EP4623075A1 - Sécrétion de caséine améliorée par des cellules hôtes non mammifères - Google Patents

Sécrétion de caséine améliorée par des cellules hôtes non mammifères

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Publication number
EP4623075A1
EP4623075A1 EP23812888.8A EP23812888A EP4623075A1 EP 4623075 A1 EP4623075 A1 EP 4623075A1 EP 23812888 A EP23812888 A EP 23812888A EP 4623075 A1 EP4623075 A1 EP 4623075A1
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EP
European Patent Office
Prior art keywords
casein
expression
kinase
host cell
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23812888.8A
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German (de)
English (en)
Inventor
Steven Christian Jozef Geysens
Guillaume Marc Sébastien LÉRONDEL
Robin Marcel Michael VANLUCHENE
Jeroen Joris Donald VANHECKE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Newmilkbuzz BV
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Newmilkbuzz BV
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Publication date
Application filed by Newmilkbuzz BV filed Critical Newmilkbuzz BV
Publication of EP4623075A1 publication Critical patent/EP4623075A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4732—Casein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01109—Dolichyl-phosphate-mannose-protein mannosyltransferase (2.4.1.109)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03002—Acid phosphatase (3.1.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21048—Cerevisin (3.4.21.48)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23006—Microbial carboxyl proteinases (3.4.23.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23041—Yapsin 1 (3.4.23.41)

Definitions

  • the present invention relates to the field of molecular microbiology, food technology and fermentation technology.
  • the invention relates to a non-mammalian host cell expressing a mammalian casein, wherein the expression of the casein is improved by coexpression of a kinase.
  • dairy production has an enormous impact on the environment.
  • 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.
  • greenhouse gas including methane, which is a much more harmful greenhouse gas than CO2
  • Water demand is very high as dairy operations consume large volumes of water to grow feed, water cows, manage manure and process products.
  • nitrogen emissions from e.g., manure and fertilizer
  • 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.
  • 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.
  • fungi e.g., yeast and filamentous fungi
  • 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).
  • nonmammalian host cells e.g., fungal host cells
  • a mammalian casein e.g., a mammalian casein
  • the expression of the casein is quantitatively and/or qualitatively improved, to obviate the farm animals to produce these proteins.
  • the invention in a first aspect, relates to a non-mammalian host cell comprising i) an expression construct comprising a nucleotide sequence encoding at least one casein and ii) an expression construct comprising a nucleotide sequence encoding at least one kinase.
  • the at least one kinase is a kinase that is capable of phosphorylating the at least one casein.
  • a host cell wherein co-expression of the at least one casein and the at least one kinase in the host cell, causes the casein to be phosphorylated, wherein co-expression of the at least one casein and the at least one kinase in the host cell increases the degree of phosphorylation of the at least one casein by at least 10%, as compared to the at least one casein produced in a corresponding host cell lacking co-expression of the at least one kinase, wherein preferably, the degree of phosphorylation of the at least one casein, is at least 1 % of the degree of phosphorylation of the same casein produced and post-translationally modified in a mammalian cell, more preferably as determined by mass spectrometry.
  • a host cell wherein the nucleotide sequence encodes at least one 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, dromedaris), Rangifer tarandus (reindeer), Equus caballus (horse), Sus spp.
  • 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 s
  • the nucleotide sequence encodes at least one 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 at least one of SEQ ID NO’s: 5 - 43, of which the bovine SEQ ID NO’s: 5, 15, 25 and 34 are most preferred.
  • a host cell wherein 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.
  • the host cell is a cell 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.
  • a host cell wherein the cell comprises at least one genetic modification selected from the group consisting of: a) the genetic modification reduces or eliminates the expression or activity of at least one of: i) a transcriptional activator regulating the expression of an array of protease genes, preferably, a transcriptional activator encoded by a prfT gene, or an orthologue thereof; ii) a vacuolar acid aspartyl protease, preferably a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof; iii) a cell-wall associated aspartic acid protease, preferably a cell-wall associated aspartic acid protease of the yapsin family, more preferably, encoded by a YPS1 gene and/or by a YPS’ gene or orthologues thereof; iv) a vacuolar serine-type protease, preferably a vacuolar serine-type protease, preferably
  • reduction of O-linked glycosylation is achieved by co-expression of a alpha mannosidase such as an alpha-1 ,2-mannosidase.
  • the host cell is a cell, wherein at least one of the genetic modification reduces or eliminates the expression or activity of at least one of: i) a vacuolar acid aspartyl protease, preferably a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof; and ii) at least one cell-wall associated aspartic acid protease, preferably a cell-wall associated aspartic acid protease of the yapsin family, more preferably, encoded by a YPS1 gene or YPS’ gene or orthologues thereof.
  • a vacuolar acid aspartyl protease preferably a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof
  • at least one cell-wall associated aspartic acid protease preferably a cell-wall associated aspartic acid protease of the yapsin family, more preferably, encoded by a YPS1
  • the host cell comprises a genetic modification that reduces or eliminates the expression or activity of i) a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof; ii) a cell-wall associated aspartic acid protease encoded by a YPS1 gene and iii) a cell-wall associated aspartic acid protease encoded by a YPS’ gene.
