Signal peptides for increased protein secretion

Abstract

The present invention relates to a nucleic acid molecule encoding a fusion protein comprising a secretion signal comprising (i) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and optionally (ii) an alpha mating factor (MFα) prosequence; and a protein of interest. The present invention further relates to a secretion signal as defined herein, an expression cassette comprising said nucleic acid molecule, and a recombinant eukaryotic host cell comprising said nucleic acid molecule or expression cassette. Also included are methods of producing a protein of interest in a eukaryotic host cell and methods of increasing secretion of a protein of interest from a eukaryotic host cell. Also provided is the use of the secretion signal to increase secretion of a recombinant protein of interest from a eukaryotic host cell and the use of the recombinant host cell to produce a recombinant protein of interest.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to EP patent application No. 21 156 986.8, filed on February 12, 2021, the contents of which are hereby incorporated by reference in their entirety for all purposes. TECHNICAL FIELD OF THEINVENTION The present invention relates to a nucleic acid molecule encoding a fusion protein comprising (i) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and optionally (ii) a secretion signal comprising an alpha mating factor (MFα) pro-sequence and a protein of interest. The present invention further relates to a secretion signal as defined herein, an expression cassette comprising said nucleic acid molecule, and a recombinant eukaryotic host cell comprising said nucleic acid molecule or expression cassette. Also included are methods of producing a protein of interest in a eukaryotic host cell and methods of increasing secretion of a protein of interest from a eukaryotic host cell. Also provided is the use of the secretion signal to increase secretion of a recombinant protein of interest from a eukaryotic host cell and the use of the recombinant host cell to produce a recombinant protein of interest. 2. Background of the Invention

[0002] Yeast in general, and Pichia pastoris (P. pastoris, synonym: Komagataella phaphi) in particular, are popular expression systems for secreting recombinant proteins. The first critical step in secretion is the translocation of the recombinant protein into the endoplasmic reticulum (ER). This process is directed by an N-terminal secretion signal fused to the recombinant protein. The signal sequence specifies the co-translational or post-translational targeting route to the ER over the conventional secretory pathway (Ng et al., 1996). The most commonly used secretion signal in P. pastoris is the Saccharomyces cerevisiae α-mating prepro leader (MFα) (Lin-Cereghino et al., 2013). This signal mediates post-translational translocation in S. cerevisiae and most likely also in P. pastoris (Fitzgerald and Glick, 2014; Ng et al., 1996). Other secretion signals are continually added to the repertoire and tested with different recombinant proteins.

[0003] Since cotranslational translocation may be required for the biosynthesis of many mammalian proteins, the MFα signal sequence may in fact be suboptimal and it would be preferable to use a cotranslational signal sequence (Ng et al., 1996). Today, mammalian antibodies are a major product class in the biopharmaceutical market (Ecker et al., 2015). Antibodies are known to be cotranslationally translocated in their native environment (Feige et al., 2010). There is also a clear trend towards the development of smaller antigen-binding fragments (e.g., Fab, scFv, and VHH) (Nelson and Reichert, 2009; Walsh, 2014). In particular, Fab fragments are sometimes inefficiently secreted and therefore only low production titers are reached (Looser et al., 2015; Pfeffer et al., 2011). This may be due to the post-translational signal sequence MFα, which has already been reported to cause a bottleneck in the translocation (Fitzgerald and Glick, 2014; Zahrl et al., 2018). WO2018165589A2 and WO2018165594 disclose recombinant secretion signals that contain signal peptides other than the MFα proleader derived from Saccharomyces cerevisiae and the MFα presequence derived from Saccharomyces cerevisiae. Fitzgerald et al. (Microb Cell Fact 13, 125(2014)) disclose a hybrid secretion signal consisting of the Ost1 signal sequence followed by the MFα prosequence.

[0004] Consequently, there remains a need for secretion signals that increase the secretion of various proteins, such as antibodies, etc. The technical problem is therefore to meet this need. Summary of the Invention

[0005] This technical problem is solved by the subject matter defined in the claims. The inventors have surprisingly found that the secretion of fusion proteins comprising a signal peptide sequence, signal peptide or presequence (all terms can be used interchangeably) derived from the KRE1 protein (internal name SP14) or SWP1 protein (internal name SP4), optionally in combination with the alpha mating factor (MFα) prosequence, is significantly increased. In other words, proteins comprising the secretion signal of the invention will be secreted at a higher rate, while the secretion signal will be cleaved (see examples 6-8).

[0006] Thus, the present invention relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal comprising: (I) (i) a signal peptide sequence derived from a KRE1 protein or a signal peptide sequence derived from a SWP1 protein; and (ii) α-mating factor (MFα) prosequence; or (II) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and (b) The protein of interest.

[0007] The present invention relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal comprising: (i) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and (ii) the α mating factor (MFα) prosequence; and (b) The protein of interest.

[0008] In particular, the present invention provides a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal comprising: (i) a signal peptide sequence derived from the KRE1 protein; and (ii) the α mating factor (MFα) prosequence; and (b) The protein of interest.

[0009] The present invention also relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal comprising: (i) a signal peptide sequence derived from the SWP1 protein; and (ii) the α mating factor (MFα) prosequence; and (b) The protein of interest.

[0010] The present invention further relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal comprising: (i) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and (b) The protein of interest.

[0011] In particular, the present invention further relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal comprising: (i) a signal peptide sequence derived from the KRE1 protein; and (b) The protein of interest.

[0012] The present invention further relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal comprising: (i) a signal peptide sequence derived from the SWP1 protein; and (b) The protein of interest.

[0013] The present invention further relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal consisting of: (i) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and (b) The protein of interest.

[0014] In particular, the present invention further relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal consisting of: (i) a signal peptide sequence derived from the KRE1 protein; and (b) The protein of interest.

[0015] The present invention further relates to a nucleic acid molecule encoding a fusion protein comprising, from N-terminus to C-terminus: (a) a secretion signal, the secretion signal consisting of: (i) a signal peptide sequence derived from the SWP1 protein; and (b) The protein of interest.

[0016] It is envisioned that the secretion signal results in increased secretion of the protein of interest from a eukaryotic host cell that expresses a nucleic acid molecule defined herein, but which contains a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (such as SEQ ID NO: 4) instead of a secretion signal defined herein.

[0017] The signal peptide sequence derived from the KRE1 protein may comprise SEQ ID NO:1 or a functional homologue thereof. The signal peptide sequence derived from the KRE1 protein may consist of SEQ ID NO:1 or a functional homologue thereof. Specifically, the functional homologue of SEQ ID NO:1 comprises at least 80%, or at least 85%, or at least 90%, or at least 94%, or at least 95% sequence identity with SEQ ID NO:1. Specifically, the functional homologue comprises one, two, or three point mutations compared to SEQ ID NO:1. Specifically, the functional homologue has a signal peptide function in a eukaryotic host cell, such as a fungal host cell or a yeast host cell, such as a Komaratella host cell.

[0018] The signal peptide sequence derived from SWP1 protein may comprise SEQ ID NO:2 or 52, or a functional homologue thereof. The signal peptide sequence derived from SWP1 protein may consist of SEQ ID NO:2 or 52, or a functional homologue thereof. In particular, a functional homologue of SEQ ID NO:2 or SEQ ID NO:52 comprises at least 80%, or at least 85%, or at least 90%, or at least 94%, or at least 95% sequence identity with SEQ ID NO:2 or SEQ ID NO:52. In particular, a functional homologue comprises one, two, or three point mutations compared to SEQ ID NO:2 or SEQ ID NO:52, respectively. In particular, a functional homologue has a signal peptide function in a eukaryotic host cell, such as a fungal host cell or a yeast host cell, such as a Komaratella host cell.

[0019] The MFα prosequence may comprise any one of SEQ ID NOs: 3, 53, or 74-80, or a functional homologue thereof. The MFα prosequence may consist of any one of SEQ ID NOs: 3, 53, 74-80, or a functional homologue thereof, preferably SEQ ID NO: 3 or 53 or a functional homologue thereof. Specifically, a functional homologue of any one of SEQ ID NOs: 3, 53, or 74-80 comprises at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with the respective SEQ ID NOs: 3, 53, or 74-80. Specifically, a functional homologue comprises one, two, or three point mutations compared to the respective SEQ ID NOs: 3, 53, or 74-80. Specifically, a functional homologue has the function of a prosequence in a eukaryotic host cell, such as a fungal host cell or a yeast host cell, such as a Komagataella or Saccharomyces host cell. The MFα prosequence preferably contains Ser at a position corresponding to position 23 of SEQ ID NO:53 and / or Glu at a position corresponding to position 64 of SEQ ID NO:53.

[0020] The protein of interest is selected from the group consisting of an antibody, such as a chimeric, humanized or human antibody, or a bispecific antibody, or an antigen-binding antibody fragment, such as a Fab or F(ab)2, a single chain antibody, such as a scFv, a single domain antibody, such as a camelid VHH fragment or a heavy chain antibody or a domain antibody (dAb), an artificial antigen-binding molecule, such as a DARPIN, an ibody, an affibody, a humabody, or a mutein based on a polypeptide of the lipocalin family, an enzyme, such as a processed enzyme, a cytokine, a growth factor, a hormone, a protein antibiotic, a fusion protein, such as a toxin fusion protein, a structural protein, a regulatory protein, and a vaccine antigen, preferably the protein of interest is a therapeutic protein, a food additive, or a feed additive.

[0021] In another aspect, the present invention relates to a secretion signal as defined herein. In particular, the present invention relates to a secretion signal comprising (i) a signal peptide sequence derived from a KRE1 protein or a signal peptide sequence derived from a SWP1 protein; and (ii) an alpha mating factor (MFα) prosequence. More particularly, the present invention relates to a secretion signal comprising (i) a signal peptide sequence derived from a KRE1 protein and (ii) an alpha mating factor (MFα) prosequence. More particularly, the present invention relates to a secretion signal comprising (i) a signal peptide sequence derived from a SWP1 protein; and (ii) an alpha mating factor (MFα) prosequence. The present invention more particularly relates to a secretion signal comprising a signal peptide sequence derived from a KRE1 protein. Even more particularly, the present invention relates to a secretion signal comprising a signal peptide sequence derived from a SWP1 protein. The present invention further particularly relates to a secretion signal consisting of a signal peptide sequence derived from a KRE1 protein. Even more particularly, the present invention relates to a secretion signal consisting of a signal peptide sequence derived from a SWP1 protein.

[0022] In yet another aspect, the present invention further relates to an expression cassette comprising the nucleic acid molecule of the present invention and a promoter operably linked thereto. The expression cassette may be comprised in a vector, preferably an expression vector, or may be integrated into a chromosome, in particular an artificial chromosome.

[0023] In another aspect, the present invention further provides a recombinant eukaryotic host cell comprising a nucleic acid molecule of the present invention, a vector of the present invention, or an expression cassette of the present invention. It is understood herein that a recombinant eukaryotic host cell engineered with such a nucleic acid molecule or expression cassette is genetically engineered to incorporate the respective nucleic acid molecule, vector, or expression cassette. A recombinant eukaryotic host cell may be genetically engineered to include such a nucleic acid molecule, vector, or expression cassette within the host cell genome.

[0024] The recombinant eukaryotic host cell may be a fungal host cell or a yeast host cell, preferably a yeast host cell selected from the group consisting of Komagataella phaphii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., and Schizosaccharomyces pombe, or a fungal host cell selected from Trichoderma reesei or Aspergillus niger.

[0025] Host cells may be engineered to overexpress one or more components of the signal recognition particle (SRP).

[0026] The invention further relates to a method for producing a protein of interest by culturing a host cell of the invention under conditions to express a nucleic acid molecule of the invention and to secrete the protein of interest upon cleavage of the secretion signal, as well as isolating the protein of interest from the host cell culture, and optionally purifying, and optionally modifying, and optionally formulating the protein of interest.

[0027] In another aspect, the present invention further provides a method for producing a protein of interest in a eukaryotic host cell comprising: (i) genetically engineering a eukaryotic host cell with a nucleic acid molecule of the invention or with an expression cassette or vector of the invention, and optionally genetically engineering the eukaryotic host cell to overexpress one or more components of the signal recognition particle (SRP); (ii) culturing the genetically engineered host cell under conditions that express the nucleic acid molecule and optionally one or more components of SRP and secrete the protein of interest upon cleavage of the secretion signal; (iii) optionally isolating the protein of interest from the cell culture; (iv) optionally purifying the protein of interest; (v) optionally modifying a protein of interest, and (vi) optionally formulating the protein of interest.

[0028] In yet another aspect, the present invention further relates to a method for increasing secretion of a protein of interest from a eukaryotic host cell, comprising expressing a nucleic acid molecule of the present invention in said eukaryotic host cell, and optionally engineering the eukaryotic host cell to overexpress one or more components of a signal recognition particle (SRP), thereby increasing secretion of the protein of interest compared to said host cell expressing a nucleic acid molecule of the present invention, but comprising a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (e.g., SEQ ID NO: 4) instead of a secretion signal described herein.

[0029] Methods for increasing secretion of a protein of interest from a eukaryotic host cell may additionally include: (i) engineering a host cell to incorporate an expression construct that expresses a nucleic acid molecule of the invention, and optionally genetically engineering the host cell to overexpress one or more components of the signal recognition particle (SRP); (ii) culturing a host cell under conditions to express the nucleic acid molecule, and optionally overexpress one or more components of SRP, and secrete the protein of interest upon cleavage of the secretion signal; (iii) optionally isolating the protein of interest from the cell culture; (iv) optionally purifying the protein of interest; (v) optionally modifying a protein of interest, and (vi) optionally formulating the protein of interest.

[0030] The nucleic acid molecule of the invention may be integrated into a chromosome of the host cell or may be comprised in an expression cassette, vector or plasmid, which is not integrated into the chromosome of the host cell.

[0031] In another aspect, the invention further relates to the use of a secretion signal as described herein (e.g., as part of or within a nucleic acid molecule of the invention) to increase secretion of a protein of interest from a eukaryotic host cell, in comparison to said eukaryotic host cell expressing a fusion protein as described herein, wherein the secretion signal comprises a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (e.g., SEQ ID NO: 4) instead of a secretion signal as defined by the invention.

[0032] In yet another aspect, the invention relates to the use of a recombinant host cell of the invention for producing a protein of interest.

[0033] The invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, which show: [Brief description of the drawings]

[0034] [Figure 1] Immunofluorescence anti-His staining of SPx-VHH(His6) clones. A: MFα secretion signal, B: SWP1(SP4), C: KRE1(SP14): Cells were observed under a fluorescent microscope with appropriate filter cubes. Fluorescence, DIC and merged images are shown. Images were adjusted for brightness and contrast. Detailed description of the invention

[0035] The invention is described in detail below and illustrated by the accompanying examples and figures.

[0036] The inventors have surprisingly found that the secretion and yield of fusion proteins comprising the signal peptide (signal peptide sequence below) of KRE1 (also designated herein as SP14) or SWP1 (also designated herein as SP4) is significantly increased, especially when combined with a prosequence, such as the prosequence of alpha-mating factor, thereby forming the secretion signal of the invention (see Examples 6-8), whereas other signal peptides or combinations do not improve the secretion of the protein of interest. Fusion proteins comprising a secretion signal according to the invention are thus secreted more efficiently. The inventors have further found that the secretion and yield of the protein of interest is further increased by overexpressing the protein of the signal recognition particle (SRP) with the addition of the protein (see Examples 6-8).

[0037] As outlined herein above, a fusion protein comprising a secretion signal of the invention and a protein of interest is secreted more efficiently by a recombinant host cell when compared to a eukaryotic host cell expressing a fusion protein as defined herein that comprises, instead of the secretion signal as defined herein, a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal, i.e., the protein of interest contained in the fusion protein of the invention is secreted while the secretion signal is cleaved during secretion. Thus, the present invention surprisingly demonstrates that a fusion protein comprising, from N-terminus to C-terminus, (a) a secretion signal, (i) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and (ii) optionally a secretion signal comprising an alpha-mating factor (MFα) pro-sequence; and (b) a protein of interest, provides superior properties, e.g. increased secretion of the protein of interest (see Examples 6-8).

[0038] The expression "from the N-terminus to the C-terminus" does not necessarily exclude that the secretion signal, the signal peptide sequence and the alpha mating factor (MFα) prosequence, including, as well as the protein of interest, are separated by one or more amino acids. These one or more amino acids may be linkers or linker sequences. A "linker sequence" (also referred to as a "spacer sequence" or "linker") is an amino acid sequence introduced between the secretion signal as defined herein and the protein of interest as defined herein. A "linker sequence" may also be an amino acid sequence introduced between the signal peptide sequence and the alpha mating factor (MFα) prosequence, and / or between the alpha mating factor (MFα) prosequence and the protein of interest. Preferably, however, there is no linker between the signal peptide sequence and the alpha mating factor (MFα) prosequence. There is a large variety of possible linker sequences, and it is within the knowledge of the skilled person to choose an appropriate linker sequence based, for example, on the size, sequence, and physical properties (such as hydrophobicity) of the polypeptide of the invention. The linker sequence may be composed of flexible residues, such as glycine and serine, or rather rigid residues, such as alanine-proline repeats. To ensure maximum flexibility, it would be preferred that the linker sequence does not adopt secondary structures (such as alpha helical structures or beta sheets). The linker sequence may be a protease cleavage site, such as that recognized by a particular protease, for example by members of the subtilisin / kexin-like proprotein convertase (PC) family. The term "linker sequence" or "linker" as used herein refers to any amino acid sequence that does not interfere with the function of the elements to which it is linked. The linker may, for example, connect a nucleotide sequence, or an amino acid sequence. Linkers may be used to engineer a suitable amount of flexibility. Preferably, the linker is short, for example, 1-20 nucleotides or amino acids, or even more, and is typically flexible. Commonly used amino acid linkers consist of multiple glycines, serines, and optionally alanines, in any order.Such linkers typically have a length of at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids, as needed. Preferably, the linker used herein comprises 1-12 amino acid residues, and preferably it is a short linker of up to 5 amino acids. Preferably, the linker used herein is any one of GS, GGSGG, GSAGSAAGSG, (GS)n (where "n" is any number between 1 and 10), GSGSGSG, GSG or GGGGS ("G4S") linkers, or any combination thereof. In some embodiments, the linker comprises one or more units, repeats, or copies of a motif, such as GS, GSG, or G4S.

[0039] Alternatively or additionally, the fusion protein may contain a cleavage site between the MFα pro-sequence and the protein of interest. The fusion protein may also contain one or more additional cleavage sites, for example for cleavage of a tag, as described below. The cleavage site may be a protease cleavage site, such as that recognized by a specific protease. Thus, the term "protease cleavage site" includes a recognition site for a specific protease, which is an amino acid sequence specifically recognized by a protease, and a site between two amino acids of a protein where cleavage occurs. The protease may be selected from the group consisting of TEV (tobacco etch virus) protease, chymotrypsin, enterokinase, pepsin, neutrophil elastase, proteinase K, thermolysin, thrombin, and trypsin. The site at which cleavage occurs by a particular protease is preferably between the N-terminal amino acid of the protein of interest as defined herein and the C-terminal amino acid of the N-terminal fusion tag or of the N-terminal fused alpha mating factor (MFα) pro-sequence as defined herein, or between the C-terminal amino acid of the protein of interest as defined herein and the N-terminal amino acid of the C-terminal fusion tag or of the C-terminal fused alpha mating factor (MFα) pro-sequence as defined herein. The recognition site may be adjacent to the respective site at which cleavage occurs outside (within the tag) of the protein of interest (POI) as defined herein. More particularly, proteases useful for cleavage of the tag are preferably selected from the group consisting of endopeptidases such as factor Xa, thrombin, TEV (tobacco etch virus protease), cysteinyl aspartate-specific proteases (caspases), and enterokinase. Thus, the recognition site used can be selected from the group consisting of factor Xa, thrombin, TEV (tobacco etch virus protease), cysteinyl aspartate-specific protease (caspase), and enterokinase recognition sites, but also any other protease cleavage site for a known protease is useful for cleavage of the tag from the protein. A preferred cleavage site for caspase 2 is VDVAD.

