Field of the invention

The present invention relates to the field of molecular microbiology, food technology and fermentation technology. In particular, the invention relates to fungal host cell comprising a nucleotide sequence coding for a mammalian casein.

Background of the invention

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.

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.

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.

The ability of yeast and filamentous fungi to grow on rather inexpensive substrates, as well as their capacity to produce a wide range of commercially interesting compounds ranging from simple organic acids to complex secondary metabolites, has attracted considerable interest to exploit them as production organisms in biotechnology. Furthermore, due to their exceptional high capacity to express and secrete certain hydrolytic proteins, filamentous fungi have nowadays become indispensable to produce enzymes (native or recombinant) with applications in paper and textile industry, feed production and food processing. The most commonly used fungal expression hosts for protein production are Aspergillus species and Trichoderma reesei. Via a combination of strain engineering and fermentation technology development, product titers exceeding 100 g/L are no longer an exception (Cherry & Fidantsef, 2003, Current opinion in biotechnology, 14(4), pp.438- 443).

Expression of recombinant mammalian caseins has previously been described in 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 has to this date never been successfully expressed and produced in filamentous fungal cells.

It is thus an object of the present invention to provide for microbial organisms that are capable to express milk casein proteins to obviate the farm animals to produce these proteins.

Summary of the invention

In one aspect, the invention provides for a filamentous fungal host cell comprising an expression cassette comprising a nucleotide sequence coding for a casein, operably linked to a promoter for expression in the fungal host cell. In certain embodiments, the fungal cell as described herein is a mutant of a parental fungal cell comprising an endogenous transcriptional activator gene regulating the expression of an array of protease genes and wherein in the fungal host cell the expression of the endogenous transcriptional activator gene is reduced or eliminated. In one embodiment, the endogenous transcriptional activator gene is an endogenous prtT gene. In certain embodiments the fungal cell comprises a nucleotide sequence that encodes a casein 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 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, dromedary), Rangifer tarandus (reindeer), Equus caballus (horse), Sus spp. including Sus domesticus (pig) and Homo sapiens.

In a second aspect, the invention provides for a process for producing a casein, the process comprising the steps of: a) culturing the fungal host cell as defined herein in a medium in a fermenter or bioreactor under conditions conducive to the expression of the casein; and, b) optionally, recovery of the casein.

In a third aspect, the invention provides for a use of a protease inhibitor to inactivate the enzymatic activity of at least one (acid) aspartyl protease, at least one serine-type protease, at least one cysteine-type proteases and/or at least one metalloprotease in a fungal host cell as defined in herein. Description of the invention Definitions

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.

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

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".

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.

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.

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.

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.

“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) 1 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.

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/.

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. Alternative Physical and Functional Classifications of Amino Acid Residues.

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 (3 ^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.). 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).

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.

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.

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.

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.

"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.

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.

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. The "specific activity" of an enzyme is herein understood to mean the amount of activity of a particular enzyme per amount of total host cell protein, usually expressed in units of enzyme activity per mg total host cell protein. In the context of the present invention, the specific activity of a particular enzyme may be increased or decreased as compared to the specific activity of that enzyme in an (otherwise identical) wild type host cell.

Detailed description of the invention

The present inventors have surprisingly found that it is possible to efficiently produce casein proteins in filamentous fungal host cells.

In a first aspect the invention therefore provides for a filamentous fungal host cell comprising an expression cassette comprising a nucleotide sequence coding for a casein, operably linked to a promoter for expression in the fungal host cell.

The term "casein" is art-known and represents a family of proteins that is present in mammal- produced milk and is capable of self-assembling 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.

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.

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

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.

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.

Non-limiting examples of sequences for casein protein from different mammals are provided herein (see table 1). Additional sequences for other caseins are known in the art.

In addition, the caseins for use in the invention 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.

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.

The filamentous fungal host cell preferably includes cells from genera such as Alternaria, Apophysomyces, Aspergillus, Cladosphialophora, Fonsecaea, Fusarium, Lichtheimia, Mucor, Myceliophthora, Neurospora, Penicillium, Rhizopus, Rhizomucor, Trichoderma and Trichophyton. Preferred filamentous fungal cells belong to a species of an 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, of which Aspergillus oryzae is most preferred.

