Expression of milk caseins in non-mammalian cells

Abstract

The present invention relates to the animal-free production of casein proteins for human consumption by expression of such proteins in non-mammalian host cell. The invention relates to non-mammalian host cell modified for the production of caseins, and to methods wherein these cells are used for the production of caseins.

Description

[0001] Expression of milk caseins in non-mammalian cells

[0002] Field of the invention

[0003] The present invention relates to the field of molecular microbiology, food technology and fermentation technology. In particular, the invention relates to a non-mammalian host cell comprising a nucleotide sequence coding for a mammalian casein and further genetic modifications.

[0004] Background of the invention

[0005] In 2050 the global population will be around 10 billion people. It is generally recognized that the production of food and its ingredients needs to change significantly to keep within the agreed sustainability development goals (SDGs) for the environment and climate.

[0006] Milk, and especially cow milk, is an important source of protein and is produced all around the world (total production in 2018: 843 million tons). However, diary production has an enormous impact on the environment. Currently over two-thirds of the world's agricultural land is used for maintaining livestock, including beef and dairy cows. Dairy cows and their manure generate significant amounts of greenhouse gas (including methane, which is a much more harmful greenhouse gas than CO2) emissions which contribute to climate change. Water demand is very high as dairy operations consume large volumes of water to grow feed, water cows, manage manure and process products. Additionally, nitrogen emissions (from e.g. manure and fertilizer) cause worldwide major issues. Consequently, the carbon footprint and land-use factor of milk and cheese are high, even higher than that of pigs, fish and chicken. Next to these environmental and climatological aspects, also animal welfare is quite often compromised. Concerns about sustainability and animal-welfare of milk production are two important motivations for an increasing percentage of consumers to replace animal-based proteins by (vegan) plant-based protein sources such as soy, almond, pea and coconut.

[0007] Bovine milk contains around 35 g / L of caseins (i.e. 80% of the milk protein fraction) divided over alphaSI-, alphaS2-, beta- and kappa-casein within an approximate ratio of 40, 10, 40 and 10% respectively. The four caseins are well studied in terms of amino acid composition, molecular weight, post-translational modifications (PTMs) and general physico-chemical properties. Due to the high content of prolyl residues, each casein molecule has an open and flexible conformation. Furthermore, hydrophobic and hydrophilic regions show a block distribution within the protein chain, giving each casein an amphiphilic character. Because of their nature and physico-chemical properties, caseins are unique proteins that, for many applications, cannot easily be replaced by plant-based alternatives.

[0008] Expression of recombinant mammalian caseins has previously been described in non- mammalian organisms such as the yeasts P. pastoris and S. cerevisiae (Chung, Kun-Sub, et al. Journal of Microbiology and Biotechnology 1.1 (1991): 31-36; Choi, Byung-Kwon, and Rafael Jimenez-Flores. Journal of agricultural and food chemistry 44.1 (1996): 358-364) but the levels of secreted product are very low. It is thus an object of the present invention to provide for non- mammalian host cells, e.g., fungal host cells, expressing a mammalian casein, wherein the expression of the casein is quantitatively and / or qualitatively improved, to obviate the farm animals to produce these proteins. It is in particular an object of the present invention to provide for such improved expression of caseins wherein the amount of secreted casein is improved and / or wherein the post-translational modifications of the secreted casein are more in line with the corresponding natural milk casein.

[0009] Summary of the invention

[0010] In one aspect, there is provided for a non-mammalian host cell comprising: i) an expression construct comprising a nucleotide sequence encoding at least one casein; ii) a genetic modification that reduces or eliminates the expression or activity of a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof; iii) a genetic modification that reduces or eliminates the expression or activity of a cell-wall associated aspartic acid protease of the yapsin family encoded by a YPS1 gene or an orthologue thereof; and iv) a genetic modification that reduces or eliminates the expression or activity of a cell-wall associated aspartic acid protease of the yapsin family encoded by a YPS’ gene or an orthologue thereof.

[0011] In one embodiment, the PEP4 gene encodes a vacuolar acid aspartyl protease or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 ; the YPS1 gene encodes a cellwall associated aspartic acid protease comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 2; and the YPS’ gene encodes a cell-wall associated aspartic acid protease comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 3.

[0012] In one embodiment, the host cell comprises at least one further genetic modification selected from the group consisting of: v) a genetic modification that that reduces or eliminates the expression or activity of a subtilisin-like Ser-type protease, preferably a subtilisin-like Ser-type protease encoded by a SBT100 gene or an orthologue thereof; and vi) a genetic modification that reduces or eliminates the level of O-linked glycosylation of caseins proteins produced by the cell.

[0013] In one embodiment, the SBT100 gene encodes a subtilisin-like Ser-type protease comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 45.

[0014] In one embodiment, the level of O-linked glycosylation of caseins produced by the cells is reduced by:

[0015] - eliminating or reducing the enzymatic activity of a protein-O-mannosyl transferase, preferably a protein-O-mannosyl transferase encoded by a PMT gene; and / or

[0016] - introducing into the host cell an expression construct comprising a nucleotide sequence encoding at least one alpha-1 ,2-mannosidase. In one embodiment, the nucleotide sequence encoding the at least one casein comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of a casein from a mammal selected from the group consisting of: Bos taurus (domestic cattle), Bos grunniens (yak), Bubalus bubalis (water buffalo), Capra hircus (goat), Ovis aries (sheep), Camelus spp. (camel, dromedaris), Rangifer tarandus (reindeer), Equus caballus (horse), Sus spp. including Sus domesticus (pig) and Homo sapiens.

[0017] In one embodiment, the nucleotide sequence encoding the at least one casein comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NO’s: 4 - 44, of which the bovine SEQ ID NO’s: 4, 14, 26 and 35 are preferred.

[0018] In one embodiment, the host cell is non-mammalian eukaryotic host cell, preferably a eukaryotic microbial host cell, more preferably a yeast or a filamentous fungus host cell. in one embodiment, the host cell is selected from a genus from the group consisting of Saccharomyces, Kiuyveromyces, Candida, Komagataella, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Kazachstania Debaryomyces, Naumovia, Alternaria, Apophysomyces, Aspergillus, Cladosphialophora, Fonsecaea, Fusarium, Lichtheimia, Mucor, Myceliophthora, Neurospora, Penicillium, Rhizopus, Rhizomucor, Trichoderma and Trichophyton, wherein preferably, the cell selected from a species from the group consisting of K. phaffii, K. pastoris, K. pseudopastoris S. cerevisiae, S. exiguus, S. bayanus, Kiuyveromyces lactis, Kiuyveromyces marxianus, Y. lipolytica, S. pombe, Alternaria alternata, Apophysomyces variabilis, Aspergillus spp., Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus sojae, Aspergillus terreus, Cladosphialophora spp., Fonsecaea pedrosoi, Fusarium spp., Fusarium oxysporum, Fusarium solani, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramosa, Myceliophthora spp., Myceliophthora thermophila, Neurospora crassa, Penicillium chrysogenum, Penicillium simplicissimum, Penicillium brasilianum, Rhizopus spp., Rhizopus microsporus, Rhizomucor spp., Rhizomucor pusillus, Rhizomucor miehei, Trichoderma spp., Trichoderma reesei, Trichophyton spp., Trichophyton interdigitale, and Trichophyton rubru, of which Komagataella phaffii is most preferred.

[0019] In a second aspect, there is provided for a process for producing a casein, the process comprising culturing a host cell as described herein such that one or more of the nucleotide sequences are expressed and the casein is produced, the process optionally comprising the step of recovery of the casein.

[0020] In a third aspect, there is provided for a casein as obtained or obtainable by the process as described herein.