[0040] As shown in the examples, the secretion signal increases the secretion of the fusion protein, or more precisely the protein of interest with the secretion signal cleaved, from the eukaryotic host cell in comparison to a eukaryotic host cell expressing a fusion protein that, within the context of the present invention, contains the wild-type Saccharomyces cerevisiae alpha mating factor secretion signal (such as SEQ ID NO: 4) instead of the secretion signal described herein. Thus, the wild-type S. cerevisiae MF alpha secretion signal (SEQ ID NO: 4) can be used as a control or reference for comparison.

[0041] Due to the (over)expression of the protein of interest as part of a fusion protein encoded by the nucleic acid of the invention and, optionally, also one or more components of SRP, according to the invention, the protein of interest (after cleavage of the POI, the secretion signal and the secretion itself) is obtainable in high yields, even if the biomass is kept low. Thus, the high yields, measured in mg POI / g dry biomass, can be in the range of 1-200, such as 50-200, such as 100-200, in laboratory, pilot and industrial scale. "Increased secretion" as used herein relates to a higher amount of detectable protein of interest in the supernatant or culture medium of the host cell in comparison to a control; both cultured under identical conditions (e.g. host cell type, culture medium, culture time, culture temperature, feeding and induction strategy). The control can be the same host cell, but in the same host cell the protein of interest is expressed as a fusion protein comprising the MFα secretion signal as depicted in SEQ ID NO: 4 instead of the secretion signal of the invention. This increase can be expressed as a fold change (FC) in secretion, e.g., an increase of at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold, or at least 10-fold. The amount of detectable protein of interest in the host cell supernatant or culture medium can be expressed as a volumetric titer in [g protein of interest / L cell culture] or as a yield in [mg protein of interest / g dry cell weight] or the like. The fold change in secretion is then the ratio of the volumetric titer or yield of the culture of the host cells to the volumetric titer or yield of a control culture.

[0042] The fusion protein, or protein of interest, may further comprise one or more (detectable) tags, one or more protease cleavage sites, and / or one or more linkers, for example between and connecting certain elements of the fusion protein (e.g. selected from a secretion signal, a signal peptide sequence, an alpha mating factor (MFα) pro-sequence, or a protein of interest), and / or as part of any one of the elements, in particular as part of the protein of interest. For example, a linker may be positioned between the protein of interest, the tag, and / or the cleavage site. Thus, when a fusion protein of the invention consists, from N-terminus to C-terminus, of a secretion signal as defined herein and also of a protein of interest as defined herein, such a protein of interest may optionally further comprise one or more (detectable) tags, one or more protease cleavage sites, and / or one or more linkers. In other words, one or more tags, one or more cleavage sites and / or one or more linkers as defined herein may also be fused to such a protein of interest at the N- or C-terminus, thus also consisting of the protein of interest of the invention, when the fusion protein of the invention consists, from N-terminus to C-terminus, of a secretion signal and of the protein of interest of the invention as described herein. In this context, such one or more tags, one or more cleavage sites and / or one or more linkers fused N-terminus to such a protein of interest may be part of such a protein of interest. Also, when the fusion protein of the invention consists, from N-terminus to C-terminus, of a secretion signal as defined herein and of a protein of interest as also defined herein, the secretion signal as defined herein may optionally further comprise one or more (detectable) tags, one or more protease cleavage sites and / or one or more linkers.In this context, such tag(s), protease cleavage site(s) and / or linker(s) may be fused either to the N-terminus or C-terminus of such secretion signal, or to the respective signal peptide sequence defined herein, or to the alpha mating factor (MFα) prosequence defined herein, and may then be part of such secretion signal.

[0043] Linkers are defined, for example, in paragraph

[38] . A (detectable) tag can be a tag used for purification of a protein of interest and / or to enhance expression and / or solubility or detection. There are many purification tags, expression-enhancing tags, solubility-enhancing tags, as well as tags that allow easy detection and quantification of a protein of interest, known to the skilled artisan. A "purification tag" (also called an "affinity tag") is an amino acid sequence that can be used, for example, for purification of a protein to which it is attached (e.g., a protein of interest that contains an affinity tag at its N-terminus). This tag has a high affinity for a suitable ligand of a solid support, such as a chromatography resin, or directly to the resin. By selectively binding a protein of interest that contains a purification tag to a specific resin, the protein of interest can be highly effectively purified by only one chromatography step. Purification tags are known to those skilled in the art and can be protein purification tags, preferably GST tags, FLAG tags, polyarginine tags, polyhistidine tags, such as 6His tags, MBP tags, S tags, influenza virus HA tags, thioredoxin tags, or Staphylococcus protein A tags, etc. More particularly, the purification tag sequence used herein is any one of histidine (His) tags, preferably polyhistidine tags, such as hexahistidine (6His) tags; arginine tags, preferably polyarginine tags, peptide substrates for antibodies, chitin binding domains, RNAse S peptide, Protein A, S-galactosidase, FLAG tags, Strep II tags, streptavidin binding peptide (SBP) tags, calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), S tags, HA tags, c-Myc tags, or any other tags known to be useful for efficient purification of fused proteins. Specifically, fusion proteins, especially proteins of interest that contain a poly- or hexahistidine tag (His tag), can be captured and purified by IMAC, preferably using Ni-NTA chromatography material.In a preferred embodiment of the present invention, a polyhistidine tag, or a hexahistidine tag (His tag), even more preferably a 6-His tag, is comprised by the fusion protein or by the protein of interest as defined herein above and applied herein. There are also many protease cleavage sites that can be used to cleave the tag from the protein of interest after expression and / or secretion of the protein of interest from the host cell using corresponding proteases known to those skilled in the art. An "expression and / or solubility enhancing tag", as defined herein, can be fused to the C-terminus or N-terminus of the protein of interest. The expression and / or solubility enhancing tag can increase the expression and / or titer and / or solubility and / or soluble expression and / or soluble titer of the protein of interest when expressed in a host cell, e.g., a prokaryotic or eukaryotic cell, a bacterial cell or a yeast cell, e.g., E. coli, e.g., Pichia pastoris, significantly compared to the expression of the protein of interest without the expression and / or solubility enhancing tag, e.g., expressed in the cytosol, periplasm or secreted from the host cell. The expression and / or solubility enhancing tag sequence used herein may be selected from the group consisting of calmodulin binding peptide (CBP), poly-Arg, poly-Lys, G B1 domain, protein D, Z domain of Staphylococcus aureus protein A, and thioredoxin or any other tag known to improve the expression and / or solubility of a protein to which it is fused, e.g., during expression in a host cell. The expression and / or solubility enhancing tag may be based on bacteriophage genes, e.g., highly charged peptides such as those listed in US 8,535,908 B2.Preferably, the solubility-promoting tag is selected from the group consisting of T7C, T7B, T7B1, T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11, T7B12, T7B13, T7A, T7A1, T7A2, T7A3, T7A4, T7A5, T7AC T3, N1, N2, N3, N4, N5, N6, N7, calmodulin binding peptide (CBP), poly-Arg, poly-Lys, G B1 domain, protein D, the Z domain of Staphylococcus aureus protein A, DsbA, DsbC, and thioredoxin, and variants thereof obtained by substitution of several amino acids, such as one, two, three or more. In a preferred embodiment of the present invention, the T7AC solubility tag is applied herein, which is comprised by the fusion protein or by the protein of interest as defined herein above. A "tag that allows easy detection and quantification of the protein of interest" is a tag that can be fused to the C-terminus or N-terminus of the protein of interest as defined herein. It is a tag that can be used to detect, quantitate and analyze the protein of interest throughout the production process (e.g., measuring the titer of the protein of interest in the fermentation broth or the content of POI in different solutions throughout the production process, such as in the eluate of chromatography, cell homogenate, filter retentate or filtrate, for example, directly in-line, in situ, online or at-line by spectroscopic or fluorimetric or other methods, or offline by state-of-the-art methods). The tag thereby provides POI characteristics such as, for example, fluorescence, UV-VIS absorbance and other absorbances useful for other spectroscopic or fluorimetric methods used for online, in-line, at-line or in-situ quantification methods known in the art. The tag can also be an affinity tag or other tag that can be used for quantitative affinity chromatography, for example affinity HPLC as an off-line measurement or immunoassays such as ELISA.For example, the monitoring tag as an example of a tag that allows easy detection and quantification of the protein of interest is any one of m-Cherry, GFP, or f-actin, or any other tag that is useful for detection or quantification of the protein of interest during production of the protein of interest, including fermentation, isolation, and purification by simple in situ, in-line online, or at-line detectors such as UV, IR, Raman, fluorescence, etc. The detection tag as another example of a tag that allows easy detection and quantification of the protein of interest can also be a protein A tag, an S-galactosidase tag, a FLAG tag, a Strep tag, or a streptavidin-binding peptide (SBP) tag or a Strep II tag for use in quantitative HPLC or ELISA.

[0044] In one embodiment of the present invention, a fusion protein comprising a secretion signal as defined herein (e.g. comprising a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein, and optionally comprising an alpha mating factor (MFα) prosequence) and a protein of interest as defined herein also comprises a solubility enhancing tag, even more preferably a T7AC, and / or a purification tag, even more preferably a 6His tag, and / or a protease cleavage site for a caspase, preferably for caspase-2, such as VDVAD, preferably the solubility enhancing tag as defined herein and / or the purification tag as defined herein and / or the protease cleavage site as defined herein are fused N-terminally to the protein of interest as defined herein. In particular, in one embodiment of the present invention, the fusion protein comprises a secretion signal comprising a signal peptide sequence derived from the KRE1 protein and an alpha mating factor (MFα) pro-sequence, and the fusion protein comprising a protein of interest as defined herein also comprises a solubility enhancing tag, even more preferably a T7AC, and / or a purification tag, even more preferably a 6His tag, and / or a protease cleavage site for a caspase, preferably caspase-2, such as VDVAD, preferably the solubility enhancing tag as defined herein and / or the purification tag as defined herein and / or the protease cleavage site as defined herein are fused N-terminally to the protein of interest as defined herein.In particular, in another embodiment of the invention, a fusion protein comprising a secretion signal comprising a signal peptide sequence derived from SWP1 protein and an alpha mating factor (MFα) pro-sequence and comprising a protein of interest as defined herein also comprises a solubility enhancing tag, even more preferably a T7AC, and / or a purification tag, even more preferably a 6His tag, and / or a protease cleavage site for a caspase, preferably for caspase-2, such as VDVAD, preferably the solubility enhancing tag as defined herein and / or the purification tag as defined herein and / or the protease cleavage site as defined herein are fused N-terminally to the protein of interest as defined herein. In a preferred embodiment of the present invention, a fusion protein comprising a secretion signal as defined herein (e.g. comprising a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein and comprising an alpha mating factor (MFα) pro-sequence) and a protein of interest as defined herein also comprises the solubility enhancing tags T7AC and 6His purification tag and the protease cleavage site VDVAD, and even more preferably the solubility enhancing tags T7AC and 6His purification tag and the protease cleavage site VDVAD are fused to the N-terminus of the protein of interest as defined herein. A fusion protein of the invention consists, from the N-terminus to the C-terminus, of a secretion signal as defined herein (e.g. comprising a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein, and optionally comprising an alpha mating factor (MFα) prosequence), and also of a protein of interest as defined herein, such protein of interest optionally comprising a solubility enhancing tag, even more preferably a T7AC, and / or a purification tag, even more preferably a 6His tag, and / or a protease cleavage site for a caspase, preferably for caspase-2, such as VDVAD, preferably the solubility enhancing tag as defined herein and / or the purification tag as defined herein and / or the protease cleavage site as defined herein are fused N-terminally to the protein of interest as defined herein.In this context, such tags and / or cleavage sites are part of the protein of interest. In a preferred embodiment, the fusion protein of the invention consists of, from N-terminus to C-terminus, a secretion signal comprising a signal peptide sequence derived from the KRE1 protein and an alpha mating factor (MFα) pro-sequence, and a protein of interest as defined herein, which also comprises the solubility enhancing tags T7AC and 6His purification tag and the protease cleavage site VDVAD, and even more preferably, the solubility enhancing tags T7AC and 6His purification tag and the protease cleavage site VDVAD are fused at the N-terminus to the protein of interest as defined herein. In another preferred embodiment, when the fusion protein of the invention consists from N-terminus to C-terminus of a secretion signal comprising a signal peptide sequence derived from the SWP1 protein and an alpha mating factor (MFα) pro-sequence, and also a protein of interest as defined herein, such protein of interest also comprises the solubility enhancing tags T7AC and 6His purification tag and the protease cleavage site VDVAD, and even more preferably the solubility enhancing tags T7AC and 6His purification tag and the protease cleavage site VDVAD are fused N-terminally to the protein of interest as defined herein. Again, in this context, such T7AC and 6His tags and said VDVAD cleavage site are part of the protein of interest. Secretion signal

[0045] To be secreted, a protein must travel through the intracellular secretory pathway of the cell that produces it. The protein is directed to this pathway, rather than to alternative cellular destinations, via an N-terminal secretion signal. At a minimum, the secretion signal contains a signal peptide sequence. A signal peptide sequence typically consists of 13-36 mostly hydrophobic amino acids flanked by a basic amino acid at the N-terminus and a polar amino acid at the C-terminus. The signal peptide sequence can interact with the signal recognition particle (SRP) or other transport proteins (e.g., SND, GET) that mediate the co-translational or post-translational translocation of the nascent protein from the cytosol into the lumen of the ER. In the ER, the signal peptide sequence is typically cleaved and the protein is folded and undergoes post-translational modifications. The protein is then delivered from the ER to the Golgi apparatus and then to secretory vesicles and the outside of the cell. In addition to the signal peptide sequence, a subset of nascent proteins that are naturally destined for secretion also possess secretion signals that also include leader peptides, such as the alpha-mating factor pro-sequence. Leader peptides typically consist of hydrophobic amino acids interrupted by charged or polar amino acids. Without wishing to be bound by theory, it is believed that leader peptides slow transport, ensure proper folding of the protein, and / or facilitate transport of the protein from the ER to the Golgi apparatus, where the leader peptide is typically cleaved. As used herein, "signal peptide sequence derived from KRE1 or SWP1 protein" describes an amino acid sequence, i.e., a signal peptide sequence, that is present in the KRE1 protein as defined herein or the SWP1 protein as defined herein. Since secretion signals including signal peptide sequences are usually cleaved during secretion, the signal peptide sequences described herein originate from the KRE1 protein or SWP1 protein before secretion and / or before cleavage of the signal peptide sequence. "Originating from" may be used interchangeably with "derived from".

[0046] KRE1, also known as killer toxin resistance protein 1, is a secreted yeast protein. KRE1 may be involved in the late stages of cell wall 1,6-beta-glucan synthesis and assembly. It has a structural, rather than enzymatic, function in the assembly and structure of cell wall 1,6-beta-glucan, presumably by being involved in covalently cross-linking 1,6-beta-glucan with other cell wall components, such as 1,3-beta-glucan, chitin, certain mannoproteins, etc. It also functions as a plasma membrane receptor for yeast K1 viral toxin. As such, it possesses a signal peptide sequence. KRE1 may be from any eukaryotic species, particularly any yeast. Exemplary yeasts include, but are not limited to, Komagataella phaphii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Saccharomyces kluyveri, Saccharomyces uvarum, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., and Schizosaccharomyces pombe. KRE1 can also be from Trichoderma reesei or Aspergillus niger. Preferably, KRE1 is from K. phaphii. A signal peptide sequence derived from KRE1 may comprise or consist of the first 18 amino acids of a full-length KRE1 protein (in particular a protein comprising a signal peptide translated from an mRNA encoding the KRE1 protein), such as the KRE1 protein from K. phaphi, or a functional homologue thereof. In a preferred embodiment, the KRE1 protein corresponds to UniProt database entry F2QWV3, sequence version 1 of May 31, 2011 (chromosomal location PP7435_Chr3-0933) or a functional homologue thereof, and the signal peptide sequence preferably corresponds to amino acids 1 to 18 of said database entry, which are also depicted in SEQ ID NO: 1. Thus, a signal peptide sequence derived from a KRE1 protein may comprise or consist of SEQ ID NO: 1 or a functional homologue thereof.The signal peptide sequence derived from the KRE1 protein may consist of SEQ ID NO:1 or a functional homologue thereof. The signal peptide sequence derived from the KRE1 protein may comprise at least any one of 80%, 85%, 90%, 94%, or 95% sequence identity with SEQ ID NO:1, which may refer to a functional homologue thereof as defined herein. The signal peptide sequence derived from the KRE1 protein may comprise at least 90% sequence identity with SEQ ID NO:1. The signal peptide sequence derived from the KRE1 protein may comprise at least 94% sequence identity with SEQ ID NO:1. The signal peptide sequence derived from the KRE1 protein may comprise at least 95% sequence identity with SEQ ID NO:1.

[0047] SWP1, also known as dolichyl diphosphate oligosaccharide-protein glycosyltransferase subunit SWP1, is a subunit of the oligosaccharyltransferase (OST) complex that catalyzes the initial transfer of a defined glycan (Glc3Man9GlcNAc2 in eukaryotes) from the lipid carrier dolichyl pyrophosphate to an asparagine residue within an Asn-X-Ser / Thr consensus motif in the nascent polypeptide chain, which is the first step in protein N-glycosylation. SWP1 also carries a signal peptide sequence. SWP1 can be from any eukaryotic species, particularly any yeast. Exemplary yeasts include, but are not limited to, Komagataella phaphii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Saccharomyces kluyveri, Saccharomyces uvarum, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., Komagataella pastoris, and Schizosaccharomyces pombe. SWP1 can also be from Trichoderma reesei or Aspergillus niger. Preferably, SWP1 is from K. phaphii. The signal peptide sequence derived from SWP1 may comprise or consist of the first 18 amino acids of the full-length SWP1 (particularly the protein including the signal peptide translated from the mRNA encoding the SWP1 protein) protein, such as the SWP1 protein from K. phaphi or a functional homologue thereof. In a preferred embodiment, the SWP1 protein corresponds to the UniProt database entry F2QNI3, sequence version 1 of May 31, 2011 (gene PP7435_Chr1-0255) or a functional homologue thereof, and the signal peptide sequence corresponds to amino acids 1 to 18 of said database entry, which are also preferably depicted in SEQ ID NO: 2. Thus, the signal peptide sequence derived from the SWP1 protein may comprise or consist of SEQ ID NO: 2 or a functional homologue thereof. The signal peptide sequence derived from the SWP1 protein may consist of SEQ ID NO: 2 or a functional homologue thereof.The signal peptide sequence derived from SWP1 protein may comprise at least any one of 80%, 85%, 90%, 94% or 95% sequence identity with SEQ ID NO:2, which may refer to its functional homologue as defined herein. The signal peptide sequence derived from SWP1 protein may comprise at least 90% sequence identity with SEQ ID NO:2. The signal peptide sequence derived from SWP1 protein may comprise at least 94% sequence identity with SEQ ID NO:2. The signal peptide sequence derived from SWP1 protein may comprise at least 95% sequence identity with SEQ ID NO:2. Preferably, SWP1 is from K. pastoris. Thus, the signal peptide sequence derived from SWP1 protein may comprise SEQ ID NO:52 or a functional homologue thereof. The signal peptide sequence derived from SWP1 protein may consist of SEQ ID NO:52 or a functional homologue thereof. The signal peptide sequence derived from the SWP1 protein may comprise at least any one of 80%, 85%, 90%, 94%, or 95% sequence identity with SEQ ID NO: 52, which may refer to a functional homologue thereof as defined herein. The signal peptide sequence derived from the SWP1 protein may comprise at least 90% sequence identity with SEQ ID NO: 52. The signal peptide sequence derived from the SWP1 protein may comprise at least 95% sequence identity with SEQ ID NO: 52.