  • a host cell wherein the cell comprises at least one genetic modification selected from the group consisting of: a) a genetic modification that reduces or eliminates the expression or activity of i) a vacuolar acid aspartyl protease encoded by a PEP4 gene, or an orthologue thereof; ii) a cell-wall associated aspartic acid protease encoded by a YPS1 gene, and iii) a cell-wall associated aspartic acid protease encoded by a YPS’ gene; b) a genetic modification that reduces the phosphatase activity of the cell, preferably the genetic modification reduces the extracellular phosphatase activity of the cell; and, c) a genetic modification that reduces O-linked glycosylation of proteins produced by the cell.
  • the invention in a second aspect, pertains to a process for producing a casein, the method comprising culturing a host cell as described in the first aspect, 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.
  • the invention relates to a casein having a non-native glycosylation pattern, wherein the casein is phosphorylated, wherein preferably the casein has a higher degree of phosphorylation as compared to a corresponding casein produced in a non-mammalian host cell that lacks expression of a heterologous kinase capable of phosphorylating the casein, wherein preferably, the degree of phosphorylation is determined by mass spectrometry.
  • the casein is a casein wherein the degree of phosphorylation of the casein is at least 1 % of the degree of phosphorylation of the same casein produced and post-translationally modified in a mammalian cell, preferably a mammary gland cell, more preferably as obtained from natural milk, wherein preferably, the degree of phosphorylation is determined by mass spectrometry.
  • the casein in this aspect is a casein that is obtained or obtainable in a process of the second aspect.
  • the invention pertains to a composition
  • a composition comprising a casein according to the third aspect, 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.
  • 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.
  • At least a particular value means that particular value or more.
  • 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 preferably means that the value may be the given value (of 10) more or less 10% of the value.
  • 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.
  • 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).
  • a global alignment algorithm e.g., Needleman Wunsch
  • 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.
  • 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).
  • 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
  • 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.
  • search can be performed using the BLASTn and BLASTP programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402.
  • the default parameters of the respective programs e.g., BLASTx and BLASTn
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the invention relates to a non-mammalian host cell comprising i) an expression construct comprising a nucleotide sequence encoding at least one casein; and ii) an expression construct comprising a nucleotide sequence encoding at least one kinase.
  • a host as described herein is a non-mammalian host cell comprising i) an expression construct comprising a nucleotide sequence encoding at least one casein and ii) an expression construct comprising a nucleotide sequence encoding at least one kinase, wherein co-expression of the at least one kinase and the at least one casein in the host cell causes the at least one casein to be phosphorylated.
  • the degree of phosphorylation of the at least one casein is at least 1 , 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the degree of phosphorylation of the same casein produced and post-translationally modified in a mammalian cell, preferably a mammary gland cell, more preferably as obtained from natural milk.
  • the degree of phosphorylation can be determined in a method for detecting the level of phosphorylation of caseins as described above, e.g., by mass spectrometry.
  • Co-expression of the at least one casein and the at least one kinase in the host is understood as that the at least one casein and the at least one kinase co-exist and can be into contact with each other for a sufficient amount of time to allow phosphorylation of the at least one casein to occur.
  • the at least one casein and the at least one kinase are co-expressed in the same subcellular compartment, e.g., the secretory pathway, including the Golgi apparatus.
  • the at least one casein and the at least one kinase are both directed into the secretory pathway of the host cell, preferably, a non-mammalian eukaryotic host cell.
  • a host as described herein is a non-mammalian host cell wherein the nucleotide sequence encoding the at least one casein encodes a signal sequence operably linked to the at least one casein and the nucleotide sequence encoding the at least one kinase encodes a signal sequence operably linked to the at least one kinase, and wherein co-expression of the at least one kinase and the at least one casein in the host cell increases the extracellular level of the at least one casein, as compared to the extracellular level of at least one casein produced in a corresponding host cell lacking co-expression of the at least one kinase.
  • the host cell is a non-mammalian eukaryotic host cell.
  • Such methods usually include separating the host cells from the spent culture medium and quantifying the amounts of extracellular and/or intracellular caseins, in respectively the culture medium or cells, by known means, e.g., immunological assays such as ELISAs or Western blots using antibodies that are specific for the one or more caseins (see e.g. the methods used in the Examples herein).
  • immunological assays such as ELISAs or Western blots using antibodies that are specific for the one or more caseins (see e.g. the methods used in the Examples herein).
  • the (extracellular) caseins can first be purified and quantified by standard methods for protein quantification.
  • a host as described herein is a non-mammalian host cell wherein the nucleotide sequence encoding the at least one casein encodes a signal sequence operably linked to the at least one casein and the nucleotide sequence encoding the at least one kinase encodes a signal sequence operably linked to the at least one kinase, and wherein coexpression of the at least one kinase and the at least one casein in the host cell decrease the intracellular level of the at least one casein, as compared to the intracellular level of at least one casein produced in a corresponding host cell lacking co-expression of the at least one kinase.
  • the decrease of intracellular casein level coincides with and/or corresponds to the increases in extracellular casein level.
  • the host cell is a non-mammalian eukaryotic host cell.
  • the intracellular level of the at least one casein is decreased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100%.