[0021] In a fourth aspect, there is provided a composition comprising a casein as obtained by the process of the second aspect above, wherein preferably the composition is a food product, wherein more preferably the composition is a dairy substitute product, wherein most preferably the composition is animal-free dairy substitute product. In one embodiment, the composition is substantially free of 15-20 kDa proteolytic casein fragments. In one embodiment of the composition, the casein imparts on the composition at least one dairy-like property selected from the group consisting of cohesiveness, firmness, elasticity, meltability, spreadability, texture, mouthfeel, hardness, creaminess, flexibility and taste, wherein preferably, the dairy-like property imparted by the casein on the composition is at least equivalent to a same property in an otherwise similar composition that comprises native milk-derived casein and does not comprise the casein as obtained by the process of the second aspect above.

[0022] Description of the invention

[0023] Definitions

[0024] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method.

[0025] For purposes of the present invention, the following terms are defined below.

[0026] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0027] As used herein, the term "and / or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0028] As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, ... ,etc.

[0029] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value.

[0030] The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods.

[0031] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (polynucleotides) I 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blosum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred.

[0032] Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTP programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0033] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below. Alternative conservative amino acid residue substitution classes.

[0034] Alternative Physical and Functional Classifications of Amino Acid Residues.

[0035] The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3rdedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.)-

[0036] Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

[0037] A "nucleic acid construct" or "nucleic acid vector" is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term "nucleic acid construct" therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms "expression vector" or “expression construct" refer to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism of the invention.

[0038] As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. An inducible promoter may also be present but not induced.

[0039] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

[0040] The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3- dimensional structure or origin. The term "gene" means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, exons, introns and a 3'-nontranslated sequence (3'-end) e.g. comprising a polyadenylation- and / or transcription termination site.

[0041] "Expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.

[0042] The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and / or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloned" genetically modified organisms (GMO's) (self-cloning is defined herein as in European Directive 98 / 81 / EC Annex II). When used to indicate the relatedness of two nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed earlier herein.

[0043] The terms "heterologous" and "exogenous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which it is introduced but have been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins, i.e. exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous / exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the term heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other. The terms heterologous and exogenous specifically also apply to non-naturally occurring modified versions of otherwise endogenous nucleic acids or proteins.

[0044] Filamentous fungi are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.

[0045] Yeasts are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina (Yeasts: characteristics and identification, J.A. Barnett, R.W. Payne, D. Yarrow, 2000, 3rd ed., Cambridge University Press, Cambridge UK; and, The yeasts, a taxonomic study, C.P. Kurtzman and J.W. Fell (eds) 1998, 4thed., Elsevier Science Publ. B.V., Amsterdam, The Netherlands) that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism.

[0046] The yeast genus Pichia has more recently been reassigned to the genus Komagataella (see e.g., Heistinger et al., Microbiology, 2020;166(7):614-616), which genus was split into the species K. phaffii, K. pastoris, and K. pseudopastoris. The Pichia species P. pastoris, that has been widely used in biotech industries and as used herein, has been reassigned to the Komagataella species K. phaffii (Heistinger et al., Microbiology, 2020, supra). Hence, when reference is made herein to the yeast species K. phaffii this is to be understood as equally referring to the yeast species formerly known as Pichia pastoris, and vice versa.

[0047] Detailed description of the invention

[0048] In-house experiments indicated that the yield of caseins in non-mammalian can be improved by deletion of a specific set of enzymes. Accordingly, in a first aspect there is provided for a nonmammalian host cell comprising: i) an expression construct comprising a nucleotide sequence encoding at least one casein; ii) a genetic modification that reduces or eliminates the expression or activity of a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof; iii) a genetic modification that reduces or eliminates the expression or activity of a cell-wall associated aspartic acid protease of the yapsin family encoded by a YPS1 gene or an orthologue thereof; and iv) a genetic modification that reduces or eliminates the expression or activity of a cell-wall associated aspartic acid protease of the yapsin family encoded by a YPS’ gene or an orthologue thereof.

[0049] In one embodiment, the host cell comprises a genetic modification that reduces or eliminates the expression or activity of a vacuolar acid aspartyl protease. In one embodiment, the vacuolar acid aspartyl protease, the activity or expression of which is to be reduced or eliminated in the host cell is a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof. In one embodiment, the PEP4 gene encodes a vacuolar acid aspartyl protease or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 .

[0050] In one embodiment, the host cell comprises a genetic modification that reduces or eliminates the expression or activity of at least two aspartic-type endopeptidases, preferably at least two aspartic-type endopeptidase of the yapsin family. In one embodiment, the aspartic-type endopeptidase, the activity or expression of which is to be reduced or eliminated in the host cell are at least two aspartic-type endopeptidases encoded by a YPS gene, e.g., a YPS1 , YPS2, YPS3, YPS7, MKC7, YPS’, YPS” gene or an orthologue thereof (see Wu et al., 2013, Journal of Industrial Microbiology and Biotechnology 40(6): 589-599). Preferably, the activity or expression of YPS1 and YPS’ are reduced.

[0051] In one embodiment, the YPS1 gene encodes an aspartic-type endopeptidase or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 2.

[0052] In one embodiment, the YPS’gene encodes an aspartic-type endopeptidase or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 3.

[0053] In one embodiment, a host as described herein, thus comprises an expression construct comprising a nucleotide sequence encoding at least one casein. The term "casein" is art-known and represents a family of proteins that is present in mammal-produced milk and is capable of selfassembling with other proteins in the family to form micelles and / or precipitate out of an aqueous solution at an acidic pH. Non-limiting examples of caseins include beta-casein, kappa-casein, alphaSI -casein, and alphaS2-casein.

[0054] As used herein, the term "alphaSI -casein" refers to not only the alphaSI -casein protein, but also fragments or variants thereof. AlphaSI -casein is found in the milk of numerous different mammalian species, including cow, yak, camel, dromedary, horse, water buffalo, goat, and sheep.

[0055] As used herein, the term "alphaS2-casein" refers to not only the alphaS2-casein protein, but also fragments or variants thereof. AlphaS2-casein is known as epsilon-casein in mouse, gammacasein in rat, and casein-A in guinea pig.

[0056] As used herein, the term "beta-casein" refers to not only the beta-casein protein, but also fragments or variants thereof. For example, A1 and A2 beta-casein are genetic variants of the betacasein milk protein that differ by one amino acid (at amino acid 67, A2 beta-casein has a proline, whereas A1 has a histidine). Other genetic variants of beta-casein include the A3, B, C, D, E, F, H1 , H2, I and G genetic variants.

[0057] As used herein, the term "kappa-casein" refers to not only the kappa-casein protein, but also fragments or variants thereof. Kappa-casein is cleaved by rennet, which releases the casein macropeptide from the C-terminal region. The remaining product with the N-terminus and two-thirds of the original peptide chain is referred to as para-kappa-casein.

[0058] Non-limiting examples of sequences for casein proteins from different mammals are provided in the sequence listing herewith. Additional sequences for other caseins are known in the art. In addition, the caseins for use in the embodiments and aspect herein can be defined by their amino acid sequences, e.g., by comprising an amino acid sequence with a minimal percentage sequence identity to a reference casein amino acid sequence as defined herein below.

[0059] In one embodiment, the at least one casein comprises or consists of an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity to the amino acid sequence of casein from a mammal selected from the group consisting of Bos taurus (domestic cattle), Bos grunniens (yak), Bubalus bubalis (water buffalo), Capra hircus (goat), Ovis aries (sheep), Camelus spp. (camel, dromedary), Rangifer tarandus (reindeer), Equus caballus (horse), Sus spp. including Sus domesticus (pig) and Homo sapiens.