[0048] Alpha mating factor (MFα), also known as mating factor alpha-1, alpha-1 mating pheromone, or mating factor alpha, is a hormone, the active factor (MFα without secretion signal) is secreted into the culture medium by haploid cells of the alpha mating type and acts on cells of the opposite mating type (a type). It mediates the mating process between the two types by inhibiting the initiation of DNA synthesis in a type cells and synchronizing them with the alpha type. MFα carries a secretion signal that includes a signal peptide sequence (pre-sequence) and a pro-sequence. MFα can be from any eukaryotic species, in particular from any yeast, preferably from any yeast of the genus Saccharomyces, such as Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum, or Saccharomyces kudriavzevii. Exemplary yeasts include, but are not limited to, Komagataella phafii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Saccharomyces kluyveri, Saccharomyces uvarum, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., and Schizosaccharomyces pombe. MFα can also be from Trichoderma reesei or Aspergillus niger. Preferably, the source is from S. cerevisiae. The prosequence may comprise or consist of amino acids 20-89 of a full-length MFα protein (i.e., the protein including the signal peptide and the prosequence translated from the mRNA encoding the MFα protein), such as the MFα protein from S. cerevisiae, or a functional homologue thereof. In a preferred embodiment, the full-length MFα protein corresponds to UniProt database entry P01149, sequence version 1 of April 1, 1988, or a functional homologue thereof, and the alpha mating factor (MFα) prosequence preferably corresponds to amino acids 20-89, more preferably amino acids 20-85, depicted in SEQ ID NO:53 of that database entry. The MFα prosequence may comprise SEQ ID NO:3, or a functional homologue thereof.The MFα prosequence may consist of SEQ ID NO:3 or a functional homolog thereof. The MFα prosequence may have at least any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:3, which may be referred to as a functional homolog thereof as defined herein. The MFα prosequence may have at least 90% sequence identity to SEQ ID NO:3. The MFα prosequence may have at least 95% sequence identity to SEQ ID NO:3. The MFα prosequence may have at least 98% sequence identity to SEQ ID NO:3. The MFα prosequence may comprise SEQ ID NO:53 or a functional homolog thereof. The MFα prosequence may consist of SEQ ID NO:53 or a functional homolog thereof. The MFα prosequence may have at least any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:53, which may be referred to as a functional homolog thereof as defined herein. The MF alpha prosequence may comprise at least 90% sequence identity to SEQ ID NO: 53. The MF alpha prosequence may comprise at least 95% sequence identity to SEQ ID NO: 53. The MF alpha prosequence may comprise at least 98% sequence identity to SEQ ID NO: 53.

[0049] The MFα prosequence may be derived from Saccharomyces paradoxus. The MFα prosequence may comprise SEQ ID NO:74 or a functional homologue thereof. The MFα prosequence may consist of SEQ ID NO:74 or a functional homologue thereof. The MFα prosequence may comprise at least any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:74, which may refer to a functional homologue thereof as defined herein. The MFα prosequence may comprise at least 90% sequence identity to SEQ ID NO:74. The MFα prosequence may comprise at least 95% sequence identity to SEQ ID NO:74. The MFα prosequence may comprise at least 98% sequence identity to SEQ ID NO:74. The MFα prosequence may comprise SEQ ID NO:75 or a functional homologue thereof. The MFα prosequence may consist of SEQ ID NO:75 or a functional homologue thereof. The MFα prosequence may comprise any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:75, which may be referred to as a functional homologue thereof as defined herein. The MFα prosequence may comprise at least 90% sequence identity to SEQ ID NO:75. The MFα prosequence may comprise at least 95% sequence identity to SEQ ID NO:75. The MFα prosequence may comprise at least 98% sequence identity to SEQ ID NO:75. The MFα prosequence may comprise SEQ ID NO:76 or a functional homologue thereof. The MFα prosequence may consist of SEQ ID NO:76 or a functional homologue thereof. The MFα prosequence may comprise any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:76, which may be referred to as a functional homologue thereof as defined herein. The MFα prosequence may comprise at least 90% sequence identity to SEQ ID NO:76. The MFα prosequence may comprise at least 95% sequence identity to SEQ ID NO: 76. The MFα prosequence may comprise at least 98% sequence identity to SEQ ID NO: 76. The MFα prosequence may comprise SEQ ID NO: 77 or a functional homolog thereof. The MFα prosequence may consist of SEQ ID NO: 77 or a functional homolog thereof. The MFα prosequence may comprise at least any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 77, which may refer to a functional homolog thereof as defined herein.The MFα prosequence may comprise at least 90% sequence identity to SEQ ID NO:77. The MFα prosequence may comprise at least 95% sequence identity to SEQ ID NO:77. The MFα prosequence may comprise at least 98% sequence identity to SEQ ID NO:77. The MFα prosequence may comprise SEQ ID NO:78 or a functional homologue thereof. The MFα prosequence may consist of SEQ ID NO:78 or a functional homologue thereof. The MFα prosequence may comprise at least any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:78, which may refer to a functional homologue thereof as defined herein. The MFα prosequence may comprise at least 90% sequence identity to SEQ ID NO:78. The MFα prosequence may comprise at least 95% sequence identity to SEQ ID NO:78. The MFα prosequence may comprise at least 98% sequence identity to SEQ ID NO:78.

[0050] The MFα prosequence may be derived from Saccharomyces eubayanus. The MFα prosequence may comprise SEQ ID NO:79 or a functional homologue thereof. The MFα prosequence may consist of SEQ ID NO:79 or a functional homologue thereof. The MFα prosequence may comprise at least any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:79, which may refer to a functional homologue thereof as defined herein. The MFα prosequence may comprise at least 90% sequence identity to SEQ ID NO:79. The MFα prosequence may comprise at least 95% sequence identity to SEQ ID NO:79. The MFα prosequence may comprise at least 98% sequence identity to SEQ ID NO:79.

[0051] The MFα prosequence may be derived from Saccharomyces kudriavzevii. The MFα prosequence may comprise SEQ ID NO:80 or a functional homologue thereof. The MFα prosequence may consist of SEQ ID NO:80 or a functional homologue thereof. The MFα prosequence may comprise at least any one of 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:80, which may refer to a functional homologue thereof as defined herein. The MFα prosequence may comprise at least 90% sequence identity to SEQ ID NO:80. The MFα prosequence may comprise at least 95% sequence identity to SEQ ID NO:80. The MFα prosequence may comprise at least 98% sequence identity to SEQ ID NO:80.

[0052] The MFα prosequence preferably has a Ser at position corresponding to position 23 of SEQ ID NO:53 and / or a Glu at position corresponding to position 64 of SEQ ID NO:53, preferably at both positions, which can further increase secretion. SEQ ID NO:3 already contains these mutations.

[0053] A functional homolog is a functional equivalent of a nucleic acid sequence or a peptide, polypeptide, or protein described herein. A functional homolog can be a biologically active sequence that has at least about 70%, at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity with a given sequence of a polypeptide, such as the sequence of SEQ ID NO: 1, 2, 3, 52, or 53. In some embodiments, a functional homolog is a biologically active sequence that has at least about 70%, at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity with a native sequence polypeptide. With respect to nucleic acid sequences, the degeneracy of the genetic code allows for the substitution of certain codons with other codons that specify the same amino acid and therefore may result in the same protein. Nucleic acid sequences can vary substantially because, with the exception of methionine and tryptophan, known amino acids can be coded for by more than one codon. Thus, part or all of the nucleic acid sequences described herein may be synthesized to give nucleic acid sequences which differ significantly from those shown in the depicted sequences, although the encoded amino acid sequences may be conserved.

[0054] A functional homolog may also describe a functional equivalent of an amino acid sequence or peptide, polypeptide, or protein described in this document, but which has up to five conservative variations, i.e., a functional homolog may have 1, 2, 3, 4, or 5 conservative variations. In some embodiments, particularly for functional homologs of the MF alpha prosequence, a functional homolog may also have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 conservative variations. As used herein, a "conservative variation" is preferably a variation that results in a point variation, e.g., a substitution, insertion, or deletion of a single amino acid, in particular a substitution is a replacement of an amino acid residue with a chemically similar amino acid residue. Examples of conservative substitutions are substitutions between members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. Conservative mutations can also be any one of a substitution, insertion, or deletion of one amino acid that does not affect the biological activity of the amino acid sequence of the peptide, polypeptide, or protein described herein. A functional homolog may contain up to 14 conservative mutations. A functional homolog may contain up to 13 conservative mutations. A functional homolog may contain up to 12 conservative mutations. A functional homolog may contain up to 11 conservative mutations. A functional homolog may contain up to 10 conservative mutations. A functional homolog may contain up to 9 conservative mutations. A functional homolog may contain up to 8 conservative mutations. A functional homologue may contain up to seven conservative mutations. A functional homologue may contain six conservative mutations. A functional homologue may contain up to five conservative mutations. A functional homologue may contain up to four conservative mutations. A functional homologue may contain up to three conservative mutations. A functional homologue may contain up to two conservative mutations. A functional homologue may contain one conservative mutation. [Table 1] TIFF2024506650000002.tif55170

[0055] The present invention further relates to a secretion signal as defined herein. Further disclosed herein is a secretion signal, the secretion signal comprising (i) a signal peptide sequence derived from the KRE1 protein; and (ii) an alpha mating factor (MFα) prosequence. The signal peptide sequence may comprise SEQ ID NO: 1 or a functional homolog thereof, whereas the alpha mating factor (MFα) prosequence may comprise SEQ ID NO: 3 or a functional homolog thereof. The signal peptide sequence may comprise SEQ ID NO: 1 or a functional homolog thereof, whereas the alpha mating factor (MFα) prosequence may comprise SEQ ID NO: 53 or a functional homolog thereof. The present invention further relates to a secretion signal, the secretion signal comprising (i) a signal peptide sequence derived from the SWP1 protein; and (ii) an alpha mating factor (MFα) prosequence. The signal peptide sequence may comprise SEQ ID NO: 2 or a functional homolog thereof, whereas the alpha mating factor (MFα) prosequence may comprise SEQ ID NO: 3 or a functional homolog thereof. The signal peptide sequence may comprise SEQ ID NO:2 or a functional homolog thereof, while the alpha mating factor (MFα) prosequence may comprise SEQ ID NO:53 or a functional homolog thereof. The signal peptide sequence may comprise SEQ ID NO:52 or a functional homolog thereof, while the alpha mating factor (MFα) prosequence may comprise SEQ ID NO:3 or a functional homolog thereof. The signal peptide sequence may comprise SEQ ID NO:52 or a functional homolog thereof, while the alpha mating factor (MFα) prosequence may comprise SEQ ID NO:53 or a functional homolog thereof. Protein of interest

[0056] The term "protein of interest" (POI) as used herein refers to a protein produced by recombinant techniques in a host cell. More specifically, the protein may be a polypeptide that does not naturally occur in the host cell, i.e. a heterologous protein, e.g. an artificial protein, such as a protein that is not naturally produced by a wild-type cell, or otherwise a protein that is native to the host cell, i.e. a homologous protein, but is produced, for example, by transformation with a self-replicating vector comprising a nucleic acid sequence encoding the POI, or upon recombinant integration of one or more copies of the nucleic acid sequence encoding the POI into the genome of the host cell, or by recombinant modification of one or more regulatory sequences that control the expression of the gene encoding the POI, e.g. a promoter sequence. Generally, the protein of interest referred to herein may be produced by methods of recombinant expression well known to those skilled in the art. The protein of interest may be a recombinant protein.

[0057] There is no limitation regarding the protein of interest (POI). The POI is usually a eukaryotic or prokaryotic polypeptide, a variant or derivative thereof, or an artificial polypeptide, such as a polypeptide not naturally produced by a wild-type cell. The POI can be any eukaryotic or prokaryotic protein. The protein can be a naturally secreted protein or an intracellular protein, i.e., a protein not naturally secreted. The present invention also includes biologically active fragments of a protein. In another embodiment, the POI can be an amino acid chain or can be present in a complex, such as a dimer, trimer, heterodimer, multimer, or oligomer. Any POI can be secreted by fusing the secretion signal of the present invention to the POI. The POI can be a protein that requires co-translational translocation.

[0058] The protein of interest may be a protein used as a nutritional, dietary, digestive, or supplement, such as in a food product, feed product, or cosmetic. The food product may be, for example, a bouillon, dessert, cereal bar, confectionery, sports drink, nutritional product, or other nutritional product. Preferably, the protein of interest is a food additive.

[0059] In another embodiment, the protein of interest may be used in animal feed.

[0060] Further examples of POI include antimicrobial proteins such as lactoferrin, lysozyme, lactoferricin, lactohedrin, kappa-casein, haptocorrin, lactoperoxidase, milk proteins, acute phase proteins such as proteins normally produced in production animals in response to infection, and small antimicrobial proteins such as lysozyme and lactoferrin. Other examples include bactericidal proteins, antiviral proteins, acute phase proteins (induced in production animals in response to infection), probiotic proteins, bacteriostatic proteins, and cationic antimicrobial proteins.

[0061] "Feed" means any natural or artificial diet, meal or the like, intended or suitable for being eaten, ingested or digested by a non-human animal, or a component of such a meal. "Feed additive" generally refers to a substance added to feed. It typically includes one or more compounds, such as vitamins, minerals, enzymes, and suitable carriers and / or excipients. For the present invention, the food additive may be an enzyme or other protein. Examples of enzymes that can be used as feed additives include phytase, xylanase, and β-glucanase. "Food" means any natural or artificial diet, meal or the like, intended or suitable for being eaten, ingested or digested by a human, or a component of such a meal.

[0062] "Food additive" generally refers to a substance added to food. It typically includes one or more compounds, such as vitamins, minerals, enzymes, and suitable carriers and / or excipients. For the present invention, the food additive can be an enzyme or other protein. Enzymes that can be used as food additives include proteases, lipases, lactases, pectin methylesterases, pectinases, transglutaminases, amylases, β-glucanases, acetolactate decarboxylases, and laccases.

[0063] In some embodiments, the food additive is an antimicrobial protein, such as, for example, (i) antimicrobial milk proteins (either human or non-human) lactoferrin, lysozyme, lactoferricin, lactohedrin, kappa casein, haptocorrin, lactoperoxidase, alpha-1-antitrypsin, and immunoglobulins, such as IgA, (ii) acute phase proteins, such as C-reactive protein (CRP); lactoferrin; lysozyme; serum amyloid A (SAA); ferritin; haptoglobin (Hp); complement 2-9, particularly complement 3; ceromucoid; ceruloplasmin (Cp); 15-keto-13,14-dihydro-prostaglandin F2 alpha (PGFM); fibrinogen (Fb); alpha(1) acid glycoprotein (AGP); alpha(1)-antitrypsin; mannose binding protein; lipopolysaccharide binding protein; alpha-2 macroglobulins and various defensins, (iii) antimicrobial peptides, such as cecropins, magainins, defensins, tachyplesins, parasin I, buforin I, PMAP-23, moronecidin, anopurin, gambicin, and SAMP-29, and (iv) other antimicrobial proteins, such as CAP37, granulysin, secretory leukocyte protease inhibitor, CAP18, ubixidine, bovine antimicrobial protein 1, Ace-AMP1, tachyplesins, big defensins, Ac-AMP2, Ah-AMP1, and CAP18.

[0064] The POI may be an enzyme. Preferred enzymes are those that can be used for industrial applications, e.g., in the production of surfactants, starches, fuels, fibers, pulp and paper, oils, personal care products, or for example, in baking, organic synthesis, etc. Examples of such enzymes are proteases, amylases, lipases, mannanases, and celluloses for stain removal and cleaning; pullulanases amylases and amyloglucosidases for starch liquefaction and saccharification; glucose isomerases for glucose to fructose conversion; cyclodextrin-glycosyltransferases for cyclodextrin production; xylanases for viscosity reduction in fuels and starches; amylases, xylanases, lipases, phospholipases, glucose oxidases, lipoxygenases, transglutaminases for dough stability and conditioning in baking; celluloses for denim finishing and cotton softening in textile manufacturing. Examples of enzymes that may be used include cellulose, cellulose acetate, cellulose esters ...

[0065] The POI may be a therapeutic protein. The POI may be a protein suitable as a biopharmaceutical agent, such as, but not limited to, an antibody or antibody fragment, a growth factor, a hormone, an enzyme, or a vaccine.

[0066] The POI may be a naturally secreted protein, an intracellular protein, i.e. a protein that is not naturally secreted. The present invention also provides for the recombinant production of functional homologues, functional equivalent variants, derivatives and biologically active fragments of naturally secreted or non-naturally secreted proteins. Functional homologues preferably have the same or corresponding sequence and functional characteristics.

[0067] The POI may be structurally similar to a naturally occurring protein and may be derived from the naturally occurring protein by the addition of one or more amino acids to either or both of the C-terminus and N-terminus or to the side chains of the naturally occurring protein, by the substitution of one or more amino acids at one or many different sites in the naturally occurring amino acid sequence, by the deletion of one or more amino acids at one or both termini of the naturally occurring protein or at one or several sites in the amino acid sequence, or by the insertion of one or more amino acids at one or several sites in the naturally occurring amino acid sequence. Such modifications are well known for some of the above mentioned proteins.

[0068] Preferably, the protein of interest is a mammalian polypeptide, even more preferably a human polypeptide. Preferably, the protein of interest is a therapeutic or biopharmaceutical protein. Particularly preferred therapeutic proteins refer to any polypeptide, protein, protein variant, fusion protein and / or fragments thereof that can be administered to a mammal, even more preferably to a human. The protein of interest may also be an artificial protein, or part of a natural protein, or of a natural protein, or of an artificial protein, or of a fusion protein. It is envisaged, but not required, that the therapeutic protein according to the invention is heterologous to the cell. Examples of proteins that may be produced by the cells of the invention are, but are not limited to, enzymes, regulatory proteins, receptors, peptide hormones, growth factors, cytokines, scaffold-binding proteins (e.g., muteins based on the lipocalin family), structural proteins, lymphokines, adhesion molecules, receptors, membrane proteins or transport proteins, as well as any other polypeptides that may act as agonists or antagonists and / or have therapeutic or diagnostic uses. Furthermore, the protein of interest may be an antigen used for vaccination, a vaccine, an antigen-binding protein, an immunostimulatory protein. It may also be an antigen-binding fragment of an antibody, which may include any suitable antigen-binding antibody fragment known in the art. For example, the antibody fragment may include, but is not limited to, Fv (a molecule comprising VL and VH), single chain Fv (scFV) (a molecule comprising VL and VH connected by a peptide linker), Fab, Fab', F(ab')2, single domain antibody (sdAb) (a molecule comprising a single variable domain and three CDRs), and multivalent presentations thereof. The antibody or fragment thereof may be a murine antibody, a human antibody, a humanized antibody, or a chimeric antibody, or a fragment thereof.Examples of therapeutic proteins include antibodies, polyclonal antibodies, monoclonal antibodies, recombinant antibodies, antibody fragments such as Fab', F(ab')2, Fv, scFv, di-scFv, bi-scFv, tandem scFv, bispecific tandem scFv, sdAb, VHH, VH, and VL, or synthetic binding proteins constructed using fibronectin type III domains (FN3) as a molecular scaffold, also known as human antibodies, humanized antibodies, chimeric antibodies, IgA antibodies, IgD antibodies, IgE antibodies, IgG antibodies, IgM antibodies, intrabodies, minibodies, or monobodies.