  • the kinase is a protein kinase, preferably the kinase is a serine and/or threonine kinase (EC 2.7.11 .1), more preferably the kinase is at least a serine kinase.
  • the kinase is a mammalian kinase.
  • the kinase can e.g., be a human kinase or a ruminant kinase, e.g., a kinase from a ruminant selected from Bos taurus, Bos grunniens, Bubalus bubalis, Capra hircus, and Ovis aries.
  • the kinase is mammary gland casein kinase, such as e.g., a Fam20C kinase.
  • Fam20C is a Golgi localized protein kinase, a serine kinase that phosphorylates both casein and other highly acidic proteins at the target motif SerXGlu or SerXphosphoSer.
  • Fam20C also known as DMP4, is a protein which in humans is encoded by the FAM20C gene.
  • the nucleotide sequence encoding the at least one kinase encodes 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 at least one of SEQ ID NO.’s: 1 and 3 and having casein kinase activity.
  • the nucleotide sequence encoding the at least one kinase encodes 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 at least one of SEQ ID NO.’s: 2 and 4 and having at least one of casein kinase activity and casein kinase stimulating activity.
  • a host as described herein thus comprises an expression construct comprising a nucleotide sequence encoding at least one casein.
  • 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.
  • caseins include beta-casein, kappa-casein, alpha-S1 -casein, and alpha-S2-casein.
  • alpha-S1 -casein refers to not only the alpha-S1 -casein protein, but also fragments or variants thereof.
  • Alpha-S1 -casein is found in the milk of numerous different mammalian species, including cow, yak, camel, dromedary, horse, water buffalo, goat, and sheep.
  • alpha-S2-casein refers to not only the alpha-S2-casein protein, but also fragments or variants thereof.
  • Alpha-S2-casein is known as epsilon-casein in mouse, gamma-casein in rat, and casein-A in guinea pig.
  • beta-casein refers to not only the beta-casein protein, but also fragments or variants thereof.
  • 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.
  • 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.
  • casein proteins from different mammals are provided in the sequence listing herewith (see Table 5). Additional sequences for other caseins are known in the art.
  • 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.
  • 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.
  • 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 5 - 14.
  • 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: 15 - 24.
  • 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: 25 - 33.
  • 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: 34 - 43.
  • 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.
  • the host cell comprises expression constructs for expression in the host cell of a beta-casein and an alphaSI -casein.
  • the host cell comprises expression constructs for expression in the host cell of a beta-casein and an alphaS2-casein.
  • the host cell comprises expression constructs for expression in the host cell of a beta-casein and a kappacasein.
  • the host cell comprises expression constructs for expression in the host cell of an alphaSI -casein and an alphaS2-casein.
  • 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.
  • 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.
  • 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.
  • 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.
  • the bacterial host cell is a food-grade bacterium.
  • 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.
  • the non-mammalian host cell is a eukaryotic microbial cell, such as a yeast cell or a filamentous fungal host cell.
  • yeast host cells examples include 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.
  • 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., Fus
  • 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, preferably in a host cell that co-expresses at least one kinase as described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the signal sequence that is operably linked to the at least one kinase is a mammalian signal sequence. In one embodiment, the signal sequence that is operably linked to the at least one kinase is a signal sequence from a mammary gland protein. In one embodiment, the signal sequence that is operably linked to the at least one kinase is a signal sequence from a kinase. In one embodiment, the signal sequence that is operably linked to the at least one kinase is a signal sequence that is native to the at least one kinase to which it is operably linked.
  • the signal sequence that is operably linked to the at least one kinase is a signal sequence that is heterologous to the at least one kinase. In one embodiment, the signal sequence that is operably linked to the at least one kinase 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 kinase 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.
  • the signal sequence can be derived from a K. phaffii protein such as the signal sequence of the K. phaffii Ost1 and Cwp1 proteins.
  • the signal sequence sequence that is operably linked to the at least one kinase is operably linked to the kinase through a pro-sequence.
  • the pro-sequence preferably is a pro-sequence 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, for release of the pro-sequence from the at least one kinase.
  • Suitable signal sequences and combinations of signal- and pro-sequences are provide in Table 1 and in the Examples herein.
  • operable linkage of signal- and prepro-sequences to a casein or kinase as described herein preferably means that the signal- and prepro-sequence is linked to the N- terminus of the mature casein or kinase.
  • a host cell as described herein comprises a genetic modification that reduces proteolytic activity of the cell, preferably the genetic modification reduces extracellular and/or vacuolar proteolytic activity of the cell.
  • the genetic modification reduces overall proteolytic activity of the cell, preferably the genetic modification reduces overall extracellular and/or vacuolar proteolytic activity of the cell.
  • the genetic modification reduces at least 30, 40, 50, 60, 70, 80, 90, 95, 100% of the overall proteolytic activity, preferably of its overall extracellular and/or vacuolar proteolytic activity.
  • the reduction of proteolytic activity of the cell preferably is a reduction of proteolytic activity towards the at least one casein expressed in the host cell.
  • the overall proteolytic activity against caseins within the cultivation broth of micro-organisms can be determined with substrates such as azocasein.
  • 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.