[0060] In one embodiment, a host cell as described herein comprises at least one expression construct comprising a nucleotide sequence encoding a beta-casein comprising or consisting of an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to at least one of SEQ ID NOs 4 - 13.

[0061] In one embodiment, a host cell as described herein comprises at least one expression construct comprising a nucleotide sequence encoding an alphaSI -casein comprising or consisting of an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to at least one of SEQ ID NOs: 14 - 25.

[0062] In one embodiment, a host cell as described herein comprises at least one expression construct comprising a nucleotide sequence encoding an alphaS2-casein comprising or consisting of an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to at least one of SEQ ID NOs: 26 - 34.

[0063] In one embodiment, a host cell as described herein comprises at least one expression construct comprising a nucleotide sequence encoding a kappa-casein comprising or consisting of an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to at least one of SEQ ID NOs: 35 - 44.

[0064] In a preferred embodiment, a host cell as described herein comprises at least one expression construct comprising a nucleotide sequence encoding a bovine casein comprising or consisting of an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to at least one of SEQ ID NO’s: 4, 14, 26 and 35.

[0065] In one embodiment, a host cell as described herein comprises more than one expression construct for expression of more than one type of casein in the host cell. Thus, in one embodiment, the host cell comprises expression constructs for expression in the host cell of a beta-casein and an alphaSI -casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of a beta-casein and an alphaS2-casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of a beta-casein and a kappa- casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of an alphaSI -casein and an alphaS2-casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of an alphaSI -casein and a kappa-casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of an alphaS2-casein and a kappa-casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of a beta-casein, an alphaSI -casein and an alphaS2- casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of a beta-casein, an alphaSI -casein and a kappa-casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of a beta-casein, an alphaS2-casein and a kappa-casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of an alphaSI-casein, an alphaS2-casein and a kappa-casein. In one embodiment, the host cell comprises expression constructs for expression in the host cell of a betacasein, an alphaSI -casein, an alphaS2-casein and a kappa-casein.

[0066] In one embodiment, the host cell as described herein comprises at least one further genetic modification selected from the group consisting of: v) a genetic modification that reduces or eliminates the expression or activity of a subtilisin- like Ser-type protease. Preferably the subtilisin-like Ser-type proteases is encoded by a SBT100 gene or an orthologue thereof; and vi) a genetic modification that reduces or eliminates the level of O-linked glycosylation of caseins proteins produced by the cell. .

[0067] In one embodiment, the host cell as described herein comprises a further genetic modification that reduces or eliminates the expression or activity of a subtilisin-like Ser-type protease. In one embodiment, the subtilisin-like Ser-type protease, the activity or expression of which is to be reduced or eliminated in the host cell is a subtilisin-like Ser-type protease encoded by a SBT100 gene, or an orthologue thereof. In one embodiment, the SBT100 gene encodes a subtilisin-like Ser- type protease or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 45.

[0068] In one embodiment, the host cell as described herein, comprises a further genetic modification that reduces the level of O-linked glycosylation of caseins produced by the cells, wherein the reduction is achieved by:

[0069] - eliminating or reducing the enzymatic activity of a protein-O-mannosyl transferase; and / or

[0070] - introducing into the host cell an expression construct comprising a nucleotide sequence encoding at least one alpha-mannosidase.

[0071] In one embodiment, the genetic modification comprised in the host cell that reduces or eliminates O-linked protein glycosylation, is a genetic modification that reduces or eliminates the expression or activity of at least one protein-O-mannosyl transferase in the cell. The protein-O- mannosyl transferase is preferably a dolichyl-phosphate-mannose-protein mannosyltransferase (EC 2.4.1.109). In one embodiment, the at least one protein-O-mannosyl transferase, the activity or expression of which is to be reduced or eliminated in the host cell is an protein-O-mannosyl transferase encoded by a PMT gene, e.g., a PMT1 - PMT7 gene or an orthologue thereof (see Govindappa et al., 2013, Protein Expression and Purification 88(1): 164-171 ; Nett et al., 2013, PLoS One 8(7): e68325), of which PMT1, PMT2 and PMT4 are preferred

[0072] In one embodiment, the PMT1 gene encodes a protein-O-mannosyl transferase or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 46. In one embodiment, the PMT2 gene encodes a protein-O-mannosyl transferase or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 47. In one embodiment, the PMT4 gene encodes a protein-O-mannosyl transferase or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO:48.

[0073] In one embodiment, reduction of O-linked glycosylation is achieved by co-expression of an alpha-mannosidase. In one embodiment the alpha-mannosidase is targeted to the secretion pathway and / or secreted by the host cell. In one embodiment the alpha-mannosidase is an alpha- 1 ,2-mannosidase, an alpha-1 ,6-mannosidase, an alpha-1 ,3-mannosidase or a mannosidase that is able to hydrolyse multiple types of alpha-mannose linkages. More preferably, the alpha- mannosidase is an alpha-1 ,2-mannosidase. Accordingly, in one embodiment, the host cell as described herein further comprises an expression construct comprising a nucleotide sequence encoding at least one alpha-1 ,2-mannosidase. In one embodiment, the nucleotide sequence encoding the at least one alpha-1 ,2-mannosidase encodes at least one a protein comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 49. In other embodiments, the alpha-1 ,2-mannosidase may be separately produced and added to the cell culture.

[0074] In one embodiment, the level of O-linked glycosylation of caseins is reduced and / or eliminated by a genetic modification that reduces or eliminates the expression or activity of at least one protein-O-mannosyl transferase in the cell such as a protein-O-mannosyl transferase encoded by a PMT gene, or an orthologue thereof such as preferably PMT1 , PMT2 and / or PMT4 as described herein and by additional co-expression of an alpha-mannosidase, such as an alpha-1 ,2- mannosidase in the host cell as described herein.

[0075] The non-mammalian host cell can be any suitable non-mammalian host cell, including both prokaryotic and eukaryotic host cells. A suitable prokaryotic host cell is usually a bacterial host cell and can be either a Gram-negative or a Gram-positive a bacterial host cell. Examples of suitable bacterial host cells include host cells from the genera Escherichia, Bacillus, Lactobacillus, Lactococcus and Streptococcus, or preferably bacterial host cells of the species Escherichia coll, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus coagulans, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus gasseri, Lactococcus lactis, Streptococcus salivarius and Streptococcus thermophilus. Preferably the bacterial host cell is a food-grade bacterium.

[0076] In a preferred embodiment, however, the non-mammalian host cell is a non-mammalian eukaryotic host cell, such as an insect cell, a plant cell, an algal cell or a eukaryotic microbial cell. In a more preferred embodiment, however, the non-mammalian host cell is a eukaryotic microbial cell, such as a yeast cell or a filamentous fungal host cell. Examples of suitable yeast host cells includes yeast from genera Saccharomyces, Kluyveromyces, Candida, Komagataella, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Kazachstania Debaryomyces and Naumovia, or preferably yeast host cells of the species K. phaffii, K. pastoris, K. pseudopastoris, S. cerevisiae, S. exiguus, S. bayanus, K. lactis, K. marxianus Y. lipolytica and S. pombe, of which K. phaffii is most preferred. Examples of suitable filamentous fungal host cells includes fungi from genera Alternaria, Apophysomyces, Aspergillus, Cladosphialophora, Fonsecaea, Fusarium, Lichtheimia, Mucor, Myceliophthora, Neurospora, Penicillium, Rhizopus, Rhizomucor, Trichoderma and Trichophyton, or preferably filamentous fungi cells of the species Alternaria alternata, Apophysomyces variabilis, Aspergillus spp., Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus sojae, Aspergillus terreus, Cladosphialophora spp., Fonsecaea pedrosoi, Fusarium spp., Fusarium oxysporum, Fusarium solani, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramosa, Myceliophthora spp., Myceliophthora thermophila, Neurospora crassa, Penicillium chrysogenum, Penicillium simplicissimum, Penicillium brasilianum, Rhizopus spp., Rhizopus microsporus, Rhizomucor spp., Rhizomucor pusillus, Rhizomucor miehei, Trichoderma spp., Trichoderma reesei Trichophyton spp., Trichophyton interdigitale, and Trichophyton rubru, and most preferably a species selected from Aspergillus oryzae and Aspergillus niger.