[0069] The protein of interest may further be selected from the group consisting of an antibody, such as a chimeric, humanized or human antibody, or a bispecific antibody, or an antigen-binding antibody fragment, such as a Fab or F(ab)2, a single chain antibody, such as a scFv, a single domain antibody, such as a camelid VHH fragment or a heavy chain antibody or a domain antibody (dAb), an artificial antigen-binding molecule, such as a DARPIN, an ibody, an affibody, a humabody or a mutein based on a polypeptide of the lipocalin family, an enzyme, such as a process enzyme, a cytokine, a growth factor, a hormone, a protein antibiotic, a fusion protein, such as a toxin fusion protein, a structural protein, a regulatory protein, and a vaccine antigen, but preferably the protein of interest is a therapeutic protein, a food additive or a feed additive.

[0070] Therapeutic proteins include, but are not limited to, insulin, insulin-like growth factors, hGH, tPA, cytokines such as interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega, or IFN tau, tumor necrosis factor (TNF), TNF alpha and TNF beta, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1, and VEGF.

[0071] In a preferred embodiment, the protein is an antibody. The term "antibody" is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits and recognizes an epitope, where one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope. The prototypical antibody molecule is an immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g., human, rodent, rabbit, bovine, ovine, porcine, canine, other mammals, chicken, other birds, etc., are considered to be "antibodies." Numerous antibody coding sequences have been described; and others can be generated by methods well known in the art.

[0072] For example, antibodies or antigen-binding antibody fragments can be produced by methods known in the art. Generally, antibody-producing cells are sensitized to the desired antigen or immunogen. Messenger RNA isolated from the antibody-producing cells is used as a template to generate cDNA using PCR amplification. A library of vectors, each containing one heavy chain gene and one light chain gene that retains the original antigen specificity, is produced by inserting appropriate sections of the amplified immunoglobulin cDNA into an expression vector. A combinatorial library is constructed by combining a heavy chain gene library and a light chain gene library. This results in a library of clones that co-express heavy and light chains (resembling the Fab fragment or antigen-binding fragment of an antibody molecule). The vectors carrying these genes are co-transfected into a host cell. When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self-assemble to produce active antibodies, which can be detected by screening with the antigen or immunogen.

[0073] Antibody coding sequences of interest include sequences encoded by native sequences, as well as nucleic acids that are not identical in sequence to the disclosed nucleic acids due to the degeneracy of the genetic code, and variants thereof. Variant polypeptides may include amino acid (aa) substitutions, additions, or deletions. Amino acid substitutions may be conservative amino acid substitutions, or substitutions that minimize misfolding by eliminating non-essential amino acids, for example altering glycosylation sites, or substituting or deleting one or more cysteine ​​residues that are not required for function. Variants can be designed to retain or have enhanced biological activity of specific regions of the protein (e.g., functional domains, catalytic amino acid residues, etc.). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and / or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the present invention are polypeptides that have been modified using routine molecular biology techniques to improve their resistance to proteolysis, or to optimize solubility properties, or to make them more suitable as therapeutic agents.

[0074] Chimeric antibodies can be produced by recombinant means by combining the variable light and heavy chain regions (VK and VH) obtained from an antibody-producing cell of one species with the constant light and heavy chain regions of another species. Typically, chimeric antibodies utilize rodent or rabbit variable regions and human constant regions to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art and can be accomplished by standard means (e.g., as described in U.S. Pat. No. 5,624,659).

[0075] Humanized antibodies are engineered to contain more human-like immunoglobulin domains and incorporate only the complementarity determining regions of the antibody from the animal. This is accomplished by carefully sequencing the hypervariable loops of the variable regions of a monoclonal antibody and adapting them to the structure of human antibody chains. Although superficially complex, the process is straightforward in practice. See, for example, U.S. Patent No. 6,187,287.

[0076] In addition to complete immunoglobulins (or their recombinant counterparts), immunoglobulin fragments (e.g., Fab', F(ab')2, or other fragments) that contain the epitope binding site may be synthesized. "Fragment" or minimal immunoglobulins may be engineered using recombinant immunoglobulin technology. For example, "Fv" immunoglobulins for use in the present invention may be produced by synthesizing a variable light chain region and a variable heavy chain region. Antibody combinations are also of interest, e.g., diabodies, which contain two different Fv specificities.

[0077] Immunoglobulins may be post-translationally modified to add chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties, and the like, or specific binding moieties, such as streptavidin, avidin, or biotin, and the like, may be utilized in the methods and compositions of the invention.

[0078] Further examples of therapeutic proteins are blood clotting factors (VII, VIII, IX), alkaline protease from Fusarium, calcitonin, CD4 receptor darbepoietin, DNase (cystic fibrosis), erythropoietin, eutropin (human growth hormone derivative), follicle stimulating hormone (follitropin), gelatin, glucagon, glucocerebrosidase (Gaucher disease), glucosamylase from A. niger, glucose oxidase from A. niger, gonadotropins, growth factors (GCSF, GMCSF), growth hormone (somatotropin), hepatitis B vaccine, human Ludin, human antibody fragments, human apolipoprotein AI, human calcitonin precursor, human collagenase IV, human epidermal growth factor, human insulin-like growth factor, human interleukin 6, human laminin, human proapolipoprotein AI, human serum albumin, insulin and muteins, insulin, interferon alpha and muteins, interferon beta, interferon gamma (muteins), interleukin 2, luteinizing hormone, monoclonal antibody 5T4, mouse collagen, OP-1 (osteogenesis, neuroprotective factor), oprelvekin (interleukin 11) agonists), organic phosphohydrolases, PDGF agonists, phytase, platelet derived growth factor (PDGF), recombinant plasminogen activator G, staphylokinase, stem cell factor, tetanus toxin fragment C, tissue plasminogen activator, and tumor necrosis factor (see Schmidt, Appl Microbiol Biotechnol (2004) 65:363-372).

[0079] The protein of interest may comprise or consist of the amino acid sequence depicted in SEQ ID NO: 26. The protein of interest may comprise or consist of the amino acid sequence depicted in SEQ ID NO: 27. The protein of interest may comprise or consist of the amino acid sequence depicted in SEQ ID NO: 28. The protein of interest may comprise or consist of the amino acid sequence depicted in SEQ ID NO: 29. When a fusion protein of the invention consists, from N-terminus to C-terminus, of a secretion signal as defined herein and of a protein of interest as depicted in SEQ ID NO: 26, 27, 28 or 29, such a protein of interest may optionally further comprise one or more (detectable) tags, one or more protease cleavage sites, and / or one or more linkers as defined elsewhere herein. In other words, one or more tags, one or more cleavage sites and / or one or more linkers as defined herein may also be fused N- or C-terminally to such a protein of interest as depicted in SEQ ID NO: 26, 27, 28 or 29, but thus also comprised by said protein of interest, when the fusion protein of the invention consists, from N- to C-terminus, of a secretion signal and such a protein of interest as defined in this context. In this context, such one or more tags, one or more cleavage sites and / or one or more linkers may be fused N- or C-terminally to a protein of interest as defined elsewhere herein and then be part of such a protein of interest. Also, when the fusion protein of the invention consists, from N-terminus to C-terminus, of a secretion signal as defined herein and of a protein of interest depicted in SEQ ID NO: 26, 27, 28 or 29, the secretion signal as defined herein may optionally further comprise one or more (detectable) tags, one or more protease cleavage sites and / or one or more linkers, as already defined herein. [Table 2] TIFF2024506650000004.tif245170 TIFF2024506650000005.tif245170 TIFF2024506650000006.tif244170 TIFF2024506650000007.tif242170 TIFF2024506650000008.tif221170

[0080] It is also encompassed by the present invention that the fusion proteins described herein comprise elements of a secretion signal, as further described herein, operably linked to a protein of interest as defined herein.

[0081] According to a particular embodiment, the present invention relates to a nucleic acid molecule encoding a fusion protein comprising: (a) a secretion signal, the secretion signal comprising: (I) (i) a signal peptide sequence derived from a KRE1 protein or a signal peptide sequence derived from a SWP1 protein; and (ii) α-mating factor (MFα) prosequence; or (II) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and (b) a protein of interest; The secretion signal is operably linked to the protein of interest. Nucleic Acid Molecules of the Invention

[0082] To utilize the secretion signals of the present invention, they may be fused to a protein of interest. The fusion proteins as described herein may be encoded by nucleic acid molecules. The nucleic acid molecules of the present invention may, for example, be transformed into a host cell. Thus, the present invention relates to nucleic acid molecules encoding a fusion protein comprising, from N-terminus to C-terminus, (a) a secretion signal, the secretion signal comprising (i) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and (ii) an alpha mating factor (MFα) prosequence; and (b) a protein of interest.

[0083] As used herein, "encode" means that a nucleic acid or polynucleotide that encodes a protein, when expressed, leads to the production of that protein.

[0084] As used herein, the term "nucleic acid molecule" refers to either DNA or RNA. "Nucleic acid molecule," "nucleic acid," "polynucleotide," or simply "nucleotide," all of which may be used interchangeably, refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both autonomously replicating plasmids, infectious polymers of DNA or RNA, and non-functional DNA or RNA.

[0085] An "expression cassette" as used herein refers to different components of (vector) DNA, including genes, such as the nucleic acid molecule of the invention, and regulatory sequences, such as a promoter, that are expressed by a transfected cell. An expression cassette can instruct the host cell machinery to express a protein of interest. Typically, an expression cassette is composed of one or more genes and sequences that control their expression. Thus, the present invention further relates to an expression cassette comprising a nucleic acid molecule of the invention and a promoter operably linked thereto. The expression cassette can be in the form of a vector. The expression cassette can be contained by a vector.

[0086] In other embodiments, the polynucleotides encoding the nucleic acid molecules of the invention and / or one or more components of the SRP can be incorporated into a plasmid or vector. Thus, the present invention also relates to vectors comprising the nucleic acids of the invention. The term "plasmid" refers to a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and / or expressed (e.g., plasmid, cosmid, lambda phage). A vector comprising foreign DNA is called recombinant DNA. Vectors include, but are not limited to, plasmids, viral vectors, cosmids, and artificial chromosomes, preferably plasmids. The vector itself can generally be a DNA sequence consisting of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. All vectors may be used for cloning and are therefore considered cloning vectors, however, there are also vectors specifically designed for cloning, while others may be specifically designed for other purposes, such as transcription and protein expression. Vectors specifically designed for expression of a transgene in a target cell are called expression vectors, and generally have a promoter sequence that drives the expression of the transgene. Depending on the host cell used, the skilled artisan can use an appropriate plasmid or vector.

[0087] Preferably, the vector is a eukaryotic expression vector, preferably a yeast expression vector.

[0088] Preferably, the vector is a eukaryotic expression vector, preferably a yeast expression vector. Examples of vectors using yeast as a host include YIp-type vectors, YEp-type vectors, YRp-type vectors, YCp-type vectors, pGPD-2, pAO815, pGAPZ, pGAPZα, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, pPICZ, pPICZα, pPIC3K, pHWO10, pPUZZLE and 2μm plasmid. Such vectors are known and are described, for example, in Cregg et al., Mol Biotechnol.(2000)16(1):23-52. Preferably, the vector is pPM2dZ30 vector (described in WO2008 / 128701A2). Alternatively, the vector is the Golden Gate-based GoldenPiCS, consisting of backbones BB1, BB2 and BB3aK / BB3eH / BB3rN ( Prielhofer et al., 2017 ).

[0089] Vectors can be used for the transcription of cloned recombinant nucleotide sequences, i.e., recombinant genes, and the translation of their mRNAs in suitable host organisms. Vectors can also be used for integrating target polynucleotides into host cell genomes by methods known in the art, such as those described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2001) or Stearns et al. (1990), Methods in Enzymology, 185:280-297. A "vector" usually comprises an origin for autonomous replication in a host cell, preferably both bacterial and eukaryotic origins for the host cell of the present invention, a selection marker, multiple restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, the components of which are operably linked together. The desired polypeptide coding sequence is operably linked to transcriptional and translational regulatory sequences that provide for the expression of the polypeptide in a host cell.

[0090] Many suitable plasmids or vectors are known to those of skill in the art, and many are commercially available. Examples of suitable vectors are provided in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989), and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1997).

[0091] Vectors or plasmids of the present invention include yeast artificial chromosomes, which refer to DNA constructs that can be genetically engineered to contain heterologous DNA sequences (e.g., DNA sequences as large as 3000 kb), including telomeric, centromere, and origin of replication (origin of replication) sequences.

[0092] The sequence encoding the fusion protein or secretion signal of the present invention may be operably linked to a promoter. As used herein, the term "promoter" refers to a region that promotes the transcription of a particular gene. A promoter typically increases the amount of recombinant product expressed from a nucleotide sequence compared to the amount of recombinant product expressed in the absence of the promoter. A promoter from one organism can be utilized to enhance the expression of a recombinant product from a sequence derived from another organism. A promoter can be integrated into a host cell chromosome by homologous recombination using methods known in the art (e.g., Datsenko et al, Proc.Natl.Acad.Sci.USA, 97(12):6640-6645(2000)). Also, a single promoter element can increase the amount of product expressed for multiple sequences attached in tandem. Thus, a single promoter element can enhance the expression of one or multiple recombinant products.

[0093] A promoter may be an "inducible promoter" or a "constitutive promoter." An "inducible promoter" refers to a promoter that can be induced by the presence or absence of a particular factor, while a "constitutive promoter" refers to an unregulated promoter that allows for continuous transcription of its associated gene or genes.

[0094] In a preferred embodiment, the nucleotide sequence encoding the fusion protein of the invention or the secretion signal of the invention is driven by an inducible promoter.

[0095] Many inducible promoters are known in the art. Many are described in the review by Gatz, Curr. Op. Biotech., 7:168 (1996) (see also Gatz, Ann. Rev. Plant. Physiol. Plant Mol. Biol., 48:89 (1997)). Examples include the tetracycline repressor system, the Lac repressor system, copper-inducible systems, salicylic acid-inducible systems (such as the PR1a system), glucocorticoid-inducible systems (Aoyama et al., 1997), alcohol-inducible systems, e.g., the AOX promoter, and the ecdysome-inducible system. Also included are benzenesulfonamide-inducible (US Pat. No. 5,364,780) and alcohol-inducible (WO 97 / 06269 and WO 97 / 06268) inducible systems and the glutathione S-transferase promoter.

[0096] Suitable promoter sequences for use in yeast host cells are described in Mattanovich et al., Methods Mol. Biol. (2012) 824:329-58, and includes glycolytic enzymes such as triosephosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase (GAL), P. pastoris glucose-6-phosphate isomerase promoter (PPGI), 3-phosphoglycerate kinase promoter (PPGK), glycerolaldehyde phosphate dehydrogenase promoter (PGAP), translation elongation factor promoter (PTEF), and promoters of P. pastoris enolase 1 (PENO1), triosephosphate isomerase (PTPI), ribosomal subunit proteins (PRPS2, PRPS7, PRPS31, PRPL1), alcohol oxidase promoter (PAOX) or variants thereof with modified features, formaldehyde dehydrogenase promoter, -(PFLD), isocitrate lyase promoter (PICL), alpha-ketoisocaproate decarboxylase promoter (PTHI), heat shock protein family members (PSSA1, PHSP90, PKAR2), 6-phosphogluconate dehydrogenase (PGND1), phosphoglycerate mutase (PGPM1), transketolase (PTKL1), phosphatidylinositol synthase (PPIS1), ferro-O2-oxidoreductase (PFET3), high affinity iron permease (PFTR1), repressible alkaline phosphatase (PPHO8), N-myristoyltransferase (PNMT1), pheromone responsive transcription factor (PMCM1), ubiquitin (PUBI4), single-stranded DNA endonuclease (PRAD2), the major ADP / ATP carrier of the inner mitochondrial membrane (PPET9) (WO2008 / 128701), and the formate dehydrogenase (FMD) promoter. Particularly preferred are the GAP promoter, the AOX promoter, or promoters derived from the GAP or AOX promoters.The AOX promoter is induced by methanol and repressed by glucose. Promoters derived from the AOX promoter for methanol-inducible as well as methanol-free production are described in WO2006 / 089329, EP1851312B1, and EP2199389B1. Carbon source-regulatable promoters may also be used, such as derepressible promoters as described in WO2013050551 (e.g., pG1 to pG8, fragments of pG1, designated pG1a to pG1f) and WO2017021541 (e.g., pG1-D1240, or pG1-D1427). Further examples are constitutive promoters such as MDH3, POR1, PDC1, FBA1-1, or GPM1 (Prielhofer et al. 2017, BMC Sys Biol. 11:123), or as disclosed in WO2014139608 (e.g., pCS1).

[0097] Further examples of suitable promoters include Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase (PGK), as well as the maltase gene promoter (MAL).

[0098] Other useful promoters for yeast host cells are described in Romanos et al, 1992, Yeast 8:423-488. Host Cells of the Invention

[0099] As used herein, "host cell" refers to a cell capable of protein expression and protein secretion. Such host cells can be applied in the methods of the present invention. For that purpose, a nucleic acid molecule encoding a fusion protein is present in or introduced into the cell so that the host cell expresses the polypeptide. The host cell provided by the present invention can be a eukaryote. As will be understood by those skilled in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus. Examples of eukaryotic cells include, but are not limited to, vertebrate cells, mammalian cells, human cells, animal cells, invertebrate cells, plant cells, nematode cells, insect cells, stem cells, fungal cells or yeast cells. Preferably, the host cell is a yeast cell.

[0100] Thus, the present invention relates to a host cell comprising a nucleic acid molecule of the present invention. Additionally, the present invention relates to a host cell comprising an expression cassette of the present invention. The present invention further relates to a host cell comprising a vector of the present invention.

[0101] Examples of yeast cells include, but are not limited to, Saccharomyces (e.g., Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces kudriavzevii), Komagataella (Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaphi), Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), Candida (e.g., Candida utilis, Candida cacaoi), Geotrichum (e.g., Geotrichum fermentans), as well as Hansenula polymorpha and Yarrowia lipolytica. Thus, the eukaryotic host cell of the invention or the eukaryotic host cell used in the methods and uses of the invention may be a fungal host cell or a yeast host cell, preferably a yeast host cell, selected from the group consisting of Komagataella phafi (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., and Schizosaccharomyces pombe, or a fungal host cell, such as Trichoderma reesei, Aspergillus niger, etc.

[0102] Of particular interest is the genus Pichia, which includes many species, including Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta. Most preferred is the species Pichia pastoris.

[0103] The former species Pichia pastoris was split and renamed Komagataella pastoris and Komagataella phafii. Thus, Pichia pastoris is a synonym for both Komagataella pastoris and Komagataella phafii, and preferably for Komagataella phafii.