In certain embodiments, the fungal host cell according to the invention is a mutant of a parental fungal cell which mutant has lost at least 30, 40, 50, 60, 70, 80, 90, 95, 100% of its overall proteolytic activity, preferably of its overall extracellular proteolytic activity. The overall proteolytic activity against caseins within the cultivation broth of micro-organisms can be determined with substrates such as azocasein. The latter is prepared by dyeing casein with sulphanilic acid. The analytical procedure is based on the cleavage of azocasein by proteases, hereby generating the free azo-dye. Precipitation and centrifugation of the proteins and larger peptide fragments allow the free azo-dye to be measured under alkaline conditions, providing an indication of the proteolytic activity. The absorbance of this product is measured at OD 440 nm and this value is directly proportional to the level of the casein-specific protease activity. One unit of protease activity is described as the amount of enzyme that catalyzes the hydrolysis of 1 mg of azocasein per hour at 37 °C within the applied assay condition. In one embodiment, the fungal host cell according the invention is a mutant of a parental fungal cell which has lost at least 30, 40, 50, 60, 70, 80, 90, 95, 100% of its overall endoproteolytic activity, preferably of its overall extracellular endoproteolytic activity. Endoproteolytic activity can be measured by use offer example the azocasein assay which has been described in the art.

In one embodiment, the fungal host cell according to the invention comprises a genetic modification that reduces or eliminates the enzymatic activity of at least one serine-type protease and/or at least one metalloprotease and/or at least one aspartic protease and/or at least one cysteine protease and/or at least one glutamic protease and/or at least one threonine protease by deletion of at least a part of at least one of the promoter and the coding sequence of the gene encoding the proteases.

In one embodiment, the fungal host cell according to the invention comprises a genetic modification that reduces or eliminates the enzymatic activity of at least one protease selected from the group consisting of: pepA (Gene ID number: An14g04710), pepB (Gene ID number: An01g00530), pepN (Gene ID number: An01g00370), pepAa, pepAb, pepAc, pepAd (as described in Li et al., 2023. J. Fungi 9(5), 528, Burggraaf et al., 2016, FEMS Microbiol Lett 363(15), and Wang et al. 2008 Fungal Genet Biol 45(1):17-27), tppA (Gene ID number: A0090011000235), pepE (Gene ID number: A0090003000693), nptB (Gene ID number: A0090010000493), dppIV (Gene ID number: AG090023000602), dppV (Gene ID number: AG090011000795), alpA/1 (Gene ID number: A0090003001036), pepA (Gene ID number: A0090120000474), AopepAa (Gene ID number: A0090012000209), AopepAd (Gene ID number: A0090023000872), epi (Gene ID number: A0090103000001) (as described in Yoon et al., 201 1 . Appl Microbiol Biotechnol 89: 747- 759), Npl (Gene ID number: AG090011000036), Npll/NptB (Gene ID number: AG090010000493), Nplll (Gene ID number: A0090138000160) (as described in Nakadai et al., 1973. Agr. Biol. Chem 37(12), 2695-2701 , Nakadai et al., 1973. Agr. Biol. Chem 37(12), 2703-2708, and Kimura et al., 2008. Biosci. Biotechnol. Biochem. 72(2), 499-505), pep1 (Gene ID number: tre74156), tsp1 (Gene ID number: tre73897), slp1 (Gene ID number: tre51365), gap1 (Gene ID number: tre69555), gap2 (Gene ID number: tre106661), pep4 (Gene ID number: tre77579), pep5 (Gene ID number: tre81004), pep3 (Gene ID number: tre121133), sep1 (Gene ID number: tre124051), amp1 (Gene ID number: tre81070), pep8 (Gene ID number: tre122076), pep9 (Gene ID number: tre79807), pep11 (Gene ID number: tre121306), slp7 (Gene ID number: tre123865), amp2 (Gene ID number: tre108592) (as described in Landowski et al., 2015. PLOSone Aug 26;10(8):e0134723 and Landowski et al., 2015 Microb Cell Fact.Jun 10;15(1 ):104), Prolyl endopepidases from A. niger as described in (Edens et al., 2005. J Agric Food Chem. 53(20)7950-7957 and Kang et al., 2013 J Ind Microbiol Biotechnol 40:855-864), Prolyl endopepidases AoS28A, AoS28B, AoS28C and AoS28D (as described in Eugster, Philippe J., et al. Microbiology 161 .Pt_12 (2015): 2277-2288).