[0077] In one embodiment, the nucleotide sequence encoding the at least one casein encodes a signal sequence operably linked to the nucleotide sequence encoding the at least one casein, wherein the signal sequence effects extracellular expression of the at least one casein. Extracellular expression of the at least one casein can be detected as described above.

[0078] In one embodiment, the signal sequence that is operably linked to the at least one casein is a mammalian signal sequence. In one embodiment, the signal sequence that is operably linked to the at least one casein is a signal sequence from a mammary gland protein. In one embodiment, the signal sequence that is operably linked to the at least one casein is a signal sequence from a casein. In one embodiment, the signal sequence that is operably linked to the at least one casein is a signal sequence that is native to the at least one casein to which it is operably linked.

[0079] In one embodiment, the signal sequence that is operably linked to the at least one casein is a signal sequence that is heterologous to the at least one casein. In one embodiment, the signal sequence that is operably linked to the at least one casein is a signal sequence derived from a secreted protein that is endogenous to a cell of the same Kingdom, Division, Class, Order or Family as the non-mammalian host cell. In one embodiment, the signal sequence that is operably linked to the at least one casein is a signal sequence derived from a protein that is endogenous to a cell of the same genus or species as the non-mammalian host cell. For example, if non-mammalian host cell is a K. phaffii cell, the signal sequence can be derived from a K. phaffii protein such as the signal sequence of the K. phaffii Ost1 and Cwp1 proteins.

[0080] In one embodiment, the signal sequence that is operably linked to the at least one casein is operably linked to the casein through a pro-sequence. The pro-sequence preferably is a prosequence that natively associated with the signal sequence. The pro-sequence preferably comprises a furin-like proprotein convertase cleavage site, i.e., a paired basic amino acid processing site (also known as KEX2 cleavage site), for release of the pro-sequence from the at least one casein.

[0081] In another aspect there is provided nucleic acid constructs, such as vectors, including cloning and expression vectors, comprising a polynucleotide or a nucleotide sequence encoding the at least one casein and methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of the polypeptide(s) occurs. As used herein, the terms “vector” and “construct” are used interchangeably and refers to a constructed nucleic acid molecule comprising and preferably capable of transporting the polynucleotide encoding the at least one casein.

[0082] Polynucleotides encoding the at least one casein can be incorporated into a recombinant replicable vector, for example a cloning or expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus, in a further embodiment, there is provided a method of making polynucleotides encoding the at least one casein by introducing the polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described above.

[0083] The vector into which the expression cassette or polynucleotide encoding the at least one casein is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced.

[0084] The vector can be an autonomously replicating vector, i.e., a vector which exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Such vectors can include an element which ensures that they are stably maintained at a single copy in each cell (e.g., a centromere-like sequence such as "CEN"). Alternatively, the autonomously replicating vector may optionally comprise an element which enables the vector to be replicated to higher than one copy per host cell (e.g., an autonomously replicating sequence or "ARS"), Methods in Enzymology, Vol. 350: Guide to yeast genetics and molecular and cell biology, Part B., Guthrie and Fink (eds.), Academic Press (2002).

[0085] In another embodiment, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. In one embodiment, the integration vector comprises a heterologous nucleic acid fragment to be integrated in the host cell’s genome, flanked on the 5' end with a nucleic acid sequence from the 5' region of the locus from that genome and on the 3' end with a nucleic acid sequence from the 3' region of the locus. The integration vector is capable of integrating into the genome by double-crossover homologous recombination.

[0086] One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms “plasmid” and “vector” can be used interchangeably herein as the plasmid is the most commonly used form of vector. However, it is intended to include such other forms of expression vectors, such as cosmid, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) and phage vectors which serve equivalent functions.

[0087] In one embodiment, vectors can be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

[0088] In one embodiment, a vector can comprise two or more, for example three, four or five, copies of the polynucleotides encoding the at least one casein, for example for overexpression.

[0089] A vector or expression construct for a given host cell may thus comprise the following elements operably linked to each other in a consecutive order from the 5'-end to 3'-end relative to the coding strand of the sequence encoding the at least one casein: (1) a promoter sequence capable of directing transcription of the nucleotide sequence encoding the at least one casein in the given host cell; (2) translation initiation sequences, such as the eukaryotic Kozak consensus sequence or the prokaryotic Ribosome Binding Site I Shine-Dalgarno sequence, (3) optionally, a signal sequence capable of directing secretion of the polypeptide from the given host cell into a culture medium; (4) a DNA sequence encoding a mature and preferably active form of the at least one casein; and preferably also (5) a transcription termination region (terminator) capable of terminating transcription downstream of the nucleotide sequence encoding the at least one casein.

[0090] Downstream of the nucleotide sequence encoding the at least one casein there may be a 3' untranslated region containing one or more transcription termination sites (e. g. a terminator). The origin of the terminator is less critical. The terminator can, for example, be native to the DNA sequence encoding the polypeptide. However, preferably a yeast terminator is used in yeast host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell (in which the nucleotide sequence encoding the polypeptide is to be expressed). In the transcribed region, a ribosome binding site for translation may be present. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

[0091] Enhanced expression of the polynucleotide encoding the at least one casein can also be achieved by the selection of heterologous regulatory regions, e. g. promoter, secretion leader and / or terminator regions, which may serve to increase expression and, if desired, secretion levels of the protein of interest from the expression host and / or to provide for the inducible control of the expression of the at least one casein.

[0092] It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The vectors, such as expression vectors, can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein. A stably transformed microorganism is one that has had one or more DNA fragments introduced such that the introduced molecules are maintained, replicated and segregated in a growing culture. Stable transformation may be due to multiple or single chromosomal integration (s) or by (an) extrachromosomal element(s) such as (a) plasmid vector(s). A plasmid vector is capable of directing the expression of polypeptides encoded by particular DNA fragments.

[0093] Expression may be constitutive or regulated by inducible (or repressible) promoters that enable high levels of transcription of functionally associated DNA fragments encoding specific polypeptides. The promoters selected are those which would be expected to be operable in the particular host system selected. For example, yeast promoters are used when a yeast such as S. cerevisiae, Kluyveromyces lactis, or K. phaffii is the host cell whereas fungal promoters would be used in host cells such as A. niger, Neurospora crassa, or Trichoderma reesei. Examples of yeast promoters include but are not limited to the GAPDH / GAP, AOX1, SEC4, HE I, PMA1, OCH1, GAL1, GAL10, hybrid GAL10 / CYC1, PGK, GAP, TPI, CYC1, ADH2, PHO5, CUP1, MFa1, FLD1, PMA1, PDI, TEF, RPL10, and GUT1 promoters. Romanos et at, Yeast 8: 423-488 (1992) provide a review of yeast promoters and expression vectors. Hartner et al., Nucl. Acid Res. 36: e76 (pub on-line 6 June 2008) describes a library of promoters for fine-tuned expression of heterologous proteins in K. phaffii.

[0094] In certain embodiments, the expression of the casein is placed under the control of an inducible promotor, preferably a methanol induced promotor such as for example an AOX1 promotor.