  • the host cell as described herein comprises a genetic modification that reduces at least 30, 40, 50, 60, 70, 80, 90, 95, 100% of the overall endoproteolytic activity of the cell, preferably of the overall extracellular and/or vacuolar endoproteolytic activity. Endoproteolytic activity can be measured by use of for example the azocasein assay which has been described in the art.
  • the genetic modification comprised in the host cell that reduces proteolytic activity as described above is a genetic modification that reduces or eliminates the expression or activity of an (endogenous) transcriptional activator gene regulating the expression of an array of protease genes.
  • the transcriptional activator regulating the expression of an array of protease genes, the activity or expression of which is to be reduced or eliminated in the host cell is encoded by a prfT gene, or an orthologue thereof. 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.
  • 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 by a fungal cell, wherein the polypeptide is sensitive for protease degradation. When the prfT gene is partially or completely inactivated, the host cell will produce less or no proteases that are under transcriptional control of prfT.
  • the prtT gene encodes a transcriptional activator 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’s: 44 or 45.
  • the genetic modification comprised in the host cell that reduces proteolytic activity as described above is a genetic modification that reduces or eliminates the expression or activity of a vacuolar acid aspartyl protease.
  • 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.
  • 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: 46.
  • the genetic modification comprised in the host cell that reduces proteolytic activity as described above is a genetic modification that reduces or eliminates the expression or activity of at least one aspartic-type endopeptidase, preferably at least one aspartic- -type endopeptidase of the yapsin family.
  • the genetic modification comprised in the host cell that reduces proteolytic activity as described above is a genetic modification that reduces or eliminates the expression or activity of at least one vacuolar serine-type protease.
  • the vacuolar serine-type protease, the activity or expression of which is to be reduced or eliminated in the host cell is a vacuolar serine-type protease encoded by a PRB1 gene, or an orthologue thereof (see e.g. Gleeson et al., 1998, Methods Mol Biol.; 103:81-94).
  • the PRB1 gene encodes a vacuolar serine-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: 87.
  • the genetic modification comprised in the host cell that reduces proteolytic activity as described above is a genetic modification that reduces or eliminates the expression or activity of at least one secreted subtilisin-type protease.
  • the secreted subtilisin-type protease, the activity or expression of which is to be reduced or eliminated in the host cell is a secreted subtilisin-type protease encoded by a SUB2 gene, or an orthologue thereof (see e.g. Salamin et al., 2010, Appl Environ Microbiol.; 76(13): 4269-4276).
  • the SUB2 gene encodes a secreted subtilisin-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: 88.
  • the genetic modification comprised in the host cell that reduces proteolytic activity as described above is a genetic modification that reduces or eliminates the expression or activity of at least one subtilisin-like Ser-type proteases.
  • the serine-type protease, the activity or expression of which is to be reduced or eliminated in the host cell is a subtilisin-like Ser-type proteases encoded by a SBT100 gene, or an orthologue thereof (see e.g. Mudassar, et al., Yeast 36.9 (2019): 557-570 - labelled kpx10 in this document).
  • the PHO1 gene encodes an extracellular phosphatase 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.
  • a host cell as described herein comprises a genetic modification that reduces the level of O-linked glycosylation of proteins produced by the cell.
  • the genetic modification comprised in the host cell that reduces O-linked protein glycosylation is a genetic modification that reduces or eliminates the expression or activity of at least one proteinO- mannosyl transferase in the cell.
  • the protein-O-mannosyl transferase is preferably a dolichyl- phosphate-mannose-protein mannosyltransferase (EC 2.4.1.109).
  • 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: 91.
  • the host cell as described herein further comprises an expression construct comprising a nucleotide sequence encoding at least one alpha-1 ,2-mannosidase.
  • 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: 92.
  • the alpha-mannosidase may be separately produced and added to the cell culture.
  • the vector into which the expression cassette or polynucleotide of the invention 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.
  • a vector according to the invention may 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").
  • 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).
  • ARS autonomously replicating sequence
  • expression vectors are referred to herein as “expression vectors”.
  • 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.
  • the invention 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.
  • Vectors according to the invention may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • a vector of the invention may comprise two or more, for example three, four or five, polynucleotides of the invention, for example for overexpression.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • a regulatory sequence such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences or the sequences are arranged so that they function in concert for their intended purpose, for example transcription initiates at a promoter and proceeds through the DNA sequence encoding the polypeptide.
  • Enhanced expression of the polynucleotide of the invention may 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 a polypeptide of the invention.
  • heterologous regulatory regions e. g. promoter, secretion leader and/or terminator regions
  • control sequences or “regulatory sequences” is defined herein to include at least any component which may be necessary and/or advantageous for the expression of a polypeptide.
  • Any control sequence may be native or foreign to the nucleic acid sequence of the invention encoding a polypeptide.
  • control sequences may include, but are not limited to, a promoter, a leader, optimal translation initiation sequences (as described in Kozak, 1991 , J. Biol. Chem. 266:19867-19870) or the prokaryotic Shine-Dalgarno sequences, a secretion signal sequence, a pro-peptide sequence, a polyadenylation sequence, a transcription terminator.
  • the control sequences typically include a promoter and translational initiation and stop signals.
  • 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.
  • 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.
  • 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.
  • 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.
  • an inducible promotor preferably a methanol induced promotor such as for example an AOX1 promotor.
