and

Claims

1 . A nucleic acid construct comprising: a first polynucleotide encoding a signal peptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to any of the amino acid sequences of SEQ ID NOs: 3, 6, 9, or 11 ; and a second polynucleotide encoding a polypeptide of interest; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion, and wherein the first polynucleotide and the second polynucleotide are heterologous to another.

2. The nucleic acid construct according to claim 1 , wherein the signal peptide comprises or consists of an amino acid sequence having at least 85%, e.g., 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 to any of the amino acid sequences of SEQ ID NOs: 3, 6, 9, or 11.

3. The nucleic acid construct according to any one of the preceding claims, wherein the first polynuclotide comprises or consists of a polynucleotide having a sequence identity of at least 85%, e.g., 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%, to any one of SEQ ID NOs: 1 , 2, 4, 5, 7, 8, or 10.

4. The nucleic acid construct according to any one of the preceding claims, wherein the polypeptide of interest comprises or consists of a lactoferrin polypeptide.

5. The nucleic acid construct according to any one of the preceding claims, wherein the polypeptide of interest comprises or consists of an amino acid sequence having a sequence identity of at least 85%, e.g. 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%, to SEQ ID NO: 13.

6. An expression vector comprising a nucleic acid construct according to any of claims 1 to 5.

7. A fungal host cell comprising in its genome: a) a nucleic acid construct according to any of claims 1 to 5; and/or b) an expression vector according to claim 6.

8. The fungal host cell of claim 7, wherein the fungal host cell is a filmentous fungal host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum , Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides , Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

9. The fungal host cell of any of claims 7 -8, wherein the cell is an Aspergillus cell, e.g., an Aspergillus niger cell, or an Aspergillus oryzae cell.

10. A method of producing a polypeptide of interest, the method comprising: a) cultivating a host cell according to any one of claims 7 -9 under conditions conducive for production of the polypeptide of interest; and optionally b) recovering the polypeptide of interest.

11. The method according to claim 10, wherein the polypeptide of interest comprises or consists of a lactoferrin polypeptide.

12. A fusion polypeptide, comprising: a signal peptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 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 to any one of SEQ ID NOs: 3, 6, 9, or 11 , and a lactoferrin polypeptide comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 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 to SEQ ID NO: 13.