[0104] Examples of Pichia pastoris strains useful in the present invention are X33 and its subtypes GS115, KM71, KM71H; X33 and its subtypes CBS7435 muts, CBS7435 mutsΔArg, CBS7435 mutsΔHis, CBS7435 mutsΔArg, ΔHis, CBS7435 muts PDI+, CBS 704 (=NRRL Y-1603 = DSMZ 70382), CBS 2612 (=NRRL Y-7556), CBS 9173-9189, and DSMZ 70877 and mutants thereof. Preferably, the host cell is P. pastoris CBS7435mutS or a subtype thereof, more preferably P. pastoris CBS7435mutS.

[0105] According to a further preferred embodiment, the host cell is Pichia pastoris, Hansenula polymorpha, Trichoderma reesei, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, and Komagataella, and Schizosaccharomyces pombe. It may also be a host cell from Ustilago maydis.

[0106] As used herein, "recombinant" refers to the modification of genetic material by human intervention. Typically, recombinant refers to the manipulation of DNA or RNA in a virus, cell, plasmid, or vector by molecular biology (recombinant DNA technology) methods, including cloning and recombination. A recombinant cell, polypeptide, or nucleic acid can typically be described with reference to how it differs from its naturally occurring counterpart ("wild type"). A "recombinant cell" or "recombinant host cell" refers to a cell or host cell that has been genetically modified to contain a nucleic acid sequence that is not native to the cell.

[0107] As currently used, the term "produce" or "producing" refers to the process by which a protein of interest is expressed. A "host cell for producing a protein of interest" refers to a host cell into which a nucleic acid sequence encoding a protein of interest may be introduced. A recombinant host cell within the present invention does not necessarily contain a nucleic acid sequence encoding a protein of interest. It will be understood by those skilled in the art that the host cell can be provided, for example, in a kit, for inserting a desired nucleotide sequence into the host cell.

[0108] The terms "polypeptide" and "protein" are used interchangeably. The term "polypeptide" refers to a protein or peptide that comprises two or more amino acids, typically at least three, preferably at least 20, more preferably at least 30, e.g., at least 50 amino acids. Thus, a polypeptide comprises an amino acid sequence, and thus, a polypeptide comprising an amino acid sequence is sometimes referred to herein as a "polypeptide comprising a polypeptide sequence." Thus, herein, the term "polypeptide sequence" is used interchangeably with the term "amino acid sequence."

[0109] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the natural amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bound to hydrogen, carboxyl groups, amino groups, and R groups (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Systems for expressing proteins of interest that contain synthetic amino acids or amino acid analogs are known to those of skill in the art and include, but are not limited to, the use of an expanded genetic code. The key prerequisites for expanding the genetic code are a non-standard amino acid to code for, an unused codon to employ, a tRNA that recognizes this codon, and a tRNA synthetase that recognizes only the tRNA and the non-standard amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0110] To further increase secretion of the fusion protein or protein of interest following cleavage of the secretion signal described herein (see Examples 4-9), the (recombinant) host cell can additionally be engineered to overexpress one or more components of the signal recognition particle (SRP).

[0111] Signal recognition particle (SRP) as used herein refers to an abundant cytosolic universally conserved ribonucleoprotein (protein-RNA complex) that recognizes and targets specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes. In eukaryotes, SRP binds to the signal peptide sequence of a newly synthesized peptide as it emerges from the ribosome, such as the signal peptide sequence derived from SWP1 or KRE1 protein. This binding leads to a delay in protein synthesis known as "elongation arrest", a conserved function of SRP that facilitates the coupling of the protein translation and protein translocation processes. SRP then targets this entire complex (ribosome-nascent chain complex) to a protein-conducting channel, also known as the translocon, in the ER (endoplasmic reticulum) membrane. This occurs through the interaction and docking of SRP with its cognate SRP receptor, which is located in close proximity to the translocon.

[0112] In eukaryotes, there are three domains between SRP and its receptor that function in guanosine triphosphate (GTP) binding and hydrolysis. These are located in the SRP receptor (SRα and SRβ) and two related subunits in the SRP protein SRP54. Upon docking, the nascent peptide chain is inserted into the translocon channel, where it enters the ER. Protein synthesis resumes as SRP is released from the ribosome. The SRP-SRP receptor complex dissociates via GTP hydrolysis, and the cycle of SRP-mediated protein translocation continues. Thus, SRP-dependent translocation can in some cases also be viewed as a co-translational mode of translocation. In a particular embodiment, translocation of the fusion protein of the invention into the ER is co-translational.

[0113] Once inside the ER, the signal peptide sequence is cleaved from the core protein by a signal peptidase and is therefore not part of the mature protein, such as the secreted protein of interest after cleavage of the secretion signal from a fusion protein of the invention.

[0114] SRP may comprise SRP68, SRP72, SRP9-21, SRP54, SRP14, Sec65, and 7SL RNA. Thus, "one or more components of SRP" may relate to at least one selected from the group consisting of SRP68, SRP72, SRP9-21, SRP54, SRP14, Sec65, and 7SL RNA. In a preferred embodiment, all components of SRP, i.e. SRP68, SRP72, SRP9-21, SRP54, SRP14, Sec65, and 7SL RNA, are overexpressed in the host cell. Advantageously, the one or more components of SRP overexpressed in the host cell are derived from the same species as the host cell. However, it is also encompassed by the present invention that a heterologous one or more components of SRP may be overexpressed. Further encompassed is the overexpression of a functional homolog of another component of SRP, including but not limited to, preferably a functional homolog of one or more of SRP68, SRP72, SRP9-21, SRP54, SRP14, Sec65, and 7SL RNA of Komagataella phaphi; or preferably a functional homolog of SRP68, SRP72, SRP9-21, SRP54, SRP14, Sec65, and 7SL RNA of Komagataella phaphi.

[0115] Exemplary sequences for each of the SRP components derived from Komagataella phafii are listed in Table 3 below. [Table 3] TIFF2024506650000010.tif241170 TIFF2024506650000011.tif242170 TIFF2024506650000012.tif241170 TIFF2024506650000013.tif242170 TIFF2024506650000014.tif242170 TIFF2024506650000015.tif242170 TIFF2024506650000016.tif241170 TIFF2024506650000017.tif248170 TIFF2024506650000018.tif185170

[0116] Thus, the host cell of the invention, preferably a Pichia pastoris host cell, may be engineered to overexpress one or more of SEQ ID NOs: 5-11 or functional homologues thereof. The host cell of the invention, preferably a Pichia pastoris host cell, may be engineered to overexpress one or more of SEQ ID NOs: 5-11. The host cell of the invention, preferably a Pichia pastoris host cell, may be engineered to overexpress one or more of SEQ ID NOs: 5-11 or functional homologues thereof. The host cell of the invention, preferably a Pichia pastoris host cell, may also be engineered to overexpress SEQ ID NOs: 5-11. Alternatively, the host cell of the invention, preferably a Pichia pastoris host cell, may be engineered to overexpress one or more of SEQ ID NOs: 11-17 or functional homologues thereof. The host cell of the invention, preferably a Pichia pastoris host cell, may be engineered to overexpress one or more of SEQ ID NOs: 11-17. The host cells of the invention, preferably Pichia pastoris host cells, may be engineered to overexpress SEQ ID NO: 11-17 or a functional homologue thereof. The host cells of the invention, preferably Pichia pastoris host cells, may be engineered to overexpress SEQ ID NO: 11-17.

[0117] As used herein, an "engineered" host cell is one that has been engineered using genetic engineering, i.e., by human intervention. When a host cell is "engineered to overexpress" a given protein, the host cell is engineered to have an increased ability to express the protein or a functional homologue, in comparison to a non-engineered host cell, whereby expression of the given protein, e.g., a fusion protein of the invention or, in addition, one or more components of the signal recognition particle (SRP), is increased, in comparison to a host cell under the same conditions prior to engineering.

[0118] "Pre-engineered", when used in the context of a host cell of the invention, means that such a host cell has not been engineered to overexpress a protein, such as a fusion protein of the invention and / or a polynucleotide encoding one or more components of SRP or a functional homolog thereof.

[0119] As used herein, the term "recombinant" means that the host cell of the invention is equipped with a heterologous polynucleotide encoding a protein of interest, i.e., the host cell of the invention is engineered to contain a heterologous polynucleotide encoding a protein of interest. This can be accomplished, for example, by transformation or transfection, or any other suitable technique known in the art for the introduction of polynucleotides into a host cell.

[0120] Overexpression can be achieved in any manner known to those skilled in the art, as described in detail below. In general, it can be achieved by increasing transcription and / or translation of the gene, e.g., increasing the copy number of the gene, or by altering or modifying regulatory sequences or sites associated with the expression of the gene. For example, overexpression can be achieved by introducing, by transformation, one or more copies of a polynucleotide encoding a protein, e.g., a protein of interest and / or one or more components of the signal recognition particle (SRP), or a functional homologue operably linked to a regulatory sequence (e.g., a promoter), into a host cell, or even into the host cell's respective genome, chromosome. For example, the gene can be operably linked to a constitutive promoter and / or a ubiquitous promoter or an inducible promoter to reach a high expression level. Such a promoter can be an endogenous promoter or a recombinant promoter. Alternatively, it is possible to remove the regulatory sequence such that expression becomes constitutive and / or increases when the negative regulatory sequence is removed. The native promoter of a given gene can be replaced with a heterologous promoter in the genome, chromosome of the host cell that increases the expression of the gene or leads to constitutive expression of the gene. For example, the promoter can be a strong constitutive promoter and / or a strong ubiquitous promoter or a strong inducible promoter to reach a higher expression level than the native promoter. In the case where one or more components of the POI and / or the signal recognition particle (SRP) foreign to the host cell are expressed, the terms "expression" or "expressing" and "overexpressing" or "overexpressing" and "(over)expressing" and "(over)expressing" are used interchangeably. For example, a protein of interest and / or one or more components of the signal recognition particle (SRP) may be (over)expressed by the host cell by more than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more than 300% compared to the host cell prior to manipulation and culture under the same conditions.As used herein, "overexpression" may relate to increased expression of a protein (e.g., one or more components of a signal recognition particle (SRP) or a protein of interest) by a host cell in comparison to a host cell (control) that naturally expresses the protein but has not been engineered to overexpress the protein, although protein expression may be increased by any or more of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300%. Additionally, as used herein, "overexpression" may relate to expression of a protein (e.g., a protein of interest) in comparison to a host cell that does not express the protein when not engineered to express the protein. The use of an inducible promoter additionally allows for increased expression over the course of culturing the host cell. Moreover, overexpression can also be achieved, for example, by altering the chromosomal location of a particular gene, by changing the nucleic acid sequence adjacent to a particular gene, such as a ribosome binding site or a transcription terminator, by modifying the proteins (e.g., regulatory proteins, suppressors, enhancers, transcription activators, and the like) involved in the transcription of the gene and / or the translation of the gene product, or by any other conventional means in the art of deregulating the expression of a particular gene routinely, including, but not limited to, the use of antisense nucleic acid molecules to block the expression of repressor proteins, or by deleting or mutating the gene for a transcription factor that normally suppresses the expression of the gene desired to be overexpressed. The level of expression can also be improved by extending the lifespan of the mRNA. For example, the half-life of the mRNA can be extended using a particular terminator region (Yamanishi et al., Biosci.Biotechnol.Biochem.(2011)75:2234 and US 2013 / 0244243). If multiple copies of the gene are included, the gene is located in a plasmid of variable copy number or integrated and amplified in the chromosome. If the host cell does not contain the gene product, it is possible to introduce the gene product into the host cell for expression.In this case, "overexpression" means expressing a gene product using any method known to the skilled artisan. Overexpression in comparison to a control as described above, for example of one or more components of the signal recognition particle (SRP) or its functional homologues, can be measured by methods known to the skilled artisan, for example by SDS Page, SDS Page / Western blot, ELISA, or peptide map with subsequent mass spectrometry (proteomics). For example, the content of one or more components of the signal recognition particle (SRP) in the cells can be measured and compared to the content in the control. Thus, the cells must be disintegrated and the content is then measured in the cell lysate. Overexpression of the protein of interest, respectively, can be measured, for example, by measuring the content of the protein of interest in the supernatant of the respective host cell and the control, which contents are then compared. The content of the protein of interest in the supernatant can be measured by methods known to the skilled artisan, for example by SDS Page, SDS Page / Western blot, ELISA, RP (reverse phase) HPLC, ion exchange HPLC, and the like.

[0121] Those skilled in the art will be familiar with Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), Eikmanns et al. (Gene 102, 93-98(1991)), EP 0 472 869, US 4,601,893, Schwarzer and Puehler(Bio / Technology 9, 84-87(1991)), Reinscheid et al.(Applied and Environmental Microbiology 60, 126-132(1994)), LaBarre et al.(Journal of Bacteriology 175, 1001- 1007 (1993)), WO 96 / 15246, Malumbres et al. (Gene 134, 15- 24 (1993)), JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), and Makrides (Microbiological Reviews 60, 512-538 (1996)), among others, will find relevant instructions in well-known textbooks on genetics and molecular biology.

[0122] The nucleic acid or vector encoding the fusion protein of the invention and / or the polynucleotide encoding one or more components of SRP are preferably integrated into the genome, more preferably into a chromosome or a non-chromosomal genomic site of the host cell. The term "genome" generally refers to the entire genetic information of an organism, encoded in DNA (or for certain viral species, RNA). It may be present in a chromosome, on a plasmid or vector, or both. The vector or nucleic acid encoding one or more components of SRP are preferably integrated into the genome of the host cell.

[0123] Polynucleotides encoding the fusion proteins of the invention and / or polynucleotides encoding one or more components of SRP may be recombined in a host cell by ligating each of the relevant genes into a vector. It is possible to construct a single vector carrying the genes, or two separate vectors, one carrying the fusion protein of the invention and the other carrying one or more components of SRP. These genes can be integrated into the host cell genome by transforming the host cell with such a nucleic acid, such as a nucleic acid of the invention or a vector described herein. In some embodiments, a gene encoding a fusion protein of the invention is integrated into the genome and a gene encoding one or more components of SRP is integrated into a plasmid or vector. In some embodiments, a gene encoding one or more components of SRP is integrated into the genome and a gene encoding a fusion protein is integrated into a plasmid or vector. In some embodiments, a gene encoding a fusion protein of the invention and a polynucleotide encoding one or more components of SRP are integrated into the genome. In some embodiments, a gene encoding a fusion protein of the invention and a polynucleotide encoding one or more components of SRP are integrated into the genome. When multiple genes encoding fusion proteins are used, some of the genes encoding the fusion proteins can be integrated into the genome while others can be integrated into the same or different plasmids or vectors.When multiple genes encoding one or more components of SRP are used, some of the genes encoding one or more components of SRP can be integrated into the genome while others can be integrated into the same or different plasmids or vectors.

[0124] Overexpression of one or more components of SRP can also be achieved by replacing the regulatory elements of the host cell's endogenous SRP protein with regulatory elements that lead to higher transcription of the host cell's endogenous SRP protein. Methods of the Invention

[0125] The present invention further relates to a method for producing a protein of interest in a eukaryotic host cell, comprising: (i) genetically engineering a recombinant host cell with a nucleic acid molecule of the invention or with an expression cassette of the invention, optionally genetically engineering the recombinant host cell to overexpress one or more components of the signal recognition particle (SRP); (ii) culturing the genetically engineered host cell under conditions that express the nucleic acid molecule and optionally one or more components of SRP and secrete the protein of interest upon cleavage of the secretion signal; (iii) optionally isolating the protein of interest from the cell culture; (iv) optionally purifying the protein of interest; (v) optionally modifying a protein of interest, and (vi) optionally formulating the protein of interest.

[0126] Step (i) of the method of production may also be understood as genetically engineering the recombinant host cell to overexpress the fusion protein of the invention and optionally one or more components of SRP (see also the relevant disclosure in section "Host Cells of the Invention"). When a host cell is "engineered to overexpress" a given protein, the host cell is engineered such that it has the ability to express, preferably overexpress, the nucleic acid molecule of the invention and optionally one or more components of SRP, whereby the expression of the given protein, e.g. the fusion protein of the invention and optionally one or more components of SRP, is increased compared to the host cell under the same conditions before engineering. In one embodiment, "engineered to overexpress" means that a genetic modification is made to the host cell to overexpress or express the expression of a protein, i.e. the cell is (intentionally) genetically engineered to overexpress such a protein. Engineering to overexpress may include exchanging the promoter of the endogenous one or more components of SRP with a promoter leading to higher transcription. Engineering to overexpress may include genetically engineering a host cell with a nucleic acid molecule, expression cassette, or vector encoding one or more components of the fusion proteins and / or SRP described herein.

[0127] The invention further relates to a method for producing a protein of interest by culturing a recombinant eukaryotic host cell of the invention under conditions that allow for expression of a nucleic acid molecule of the invention and for secretion of the protein of interest after cleavage of the secretion signal, and isolating the protein of interest from the host cell culture upon cleavage of the secretion signal, and optionally purifying, and optionally modifying, and optionally formulating the protein of interest.

[0128] "Prior to engineering" or "prior to manipulation" as used in the context of host cells disclosed herein means that such host cells have not been engineered with a nucleic acid encoding a fusion protein of the invention. The term thus also means that the host cells do not overexpress a nucleic acid encoding a fusion protein of the invention and / or have not been engineered to overexpress one or more components of SRP.

[0129] For example, the procedures used to engineer nucleic acid molecule sequences encoding one or more components of the fusion proteins and / or SRP of the invention, promoters, enhancers, leaders, etc., are well known to those of skill in the art and are described, for example, by J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2001).

[0130] Foreign or target polynucleotides, such as the nucleic acid molecules of the present invention, can be inserted into a chromosome by various means, for example, by homologous recombination or by using a hybrid recombinase that specifically targets the sequence of the integration site. The foreign or target polynucleotides described above are typically present in a vector ("insertion vector"). These vectors are typically circular and linearized before being used for homologous recombination. As an alternative, the foreign or target polynucleotides can be DNA fragments linked by fusion PCR, or synthetically constructed DNA fragments that are then recombined into the host cell. In addition to the homology arms, the vectors can also contain appropriate markers for selection or screening, origins of replication, and other elements. It is also possible to use heterologous recombination, which results in random or non-targeted integration. Heterologous recombination refers to recombination between DNA molecules with significantly different sequences. Methods of recombination are known in the art and are described, for example, in Boer et al., Appl Microbiol Biotechnol (2007) 77:513-523. For genetic manipulation of yeast cells, reference may also be made to Principles of Gene Manipulation and Genomics by Primrose and Twyman (7th edition, Blackwell Publishing 2006).

[0131] The nucleic acid molecules of the invention and / or one or more components of the SRP may also be present on a vector, such as an expression vector. Such vectors are known in the art. In an expression vector, a promoter is placed upstream of a gene encoding a heterologous protein to regulate the expression of the gene. Multicloning vectors are particularly useful due to their multiple cloning sites. For expression, a promoter is generally placed upstream of the multiple cloning site. Vectors for the incorporation of nucleic acid molecules encoding a fusion protein of the invention or one or more components of the SRP may be constructed by first preparing a DNA construct containing the entire DNA sequence encoding the fusion protein of the invention or one or more components of the SRP and then inserting this construct into an appropriate expression vector, or by sequential insertion of DNA fragments containing the genetic information for the individual elements, e.g. DNA binding domains, activation domains, etc., followed by ligation. As an alternative to restriction and ligation of fragments, recombination methods based on attachment sites (att) and recombinases may be used to insert DNA sequences into the vector. Such methods are described, for example, by Landy (1989) Ann. Rev. Biochem. 58:913-949; and are known to those of skill in the art.