  • polypeptides of the invention Regardless of the exact mechanism utilized for expression of polypeptides of the invention, 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).
  • the recombinant DNA utilized for transforming the host cell in accordance with this invention 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.
  • expression control sequences including promoters, repressors and enhancers, that act to control expression or derepression of coding sequences for proteins, apoproteins or antisense RNA.
  • 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
  • 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.
  • 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.
  • Means for introducing genetic elements into a host cell are well known to the skilled artisan.
  • 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.
  • infection e.g., packaging in phage lambda
  • other mechanism for plasmid transfer e.g., electroporation, microinjection, etc.
  • Plasmids suitable for insertion of genetic elements into the host cell are well known to the skilled artisan.
  • genes 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.
  • the nucleotide sequence encoding the casein or kinase in the expression construct of the invention preferably is adapted to optimize its codon usage to that of the nonmammalian 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).
  • CAI codon adaptation index
  • 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 process further comprises recovering the casein.
  • the recovery is during fermentation. This may be carried out in a continuous mode to increase productivity.
  • the recovery is post fermentation.
  • 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.
  • other recovery methods are suitable, such as e.g., acid or salt precipitation and solvent extraction, as known in the art.
  • the casein has 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 mature amino acid sequence of at least one of SEQ ID NO’s: 5 - 43. In one embodiment, the casein has an amino acid sequence that is comprised in 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: 5, 15, 25 and 34.
  • the casein has 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 mature amino acid sequence of at least one of SEQ ID NO’s: 5, 15, 25 and 34.
  • non-native glycosylation pattern means that the protein is unglycosylated, or that the protein has a “non-mammalian glycosylation pattern”, which means one of a difference in one or more location(s) of glycosylation in a protein, and/or a difference in the amount of and/or type of glycosylation at one or more location(s) in a casein produced and post- translationally modified in a non-mammalian host cell as compared to a corresponding reference casein, i.e. the same casein produced and post-translationally modified in a mammalian cell, preferably a mammary gland cell, more preferably as obtained from natural milk.
  • the degree of phosphorylation of the casein having a non-native glycosylation pattern is higher than the degree of phosphorylation of a corresponding casein produced in a non-mammalian host cell that lacks expression of a kinase capable of phosphorylating the casein, preferably a non-mammalian host cell that lacks expression of a heterologous kinase capable of phosphorylating the casein, such as e.g. mammary gland kinases, kinases from the FAM20 family of kinases or Fam20C kinase and Fam20A kinases.
  • a “heterologous kinase” is herein understood as a kinase that is heterologous or foreign to the non-mammalian host cell.
  • the degree of phosphorylation of the casein having a non-native glycosylation pattern is higher than the degree of phosphorylation of a corresponding casein produced in a K. phaffii host cell that lacks expression of a heterologous kinase capable of phosphorylating the casein, such as e.g., mammary gland kinases, kinases from the FAM20 family of kinases or Fam20C kinase and Fam20A kinases.
  • the degree of phosphorylation can be determined in a method for detecting the level of phosphorylation of caseins as described above, e.g., by mass spectrometry.
  • the degree of phosphorylation of the casein having a non-native glycosylation pattern is at least 1 , 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the degree of phosphorylation of the same casein produced and post-translationally modified in a mammalian cell, preferably a mammary gland cell, more preferably as obtained from natural milk.
  • the degree of phosphorylation can be determined in a method for detecting the level of phosphorylation of caseins as described above, e.g., by mass spectrometry.
  • the casein having a non-native glycosylation pattern is a casein that is obtained or obtainable by a process for producing a casein as herein described.
  • the invention relates to compositions comprising a casein having a non- native glycosylation pattern and that is phosphorylated, as described herein, or a casein that is obtained or obtainable by a process for producing a casein as herein described.
  • the composition is a dairy substitute product comprising at least one casein having a non-native glycosylation pattern and that is phosphorylated, as described herein, or a casein that is obtained or obtainable by a process for producing a casein as herein described.
  • 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.
  • the composition is a composition as described above, wherein at least one casein selected from the group consisting of alpha-S1 casein, alpha-S2 casein, beta casein and kappa casein is not present.
  • 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.
  • the composition is a composition as described above, wherein at least two caseins selected from the group consisting of alpha-S1 casein, alpha- 82 casein, beta casein and kappa casein are not present.
  • the composition is a composition as described above, wherein at least three caseins selected from the group consisting of alpha-S1 casein, alpha-S2 casein, beta casein and kappa casein are not present.
  • 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.
  • the invention pertains to a method for producing a composition
  • a method for producing a composition comprising a casein having a non-native glycosylation pattern and that is phosphorylated, as described herein, or 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.
  • a highly desirable advantage of the present invention is that co-expression of the kinase with the casein in a non-mammalian host cell not only increases the degree of phosphorylation of the casein but surprisingly also drastically improves secretion of the phosphorylated casein for the host cell, thus facilitating its recovery.
  • Further advantages of the invention are environmental in nature such as 8 times more energy efficient, 260 times more water efficient than conventional milk product.
  • Other environmental advantages include less water usage than conventional milk production, which is estimated to be about 1000 L/L, reduced land usage compared to conventional milk production which typically requires grazing and crop land, and ability to reduce the 600 billion kg of carbon dioxide per year that is emitted from conventional milk production.