[0095] Regardless of the exact mechanism utilized for expression of the at least one casein, it is contemplated that such expression is transferable by the introduction of genes encoding these polypeptides into another host cell by methods known in the art. Genetic elements as herein defined include nucleic acids (generally DNA or RNA) having expressible coding sequences for products such as proteins, including enzymes, apoproteins or antisense RNA, which express or regulate expression of relevant polypeptides. The expressed proteins can be structural proteins, can function as enzymes, repress or derepress enzyme activity or control expression of enzymes or function as transporter of compounds, e.g., metabolites. Recombinant DNA encoding these expressible sequences can be either chromosomal (integrated into the host cell chromosome by, for example, homologous recombination) or extra-chromosomal (for example, carried by one or more plasmids, cosmids and other vectors capable of self-replication). It is understood that the recombinant DNA utilized for transforming the host cell can include, in addition to structural genes and transcription factors, expression control sequences, including promoters, repressors and enhancers, that act to control expression or derepression of coding sequences for proteins, apoproteins or antisense RNA. For example, such control sequences can be inserted into wild-type host cells to promote overexpression of selected polypeptides already encoded in the host cell genome, or alternatively they can be used to control synthesis of extrachromosomally encoded polypeptides. Recombinant DNA can be introduced into the host cell by any means, including, but not limited to, plasmids, cosmids, phages, yeast artificial chromosomes or other vectors that mediate transfer of genetic elements into a host cell. These vectors can include an origin of replication, along with cis-acting control elements that control replication of the vector and the genetic elements carried by the vector. Selectable markers can be present on the vector to aid in the identification of host cells into which genetic elements have been introduced.

[0096] Means for introducing genetic elements into a host cell (e.g., cloning) are well known to the skilled artisan. One can utilize an extrachromosomal multi-copy plasmid vector to insert the genetic elements in accordance with the present invention. Plasmid-borne introduction of the genetic element into host cells involves an initial cleaving of a plasmid vector with a restriction enzyme, followed by ligation of the plasmid and genetic elements encoding for the targeted enzyme species in accordance with the invention. Upon recircularization of the ligated recombinant plasmid, infection (e.g., packaging in phage lambda) or other mechanism for plasmid transfer (e.g., electroporation, microinjection, etc.) is utilized to transfer the plasmid into the host cell. Plasmids suitable for insertion of genetic elements into the host cell are well known to the skilled artisan.

[0097] Other gene cloning methods include, but are not limited to, direct integration of the genetic material into the chromosome. This can occur by a variety of means, including cloning the genetic elements described herein on non-replicating plasmids flanked by homologous DNA sequences of the host chromosome; upon transforming said recombinant plasmid into a host the genetic elements can be introduced into the chromosome by DNA recombination. Such recombinant strains can be recovered if the integrating DNA fragments contain a selectable marker, such as antibiotic resistance. Alternatively, the genetic elements can be directly introduced into the chromosome of a host cell without use of a non-replicating plasmid. This can be done by synthetically producing DNA fragments of the genetic elements in accordance to the present invention that also contain homologous DNA sequences of the host chromosome. Again, if these synthetic DNA fragments also contain a selectable marker, the genetic elements can be inserted into the host chromosome.

[0098] In one embodiment, the nucleotide sequence encoding the casein in the expression construct of the invention preferably is adapted to optimize its codon usage to that of the non-mammalian host cell in question. The adaptiveness of a nucleotide sequence encoding an enzyme to the general codon usage of a host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1 , with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen et al, 2003, Nucleic Acids Res. 3J (8):2242-51). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9.

[0099] In a second aspect, the invention provides for a process for producing a casein, the process comprising culturing a non-mammalian host cell as described herein in a medium in a fermenter or bioreactor under conditions conducive to the expression of casein. Thus, in one embodiment, the invention provides for a process for producing a casein, the process comprising culturing the non- mammalian host cell comprising i) an expression construct comprising a nucleotide sequence encoding at least one casein; ii) a genetic modification that reduces or eliminates the expression or activity of a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof; iii) a genetic modification that reduces or eliminates the expression or activity of a cell-wall associated aspartic acid protease of the yapsin family encoded by a YPS1 gene or an orthologue thereof; and iv) a genetic modification that reduces or eliminates the expression or activity of a cellwall associated aspartic acid protease of the yapsin family encoded by a YPS’ gene or an orthologue thereof such that one or more of the nucleotide sequences are expressed and the casein is produced, the method optionally comprising the step of recovery of the casein.

[0100] Media for growth of non-mammalian host cells, e.g., fungal host cells are generally known in the art. In a preferred embodiment, the medium for culturing a host cell of the invention is a chemically defined medium. Typical compositions of the chemically defined media for growth of filamentous fungi and yeasts are e.g., described in US 20140342396 A1 , incorporated by reference herein. The pH in the fermenter or bioreactor may be controlled using ammonia as titrant.

[0101] In certain embodiments, the host cell is cultured in a medium comprising glucose, glycerol, methanol or maltose as a carbon source. In preferred embodiments, glucose, glycerol, methanol or maltose is the sole carbon source.

[0102] Optionally, the process further comprises recovering the casein.

[0103] In one embodiment, the recovery is during fermentation. This may be carried out in a continuous mode to increase productivity. In one embodiment, the recovery is post fermentation. In one embodiment, the recovery is both during and post fermentation. The recovery of casein preferably at least includes separation of the host cell’s biomass from the medium comprising the (dissolved) casein. One of the possibilities to separate the microbial biomass is by centrifugation. Even more preferred the fermented broth may be set for release of the casein at the end of the fermentation, which may be following the separation of the released protein directly by centrifugation of the biomass. Therefore, in one embodiment, the recovery is by centrifugation. However, other recovery methods are suitable, such as e.g., acid or salt precipitation and solvent extraction, as known in the art.

[0104] In another embodiment the process of the second aspect is a process for the enrichment of a casein in a composition comprising the casein and other biomolecules, the process comprising: i) providing the composition comprising casein; ii) reducing the pH of the composition to a pH lower than 3.0 so as to reduce the amount of the other biomolecules in the soluble fraction of the composition; and, iii) recovering the soluble fraction of the composition, thereby enriching casein in the soluble fraction of the composition.

[0105] In certain embodiments, the pH in step ii) is reduced to pH 1 .0 to 3.0, preferably to pH 1 .5 to 2.8, more preferably to 1.8 to 2.5, or even more preferably to a pH of 2.0 to 2.4. In some embodiments the pH of the composition is reduced to 2.0. In certain embodiments, the pH can be reduced by addition of an acid to the composition. Suitable acids are preferably acids that are food safe and / or acids that are commonly used in an industrial scale such as hydrochloric acid, sulfuric acid, phosphorous acid, citric acid and acetic acid. The process to enrich casein may comprise an additional thermal lysis as the soluble other biomolecules of the host cells will precipitate or degrade at high temperature. Accordingly, in certain embodiments, the composition is heated before step ii). More specifically, the composition is subjected to a thermolysis step before step ii). In certain embodiments, the thermolysis step is performed at about 50°C to 100°C, preferably at about 60°C to 90°C.

[0106] In certain embodiments, the treatment at low pH itself may be combined with thermal lysis. Thus, step ii) may be performed at about 50°C to 100°C or at about 60°C to 90°C. The duration of such thermal lysis may be as long as the treatment at low pH but may also be shorter, for example from 1 to 60 min, or from 2 to 30 min, or from 3 to 20 min.

[0107] In certain embodiments, the soluble fraction of step iii) is recovered by at least one of filtration, decantation, centrifugation and chromatography methods.

[0108] In a third aspect, there is provided a composition comprising a casein that is obtained or obtainable by a process for producing a casein as herein described.