In an alternative embodiment, the fungal host cell is a mutant of a parental fungal cell comprising an endogenous transcriptional activator gene regulating the expression of an array of protease genes and wherein in the fungal host cell the expression of the endogenous transcriptional activator gene is reduced or eliminated.

In certain embodiments, the fungal host cell according the invention is a mutant of a parental fungal cell wherein the endogenous transcriptional activator gene is an endogenous prfT gene and wherein in the fungal host cell the expression of the endogenous prfT gene is reduced or eliminated. prfT is a transcriptional activator of proteases in eukaryotic cells. Several fungal transcriptional activators of proteases have been recently described in WO 00/20596, WO 01/68864, WO 2006/040312 and WO 2007/062936. These transcriptional activators were isolated from A. niger, A. fumigatus, P. chrysogenum and A. oryzae. These transcriptional activators of protease genes can be used to improve a method for producing a polypeptide in a fungal cell, wherein the polypeptide is sensitive for protease degradation. When the endogenous prfT gene is partially or completely inactivated, the host cell will produce less or no proteases that are under transcriptional control of prfT.

In certain embodiments, the endogenous prtT gene encodes a transcriptional activator 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’s: 1 or 2.

In certain embodiments, the fungal host cell according to the invention further comprises a genetic modification that reduces or eliminates the enzymatic activity of at least one serine-type protease and/or at least one metalloprotease and/or at least one aspartic protease and/or at least one cysteine protease and/or at least one glutamic protease and/or at least one threonine protease by deletion of at least a part of at least one of the promoter and the coding sequence of the gene encoding the proteases. Preferably the genetic modification reduces or eliminates the specific activity or amount of the at least one protease to no more than 90, 75, 50, 20, 10, 5, 2 or 1 % of the specific activity or amount of the enzyme or protein compared to a corresponding host cell lacking the genetic modification, when cultivated under identical conditions. More preferably, the genetic modification completely eliminates the specific activity or amount of the enzyme in the host cell.

The advantage of reducing or eliminating the expression in the host cell of at least one serine- type protease and/or at least one metalloprotease and/or at least one aspartic protease and/or at least one cysteine protease and/or at least one glutamic protease and/or at least one threonine protease is that it reduces the negative impact of host cell proteases on the yield and quality of the casein.

In certain embodiments the fungal host cell according to the invention is a mutant of a parental fungal cell comprising an endogenous a/p1 gene encoding an alkaline serine-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: 3 and wherein in the fungal host cell the enzymatic activity of the a/p1 encoded alkaline serine-type protease is reduced or eliminated.