  • the present invention also provides reduction or elimination of costs of feed, operations, labour, animals and marketing. Preferably, substantially reduce feed cost by a factor of 8.
  • kits for food safety include reduction or removal of antibiotic residues, heavy metals, bacteria, adulterations.
  • certain aspects of the present invention provide animal-free dairy substitute products that are bacteria-free, require no pasteurization or cold shipping yet have an increased shelf-life and exhibit a number of characteristics such as taste, appearance, handling and mouth feel properties which are identical or at least closely similar to their traditional dairy counterparts.
  • the dairy substitute products are essentially free of bacteria such as Brucella, Campylobacter, Listeria, Mycobacterium, Salmonella, Shigella, Yersinia, Giardia and noro viruses, and thus are safer for consumption.
  • Further advantages include minimal or no pasteurization and/or homogenization.
  • the dairy substitute is shelf stable for relatively long periods (e.g., at least three weeks and preferably longer) for production and distribution. Even more preferably, the dairy substitute product has a lower environmental impact.
  • Figure 2 Immunodetection (western blot) of intracellularly accumulated His-tagged bovine Betacasein variant A2, upon expression by K. phaffi using a collection of yeast-specific signal peptides.
  • the identity of the different signal peptides (indicated by the numbers on top of the blot) is represented in Table 1 .
  • P20 intracellular analysis on strain Pp0020 (multicopy Beta-casein strain; pAOX1 ; beta-casein signal peptide); 115: K. phaffi GS115 strain; Bo: reference beta-casein from bovine milk.
  • the applied CRISPR KO strategy was based on the Cas12a system, first described for mammalian cells and adapted in-house for use in K. phaffi.
  • the human codon-optimized Cas12a coding sequence from Lachnospiraceae bacterium (hLbCas12a), the nuclear localization signal from SV40 (NLS-2xSV40) as well as a guideRNA (gRNA) cloning site with GFP dropout cassette (pGAP-gRNA-FBP1tt) were generated via gene synthesis (gBIock - IDT) or oligonucleotide annealing and each subcloned via BsmBI assembly into the pPTK081_0_Empty entry vector of the OPENPichia system.
  • an ‘empty’ destination CRISPR/Cas12a expression vector was constructed through Bsal-mediated Golden Gate Assembly of the required entry vectors belonging to or compatible with the OPENPichia toolkit (VIB/GeneCorner).
  • the resulting plasmid expresses the Cas12a with in-frame C-terminal nuclear localization signal under the transcriptional control of the constitutive pGPM1 promotor and AOX1 transcription terminator and further contains the empty expression cassette (GFP dropout) for the required gRNA sequences as well as the hygromycin resistance marker (HygR) and an autonomous replicating sequence (ARS) for transient propagation of the plasmid within the K. phaffi cells.
  • GFP dropout for the required gRNA sequences
  • HygR hygromycin resistance marker
  • ARS autonomous replicating sequence
  • the gRNA (or an array of gRNAs) for the genes of interest were designed as annealed oligonucleotides according to Zetsche et al. (2017, Nature Biotechnology 35(1): 31- 34) and subsequently cloned into the destination vector through Bbsl-mediated Golden Gate Assembly. Due to the corresponding loss of the GFP dropout cassette, a green-white screen allowed for easy identification of correctly assembled clones.
  • the CRISPR construct to knock-out K. phaffi PMT1 was generated by introducing into the
  • ‘empty’ CRISPR/Cas12a destination vector an array forthe simultaneous expression of a first gRNA (5’-TGGCACCTCTTGAGGACTTG-3’ SEQ ID NO: 68) and a second gRNA sequence (5’- ACTGTAACACCTTCCCCAGT-3’ SEQ ID NO: 69) targeting resp. a 5’ and 3’ sequence of the PMT1 open reading frame.
  • the plasmid was initially transformed towards strain Pp0020, the high-copy expression strain for His-tagged bovine beta-casein variant A2. Transformants were selected on YPD plates containing zeocin (100 pg/mL) and hygromycin (200 pg/mL).
  • phaffi PEP4 was generated by introducing into the ‘empty’ CRISPR/Cas12a destination vector an array for the simultaneous expression of a first gRNA (5’-GCTTCAGCACCAATACCTAG-3’ SEQ ID NO: 70) and a second gRNA sequence (5’-GACCTAGGCAAAGATGCAG-3’ SEQ ID NO: 71) targeting resp. a 5’ and 3’ sequence of the PEP4 open reading frame.
  • the plasmid was transformed towards strain Pp0070, co-expressing the His-tagged bovine beta-casein variant A2 and human Fam20C/Fam20A.
  • Transformants were selected on YPD plates containing zeocin (100 pg/mL), nourseothricin (100 pg/mL) and hygromycin (200 pg/mL). Deletion of the PEP4 gene was confirmed via colony PCR, after which the pep4 deletion clones were further purified on YPD plates containing zeocin (100 pg/mL) and nourseothricin (100 pg/mL) to lose the episomal Crispr plasmid containing the hygromycin resistance marker.