[0109] In one embodiment, the composition is substantially free of 15-20 kDa proteolytic casein fragments, preferably the composition is substantially free of 15-20 kDa proteolytic alphaSI -casein fragments, more preferably, the composition is substantially free of 15-20 kDa proteolytic alphaSI - casein variant A fragments. “Substantially free” is herein understood to mean that the composition comprises no more than 10, 5, 2, 1 , 0.5, 0.2 or 0.1 weight % of the indicated proteolytic fragments.

[0110] In one embodiment, the composition is a dairy substitute product comprising at least one casein that is obtained or obtainable by a process for producing a casein as herein described. In one embodiment, the composition is a dairy substitute product that is animal-free. Such an animal- free dairy substitute product is e.g., a composition comprising animal-free milk fats and proteins. A wide variety of dairy substitute products can be made using the methods and compositions described herein. Methods for producing animal-free dairy substitute products are inter alia described in WO2016 / 029193, which is herein incorporated by reference. Such products include without limitation, milk, whole milk, buttermilk, skim milk, infant formula, condensed milk, dried milk, evaporated milk, butter, clarified butter, cream, cottage cheese, cream cheese, creme fraiche, skyr, yogurt and various types of cheese. The dairy substitute products can also be incorporated into various food applications as a replacement for dairy products, which include ice cream, frozen custard, frozen yogurt, cookies, chocolate and cakes.

[0111] In one embodiment, the composition is a composition as described above, wherein at least one casein selected from the group consisting of alphaSI -casein, alphaS2-casein, beta casein and kappa casein is not present. As will be understood a composition wherein at least one of the caseins is not present will usually be a composition during the manufacture of which the particular casein has not been added. In one embodiment, the composition is a composition as described above, wherein at least two caseins selected from the group consisting of alphaSI -casein, alphaS2-casein, beta casein and kappa casein are not present. In one embodiment, the composition is a composition as described above, wherein at least three caseins selected from the group consisting of alphaSI - casein, alphaS2-casein, beta casein and kappa casein are not present. Advantageously, a composition wherein at least one of the caseins is not present is comparably simple to produce. The absence of the particular casein can e.g., be established by immunological means (see above) using antibodies specific for the particular casein.

[0112] In one embodiment, the composition is a composition as described above, wherein the casein that is obtained or obtainable by a process for producing a casein as herein described, imparts on the composition at least one dairy-like property selected from the group consisting of cohesiveness, firmness, elasticity, meltability, spreadability, texture, mouthfeel, hardness, creaminess, flexibility and taste. In one embodiment, the composition is a composition wherein the dairy-like property imparted by the casein on the composition is at least equivalent to a same property in an otherwise similar composition that comprises native milk-derived casein and does not comprise the casein as obtained or obtainable by a process for producing a casein as herein described.

[0113] In one embodiment, the at least one dairy-like property of the composition can be tested, and / or compared with the otherwise similar composition comprising a native milk-derived casein, using a trained panel of human subjects. In another embodiment, the at least one dairy-like property of the composition can be tested and / or compared, using a machine or apparatus. For example, cohesiveness, firmness, elasticity, texture, hardness, creaminess and flexibility can be analysed using a texture analyzer. Meltability and spreadability can be analysed using a melting station and / or oven and computer imaging or e.g. as described in co-pending application EP 24203511 .1 .

[0114] In a fourth aspect, there is provided a method for producing a composition comprising a casein that is obtained or obtainable by a process for producing a casein as herein described, the method comprising the step of combining the casein with further ingredients, preferably animal-free ingredients, to obtain the composition, e.g. a food product or a dairy substitute product, preferably an animal-free dairy substitute product.

[0115] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0116] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0117] Description of the figures

[0118] Figure 1 : Immunodetection (western blot) of extracellular bovine alphaSI -casein variant A (24 deepwell cultivation) upon expression by a protease gene deleted Pichia strain. A) effect of the additional knock-out of the yapsin gene YPS’ B) effect of the additional knock-out of a yapsin gene - comparison between YPS’ versus YPS3, YPS" or MKC7 gene deletions. Pp0357: parental expression strain (pep4- yps1-). -P / +P: cultivation without / with addition of pepstatin A. Black arrow: position of secreted alphaSI-casein; grey arrows: alphaSI -casein degradation fragments.

[0119] Figure 2: Immunodetection (western blot) of extracellular bovine alphaSI -casein variant A (24 deepwell cultivation) upon expression by a protease gene deleted Pichia strain: effect of the knock-out of Ser-protease genes (SBT100). Pp0487: parental expression strain (pep4- yps1- yps’-). - or +: cultivation without / with addition of PMSF. Black arrow: position of secreted alphaSI-casein; grey arrows: alphaSI-casein degradation fragment.

[0120] Figure 3: Detection of extracellular bovine alphaSI-casein variant A (24 deep-well cultivation) upon expression by a protease gene deleted Pichia strain. A) Western blot analysis on effect of the knockout of PMT1 and / or alpha-1 ,2-mannosidase co-expression. Pp0487: parental expression strain (pep4- yps1- yps’-; 6 copies of the casein expression cassette); Pp0520: Pp0487 with PMT1 gene deletion. B) Coomassie stain analysis on effect of a PMT1 versus PMT4 gene knock-out. Parental expression strain (pep4- yps1- yps’-; >30 copies of the casein expression cassette). Black arrow: position of secreted alphaSI-casein.

[0121] Examples

[0122] Example 1 : Reduction of proteolysis of bovine alphaSI-casein upon secretion by Pichia pastoris

[0123] Direct gel analysis on cell-free medium samples, as well as LC-MS / MS analysis (data not shown) on bovine alphaSI-casein isolated from the Pichia cultivation broth, indicate the occurrence of one or more proteolytic events that result in alphaSI-casein molecules with a significant N- terminal truncation. This N-terminal truncation can be partially inhibited by cultivating the Pichia strains in the presence of pepstatin A, which suggests that acid aspartyl protease activity is responsible for the observed degradation of alphaSI-casein. Given that the described strains are already gene-deleted for PEP4 and YPS1, the major vacuolar resp. cell-wall associated acid aspartyl proteases, additional gene knock-out studies were initiated in strain Pp0357. This strain was derived via genomic integration of plasmid P0532 into the strain Pp0096 (Apep4, Aypsl). As a result, this strain expresses bovine alphaSI-casein fused with a Gly-Ser linker to the S. cerevisiae alpha-mating factor prepro-region (alphaMFprepro) for secretion, under the control of the methanolinducible AOX1 promoter.

[0124] CRISPR-based knock-out plasmids were generated according to the methods described above to delete additional genes encoding acid aspartyl proteases, such as YPS2 (PP7435_Chr3- 0919), YPS3 (PP7435_Chr3-0913), YPS7 (PP7435_Chr3-0819), MKC7 (PP7435_Chr1-0699), YPS’ (PP7435_Chr3-1068) and YPS” (PP7435_Chr3-0313) (Guan et al. 2012, Wu et al. 2013). The resulting plasmids (P0420, P0423, P0421 , P0425, P0471 resp. P0424) were transformed into strain Pp0357, and transformants were selected and genetically characterized via colony PCR.