In one embodiment, the fungal host cell according to the invention comprises a further genetic modification that reduces or eliminates the enzymatic activity of at least one protease selected from the group consisting of: pepA (Gene ID number: An14g04710), pepB (Gene ID number: An01g00530), pepN (Gene ID number: An01g00370), pepAa, pepAb, pepAc, pepAd (as described in Li et al., 2023. J. Fungi 9(5), 528, Burggraaf et al., 2016, FEMS Microbiol Lett 363(15), and Wang et al. 2008 Fungal Genet Biol 45(1):17-27), tppA (Gene ID number: A0090011000235), pepE (Gene ID number: A0090003000693), nptB (Gene ID number: A0090010000493), dppIV (Gene ID number: AG090023000602), dppV (Gene ID number: AG090011000795), alpA/1 (Gene ID number: A0090003001036), pepA (Gene ID number: A0090120000474), AopepAa (Gene ID number: A0090012000209), AopepAd (Gene ID number: A0090023000872), epi (Gene ID number: A0090103000001) (as described in Yoon et al., 2011 . Appl Microbiol Biotechnol 89: 747- 759), Npl (Gene ID number: AG090011000036), Npll/NptB (Gene ID number: AG090010000493), Nplll (Gene ID number: A0090138000160) (as described in Nakadai et al., 1973. Agr. Biol. Chem 37(12), 2695-2701 , Nakadai et al., 1973. Agr. Biol. Chem 37(12), 2703-2708, and Kimura et al., 2008. Biosci. Biotechnol. Biochem. 72(2), 499-505), pep1 (Gene ID number: tre74156), tsp1 (Gene ID number: tre73897), slp1 (Gene ID number: tre51365), gap1 (Gene ID number: tre69555), gap2 (Gene ID number: tre106661), pep4 (Gene ID number: tre77579), pep5 (Gene ID number: tre81004), pep3 (Gene ID number: tre121133), sep1 (Gene ID number: tre124051), amp1 (Gene ID number: tre81070), pep8 (Gene ID number: tre122076), pep9 (Gene ID number: tre79807), pep11 (Gene ID number: tre121306), slp7 (Gene ID number: tre123865), amp2 (Gene ID number: tre108592) (as described in Landowski et al., 2015. PLOSone Aug 26;10(8):e0134723 and Landowski et al., 2015 Microb Cell Fact. Jun 10;15(1):104), Prolyl endopepidases from A. niger as described in (Edens et al., 2005. J Agric Food Chem. 53(20):7950-7957 and Kang et al., 2013 J Ind Microbiol Biotechnol 40:855-864), Prolyl endopepidases AoS28A, AoS28B, AoS28C and AoS28D (as described in Eugster, Philippe J., et al. Microbiology 161 .Pt_12 (2015): 2277-2288).

In yet a further aspect, the invention provides for a fungal host cell wherein the specific enzymatic activity of an Aspergillus proteases that is inhibited by at least one of pepstatin A, chymostatin, SBTI (soybean trypsin inhibitor), E-64, complete protease inhibitor, PMSF, EDTA or EGTA is reduced or eliminated.

The fungal host cell as described in the various embodiments of te invention preferably comprises an expression cassette comprising a nucleotide sequence coding for a (mammalian) casein.

Means and methods for constructing the expression vectors and cassettes of the present invention are well known to one skilled in the art (see, e.g., Sambrook and Russell, supra; and Ausubel et al., Current Protocols in Molecular Biology, Wiley InterScience, NY, 1995).

The vectors (comprising the expression cassettes of the invention) preferably contain one or more selectable markers, which permit easy selection of transformed cells. Using the method of cotransformation, one vector may contain the selectable marker whereas another vector may contain the polynucleotide of interest or the nucleic acid construct of interest; the vectors are simultaneously used for transformation of the host cell. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), b/eA (phleomycin binding), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents from other species. Preferred for use in an Aspergillus host cell are the amdS (US5876988, US6548285B1) and pyrG genes of A. nidulans or A. oryzae and the bar gene of Streptomyces hygroscopicus. More preferably an amdS gene is used, even more preferably an amdS gene from A. nidulans or A. niger. A most preferred selection marker gene is the A. nidulans amdS coding sequence fused to the A. nidulans gpdA promoter (see EP 0635574 B1). AmdS genes from other filamentous fungi may also be used (US 6548285 B1). As will be understood, when the pyrG selectable marker is used, the fungal host cell according to the invention is a mutant of a parental fungal cell comprising an endogenous pyrG gene encoding an orotidine-5'-phosphate decarboxylase and the endogenous pyrG gene is inactivated wherein the endogenous pyrG gene encodes an orotidine-5'-phosphate decarboxylase 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: 4.

Preferably the nucleotide sequence coding for a casein is operably linked to at least one regulatory sequence that effects or controls expression of the encoded casein by the fungal host cell. The expression regulatory sequence preferably at least includes a transcription regulatory sequence or promoter operably linked to the coding sequence. The expression cassette further preferably includes regulatory sequences such as translation initiation sequences, secretion signal sequences, transcription termination sequences, polyadenylation signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements. In one embodiment the regulatory sequences are the regulatory sequences of a highly expressed fungal protein. In one embodiment, the expression cassette comprises a nucleotide sequence encoding a signal sequence from a highly expressed secreted fungal protein, and optionally a pro-sequence, operably linked in frame to the nucleotide sequence coding for the casein or a (functional) part of the casein.