  • strain Pp0130 was selected to test the functionality of bovine homologues of the human Fam20C and Fam20A kinase sequences. Since this background is different from the Pp0020 in which the original kinase constructs were tested, strain Pp0130 was initially transformed with the pGAP driven expression construct for human Fam20C (own prepro- sequence) or the combination construct for pGAP-driven expression of both the human Fam20C (own prepro-sequene) and the human Fam20A (Ost1 pre-sequence) to serve as positive controls when evaluating the co-expression of the bovine Fam20 kinase sequences.
  • Transformants were selected based on their ability to grown on YPD plates with 100 pg/mL nourseothricin and zeocin. Integration of the expression cassettes was confirmed via colony PCR.
  • Several PCR-positive (and negative) clones were selected for 24 deep-well cultivation and grown using the following protocol: 1) inoculation from single colony into 2 mL BMGY pH 6.5; 2) cultivation for 24 hours at 200 rpm and 28°C; 3) collection of cells via centrifugation and removal of the BMGY medium; 4) resuspension of cells in 2 mL BMMY (1 % methanol) pH 6.5, followed by cultivation for 48 hours; 5) at regular time intervals additional methanol is spiked into the cultivation to avoid longer periods of C-source starvation.
  • the nucleotide sequence of the mature bovine alphaSI -casein variant B was ordered synthetically (gBIock - IDT) and codon-optimized for expression by K. phaffi.
  • the sequence was flanked with the required BsmBI and Bsal sites to allow Golden Gate based modular cloning strategies using the commercially available OPENPichia plasmid system (distributed by GeneCorner - BCCM) and was initially subcloned via BsmBI assembly into the pPTK081_0_Empty entry vector.
  • Intracellular analysis of the presented cultivation also showed a discrete difference in gel migration between intracellular alphaSI-casein of the secreting kinase co-expression clones versus the parental strain or a clone that does not secrete part of the alphaSI-casein (no expression of active kinase) ( Figure 9, right panel).
  • the slight reduction in gel mobility for the intracellular alphaSI- casein of kinase co-expression strains can be the result of protein phosphorylation.
  • the above results indicate that kinase co-expression can stimulate the secretion of the expressed alphaSI-casein.
  • new alphaSI-casein expression strains were generated using a background strain in which both the YPS1 and PEP4 gene were knocked out.
  • the gene knock-outs were generated in the commercial K. phaffi strain PPS-9010 (ATUM) according to the same principles and work methods as described above (CRISPR- mediated deletion) and the resulting strain was designated Pp0096. Since the custom-made inhouse antibody proved to be sufficient to evaluate expression of alphaSI-casein, a C-terminal His- tag was no longer deemed to be required the expression cassette.
  • the Bsal-based modular cloning strategy was used to combine the AOX1 promotor, the subcloned alphaSI -casein with direct in-frame fusion to the N-terminal signal peptide, the AOX1 transcription terminator and the zeocin resistance marker into a new expression cassette.
  • the generated plasmid for tagless alphaSI -casein was transformed to strain Pp0096. Transformants were selected based on their ability to grow on YPD with 1000 pg/mL of zeocin, which could induce the isolation of clones with high-copy integration of the expression cassette. Integration of the expression cassettes was confirmed via colony PCR and number of integrated copies by qPCR analysis. PCR-positive multicopy strains were cultivated as described in example 6 and intracellular expression evaluated (not shown). A well expressing 9-copy clone (Pp0196) was selected for further engineering work.
  • Intracellular analysis ( Figure 10, right panel) of the presented cultivation also showed a difference in gel migration between intracellular alphaSI -casein of the secreting kinase co-expression clones versus the parental strain Pp0196, PCR-negative transformants (no secretion of the alphaSI- casein due to lack of active kinase co-expression).
  • Extracellular cell-free samples of Pp0196, Pp0163 and kinase co-expression clones of Pp0196 (human Fam20C or human Fam20C+A) were loaded for standard SDS-PAGE analysis, followed by Coomassie staining.
  • Strains Pp0121 expressing the His-tagged AlphaSI -casein variant B, and Pp0163, coexpressing the AlphaSI -casein and the human Fam20(A+C) were cultivated in 1.5 L bioreactor vessels (Dasgip). Shake flask precultivations and bioreactor inoculation were done as described above. Fermentations were performed at 24°C and pH 6.8 while the dissolved oxygen was maintained at 30%. The medium for the batch phase consisted of BMGY with 1 % glycerol.
  • K. phaffi cells were centrifuged to remove the supernatant and the wet cell pellet was then washed in 50 mM Tris-HCI pH 8 (1 .1 mL per g cells). Lysis buffer (50 mM Tris-HCI pH 8 + 4 pg/ml PepstatinA + 1 mM PMSF + 0.1 % Empigen BB + 5 U/mL nuclease) was added at 5 mL per gram wet cell weight. The resuspended cells were lysed via four consecutive passages through an Agitator bead mill DynoMill (Multi Lab) in which the lysis chamber was filled with 250 mL glass beads (425-600 pm).
  • a buffer exchange was performed to 50 mM Tris-HCI, 4 M urea, 200 mM NaCI, pH 8 (to remove remaining ammonium sulphate) via tangential flow filtration (TFF) using a 0.0264 m 2 Pellicon 3 RC membrane with 10 kDa cut-off (Merck Millipore).