[0125] For each targeted yapsin gene, at least one correct knock-out clone could be obtained within the Pp0357 strain background. These strains and the parental clone Pp0357 were cultivated (24 deep-well format) at 28°C and 200 rpm for 24 hours in standard BMGY medium pH 7, after which the cells were transferred to standard BMMY medium (1 % methanol, pH 7) and cultivated for another 48 hours at 28°C and 200 rpm. During the methanol induction phase, PMSF was added at a final concentration of 2 mM. Each strain was cultivated in duplicate in either the presence or absence of 5 pg / mL pepstatin A. At the end of the cultivation, cells were separated from the broth and the cell-free broth was analyzed via western blot for the extracellular presence of (full-size) alphaSI -casein (Fig. 1A). The results show that the knock-out of the YPS’ gene (SEQ ID NO: 3) significantly reduced the proteolysis of Pichia-secreted bovine alphaSI -casein as there is no longer a difference between cultivations with and without the presence of pepstatin A. Moreover, the LC- MS / MS analysis on isolated secreted alphaSI -casein confirmed the absence of a major N-terminal truncation event (data not shown). In contrast, western blot analysis clearly shows that proteolysis is still ongoing upon secretion by the parental Pp0357 strain or by the Pp0357-derived strains in which one of the other yapsin genes was deleted (Fig. 1 B).

[0126] Example 2: Further reduction of proteolysis of bovine alphaSI -casein upon secretion by Pichia pastoris

[0127] Despite the additional deletion of the YPS’ gene, which was generated already on top of the deletion of the PEP4 and YPS1 genes, some alphaSI -casein (variant A) proteolysis could still be observed (i.e. formation of a 15-20 kDa proteolytic fragment and a slight reduction in the amount of full-size protein), in particular when performing the cultivation at elevated pH (> 6.5) and in the absence of PMSF. These observations were indicative for Ser-type protease activity. LC-MS / MS analysis, performed on the cell-free medium of a bioreactor cultivation broth of a Pichia strain, which secretes bovine alphaSI -casein, effectively showed the extracellular presence of Ser-type proteases (data not shown). Apart from a known vacuolar Ser-type protease, encoded by the PRB1 gene (PP7435_Chr1-0540; Wu et al. 2013), and a known secreted Ser-type protease, encoded by the SUB2 gene (PP7435_Chr1-1352; Salamin et al. 2010), a third protein, termed SBT100 (PP7435_Chr2-1137), was identified, which showed some homology to subtilisin-like Ser-type proteases.

[0128] CRISPR-based knock-out plasmids were generated according to the methods described above to delete the SBT100 gene. The resulting plasmid P0631 was separately transformed into strain Pp0487 (Apep4, Aypsl, Ayps1) and transformants were selected and genetically characterized via colony PCR. Strain Pp0487 contains up to 6 copies of the P0573 expression cassette, which consists of the AOX1 promoter, a direct fusion of the alphaMFprepro secretion signal to the N-terminus of alphaSI -casein variant A, and the AOX7TT terminator. At least one correct knock-out clone could be selected within the Pp0487 strain background. These strains and the parental clone Pp0487 were cultivated (24 deep-well format) at 28°C and 200 rpm for 24 hours in standard BMGY medium at pH 7, after which the cells were transferred into BMMY (1 % methanol; pH 7) and cultivated for another 48 hours at 28°C and 200 rpm. A clone was cultivated in duplicate in either the absence or the presence of 2 mM of PMSF during the methanolinduction phase. At the end of the cultivation, cells were separated from the broth and the cell-free broth was analyzed via SDS-PAGE for the extracellular presence of alphaSI -casein (Fig. 2). The results show that the knock-out of the SBT100 gene (SEQ ID NO: 45) significantly reduced the degradation of full-size bovine alphaSI -casein as there is no longer a clear difference between cultivations with and without the presence of PMSF. Moreover, the accumulation of the 15-20 kDa proteolytic fragment is virtually abolished, even when culturing the expression strains at pH 7 and in the absence of PMSF.

[0129] Example 3: Reduction of O-glycosylation of bovine alphaSI -casein upon secretion by Pichia pastoris

[0130] The extracellular analysis on cultivation broth of Pichia secreting bovine alphaSI -casein (variant A) shows the presence of a heterogenous casein protein population. Product-related protein bands with a higher mobility are most probably the result of proteolytic events. Apart from that, a major fraction of the extracellular AlphaSI -casein shows a reduced mobility which is most probably related to post-translational modifications such as extended O-glycosylation (confirmed via LC-MS / MS analysis; results not shown). Hence, a CRISPR-based knock-out strategy was designed to eliminate the Pichia PMT1 gene (PP7435_Chr2-1095; SEQ ID NO: 46) or PMT4 gene (PP7435_Chr1-0806: SEQ ID NO: 48), coding for protein-O-mannosyltransferase involved in O- glycosylation by Pichia pastoris cells (Govindappa et al., 2013; Nett et al., 2013). The knock-out plasmids for PMT1 or PMT4 were transformed towards strain Pp0487 and transformants were selected and genetically characterized via colony PCR.

[0131] Another strategy to reduce O-glycosylation on P / ch / a-produced proteins is by co-expression of a secreted alpha-1 ,2-mannosidase as most of the Pichia O-glycan structures are mainly consisting of alpha-1 ,2-linked mannose residues (Laukens et al., 2015). Using the modular OPENPichia plasmid system (Van Herpe et al., 2022), a pGAP-driven expression construct was generated for the Trichoderma reesei alpha-1 ,2-mannosidase in which the coding sequence for the mature secreted protein was fused towards the S. cerevisiae alpha-mating factor prepro-region as described in literature (Maras et al., 1997; SEQ ID NO 49). The resulting plasmid, P0375, contains the G418 resistance marker to allow screening of mannosidase positive transformants within zeocin-resistant alphaSI -casein expression strains. Strains Pp0487 and Pp0520, a PCR-selected PMT1 KO strain derived from Pp0487, were transformed with plasmid P0375 and clones were selected on YPD agar plates (pH 8) containing 100 pg / mL of zeocin and 500 pg / mL of G418. Integration of the mannosidase expression cassette was confirmed via colony PCR analysis.

[0132] PCR-positive clones for correct PMT gene knockout and / or alpha-1 ,2-mannosidase coexpression were cultivated in 24 deep-well format as described before, in combination with cultivations of corresponding parental strains. During the methanol induction phase, PMSF was added at a final concentration of 2 mM. At the end of the cultivation, cells were separated from the broth and the cell-free broth was analyzed via SDS-PAGE / lnstant blue staining and via western blot.

[0133] The results indicate that both the PMT gene knock-out strategy as well as mannosidase coexpression can reduce the degree of O-glycosylation. Based on the gel mobility of secreted bovine alphaSI -casein variant A (Fig. 3A), the PMT1 gene knock out (replicate cultivations of Pp0520) and the alpha-1 ,2-mannosidase co-expression (different individual transformants of Pp0487) result into a relatively similar reduction in protein size and heterogeneity. The latter is due to O-glycan reduction, as confirmed by mass spectrometry (RPC-UV-MS; results not shown). On top of that, the combination of both strategies appears to have a synergistic effect, resulting in a further reduction in heterogeneity and overall size of the secreted alphaSI-casein. Similar results for the PMT1 gene knock-out strategy were obtained when performed in a strain background (pep4-, yps1 , yps’-) with more than 30 integrated copies of the alphaSI -casein (variant A) expression cassette (the parent strain). Finally, the knock-out of the PMT4 gene within the parent strain also resulted in a significant reduction of the casein heterogeneity, although in a different way compared to the PMT1 knock-out strategy (Fig. 3B). Mass spectrometry (RPC-UV-MS; results not shown) confirms that about half of the secreted alphaSI -casein contains no O-glycosylation (lowest and most intense protein band), whereas the other half still represents a heterogenous pool of O-glycosylated casein.

[0134] References

[0135] Govindappa N, Hanumanthappa M, Venkatarangaiah K, Kanojia K, Venkatesan K, Chatterjee A, Kusumanchi M, Dave N, Hazra P, Tiwari S, Sastry K (2013). PMT1 gene plays a major role in O- mannosylation of insulin precursor in Pichia pastoris. Protein Expression and Purification 88(1): 164-171.