A "signal sequence" is an amino acid sequence which when operably linked to the aminoterminus of a protein of interest (i.c. a casein) permits the secretion of such protein from the host fungus. Such signal sequences may be the signal sequence normally associated with the protein of interest (i.e., a native signal sequence) or may be derived from other sources (i.e., a signal sequence foreign or heterologous to the protein of interest). Signal sequences are operably linked to a heterologous polypeptide either by utilizing a native signal sequence or by joining a DNA sequence encoding a foreign signal sequence to a DNA sequence encoding the protein of interest in the proper reading frame to permit translation of the signal sequence and protein of interest.

In one embodiment, the expression cassette comprises an N-terminal secretion signal sequence that is operably linked to the coding sequence of the casein for directing secretion of the casein from the fungal host cell.

In a further preferred embodiment, the expression cassette encodes casein as part of a fusion protein. Preferably, the expression cassette encodes a fusion protein wherein the casein is fused at its N-terminus with at least a part of highly expressed secreted fungal protein. More preferably, the expression cassette encodes a fusion protein wherein the casein is fused at its N-terminus with at least an N-terminal part of the highly expressed secreted fungal protein, which preferably includes at least the signal sequence of the highly expressed secreted fungal protein. The fusion protein can further also comprise the pro-sequence of the highly expressed secreted fungal protein and/or or further parts of the mature highly expressed secreted fungal protein. Alternatively, the signal sequence may contain the pre-pro-sequences. In one embodiment, the N-terminal part of the highly expressed secreted fungal protein can be fused to the N-terminus of the mature secreted casein. Alternatively, the N-terminal may be fused to the start of the casein (e.g. after the processing to avoid possible misprocessing in the fungal cell).

In a preferred embodiment, the expression cassette encodes a fusion protein comprising in a N- to C-terminal direction: a signal sequence from a highly expressed secreted fungal protein; optionally a pro-sequence from a highly expressed secreted fungal protein; optionally, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the amino acid sequence of the mature highly expressed secreted fungal protein; optionally, a non-cleavable linker polypeptide or a cleavable linker polypeptide; a casein or a (functional) part of the casein.

In one embodiment the highly expressed secreted fungal protein is selected from the group consisting of an amylase, glucoamylase or cellobiohydrolase, preferably selected from the group consisting of acid a-amylase, a-amylase, TAKA-amylase, amylase C, glucoamylase, xylanase, cellobiohydrolase,

Preferably, at least one copy of the expression cassette is integrated in the genome of the fungal host cell.

In one embodiment, the fungal host cell comprises multiple copies of the expression cassette, preferably integrated into the genome of the fungal host cell. Preferably, the fungal host cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25 or 30 copies of the expression cassette, preferably integrated into the genome of the fungal host cell, more preferably at a predefined location, such as a locus comprising a highly expressed endogenous fungal gene.

In one embodiment, the expression cassette is integrated by (homologous) recombination via a single cross-over.

In another embodiment, the expression cassette is integrated by (homologous) gene replacement in a locus of a gene coding for a highly expressed fungal protein (i.e. double crossover).

In certain embodiments, therefore, the fungal host cell according to the various embodiments of the invention is a fungal host cell that has been modified to favour homologous recombination over non-homologous recombination. Thus, in one embodiment, the fungal host cell according to the invention is a mutant of a parental fungal cell comprising an endogenous Iig4 gene encoding a DNA ligase IV and wherein in the fungal host cell the endogenous Iig4 gene is inactivated and wherein the endogenous Iig4 gene encodes a DNA ligase IV 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: 5.

In one embodiment, the casein that is encoded in the expression cassette of the invention comprises 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.

In one embodiment, the casein that is encoded in the expression cassette of the invention 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: 6, 16, 26 and 35.

In one embodiment, the casein that is encoded in the expression cassette of the invention is a beta-casein and 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 at least one of SEQ ID NOs 6-15

In one embodiment, the casein that is encoded in the expression cassette of the invention is a AlphaSI -casein and 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 at least one of SEQ ID NOs: 16-25.