[0132] A host cell according to the invention can be obtained by introducing into the cell a vector or plasmid comprising a target polynucleotide sequence, such as a nucleic acid molecule of the invention. Techniques for transfecting or transforming eukaryotic cells or transforming prokaryotic cells are well known in the art. These may include lipid vesicle-mediated uptake, heat shock-mediated uptake, electroporation, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), viral infection, especially using modified viruses, such as modified adenoviruses, microinjection and electroporation. For prokaryotic transformation, techniques may include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection and electroporation. Techniques for plant transformation include Agrobacterium-mediated transfer, such as by A. tumefaciens, fast-propelling tungsten or gold particles, electroporation, microinjection and polyethylene glycol-mediated uptake. DNA may be single-stranded or double-stranded, linear or circular, relaxed or supercoiled DNA. For various techniques for transfecting mammalian cells, see, e.g., Keown et al. (1990) Processes in Enzymology 185:527-537.

[0133] The phrase culturing a (genetically engineered) host cell under conditions to express a nucleic acid molecule of the invention, and optionally overexpress "one or more components of SRP", refers to maintaining and / or growing a eukaryotic host cell under conditions (e.g., temperature, pressure, pH, induction, growth rate, medium, duration, supply, etc.) suitable or sufficient to obtain production of a desired protein of interest or to overexpress one or more components of SRP.

[0134] A host cell according to the invention, obtained by engineering the host cell with a nucleic acid molecule of the invention or with an expression cassette of the invention, and optionally genetically engineering the host cell to overexpress one or more components of the signal recognition particle (SRP), may be initially cultured, preferably under conditions that allow efficient growth to large numbers of cells without the burden of expressing recombinant proteins. When the cells are prepared for expression of a fusion protein, appropriate culture conditions are selected and optimized to produce the fusion protein.

[0135] By way of example, using different promoters and / or copies and / or integration sites for the fusion protein and the one or more components of SRP, the expression of the fusion protein can be controlled with respect to the time point and strength of induction relative to the expression of the one or more components of SRP. For example, the one or more components of SRP can be expressed first, prior to induction of the fusion protein. This has the advantage that the one or more components of SRP are already present at the start of translation of the fusion protein. Alternatively, the fusion protein and the one or more components of SRP can be induced simultaneously.

[0136] Inducible promoters may be used, which are activated as soon as an inducing stimulus is applied and direct the transcription of genes under their control. Under growth conditions with an inducing stimulus, cells grow slower than under normal conditions, but because the culture has already grown to a high cell number in the previous stage, the culture system produces a large amount of recombinant protein overall. The inducing stimulus is preferably the addition of a suitable agent (e.g., methanol for the AOX promoter) or the depletion of a suitable nutrient (e.g., methionine for the MET3 promoter). Also, the addition of ethanol, methylamine, cadmium or copper, as well as heat or osmolality increasing agents, can induce expression depending on the promoter operably linked to the fusion protein and one or more components of SRP.

[0137] It is preferred to cultivate the host cells according to the invention in a bioreactor under optimized growth conditions to obtain at least 1 g / L, preferably at least 10 g / L cell dry weight, more preferably at least 50 g / L cell dry weight. It is advantageous to achieve such yields of biomolecule production not only on laboratory scale, but also on pilot or industrial scale.

[0138] The host cells according to the invention can be tested for their expression / secretion capacity or yield by measuring the titer of the protein of interest in the cell culture supernatant or in the cell homogenate of the cells after cell homogenization by using standard tests, e.g. ELISA, activity assays, HPLC, surface plasmon resonance (Biacore), Western blot, capillary electrophoresis (Caliper) or SDS-Page.

[0139] Preferably, the host cells are cultured in a minimal medium with an appropriate carbon source, which further significantly simplifies the isolation process. By way of example, a minimal medium contains an available carbon source (e.g., glucose, glycerol, ethanol, or methanol), salts including macroelements (potassium, magnesium, calcium, ammonium, chloride, sulfate, phosphate) and trace elements (copper, iodide, manganese, molybdate, cobalt, zinc, and iron salts, and boric acid).

[0140] In the case of yeast cells, the cells can be transformed with one or more of the expression vectors described above and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Numerous minimal media suitable for yeast growth are known in the art. Any of these media can be supplemented as necessary with salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES, citrate, and phosphate buffers), nucleosides (e.g., adenosine and thymidine, etc.), trace elements, vitamins, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations and will be known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and are known to those skilled in the art. Cell culture conditions for other types of host cells are also known and can be easily determined by those skilled in the art. Descriptions of culture media for various microorganisms are contained, for example, in the American Society of Bacteriology handbook "Manual of Methods for General Bacteriology" (Washington, DC, USA, 1981).

[0141] The host cells can be cultured continuously or intermittently by conventional culture methods, such as test tube culture, shaking culture (e.g., rotary shake culture, shake flask culture, etc.), aerated spinner culture, fermentation, etc. In some embodiments, the cells are cultured in a shaking flask or deep well plate. In yet other embodiments, the cells are cultured in a bioreactor (e.g., in a bioreactor culture process). Culture processes include, but are not limited to, batch, fed-batch culture, and continuous culture methods. The terms "batch process" and "batch culture" refer to a closed system in which the composition of the medium, nutrients, supplementary additives, and the like is set at the beginning of the culture and is not subject to modification during the culture; however, attempts may be made to control factors such as pH and oxygen concentration to prevent excessive medium acidification and / or cell death. The terms "fed-batch process" and "fed-batch culture" refer to a batch culture, except that one or more substrates or supplements are added (e.g., added incrementally or continuously) as the culture progresses. The terms "continuous process" and "continuous culture" refer to a system in which a defined culture medium is added continuously to a bioreactor and an equal amount of spent or "conditioned" medium is simultaneously removed, e.g., for recovery of a desired product. A variety of such processes have been developed and are well known in the art.

[0142] In some embodiments, the host cells are cultured for about 12-24 hours, while in other embodiments, the host cells are cultured for about 24-36 hours, about 36-48 hours, about 48-72 hours, about 72-96 hours, about 96-120 hours, about 120-144 hours, or more than 144 hours. In still other embodiments, the culture is continued for a time sufficient to reach a desired production yield of the POI.

[0143] The methods of the invention, for example, methods of producing a protein of interest or methods of increasing secretion of a protein of interest, may further comprise a step of isolating the expressed POI. The POI is secreted from the cells and can be isolated and purified from the culture medium using state-of-the-art technology. During the process of secretion, the secretion signal is cleaved. Secretion of the POI from the cells is generally preferred because the product is recovered from the culture supernatant, rather than from a complex mixture of proteins that results when the cells are disrupted and the intracellular proteins are released. Protease inhibitors may be useful to inhibit protein degradation during purification. The composition may be concentrated, filtered, dialyzed, etc., using methods known in the art. The cell culture after fermentation / cultivation may be centrifuged using a separator or tube centrifuge to separate the cells from the culture supernatant. The supernatant may then be filtered or concentrated by using tangential flow filtration.

[0144] Isolation and purification methods for obtaining POI can be based on methods utilizing differences in solubility, such as salting out, solvent precipitation, heat precipitation, etc., methods utilizing differences in molecular weight, such as size exclusion chromatography, ultrafiltration, and gel electrophoresis, methods utilizing differences in charge, such as ion exchange chromatography, methods utilizing specific affinity, such as affinity chromatography, methods utilizing differences in hydrophobicity, such as hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography, methods utilizing differences in isoelectric point, such as isoelectric focusing, methods utilizing specific amino acids, such as IMAC (immobilized metal ion affinity chromatography), etc. If the POI is expressed as inactive, soluble inclusion bodies, the solubilized inclusion bodies need to be refolded.

[0145] The isolated and purified POI can be identified by conventional methods, such as Western blotting or specific assays for POI activity. The structure of the purified POI can be determined by amino acid analysis, amino-terminal peptide sequencing, primary structure analysis, e.g., by mass spectrometry, RP-HPLC, ion-exchange-HPLC, ELISA, and the like. The POI is preferably available in large quantities and high purity levels, thus fulfilling the necessary requirements for use as an active ingredient in pharmaceutical compositions or as a feed or food additive.

[0146] As used herein, the term "isolated" refers to a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include: (1) any non-naturally occurring substance; (2) any substance, including but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, that is at least partially removed from one or more or all of the natural components with which it is associated in nature; (3) any substance that has been modified by the hand of man relative to the substance with which it is associated in nature, such as cDNA made from mRNA; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of the gene encoding the substance; and the use of a stronger promoter than that naturally associated with the gene encoding the substance).

[0147] The term "modifying a protein of interest" means that the POI is modified chemically or enzymatically. There are many methods known in the art for modifying proteins. Proteins can be conjugated with carbohydrates or lipids. POIs can be PEGylated (POI chemically conjugated to polyethylene glycol) or HESylated (POI chemically conjugated to hydroxyethyl starch) for half-life extension. POIs can also be conjugated with other moieties, such as affinity domains for human serum albumin for half-life extension. POIs can also be treated with proteases or under hydrolytic conditions for cleavage to form active components from presequences or to cleave tags, such as affinity tags, for purification. POIs can also be conjugated with other moieties, such as toxins, radioactive moieties, or any other moieties. POIs can be further treated under conditions to form dimers, trimers, and the like.

[0148] Furthermore, the term "formulating a protein of interest" refers to bringing the POI into a condition where it can be stored for a longer period of time. Many different methods known in the art are available for stabilizing proteins. The POI can be placed under more stable conditions by exchanging the buffer in which the POI resides after purification and / or modification. Different buffer substances and additives, such as sucrose, neutral detergents, stabilizers, and the like, are known in the art and can be used. The POI can also be stabilized by lyophilization. For some POIs, formulation can be performed by forming a complex between the POI and lipids or lipoproteins, such as polyplexes, and the like. Some proteins can be co-formulated with other proteins.

[0149] Use of the secretion signal of the invention may increase secretion of a protein of interest. Thus, the invention also relates to a method for increasing secretion of a protein of interest from a eukaryotic host cell, comprising expressing a nucleic acid molecule of the invention in said eukaryotic host cell, and optionally genetically engineering the host cell to overexpress one or more components of a signal recognition particle (SRP), thereby increasing secretion of said protein of interest in comparison to a host cell expressing a fusion protein as defined herein, but comprising a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (such as SEQ ID NO: 4) instead of the secretion signal as defined in the nucleic acid molecule of the invention.

[0150] "Secretion" as used herein relates to the transfer of the protein of interest forming part of the fusion protein of the invention from the (recombinant) host cell. In this way, only the protein of interest is secreted, not the secretion signal. The signal peptide sequence is cleaved in the endoplasmic reticulum and the MFα pro sequence is cleaved in the Golgi apparatus. In this way, when secretion is increased, only the secretion of the protein of interest is increased. The titer of the protein of interest in the supernatant of the cell culture can be determined using standard tests, such as ELISA, activity assays, HPLC, surface plasmon resonance (Biacore), Western blot, capillary electrophoresis (Caliper) or SDS-Page.

[0151] Methods for increasing secretion of a protein of interest from a eukaryotic host cell can include engineering the host cell to incorporate an expression construct for expressing a nucleic acid molecule of the invention, and, optionally, further comprising genetically engineering the eukaryotic host cell to overexpress one or more components of the signal recognition particle (SRP).

[0152] Methods for increasing secretion of a protein of interest from a eukaryotic host cell can include culturing said host cell under conditions for expression of a nucleic acid molecule of the invention, and, optionally, further comprising genetically engineering the host cell to overexpress one or more components of SRP and to secrete the protein of interest upon cleavage of the secretion signal.

[0153] The method of increasing secretion of a protein of interest from a eukaryotic host cell may further comprise isolating the protein of interest from the cell culture.

[0154] The method of increasing secretion of a protein of interest from a eukaryotic host cell may further comprise purifying the protein of interest.

[0155] The method of increasing secretion of a protein of interest from a eukaryotic host cell may further comprise modifying the protein of interest.

[0156] The method of increasing secretion of a protein of interest from a eukaryotic host cell may further include formulating the protein of interest.

[0157] In methods for increasing secretion of a protein of interest from a eukaryotic host cell, the nucleic acid molecule of the invention can be integrated into the chromosome of the host cell or can be contained in a vector or plasmid that is not integrated into the chromosome of the host cell. Uses of the invention

[0158] A person skilled in the art will easily recognize that the secretion signal described herein can be used to increase the secretion of a recombinant protein of interest. Thus, the present invention also relates to the use of the secretion signal defined herein to increase the secretion of a protein of interest from a eukaryotic host cell. As already described herein, the secretion signal may comprise, from the N-terminus to the C-terminus, a signal peptide sequence derived from the KRE1 protein, optionally followed by an alpha mating factor (MFα) pro-sequence. Thus, the present invention also relates to the use of the secretion signal defined herein to increase the secretion of a protein of interest from a eukaryotic host cell, in which the secretion signal comprises, from the N-terminus to the C-terminus, a signal peptide sequence derived from the KRE1 protein, followed by an alpha mating factor (MFα) pro-sequence. The .... As already described herein, the secretion signal may comprise, from N-terminus to C-terminus, a signal peptide sequence derived from the SWP1 protein, optionally followed by an alpha mating factor (MFα) pro-sequence. Thus, the present invention also relates to the use of a secretion signal as defined herein for increasing secretion of a protein of interest from a eukaryotic host cell, in which the secretion signal comprises, from N-terminus to C-terminus, a signal peptide sequence derived from the SWP1 protein, followed by an alpha mating factor (MFα) pro-sequence. The present invention also relates to the use of a secretion signal as defined herein for increasing secretion of a protein of interest from a eukaryotic host cell, in which the secretion signal comprises a signal peptide sequence derived from the SWP1 protein.

[0159] The secretion signal may increase secretion of the protein of interest from a eukaryotic host cell in comparison to a eukaryotic host cell expressing a fusion protein of the invention that includes a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (e.g., SEQ ID NO: 4) in place of the secretion signal defined herein.

[0160] As one skilled in the art will readily appreciate, the recombinant host cells of the present invention are useful for the production of various proteins of interest because they are efficiently secreted into the supernatant, thereby avoiding lysis of the host cells. Thus, the present invention further relates to the use of the recombinant host cells of the present invention to produce a protein of interest. item 1. A nucleic acid molecule encoding a fusion protein comprising from N-terminus to C-terminus: (a) a secretion signal, the secretion signal comprising: (I) (i) a signal peptide sequence derived from a KRE1 protein or a signal peptide sequence derived from a SWP1 protein; and (ii) α-mating factor (MFα) prosequence; or (II) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein; and (b) The protein of interest. 2. The nucleic acid molecule of claim 1, which expresses the nucleic acid molecule of claim 1 with a secretion signal, but which results in increased secretion of the protein of interest from a eukaryotic host cell compared to a eukaryotic host cell that contains a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (such as SEQ ID NO: 4) instead of the secretion signal defined in claim 1. 3. The nucleic acid molecule of item 1 or 2, wherein the signal peptide sequence derived from the KRE1 protein comprises SEQ ID NO: 1 or a functional homologue thereof. 4. The nucleic acid molecule of item 1 or 2, wherein the signal peptide sequence derived from the SWP1 protein comprises SEQ ID NO: 2 or 52 or a functional homologue thereof. 5. The nucleic acid molecule of any one of items 1 to 4, wherein the MFα prosequence comprises SEQ ID NO: 3 or 53 or a functional homologue thereof and / or the MFα prosequence comprises Ser at a position corresponding to position 23 of SEQ ID NO: 53 and / or Glu at a position corresponding to position 64 of SEQ ID NO: 53. 6. The nucleic acid molecule according to any one of items 1 to 5, wherein the protein of interest is selected from the group consisting of an antibody, such as a chimeric, humanized or human antibody, or a bispecific antibody, or an antigen-binding antibody fragment, such as Fab or F(ab)2, a single chain antibody, such as scFv, a single domain antibody, such as a camelid VHH fragment or a heavy chain antibody or a domain antibody (dAb), an artificial antigen-binding molecule, such as DARPIN, an ibody, an affibody, a humabody, or a mutein based on a polypeptide of the lipocalin family, an enzyme, such as a processed enzyme, a cytokine, a growth factor, a hormone, a protein antibiotic, a fusion protein, such as a toxin fusion protein, a structural protein, a regulatory protein, and a vaccine antigen, preferably the protein of interest is a therapeutic protein, a food additive, or a feed additive. 7. A secretion signal as defined in any one of items 1 to 6. 8. An expression cassette or vector comprising the nucleic acid molecule of any one of items 1 to 6 and a promoter operably linked thereto. 9. A recombinant eukaryotic host cell comprising a nucleic acid molecule according to any one of items 1 to 6, or an expression cassette or vector according to item 8, preferably (a) the host cell may be a fungal or yeast host cell selected from the group consisting of Komagataella phaphi (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., and Schizosaccharomyces pombe, preferably a yeast host cell, or a fungal host cell, such as Trichoderma reesei or Aspergillus niger; and / or (b) Host cells are engineered to overexpress one or more components of the signal recognition particle (SRP). 10. A method for producing a protein of interest in a eukaryotic host cell, comprising: (i) genetically engineering a eukaryotic host cell with a nucleic acid molecule according to any one of items 1 to 6, or with an expression cassette or vector according to item 8, and optionally genetically engineering the eukaryotic host cell to overexpress one or more components of the signal recognition particle (SRP); (ii) culturing the genetically engineered host cell under conditions to express the nucleic acid molecule, and optionally overexpress one or more components of SRP, and secrete the protein of interest upon cleavage of the secretion signal; (iii) optionally isolating the protein of interest from the cell culture; (iv) optionally purifying the protein of interest; (v) optionally modifying a protein of interest, and (vi) optionally formulating the protein of interest. 11. A method for increasing secretion of a protein of interest from a eukaryotic host cell, comprising expressing in said eukaryotic host cell a nucleic acid molecule according to any one of items 1 to 6, and optionally engineering said eukaryotic host cell to overexpress one or more components of a signal recognition particle (SRP), thereby increasing secretion of said protein of interest compared to said host cell expressing the nucleic acid molecule of items 1 to 6, except that said host cell comprises a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (e.g., SEQ ID NO: 4) instead of a secretion signal as defined in any one of items 1 to 6. 12. The method according to item 10 or 11, (a) the method comprises: (i) engineering said host cell to incorporate an expression construct that expresses the nucleic acid molecule of any one of items 1 to 6, and optionally genetically engineering the host cell to overexpress one or more components of the signal recognition particle (SRP); (ii) culturing the host cell under conditions to express the nucleic acid molecule, and optionally overexpress one or more components of SRP, and secrete the protein of interest upon cleavage of the secretion signal; (iii) optionally isolating the protein of interest from the cell culture; (iv) optionally purifying the protein of interest; (v) optionally modifying a protein of interest, and (vi) optionally formulating the protein of interest; and / or (b) the nucleic acid molecule is contained in an expression cassette, vector, or plasmid that is integrated into a chromosome of the host cell or that is not integrated into a chromosome of the host cell; and / or (c) the eukaryotic host cell is a fungal host cell or a yeast host cell, preferably a fungal host cell or a yeast host cell selected from the group consisting of Komagataella phafii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Saccharomyces kluyveri, Saccharomyces uvarum, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., and Schizosaccharomyces pombe, preferably a yeast host cell, or a fungal host cell, such as Trichoderma reesei or Aspergillus niger; and / or (d) the protein of interest is selected from the group consisting of an antibody, such as a chimeric, humanized or human antibody, or a bispecific antibody, or an antigen-binding antibody fragment, such as a Fab or F(ab)2, a single chain antibody, such as a scFv, a single domain antibody, such as a camelid VHH fragment or a heavy chain antibody or a domain antibody (dAb), an artificial antigen-binding molecule, such as a DARPIN, an ibody, an affibody, a humabody, or a mutein based on a polypeptide of the lipocalin family, an enzyme, such as a processed enzyme, a cytokine, a growth factor, a hormone, a protein antibiotic, a fusion protein, such as a toxin fusion protein, a structural protein, a regulatory protein, and a vaccine antigen, preferably the protein of interest is a therapeutic protein, a food additive, or a feed additive. 13. Use of a secretion signal as defined in any one of items 1 to 7 for increasing secretion of a protein of interest from a eukaryotic host cell, in comparison to said eukaryotic host cell expressing a fusion protein as defined in item 1, which preferably comprises a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (such as, for example, SEQ ID NO: 4) instead of the secretion signal as defined in item 1. 14. Use of the recombinant eukaryotic host cell of item 9 for producing a protein of interest. 15. A method for producing a protein of interest, by culturing a recombinant eukaryotic host cell of item 9 under conditions in which the nucleic acid molecule of any one of items 1 to 6 is expressed and the protein of interest is secreted upon cleavage of the secretion signal, and isolating the protein of interest from the host cell culture, and optionally purifying, and optionally modifying, and optionally formulating the protein of interest.