  • THF tangential flow filtration
  • a chromatography separation by IMAC was performed on a 20 mL column with Chelating Sepharose FF (Cytiva), loaded with nickel chloride. After loading, the column was wash with 10 mM imidazole and the His-tagged caseins eluted with 200 mM imidazole.
  • the product was concentrated and diafiltrated by TFF using a 0.0264 m 2 Pellicon 3 RC membrane with 10 kDa cut-off (Merck Millipore). Finally, the product was concentrated and diafiltered with at least 5 diavolumes 50 mM Tris-HCI pH 8, followed by 0.45 pm filtration. Protein concentration, purity and identity were determined using resp. OD280 measurement, SDS-PAGE + Coomassie staining and Western blot.
  • Example 9 Further reduction of proteolysis of bovine aS1 -casein upon secretion by a Pichia pastoris strain which co-expresses the HuFam20c kinase
  • the Pp0196 contains up to nine genomically integrated copies of an expression cassette consisting of the AOX1 promoter, the coding sequence for the SP13 secretion signal in direct fusion to the N-terminus of the mature bovine AlphaSI -casein variant B, and the AOX1TT terminator.
  • a plasmid was designed (P0626) in which the pGAP-driven HuFam20C (own prepro-sequence) expression cassette was flanked by a 581 bp and 659 bp sequence located 5’ resp. 3’ of the Pichia YPS’ coding sequence.
  • the linearized knock-out/knock-in cassette from P0626 was co-transformed with a YPS’-specific CRISPR-based knock-out plasmid P0471.
  • the latter plasmid was generated according to the methods as previously described for the PEP4 and YPS1 CRISPR-based knock-out plasmids. Pichia transformants were selected on hygromycin-containing medium to select for the presence of the CRISPR plasmid and single clones were then screened by cPCR to identify those in which the correct insertion of the HuFam20c kinase co-expression cassette resulted into the simultaneous deletion of the YPS’ ORF.
  • Colony PCR indicated that several correct knock-out/knock-in clones could be obtained within the Pp0196 strain background, and two pure single clones were then stored as strains Pp0587 and Pp0588. These two strains, as well as strain Pp0196 (the parental strain), strain Pp021 1 (a strain with pGAP-driven HuFam20c and HuFam20a co-expression but without a deletion of the YPS’ gene) and strain Pp0213 (a strain with pGAP-driven HuFam20c co-expression but without a deletion of the YPS’ gene) were selected for cultivation. The cultivation was performed in a 24 deepwell plate at 28°C and 200 rpm for 24 hours in standard BMGY medium pH 7.
  • the cells were then 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. At the end of the cultivation, cells were separated from the broth and the cell-free medium was analyzed via western blot for the extracellular presence of (full-size) AlphaSI -casein variant B ( Figure 12).

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Abstract

La présente invention concerne des améliorations dans la production de caséines dans des cellules hôtes non mammifères, telles que des levures ou des champignons filamenteux. En particulier, l'invention concerne des cellules hôtes non mammifères dans lesquelles au moins une caséine est co-exprimée avec au moins une kinase capable de phosphoryler la caséine. La kinase peut être une kinase de la famille des kinases FAM20, de préférence une Fam20C et/ou une Fam20A kinase. La co-expression de la kinase avec la caséine augmente non seulement le degré de phosphorylation de la caséine mais, étonnamment, améliore aussi considérablement la sécrétion de la caséine phosphorylée pour la cellule hôte, facilitant ainsi sa récupération. L'invention concerne en outre des procédés de production d'une caséine à l'aide des cellules hôtes non mammifères de l'invention, ainsi que des caséines phosphorylées ayant un motif de glycosylation non natif, et des compositions comprenant de telles caséines phosphorylées, par exemple des produits alimentaires tels que des produits de substitution de produits laitiers sans animaux.
EP23812888.8A 2022-11-22 2023-11-22 Sécrétion de caséine améliorée par des cellules hôtes non mammifères Pending EP4623075A1 (fr)

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CZ299290B6 (cs) 1997-02-20 2008-06-11 Dsm Ip Assets B.V. Zpusob výroby beta-laktamové slouceniny, zpusob prípravy a/nebo zlepšení vláknitého mikrobiálního kmene a použití chemicky definovaného fermentacníhomédia
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DE60142226D1 (de) 2000-03-14 2010-07-08 Novozymes As Pilz transkriptionsaktivator zur verwendung in verfahren zur herstellung von polypeptiden
CN101094919B (zh) 2004-10-12 2014-09-03 帝斯曼知识产权资产管理有限公司 在生产多肽的方法中有用的真菌转录活化因子
EP1954812B1 (fr) 2005-11-29 2012-11-21 DSM IP Assets B.V. Site de liaison a l'adn d'un activateur transcriptionnel utile dans l'expression genetique
CA3222886A1 (fr) 2014-08-21 2016-02-25 Perfect Day, Inc. Compositions comprenant une caseine et procedes de production de celles-ci
CN113301812A (zh) * 2018-10-17 2021-08-24 完美日股份有限公司 用于食物产品的重组组分和组合物
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EP4122950A1 (fr) * 2021-07-23 2023-01-25 Redbiotec AG Cellules hôtes recombinantes et procédés de production de protéines de caséine
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