In one embodiment, the casein that is encoded in the expression cassette of the invention is a AlphaS2-casein and 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 at least one of SEQ ID NOs: 26-34

In one embodiment, the casein that is encoded in the expression cassette of the invention is a Kappa-casein and 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 at least one of SEQ ID NOs: 35-44.

Table 1 : casein sequences.

In one embodiment, the nucleotide sequence encoding the protein of interest in the expression cassette of the invention preferably is adapted to optimize its codon usage to that of the 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.

In certain embodiments, the nucleotide sequence coding for the casein is codon optimized with reference to the native codon usage of highly expressed fungal proteins. More preferably, the nucleotide sequence encoding the casein in the expression cassette of the invention is adapted to optimize its codon usage to that of a highly expressed fungal protein selected from the group consisting of acid a-amylase, a-amylase, TAKA-amylase, amylase C, glucoamylase, xylanase, cellobiohydrolase, pyruvate kinase, pyruvate decarboxylase, glyceraldehyde-phosphate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, sucrase, acetamidase, chitinase, enolase, translation elongation factor 1 , hexagonal (HEX-1) protein of the fungal Woronin body and superoxide dismutase.

The promoter that is operably linked to the coding sequence in the expression cassette according to the invention can be a constitutive promoter, an inducible promoter or a hybrid promoter. Examples of preferred inducible promoters that can be used are a starch-, maltose, cellulose-, lactose-inducible promoters.

A preferred promoter is a promoter of a highly expressed fungal protein or a highly expressed synthetic promoter. Preferred promoters from highly expressed fungal genes include the promoters of acid a-amylase, a-amylase, TAKA-amylase, amylase C, glucoamylase, xylanase, cellobiohydrolase, pyruvate kinase, pyruvate decarboxylase, glyceraldehyde-phosphate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, sucrase, acetamidase, chitinase, enolase, translation elongation factor 1 , hexagonal (HEX-1) protein of the fungal Woronin body and superoxide dismutase, or the Trichoderma cDNA1 promotor. Another preferred promotor is the synthetic SES promoter. The synthetic SES promotor has been described in WO 2017/144777 which is incorporated by reference herein.

In a further aspect, the invention provides for a process for producing a casein, the process comprising culturing the fungal host cell as defined herein in a medium in a fermenter or bioreactor under conditions conducive to the expression of casein.

Media for growth of fungal host cells of the invention are generally known in the art. In a preferred embodiment, the medium for culturing a fungal host cell of the invention is a chemically defined medium. Typical composition of the chemically defined media for growth of (filamentous) fungi 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.

In certain embodiments, the fungal cell is cultured in a medium comprising glucose or maltose as a carbon source. In preferred embodiments, glucose or maltose is the sole carbon source.

Optionally, the process further comprises recovering the casein.

In one embodiment, the recovery is during fermentation. 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 fungal 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.

In yet a further aspect, the invention provides for a use of a protease inhibitor to inactivate the enzymatic activity of at least one (acid) aspartyl protease, at least one (alkaline) serine-type protease, at least one cysteine-type proteases and/or at least one metalloprotease in a fungal host cell as defined herein. In preferred embodiments, the protease inhibitor is selected from the group consisting of pepstatin A, chymostatin, SBTI (soybean trypsin inhibitor), E-64, complete protease inhibitor, PMSF, EDTA or EGTA.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Description of the figures

Figure 1 : Western blot analysis of cell-free cultivation broth (up to 6 days of cultivation in AoMM) of Ao0025 transformants with different AlphaSI -casein expression plasmids. Dashed arrow: aspecific detection of Amylase; grey arrow: position of full-size AlphaSI -casein; white arrow: casein degradation fragments; black solid arrow: products related to the secretion of the Amylase-casein fusion protein. P = parental Ao0025 strain; M = protein marker; R: Alpha-casein (S1 +S2) purified from milk. P120: pSES-AoAmyC-Kex2_AlphaS1-casein-glaTT-PyrG; P124: pSES-AoAmyC- AlphaS1-casein-glaTT-PyrG; P122: pAmyC-AoAmyC-Kex2_AlphaS1-casein-glaTT-PyrG; P126: pAmyC-AoAmyC-AlphaS1-casein-glaTT-PyrG.