[0161] It is noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such different reagents, and reference to a "method" includes reference to equivalent steps and methods known to those of skill in the art that may modify or substitute for the method described herein.

[0162] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0163] The term "and / or," whenever used in this specification, is inclusive of the meaning of "and," "or," and "all or any other combination of the elements connected by said term."

[0164] The term "about" means plus or minus 20%, preferably plus or minus 10%, more preferably plus or minus 5%, and most preferably plus or minus 1%.

[0165] The terms "less than" or "more than" do not include specific numbers.

[0166] For example, less than 20 means less than the stated number. Similarly, "more than" or "greater than" means more than or greater than a stated number, for example, "more than 80%" means more than or greater than 80% of the stated number.

[0167] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" may be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps. As used herein, the term "comprising" can be substituted with the terms "containing" or "including," and occasionally, as used herein, with the term "having." As used herein, "consisting of" excludes unspecified elements, steps, or ingredients.

[0168] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0169] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0170] All publications cited throughout the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manuals, etc., are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference is contradictory or inconsistent with the present specification, the present specification supersedes any such material.

[0171] The contents of all publications and patent documents cited herein are incorporated by reference in their entirety. Working Example

[0172] A better understanding of the present invention and its advantages will be apparent from the following examples, which are provided for illustrative purposes only. The examples are not intended to limit the scope of the invention in any manner. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, concentrations, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius; and pressure is at or near atmospheric pressure.

[0173] The following examples demonstrate that the newly identified signal peptide, in combination with the pro-sequence of the Saccharomyces cerevisiae alpha mating factor, increases the titer (product per volume (mg / g)) and yield (product per biomass measured as wet cell weight (mg / L)) of secreted recombinant proteins compared to known secretion leaders, including the commonly used S. cerevisiae alpha mating factor pre-pro leader. As an example, the use of the novel signal peptide led to an increase in the yield of different recombinant proteins and antibody derivatives in the yeast Pichia pastoris, including single chain variable fragments, single domain antibodies, antigen binding fragments, and easy to detect proteins (scR, VHH, SDZ-Fab, m-Cherry). The positive effects were shown in shaking cultures (performed in 24 deep-well plates) and in fed-batch bioreactor cultures. Example 1: Selection of novel signal peptides.

[0174] The most commonly used secretion signal yeast, including Pichia pastoris (syn. Komagataella), is the Saccharomyces cerevisiae alpha mating factor (MFα) pre-pro leader, i.e., the MFα secretion signal. Some recombinant proteins are inefficiently secreted using the MFα secretion signal and thus reach only low production titers. Thus, there is an urgent need for novel signal peptides and secretion signals to increase secretion efficiency.

[0175] The first critical step in secretion is the translocation of recombinant proteins into the endoplasmic reticulum (ER). This process is directed by an N-terminal cleavable secretion signal fused to the recombinant protein. For secretion of recombinant proteins, at least an N-terminal cleavable signal peptide sequence is required. These can be predicted from the amino acid sequence using the most widely used program SignalP (Nielsen, 2017). Based on sequence data of P. pastoris, 241 proteins with cleavable signal peptides (SPs) were predicted by SignalP 4.1 (Valli et al., 2016). However, no distinction is made between co-translational or post-translational SPs, i.e., the predicted SPs can act co-translationally or post-translationally, but the amino acid sequence itself does not give information on whether the predicted signal peptide sequence is capable of secreting a recombinant fusion protein (i.e., a protein different from the protein naturally secreted by this signal peptide sequence). To date, no sufficiently distinct characteristics have been found with regard to key physicochemical properties, such as hydrophobicity of signal peptide sequences, putative binding motifs, or other sequence patterns, that would describe efficient signal peptides for co-translational or post-translational translocation of recombinant proteins (Janda et al., 2010; Pechmann et al., 2014; Massahi et al. (2016),. J Theor Biol., 408:22-33.).

[0176] We used an in silico approach to select for new efficient SPs. First, the hydrophobicity average was calculated using the hydrophobicity index normalized to the number of amino acids in the signal peptide (Kyte and Doolittle, 1982). Then, we searched for the most hydrophobic stretch of eight amino acids in the SP and assigned it a maximum score of 1, representing the most hydrophobic stretch in the entire collection. Furthermore, we evaluated the average relative fitness of codons 43-48 of naturally associated proteins (Sharp and Li, 1987) and assigned it a maximum score of 1, representing the average with the "latest" codon in this stretch. In other words, a score of 0 was given if only optimal codons were present in this stretch. The two scores generated were summed and candidates representing different rankings were selected (Table 4:). Among the scored candidates, SP4 (the first 18 amino acids of SWP1, PP7435_Chr1-0255) and SP14 (the first 18 amino acids of KRE1, PP7435_Chr3-0933) represented the signal peptides with the highest and lowest total scores, respectively. [Table 4] Example 2: Construction and selection of P. pastoris strains secreting recombinant secreted proteins using novel signal peptide sequences. Construction of plasmids carrying genes of interest.

[0177] P. pastoris CBS7435 mutS mutant (genome sequenced by Sturmberger et al. 2016) was used as the host strain. Genes encoding POIs (e.g., SDZ-Fab-LC, SDZ-Fab-HC, scR, VHH, and mCherry) were codon-optimized by Geneart or DNA2.0 (now ATUM) and obtained as synthetic DNA. A His6 tag was fused C-terminally to the scR and VHH genes for detection, while a FLAG tag was added to the C-terminus of mCherry. The sequences of these proteins are shown in Table 2 in the Description.

[0178] To construct expression vectors with novel signal peptide sequences (see Tables 4 and 7), the fragments selected for cloning were amplified by PCR (Q5® High-Fidelity DNA polymerase, New England Biolabs). Genomic DNA from P. pastoris strain CBS7435 mutS, synthetic genes, or gBlocks (Integrated DNA Technologies) served as PCR templates. The amplified coding sequences were cloned into either the pPUZZLE-based expression plasmid pPM2dZ30 (described in WO2008 / 128701A2) or the GoldenPiCS vector (Prielhofer et al., 2017). The signal peptide (SP) was directly assembled into the expression plasmid with the promoter, terminator, and product coding sequence.

[0179] The GoldenPiCS system (Prielhofer et al. 2017. BMC Systems Biol. doi:10.1186 / s12918-017-0492-3) requires the introduction of silent mutations in some coding sequences. Alternatively, gBlock or synthetic codon-optimized genes were obtained from commercial providers, including Integrated DNA Technology IDT, Geneart, and ATUM. The amplified coding sequences were cloned into either pPUZZLE-based expression plasmids pPM2aK21 or pPM2eH21, or into the GoldenPiCS system (consisting of backbones BB1, BB2, and BB3aZ / BB3aK / BB3eH / BB3rN). The gene fragments listed in Tables 1 and 2 were introduced into the expression plasmids by using restriction enzymes. All promoters and terminators used to construct expression cassettes in the BB2 or BB3 backbone are described in Prielhofer et al. 2017 (BMC Systems Biol. doi:10.1186 / s12918-017-0492-3). pPM2aK21 and BB3aK allow integration into the 3'-AOX1 genomic region and contain a KanMX selection marker cassette for selection in E. coli and yeast. pPM2eH21 and BB3eH contain a 5'-ENO1 genomic integration region and a HphMX selection marker cassette for selection with hygromycin. BB3rN contains a 5'-RGI1 genomic integration region and a NatMX selection marker cassette for selection with nourseothricin. The BB3aZ plasmid contains a Zeocin selection marker cassette and was linearized with AscI to allow integration into the 3'-AOX1 genomic region. All plasmids contain an origin of replication for E. coli (pUC19).

[0180] After electroporation (Example 3), transformants were selected on YPD (yeast extract, peptone, dextrose) plates containing the required antibiotic for the integrated selection marker (50 μg / mL Zeocin for Zeocin resistance marker with Sh ble gene, 500 μg / mL G418 for KanMX, and 100 μg / mL nourseothricin for NatMX) after incubation at 30° C. for 48 hours and homogenized under the same conditions.

[0181] The pPM2d_pGAP and pPM2d_pAOX expression plasmids are derivatives of the pPuzzle_ZeoR plasmid backbone described in WO2008 / 128701A2 and consist of a pUC19 bacterial replication origin and a Zeocin antibiotic resistance cassette. Expression of the heterologous genes is mediated by the P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) or alcohol oxidase (AOX) promoters, respectively, and the S. cerevisiae CYC1 transcription terminator. Some plasmids already contained the N-terminal S. cerevisiae alpha mating factor pre-pro leader sequence. After restriction digestion with XhoI and BamHI (for scR) or EcoRV (for VHH), each gene was ligated into both plasmids pPM2d_pGAP and pPM2d_pAOX digested with XhoI and BamHI or EcoRV.

[0182] Plasmids were linearized using AvrII (for pPM2d_pGAP) or PmeI (for pPM2d_pAOX) restriction enzymes, respectively, before electroporation into P. pastoris (using the standard transformation protocol described in Gasser et al. 2013. Future Microbiol. 8(2):191-208 - see Example 3). Selection of positive transformants was performed on YPD plates (per liter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar-agar) containing 50 μg / mL Zeocin.

[0183] In the following examples, different heterologous proteins were used as reporters: the variable vH region of a bivalent camelid antibody (VHH), a signal chain variable fragment antibody (scFv, called scR), an antigen-binding fragment of human IgG (SDZ-Fab) and the fluorescent protein mCherry. Expression of the heterologous POI was mediated by an appropriate P. pastoris promoter, for example the P. pastoris alcohol oxidase (AOX1) promoter (VHH, scR, SDZ-Fab-HC, mCherry) or the dihydroxyacetone synthase (DAS1) promoter (SDZ-Fab-LC) and the S. cerevisiae CYC1 (VHH, scR, mCherry, and SDZ-Fab-HC) or the P. pastoris TDH1 (SDZ-Fab-LC) transcription terminator. Plasmid construction was performed using the GoldenPiCS kit using Golden Gate Assembly as described in Prielhofer et al. (2017). POI genes were amplified from vector DNA templates (carrying the gene of interest) by PCR using primers containing SP sequences, and each was ligated into BB1 plasmid using the restriction enzyme BsaI, thereby generating expression vectors carrying the tested signal peptide. For multimeric proteins, such as Fabs, the individual expression vectors BB1_SDZ-Fab-HC and BB1_SDZ-Fab-LC were then assembled into BB2 plasmid using the restriction enzyme BpiI, resulting in BB2_ab_pAOX1_SDz-Fab-HC-CYC1tt and BB2_bc_pDAS1_SDZ-Fab-LC-TDH1tt plasmids. These BB2 plasmids were finally combined to generate BB3aZ_SDZ-Fab plasmids containing expression cassettes for both the HC and LC of the Fab fragment. For VHH and scR, the BB1 ​​plasmid was directly constructed with the BB3aZ plasmid, resulting in BB3aZ_VHH and BB3aZ_scR, respectively. New signal peptides were added as part of the 5' primer sequence by using fusion PCR to amplify them by PCR followed by Golden Gate assembly.After sequence verification, expression cassettes for all proteins were combined on one vector by using compatible restriction enzymes BpiI and BsaI. The coding DNA sequences of the recombinant proteins are given in Table 2. For secretion, the newly identified signal peptide alone (sequences see Table 7), the newly identified signal peptide in combination with the prosequence of the S. cerevisiae alpha mating factor (MFα) secretion leader (SEQ ID NO: 3), or the entire secretion signal of S. cerevisiae MFα (SEQ ID NO: 4) was used. Example 3: Generation of P. pastoris strains producing recombinant secreted proteins with novel signal peptide sequences.

[0184] The POI expression plasmid (up to 3 μg) was linearized with AscI before electroporation into P. pastoris. For this purpose, P. pastoris strains were made electrocompetent. Strains were inoculated in 100 mL YPD medium (main culture) for 16–20 h (25°C, 180 rpm) and harvested at an optical density (OD600) of 1.8–3 by centrifugation (5 min, 1500 g, 4°C) in two 50 mL Falcon tubes. The cell pellet was resuspended in 10 mL YPD + 20 mM HEPES + 25 mM DTT and incubated (30 min, 25°C, 180 rpm). After the incubation period, the Falcon tubes were filled with 40 mL ice-cold sterile distilled water and centrifuged (5 min; 1500 g; 4°C) (Eppendorf AG, Germany). The cell pellet was resuspended in ice-cold sterile 1 mM HEPES buffer (pH 8) and centrifuged (30 min; 25° C.; 180 rpm). The cell pellet was resuspended in 45 mL ice-cold 1 M sorbitol and centrifuged (30 min; 25° C.; 180 rpm). The pellet was resuspended in 500 μL ice-cold 1 M sorbitol and 80 μL was dispensed into ice-cold 1.5 mL Eppendorf tubes. The dispensed electrocompetent cells were kept at −80° C. until use.

[0185] Electroporation was performed at 2 kV for 4 ms (Gene Pulser, Bio-Rad Laboratories, Inc, USA). After transformation, electroporated cells were suspended in 1 mL YPD medium and allowed to recover for 1.5-3 h at 30° C. with shaking at 650 rpm on a thermoshaker (Eppendorf AG, Germany). Then, 20 μL and 200 μL of cell suspension were plated on YPD plates (per liter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar) containing 50 μg / mL Zeocin (CBS7435 mutS background) or 50 μg / mL Zeocin and 100 μg / mL nourseothricin (SRP co-expressing CBS7435 mutS, see Example 5) for selection and incubated at 30° C. for 48 h. Emerging colonies were restreaked onto fresh YPD plates containing the appropriate antibiotic. Example 4: Small scale cultivation (screening) of P. pastoris strains producing recombinant secreted proteins with novel signal peptide sequences. culture

[0186] 24 deep well plates (DWPs) sealed with air permeable membranes were used for screening. Single colonies (20 colonies from each transformation) were picked from the transformation plates for pre-culture and used to inoculate 2 mL of YPD with the appropriate antibiotic based on the antibiotic resistance used for selection. Precultures were grown in 24-DWP at 25 °C and 280 rpm for approximately 24 h, then cultured in 2 mL of synthetic screening medium ASMv6 (per liter: 22.0 g citric acid monohydrate, 6.3 g (NH4)2HPO4, 0.49 g MgSO4*7H2O, 2.64 g KCl, 0.054 g CaCl2*2H2O, 1.47 mL PTM0 trace salts stock solution (per liter: 6.0 g CuSO4*5H2O, 0.08 g NaI, 3.36 g MnSO4*H2O, 0.2 g Na2MoO4*2H2O, 0.02 g H3BO3, 0.82 g CoCl2*2H2O, 20.0 g H3BO3, 0.05 ... ZnCl2, 65.0 g FeSO4*7H2O and 5.0 ml H2SO4 (95%-98%), 4 mg biotin; pH set to 6.5 with KOH (solid). The main culture was continued for 48 hours and methanol was added four times for induction (0.5% at t=4 hours, 1% at t=20 hours, t=28 hours, and t=44 hours, respectively). Recovery and analysis

[0187] After 48 hours, 1 mL of each culture was removed and centrifuged in a pre-weighed Eppendorf tube. The wet cell weight (WCW) was determined by weighing the Eppendorf tube with the cell pellet and calculated as follows: Weight (full) - Weight (empty) = Wet Cell Weight (WCW) (g / L). The supernatant was used to quantify the recombinant secreted protein concentration using microfluidic capillary electrophoresis (mCE), ELISA, or fluorescence spectroscopy, as described below. From this data, the yield was calculated: Yield (μg / mg) = Protein concentration / Wet Cell Weight. The volumetric titer (also referred to as "titer") is the content (g / L or mg / L and the like) of the protein of interest (POI) in the supernatant of the culture described in Examples 4 and 6. Only clones with one copy of the gene of interest were included in the analysis. The gene copy number of the clones selected for bioreactor cultivation was determined by qPCR (Example 4). Fold changes in titer and yield are each given relative to a reference clone carrying one copy of the gene of interest secreted by using the S. cerevisiae MFα secretion signal (SEQ ID NO: 4). Quantification by microfluidic capillary electrophoresis (mCE).

[0188] The "LabChip GX / GXII system" (PerkinElmer) was used for quantitative analysis of secreted protein titers in culture supernatants. The consumables "Protein Express Lab Chip" (760499, PerkinElmer) and "Protein Express Reagent Kit" (CLS960008, PerkinElmer) were used. Briefly, 6 μL of culture supernatant was mixed with 21 μL of non-reducing sample buffer. This mixture was denatured at 100° C. for 5 min, briefly centrifuged, and further mixed with 105 μL of water (Milli-Q® or equivalent). The sample was then centrifuged at 1200 g for 2 min and applied to the instrument. Fluorescently labeled samples were analyzed according to protein size in the instrument using a microfluidics-based electrophoresis system. An internal standard allowed the approximate assignment of size in kDa and approximate concentration of the detected signal. Quantification of Fab by ELISA.

[0189] Quantification of intact Fab by ELISA was performed using anti-human IgG antibody (ab7497, Abcam) as coating antibody and goat anti-human IgG (Fab specific)-alkaline phosphatase conjugated antibody (Sigma A8542) as detection antibody. Human Fab / kappa, IgG fragment (Bethyl P80-115) was used as standard at a starting concentration of 100 ng / mL and supernatant samples were diluted accordingly. Detection was performed with pNPP (Sigma S0942). Coating, dilution and washing buffers were based on PBS (2 mM KH2PO4, 10 mM Na2HPO4.2H2O, 2.7 mM g KCl, 8 mM NaCl, pH 7.4) and completed with BSA (1% (w / v)) and / or Tween20 (0.1% (v / v)) accordingly. Quantification by fluorescence spectroscopy.