[0136] Guan B, Lei J, Su S, Chen F, Duan Z, Chen Y, Gong X, Li H, Jin J (2012). Absence of Yps7p, a putative glycosylphosphatidylinositol-linked aspartyl protease in Pichia pastoris, results in aberrant cell wall composition and increased osmotic stress resistance. FEMS Yeast Res. 2012 Dec;12(8):969-79.

[0137] Laukens B, De Wachter C, Callewaert N (2015). Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology. Methods Mol Biol. 2015; 1321 : 103-22.

[0138] Maras M, Callewaert N, Piens K, Claeyssens M, Martinet W, Dewaele S, Contreras H, Dewerte I, Penttila M, Contreras R (1997). Molecular cloning and enzymatic characterization of a Trichoderma reesei 1 ,2-alpha-D-mannosidase. J Biotechnol. 2000 Feb 17;77(2-3):255-63.

[0139] Nett JH, Cook WJ, Chen MT, Davidson RC, Bobrowicz P, Kett W, Brevnova E, Potgieter TI, Mellon MT, Prinz B, Choi BK, Zha D, Burnina I, Bukowski JT, Du M, Wildt S, Hamilton SR (2013). Characterization of the Pichia pastoris protein O-mannosyltransferase gene family. PLoS One 8(7):e68325.

[0140] Salamin K, Sriranganadane D, Lechenne B, Jousson O, Monod M. (2010). Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71. Appl Environ Microbiol. 2010 Jul;76(13):4269-76.

[0141] Van Herpe D, Vanluchene R, Vandewalle K, Vanmarcke S, Wyseure E, Van More B, Eeckhaut H, Fijalkowska D, Grootaert H, Lonigro C, Meuris L, Michielsen G, Naessens J, Roles C, van Schie L, De Rycke R, De Bruyne M, Borghgraef P, Claes K, Callewaert N (2022). OPENPichia: building a free-to-operate Komagataella phaffii protein expression toolkit. bioRxiv preprint doi: https: / / doi.Org / 10.1 101 / 2022.12.13.519130.

[0142] Wu M, Shen Q, Yang Y, Zhang S, Qu W, Chen J, Sun H, Chen S (2013). Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA / PTH in Pichia pastoris GS115.

[0143] Journal of Industrial Microbiology and Biotechnology 40(6): 589-599.

Claims

Claims1 . A non-mammalian host cell comprising: i) an expression construct comprising a nucleotide sequence encoding at least one casein; ii) a genetic modification that reduces or eliminates the expression or activity of a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof; iii) a genetic modification that reduces or eliminates the expression or activity of a cellwall associated aspartic acid protease of the yapsin family encoded by a YPS1 gene or an orthologue thereof; and iv) a genetic modification that reduces or eliminates the expression or activity of a cellwall associated aspartic acid protease of the yapsin family encoded by a YPS’ gene or an orthologue thereof.

2. A host cell according to claim 1 , wherein: a) the PEP4 gene encodes a vacuolar acid aspartyl protease or an orthologue thereof comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 ; b) the YPS1 gene encodes a cell-wall associated aspartic acid protease comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 2; and c) the YPS’ gene encodes a cell-wall associated aspartic acid protease comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 3.

3. A host cell according to claim 1 or 2, wherein the host cell comprises at least one further genetic modification selected from the group consisting of: v) a genetic modification that that reduces or eliminates the expression or activity of a subtilisin-like Ser-type protease, preferably a subtilisin-like Ser-type protease encoded by a SBT100 gene or an orthologue thereof; and vi) a genetic modification that reduces or eliminates the level of O-linked glycosylation of caseins proteins produced by the cell.

4. A host cell according to claim 3, wherein the SBT100 gene encodes a subtilisin-like Ser-type protease comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 45.

5. A host cell according to claim 3 or 4, wherein the level of O-linked glycosylation of caseins produced by the cells is reduced by:- eliminating or reducing the enzymatic activity of a protein-O-mannosyl transferase, preferably an protein-O-mannosyl transferase encoded by a PMT gene; and / or- introducing into the host cell an expression construct comprising a nucleotide sequence encoding at least one alpha-mannosidase such as an alpha-1 ,2-mannosidase.

6. A host cell according to any one of the preceding claims, wherein the nucleotide sequence encoding the at least one casein comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of a casein from a mammal selected from the group consisting of: Bos taurus (domestic cattle), Bos grunniens (yak), Bubalus bubalis (water buffalo), Capra hircus (goat), Ovis aries (sheep), Camelus spp. (camel, dromedaris), Rangifer tarandus (reindeer), Equus caballus (horse), Sus spp. including Sus domesticus (pig) and Homo sapiens.

7. A host cell according to claim 6, wherein the nucleotide sequence encoding the at least one casein comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of at least one of SEQ ID NO’s: 4 - 44, of which the bovine SEQ ID NO’s: 4, 14, 26 and 35 are preferred.

8. A host cell according to any one of the preceding claims, wherein the host cell is nonmammalian eukaryotic host cell, preferably a eukaryotic microbial host cell, more preferably a yeast or a filamentous fungus host cell.

9. A host cell according to claim 8, wherein the cell is selected from a genus from the group consisting of Saccharomyces, Kiuyveromyces, Candida, Komagataella, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Kazachstania Debaryomyces, Naumovia, Alternaria, Apophysomyces, Aspergillus, Cladosphialophora, Fonsecaea, Fusarium, Lichtheimia, Mucor, Myceliophthora, Neurospora, Penicillium, Rhizopus, Rhizomucor, Trichoderma and Trichophyton, wherein preferably, the cell selected from a species from the group consisting of K. phaffii, K. pastoris, K. pseudopastoris S. cerevisiae, S. exiguus, S. bayanus, Kiuyveromyces lactis, Kiuyveromyces marxianus, Y. lipolytica, S. pombe, Alternaria alternata, Apophysomyces variabilis, Aspergillus spp., Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus sojae, Aspergillus terreus, Cladosphialophora spp., Fonsecaea pedrosoi, Fusarium spp., Fusarium oxysporum, Fusarium solani, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramosa, Myceliophthora spp., Myceliophthora thermophila, Neurospora crassa, Penicillium chrysogenum, Penicillium simplicissimum, Penicillium brasilianum, Rhizopus spp., Rhizopus microsporus, Rhizomucor spp., Rhizomucor pusillus, Rhizomucor miehei, Trichoderma spp.,Trichoderma reesei, Trichophyton spp., Trichophyton interdigitale, and Trichophyton rubru, of which Komagataella phaffii is most preferred.

10. A process for producing a casein, the process comprising culturing a host cell according to any one of claims 1-9 such that one or more of the nucleotide sequences are expressed and the casein is produced, the process optionally comprising the step of recovery of the casein.

11. A composition comprising a casein as obtained by the process of claim 10, wherein preferably the composition is a food product, wherein more preferably the composition is a dairy substitute product, wherein most preferably the composition is animal-free dairy substitute product.

12. A composition according to claim 10, wherein the composition is substantially free of 15-20 kDa proteolytic casein fragments.

13. A composition according to claim 11 or 12, wherein the casein imparts on the composition at least one dairy-like property selected from the group consisting of cohesiveness, firmness, elasticity, meltability, spreadability, texture, mouthfeel, hardness, creaminess, flexibility and taste.

14. A composition according to claim 13, wherein the dairy-like property imparted by the casein on the composition is at least equivalent to a same property in an otherwise similar composition that comprises native milk-derived casein and does not comprise the casein as obtained by the process of claim 10.

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