Figure 2: Western blot analysis of cell-free cultivation broth (up to 4 or 5 days of shake flask cultivation in AoMM) of a single Ao0025 transformant with the P120 AlphaSI -casein expression plasmid (pSES + Kex2). A): cultivation in the absence/ presence of a protease inhibitor cocktail; B) and C): cultivation in the absence/ presence of individual protease inhibitors. Days of cultivation are indicated on top. Grey arrow: position of full-size AlphaSI-casein; white arrow: casein degradation fragments. M = protein marker; R/Ref: Alpha-casein (S1 +S2) purified from milk.

Figure 3: Western blot analysis of cell-free cultivation (up to 5 days of cultivation in AoMM) of Ao0025 transformed with different AlphaS2-casein expression plasmids (P121 , P123, P125). Clone identification and days of cultivation are indicated on top. The AlphaSI expression clone P120 (pSES + Kex2) 8 was co-cultivated as reference. Grey horizontal arrow: position of full-size AlphaSI-casein; white arrow: AlphaSI-casein degradation fragments; gray vertical arrow: full-size AlphaS2-casein, black solid arrow: products related to the secretion of the Amylase + AlphaS2- casein fusion protein; dashed arrow: aspecific detection of Amylase. R = Alpha-casein (S1 +S2) purified from milk; M = protein marker.

Examples

Example 1 : Construction of plasmids for the expression of caseins by filamentous fungi

Codon-optimized bovine casein gene sequences for expression in Aspergillus (oryzae) were generated synthetically and cloned into suitable expression plasmids under the transcriptional control of an inducible AmyC or GlaA promotor or a constitutive synthetic SES promotor (Rantasalo et al., 2018). The casein sequences were integrated in the expression constructs via in-frame cloning with a well-secreted N-terminal fusion partner, such as the amylase C or gluco-amylase core protein. To evaluate the effect of the N-terminal fusion partner on the casein stability after secretion of the protein into the cultivation broth, casein expression constructs were generated with or without a Kex2 proteolytic processing site in between the C-terminal casein and the N-terminal fusion partner. Use of the Kex2 site facilitates cleavage between the C-terminal casein and the N- terminal fusion partners shortly before secretion. The prepared vectors contain either an amyC 5’ and amyC 3’ untranslated region (UTR) or the P amyC and amyC 3’ UTR to promote double homologous recombination into the AmyC (and equally the AmyB) locus. The amyC 3’ flank loop- out region in front of the PyrG marker allows for easy homologous out-recombination (with part of the 3’ UTR) of the selection marker under non-selective growth conditions (in combination with screening on FOA plates). An overview of the transformed casein expression plasmids, with corresponding reference numbers, is shown in table 2.

Table 2: AlphaSI- and AlphaS2-casein expression constructs and their corresponding reference numbers.

Example 2: Expression of AlphaSI -casein by Aspergillus oryzae

The A. oryzae strain Ao0025 (AprtT Alig4 AoryA(Alp1) ApyrG) was transformed with plasmids for the expression of alphaSI -casein under the control of either the synthetic pSES or the inducible pAmyC promotor. The coding sequence for alphaSI -casein was fused in frame to that of the amylaseC N-terminal fusion partner, in the presence or absence of an intermediate linker containing the Kex2 recognition site. Transformants were selected based on their ability to grown on medium without supplemented uracil/uridine. Several clones scoring positive upon an initial alphaSI -casein specific PCR screening were cultivated in 24 deep-well plates at 28 °C in 2 ml of AoMM (comprising per liter: 6 g NaNOs; 1 .5 g KH2PO4; 0.5 g KOI; 0.4 g/L MgSO4; 2% glucose or maltose; 1 ml 1000x Hutner’s trace elements having a pH of 6.0) with glucose as carbon source. Transformants where the casein expression is under the control of the AmyC promotor were supplemented with maltose as well. At several time points during cultivation, samples were taken for protein analysis. By the end of the cultivation, the pH of the medium had raised from 6 towards 8 - 8.5 for most of the selected clones.