[0190] Secreted mCherry fluorescence was measured directly by transferring 100 μL of each screening supernatant into a FluoroNunc™ / LumiNunc™ 96-well plate (ref. 10366281, Thermo Fischer Scientific, Waltham, USA). mCherry standards (ref. TP790040, OriGene Technologies, Herford, Germany) were diluted in PBSG (1× PBS in 10% glycerol) in the same plate to obtain a standard curve from 0 to 80 ng / μL. The plate was loaded into an Infinite M200 device and controlled by i-control v.1.6 software. Before the measurement, the samples were shaken for 5 s in linear mode with an amplitude of 1 mm. The measurement was performed in fluorescence top read mode, consisting of 25 flashes of 20 μs each at 587 nm (bandwidth 9 nm) and the emission was read at 640 nm (bandwidth 20 nm) without any delay time. Determination of gene copy number by quantitative real-time PCR (qPCR).

[0191] Cell pellets from 1 mL cultures were first prepared for lysis with lyticase (5 U / μL with the addition of 50 mM 2-mercaptoethanol) and genomic DNA was then further extracted with DNeasy® Blood & Tissue Kit (Qiagen). qPCR reactions consisted of 0.25 μL of forward primer and 0.25 μL of reverse primer (Table 5), 5 μL of 2x SensiMix SYBR Hi-ROX Kit (Bioline), 3.5 μL of nuclease-free water, and 1 μL of sample. qPCR was performed under the following conditions: hot start at 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 15 s on a Rotorgene 6000 (Qiagen). GCN was determined by normalizing the fluorescent signal of each sample to a selected control sample by using the Rotor-Gene software comparative quantification method. This ratio was further normalized to the ACT1 signal of the same sample to compensate for differences in initial concentrations. [Table 5] Example 5: Generation of signal recognition particle (SRP)-overexpressing P. pastoris strains.

[0192] We further decided to test the SP in an SRP overexpressing strain to test whether the secretory pathway was primed to handle a high load of recombinant proteins fused to the novel SP.

[0193] To generate the SRP expression plasmid 236_BB3rN, expression cassettes for all seven subunits of SRP (six protein subunits and one non-coding RNA) were assembled into one single plasmid using Golden Gate-based cloning as described in Prielhofer et al. (2017) to ensure equal gene copy numbers. Genes for all subunits were amplified by PCR from the genome of CBS7435 and cloned into the BB1 ​​plasmid, which were then combined with their respective promoters and terminators in the BB2 plasmid. The promoters and transcriptional terminators used for expression of the SRP subunits are given in Table 6. Non-coding RNAs were overexpressed with hammerhead and HDV ribozymes to remove mRNA traits after RNA polymerase II transcription. [Table 6]

[0194] To generate background strains overexpressing SRP, CBS7435 mutS was transformed with plasmid 236_BB3rN carrying all seven SRP subunits (Example 3). Twenty transformants and four control strains were cultured according to the 24-deep-well plate screening procedure (Example 4). The wet cell weights of the transformed clones and the four untransformed clones did not show any significant differences. Next, gene copy number (GCN) analysis (Example 4) of the seven SRP protein components was performed on three randomly selected clones (clones #3, #7, and #10). For #3 and #10, the GCN of the SRP gene was determined to be 2, indicating the integration of one overexpression cassette into the genome. Clone #7 (designated CBS7435 mutS SRP#7) has three copies of the gene, indicating the integration of two overexpression cassettes. This clone was selected as the background SRP-overexpressing P. pastoris strain for the expression of heterologous proteins. Example 6: Bioreactor cultivation of P. pastoris strains producing recombinant secreted proteins with novel signal peptide sequences.

[0195] Clones expressing proteins of interest (POIs, e.g., VHH, scR, SDZ-Fab, mCherry) by using the novel signal peptide sequences, as well as a control strain using the full-length MFα secretion signal (SEQ ID NO: 4) (Example 2), were selected after small-scale screening cultures (Example 4). Selected clones were further evaluated in larger culture volumes by fed-batch bioreactor cultivation. Preculture

[0196] The clone was inoculated into a wide-mouthed, baffled, covered 300 mL shake flask filled with 50 mL of YPhyG (per liter: 20.0 g phytone-peptone, 10.0 g bacto yeast extract, 20.0 g glycerol) and shaken overnight at 28° C. and 110 rpm (preculture 1). Preculture 2 (200 mL YPhyG in a 2000 mL wide-mouthed, baffled, covered shake flask) was inoculated from preculture 1 in such a way that the OD600 (optical density measured at 600 nm) reached approximately 20 (measured against YPhyG medium) in the late afternoon. Incubation of preculture 2 was also carried out at 28° C. and 110 rpm. Production batch phase.

[0197] Fermentations (bioreactor cultivations; all stages) were carried out in a fully instrumented and controllable Infors Multifors 1 L reactor (750 mL working volume). 400 mL of BSM medium (per liter: 13.5 mL H3PO4 (85%), 0.5 g CaCl 2H2O, 7.5 g MgSO4 7H2O, 9.0 g K2SO4, 2.0 g KOH, 40 g glycerol, 0.25 g NaCl, 0.1 mL antifoam (Glanapon 2000), 4.35 mL PTM1 (per liter: 0.2 g biotin, 6.0 g CuSO4 5H2O, 0.09 g KI, 3.0 g MnSO4 H2O, 0.2 g Na2MoO4 2H2O, 0.02 g H3BO3, 0.5 g CoCl2, 42.2 g ZnSO4 7H2O, 65.0 g Fe(II)SO4 7H2O, 5 mL All fermenters filled with 1000 ml H2SO4) were inoculated individually with preculture 2 to an OD600 of 2.0 at a pH of about 5.5. P. pastoris was grown on glycerol to produce biomass and the cultures were subsequently subjected to a glycerol feed (60% w / w + 12 ml / L PTM1), followed by a mixed methanol / glycerol feed and then a methanol feed. Ammonia solution (25%) was used for pH control.

[0198] In the first batch phase, the temperature was set at 28°C. Over the final hour period before starting the production phase, it was reduced to 24°C and kept at this level throughout the rest of the process, while the pH dropped to 5.0 and was kept at this level. Oxygen saturation was set at 30% throughout the entire process (cascade control: agitator, flow, oxygen supplement). Agitation was applied between 700-1200 rpm and a flow rate range (air) of 1.0-2.0 L / min was selected.

[0199] During the batch phase, biomass is produced (μ~0.30 / hr) to about 110-120 g / L wet cell weight (WCW). A classical batch phase (biomass production) can last about 14 hours. Fed-batch production stage

[0200] Glycerol was fed at a rate defined by the equation 2.6+0.3*t (g glycerol (60%) / h), where t=time in hours, so that a total of 30 g glycerol (60%) was replenished within 8 hours. The first sampling time point was chosen to be 20 hours. During the next 18 hours (treatment time 20-38 hours), a mixed glycerol / methanol feed was applied: - Glycerol feed rate defined by the equation: 2.5+0.13*t(g glycerol(60%) / hour), delivering 66 g glycerol(60%). - Methanol feed rate defined by the equation: 0.72+0.05*t(g methanol (100%) / hr), 21 g of methanol is added.

[0201] During the next about 72.5 hours (from 38 hours to about 110.5 hours process time), methanol was fed according to equation 2.2+0.016*t (g methanol (100%) / hour), resulting in a feed of about 223 g of methanol. Sampling and analysis

[0202] Samples were taken at the indicated time points with the following procedure: the first 3 mL of sampled fermentation broth was discarded (using a syringe). 1 × 1 mL of the freshly taken sample (3-5 mL) was transferred into a 1.5 mL centrifuge tube and spun at 13200 rpm (16100 g) for 5 min. The supernatants, together with the corresponding wet pellet fractions, were carefully transferred into separate vials and immediately frozen. To determine WCW, 1 mL of fermentation broth was centrifuged in a tared Eppendorf vial at 13200 rpm (16100 g) for 5 min and the resulting supernatant was accurately removed. The vials were weighed (with an accuracy of 0.1 mg) and the tare weight of the empty vial was subtracted to obtain the wet cell weight. Protein quantification was performed as described in Example 4. Example 7: Screening results for P. pastoris strains producing recombinant secreted proteins with novel signal peptide sequences.

[0203] Transformation of P. pastoris strains was carried out as described in Example 3. Secretion improvements obtained with the new SP (Example 4) are measured by titer and yield fold change values ​​(also called FC titer or titer FC, FC yield or yield FC, respectively), which refer to the respective control clones secreting POI using the MFα secretion signal (SEQ ID NO: 4) (Example 1) in the same strain background (CBS7435 mutS WT or CBS7435 mutS SRP#7, Example 5). Titer fold change is understood as the quotient of the titer of the respective fermentation or small scale culture divided by the titer of the control. Yield change is understood as the quotient of the yield of the respective fermentation or small scale culture divided by the yield of the control.

[0204] Screening of the performance of novel signal peptides (SPs) for production of proteins of interest (POIs) in comparison with the MFα secretion signal was carried out as described in Example 4.

[0205] The results of screening the SP candidates shown in Tables 4 and 7 using VHH as reporter proteins for secretion are shown in Table 7. [Table 7]

[0206] As can be seen in Table 7, SP4 and SP14 represent highly effective SPs when used as the sole secretion signal for secretion of POI, exceeding the secretion product titer by approximately 1.3-1.4 fold in comparison to the MFα secretion signal.

[0207] Next, we decided to test whether fusing the novel SP to a prosequence, such as the MFα prosequence, would have any further effect.

[0208] A large effect was observed when the signal peptide was fused to the MFα prosequence (see Table 8). From these screens, we concluded that the addition of a prosequence, such as the MFα prosequence, would be even more beneficial for recombinant protein production. [Table 8]

[0209] Fusion of different signal peptides (SPs) to other Pichia pastoris (Comagataella phaphi) derived prosequences resulted in lower or even no secretion of the POI compared to the combination with the MFα prosequence (see Table 9). [Table 9] Example 8. Fed-batch culture of P. pastoris strains producing recombinant secreted proteins with novel signal peptide sequences.

[0210] Bioreactor cultivation was carried out as described in Example 6. Firstly, single copy clones secreting POI (see Table 2) obtained and screened as described in Examples 3 and 4 were selected for bioreactor cultivation to further confirm the production performance of SP4 and SP14 without (Table 7) or with (Table 8) MFα pro-sequence in comparison with MFα secretion signal as control.

[0211] For the purpose of testing the production of POI and the secretion-increasing effect of the signal peptides of the invention (SP4 (SEQ ID NO: 2) and SP14 (SEQ ID NO: 1), which are the best performing strains in the screening for POI titer), the best performing strains in the screening for POI titer, POI yield, and growth are selected for bioreactor cultivation, which may also be multicopy strains (i.e., they contain multiple copies of the POI expression cassette). Thus, for the expression and secretion of POI by using SP4 or SP14 alone without the prosequence or by using SP4 or SP14 with the MFα prosequence, vectors and strains are generated and selected as described in Examples 2, 3, 4, and 5, and cultured as described in Example 6, except that after screening as described in Example 4, the best performing strains are selected for fermentation as described in Example 6. This also applies to the respective control strains expressing the respective POI by using the MFα secretion signal (SEQ ID NO: 4). Titers and yield fold changes for expression / secretion of the POI using SP4 or SP14 with or without the MFα pro-sequence relative to expression / secretion using the MFα secretion signal (SEQ ID NO: 4) are calculated.

[0212] Using bioreactor cultivation, we confirmed that SP4 and SP14 combined with the MFα pro sequence clearly improved secretion capacity as seen in POI titer and yield compared to the MFα secretion signal (SEQ ID NO: 4) (Table 10). Again, despite there being no strong difference between the WT and SRP overexpression strain backgrounds, scR secretion levels with SP4 were higher in the SRP overexpression strain than in the WT, indicating that SRP overexpression provides benefits for some POIs. [Table 10] Example 9: Confirmation of the cotranslational mode of translocation when using novel signal peptide sequences.

[0213] Immunofluorescence microscopy was used to confirm the cotranslational transport mode of SP4 and SP14. P. pastoris strains producing VHH with SP4 or SP14 were used for microscopic analysis (see Example 2 for strain generation). Mouse anti-6XHis antibody (abcam, ab18184) and appropriate fluorescently labeled secondary antibodies were used for immunofluorescence microscopy. While clones using the standard MFα secretion signal showed a predominantly cytosolic pattern, indicative of posttranslational transport (Figure 1A), both SP4-VHH and SP14-VHH led to a typical ER pattern (perinuclear ring), indicative of a cotranslational transport mode (Figure 1B-C). This confirms that they are preferentially transported by a cotranslational mechanism. References [Table 11]

Claims

1. From N-terminus to C-terminus (a) a secretion signal, (I) (i) a signal peptide sequence derived from a KRE1 protein or a signal peptide sequence derived from a SWP1 protein; and (ii) α-mating factor (MFα) prosequence; or (II) a signal peptide sequence derived from the KRE1 protein or a signal peptide sequence derived from the SWP1 protein a secretion signal comprising and (b) a protein of interest A nucleic acid molecule encoding a fusion protein comprising: where The signal peptide sequence derived from the KRE1 protein comprises SEQ ID NO:1 or a functional homolog thereof that comprises at least 80% sequence identity with SEQ ID NO:1; and The signal peptide sequence derived from the SWP1 protein comprises SEQ ID NO:2 or a functional homolog thereof having at least 80% sequence identity with SEQ ID NO:2; Nucleic acid molecule.

2. 2. The nucleic acid molecule of claim 1, wherein the secretion signal increases secretion of the protein of interest from a eukaryotic host cell in comparison to a eukaryotic host cell expressing a nucleic acid molecule defined in claim 1, but containing a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (e.g., SEQ ID NO: 4) instead of the secretion signal defined in claim 1.

3. The nucleic acid molecule according to claim 1 or 2, wherein the MFα prosequence comprises any one of SEQ ID NO: 3, 53 or 74-80 or a functional homologue thereof comprising at least 80% sequence identity with any one of SEQ ID NO: 3, 53 or 74-80, preferably SEQ ID NO: 3 or 53.

4. 4. The nucleic acid molecule of any one of claims 1 to 3, wherein the MFα prosequence comprises Ser at a position corresponding to position 23 of SEQ ID NO:53 and / or comprises GIu at a position corresponding to position 64 of SEQ ID NO:

53.

5. The protein of interest is an antibody, e.g. a chimeric, humanized or human antibody, or a bispecific antibody; Antigen-binding antibody fragments, such as Fab or F(ab)2; Single chain antibodies, such as scFv; Single domain antibodies, such as camelid VHH fragments or heavy chain antibodies or domain antibodies (dAbs); Artificial antigen-binding molecules, such as DARPINs, ibodies, affibodies, humabodies, or muteins based on polypeptides of the lipocalin family; Enzymes, such as process enzymes, cytokines, growth factors, hormones, or protein antibiotics; Fusion proteins, such as toxin fusion proteins, structural proteins, regulatory proteins, etc.; and Vaccine antigens is selected from the group consisting of Preferably, the protein of interest is a therapeutic protein, a food additive, or a feed additive; A nucleic acid molecule according to any one of claims 1 to 4.

6. A secretion signal as defined in any one of claims 1 to 5.

7. An expression cassette or vector comprising the nucleic acid molecule of any one of claims 1 to 5 and a promoter operably linked thereto.

8. A recombinant eukaryotic host cell comprising a nucleic acid molecule according to any one of claims 1 to 5, or an expression cassette according to claim 7, or a vector according to claim 7, Preferably a) the host cell is a fungal host cell or a yeast host cell; More preferably, the yeast host cell is selected from the group consisting of Komagataella phafi (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces kudriavzevi, Saccharomyces kluyveri, Saccharomyces uvarum, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., and Schizosaccharomyces pombe, or the fungal host cell is selected from Trichoderma reesei or Aspergillus niger, and / or b) the host cell has been engineered to overexpress one or more components of the signal recognition particle (SRP); Eukaryotic host cell.

9. 1. A method for producing a protein of interest in a eukaryotic host cell, comprising: (i) genetically engineering a eukaryotic host cell with a nucleic acid molecule according to any one of claims 1 to 5, or with an expression cassette or vector according to claim 7; (ii) culturing the genetically engineered host cell under conditions to express the nucleic acid molecule and secrete the protein of interest upon cleavage of the secretion signal; (iii) optionally isolating said protein of interest from said cell culture; (iv) optionally purifying said protein of interest; (v) optionally modifying said protein of interest; and (vi) optionally formulating said protein of interest.

10. 10. A method for increasing secretion of a protein of interest from a eukaryotic host cell, comprising expressing in said eukaryotic host cell a nucleic acid molecule according to any one of claims 1 to 5, thereby increasing secretion of said protein of interest in comparison to said host cell expressing a nucleic acid molecule according to any one of claims 1 to 5 comprising a wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (such as e.g. SEQ ID NO: 4) instead of a secretion signal as defined in any one of claims 1 to 5.

11. 11. The method according to claim 9 or 10, (i) engineering said host cell to incorporate an expression construct that expresses the nucleic acid molecule of any one of claims 1 to 5, and genetically engineering said host cell to overexpress one or more components of a signal recognition particle (SRP); (ii) culturing said host cell under conditions suitable to express said nucleic acid molecule and to overexpress one or more components of said SRP and to secrete said protein of interest upon cleavage of the secretion signal; (iii) optionally isolating said protein of interest from said cell culture; (iv) optionally purifying said protein of interest; (v) optionally modifying said protein of interest; and (vi) optionally formulating said protein of interest.

12. (a) the nucleic acid molecule is comprised in an expression cassette, vector, or plasmid that is integrated into a chromosome of the host cell or that is not integrated into the genome of the host cell. (b) the eukaryotic host cell is a fungal host cell or a yeast host cell; Preferably, wherein said yeast host cell is selected from the group consisting of Komagataella phafii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp., and Schizosaccharomyces pombe, or said fungal host cell is selected from Trichoderma reesei or Aspergillus niger. and / or (c) the protein of interest is Antibodies, such as chimeric, humanized or human antibodies, or bispecific antibodies; Antigen-binding antibody fragments, such as Fab or F(ab)2; Single chain antibodies, such as scFv; Single domain antibodies, such as camelid VHH fragments or heavy chain antibodies or domain antibodies (dAbs); Artificial antigen-binding molecules, such as DARPINs, ibodies, affibodies, humabodies, or muteins based on polypeptides of the lipocalin family; Enzymes, such as process enzymes, cytokines, growth factors, hormones, protein antibiotics, etc.; Fusion proteins, such as toxin fusion proteins, structural proteins, regulatory proteins, etc.; and Vaccine antigens Preferably, the protein of interest is a therapeutic protein, a food additive, or a feed additive; The method according to any one of claims 9 to 11.

13. Use of a secretion signal according to any one of claims 1 to 5 for increasing secretion of a protein of interest from a eukaryotic host cell, preferably 2. The method of claim 1, further comprising: increasing secretion of the protein of interest from a eukaryotic host cell in comparison to a eukaryotic host cell expressing a fusion protein as defined in claim 1, wherein the secretion signal comprises the wild-type Saccharomyces cerevisiae alpha-mating factor secretion signal (e.g., SEQ ID NO: 4) instead of the secretion signal as defined in claim 1.

14. Use of a recombinant eukaryotic host cell according to claim 8 for producing a protein of interest.

15. 10. A method for producing a protein of interest by culturing a recombinant eukaryotic host cell according to claim 8 under conditions which express a nucleic acid molecule according to any one of claims 1 to 5 and secrete said protein of interest upon cleavage of said secretion signal, and isolating said protein of interest from said host cell culture, and optionally purifying, and optionally modifying, and optionally formulating said protein of interest.