A multi-day western blot analysis was performed on the parental strain and of transformants of P120 (pSES-driven expression of fusion with Kex2 site - clone 10), of P124 (pSES-driven expression of fusion without Kex2 site - clones 11 and 13) and of P126 (pAmyC-driven expression of fusion without Kex2 site - clones 16 and 18). Cell-free cultivation samples were denatured and loaded for a reducing SDS-PAGE analysis, followed by western blotting and immunodetection using a rabbit polyclonal antibody against bovine caseins (Abeam, #ab166596) (Figure 1).

The results show the presence of full-size AlphaSI -casein and casein-specific degradation products at all the analysed time points for all transformants. Likewise, all potential AlphaSI -casein specific protein bands for the P124 transformants, previously identified via Coomassie staining, were confirmed via immunostaining (solid black, grey and white arrows). The most intense bands for the P124 transformants were also observed for the selected P126 transformants, but at weaker intensity.

Example 3: Effect of protease inhibitors on the expression of caseins

Next P120 transformants (results shown for clone 10) were cultivated at 28°C in small shake flasks (250 ml volume) using 20 ml of AoMM (+ 2% glucose). Cultivation was performed for up to 4 or 5 days and the expression of the amylase was monitored via SDS-PAGE, followed by Coomassie staining (not shown). In contrast to the 24 deep-well cultivations, the glucose content in the shake flask cultures was measured daily and where needed, a shot of concentrated glucose was added to approximate again a 2% final glucose concentration. The pH was measured daily and shown to raise gradually from 6 towards 7.5 - 8. After 1 day of cultivation, 5 ml extra AoMM medium was added with or without a mixture of protease inhibitors (Figure 2A). The final concentration of the protease inhibitors was: 4 pg/ml pepstatin A, 2.5 pM E-64, 25 pM chymostatin and complete protease inhibitor without EDTA (Ix final concentration, according to the recommendations of the manufacturer (Roche)). The choice of the inhibitors was an attempt to inhibit a whole spectrum of protease families, including acid aspartyl proteases as well as serine- and cysteine-type proteases. Western bot analysis on cultivation samples taken each day, followed by the anti-casein immunodetection, indicates that the mixture of protease inhibitors is partially stabilizing the full-size AlphaSI-casein. In the absence of the inhibitor cocktail a degradation fragment of around 20 kDa is observed at all analyzed time points.

The above experiment was repeated, but this time with the addition of only one of the protease inhibitors per shake flask (Figure 2B-C). The cultivation was run over 5 days and samples taken at day 3, 4 and 5 were analyzed. Full-size product was only detected at day 3 (and slightly at day 4) in the presence of pepstatin A, indicating the involvement of (acid) aspartyl proteases in the degradation of AlphaSI-casein. The 3 other individual protease inhibitors only resulted in the presence of the AlphaSI-casein degradation products (largest one running around 20 kDa), like the ones observed when no protease inhibitor was added at all. The presence of chymostatin seems to result into a longer stabilization of the observed degradation products, which implies that Serine- type proteases are also involved in the degradation of AlphaSI -casein. The differential degradation pattern at day 3 of the pepstatin cultivation and the almost complete lack of casein-related signals at days 4 and 5 may indicate that at a certain stage during cultivation, the presence of pepstatin A is triggering a change within the extracellular protease profile (either by type or by ratio, or a combination of both), resulting into a fast degradation of the full-size product into smaller fragments. This appears not to be the case for the other protease inhibitors.

Example 4: Expression of AlphaS2-casein by Aspergillus oryzae

Similar to what has been described above, A. oryzae strain Ao0025 (AprtT Alig4 AoryA(Alp1) ApyrG) was transformed with plasmids for the expression of alphaS2-casein under the control of either the synthetic pSES or the inducible pAmyC promotor. The 5-day cultivation broth of P120 (pSES + AmyC-Kex2-alphaS1 ; cultivated as reference), P121 (pSES + AmyC-Kex2-alphaS2), P123 (pAmyC + AmyC-Kex2-alphaS2) and P125 (pSES + AmyC-alphaS2) were evaluated via western blot, followed by immunodetection with a rabbit polyclonal casein-specific antibody (Abeam, #ab166596) (Figure 3). The immunostaining confirmed the presence of the full length AlphaS2- casein protein and fragments thereof within the cultivation broth of the transformants.