WO2006026992A1 - Structure de n-glycanes modifiee dans un champignon - Google Patents

Structure de n-glycanes modifiee dans un champignon Download PDF

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WO2006026992A1
WO2006026992A1 PCT/DK2005/000569 DK2005000569W WO2006026992A1 WO 2006026992 A1 WO2006026992 A1 WO 2006026992A1 DK 2005000569 W DK2005000569 W DK 2005000569W WO 2006026992 A1 WO2006026992 A1 WO 2006026992A1
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nucleic acid
acid sequence
sequence encoding
alpha
fungal cell
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Christel Thea JØRGENSEN
Carsten Hjort
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Novozymes A/S
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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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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)
    • C12Y204/01038—Beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase (2.4.1.38)
    • 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)
    • C12Y204/01101—Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (2.4.1.101)
    • 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)
    • C12Y204/01143—Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (2.4.1.143)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01113—Mannosyl-oligosaccharide 1,2-alpha-mannosidase (3.2.1.113), i.e. alpha-1,2-mannosidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01114—Mannosyl-oligosaccharide 1,3-1,6-alpha-mannosidase (3.2.1.114)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/05—Fusion polypeptide containing a localisation/targetting motif containing a GOLGI retention signal

Definitions

  • the present invention relates to a method for altering the structure of N-glycans in a fungus and to a fungal cell having an altered N-glycan structure.
  • Glycosylation of proteins is the process of post-translational modification of eukaryotic proteins by attachment of carbohydrates to the protein. Glycosylation is generally divided into; N- glycosylation or O-glycosylation, wherein N-glycosylation is the attachment of carbohydrate(s) (also known as glycans) on the nitrogen atom of an Asparagine (Asn) residue in the protein and O-glycosylation is the attachment of carbohydrates to an oxygen atom of hydroxyls, in par ⁇ ticular of a Serine (Ser) or Threonine (Thr) residue in the protein (see e.g. pp. 91-94 of Creigh- ton TE (1993), Proteins; Structure and Molecular Properties, second edition, W.
  • Filamentous fungi are widely used to industrially produce proteins, such as enzymes. How- ever, the use of said fungi as production strains for production of pharmaceutical proteins has so far been somewhat limited. One reason for this is that the structure of glycans added to pro ⁇ teins in filamentous fungi is different to that found in mammalian cells and these differences have been shown to be able to elicit an immunological response in mammalians, such as hu ⁇ mans.
  • Man ⁇ and Man9 are further modified in the Golgi where the following sequential steps take place (Hamilton SR et al. (2003), Science, 301, 1244-1246):
  • Man ⁇ or Man9 is processed by alpha-1 ,2-mannosidase 1A, 1 B and 1C to produce Man5
  • Man ⁇ is processed by beta-1 ,2- ⁇ /-acetylglucosaminyltransferase I to produce GlcNAcMan ⁇
  • GlcNAcMan ⁇ is processed by Mannosidase Il to produce GlcNAcMan3
  • GlcNAcMan3 is processed by beta-1 ,2- ⁇ /-acetylglucosaminyltransferase Il (GnTII) to produce GlcNac2Man3
  • WO 01/25406 discloses mannosidase enzymes and use of such enzymes to alter the glycosy ⁇ lation patterns of macromolecules.
  • WO 02/00879 discloses methods for producing modified glycoproteins.
  • US 5,047,335 discloses a process for controlling the glycosylation of protein in a cell wherein the cell is genetically engineered to produce one or more enzymes which provide internal con ⁇ trol of the cell's glycosylation mechanism.
  • the present invention provides a method for altering the N-glycan structure of fungi so that it is more similar to the pattern observed in mammalians, e.g. humans.
  • the present invention relates in a first aspect to a method of altering the structure of N- glycans in a fungus comprising insertion into a fungal cell of a nucleic acid construct or a com ⁇ bination of nucleic acid constructs selected from the group consisting of:
  • a) a first nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,2-mannosidase obtained from a filamentous fungi
  • a second nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase
  • a third nucleic acid construct comprising a nucleic acid sequence encoding a mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase
  • a fourth nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase
  • nucleic acid construct comprising a nucleic acid sequence encoding a beta-
  • the present invention relates to a fungal cell comprising a nucleic acid construct or a combination of nucleic acid constructs selected from the group consisting of:
  • a) a first nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,2-mannosidase obtained from a filamentous fungus
  • a second nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetyl- glucosaminyltransferase
  • a third nucleic acid construct comprising a nucleic acid sequence encoding a mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase
  • a fourth nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase
  • nucleic acid construct comprising a nucleic acid sequence encoding a beta-1 ,4-galactosyltransferase
  • said cell has an altered N-glycan structure as compared to a parental fungal cell.
  • Figure 1 shows the stepwise altering of the N-glycan structure in a fungal cell of the present invention wherein the glycan-structure before the arrow is the substrate and the glycan-structure after the arrow is the product and wherein "Asn” is an Asparagine residue in a protein, the "square ( ⁇ )” is a GIcNAc residue, the “circle (•)” is a mannose residue and the
  • Step 1 (the conversion of Man9GlcNAc2 to Man5GlcNAc2) is catalysed by alpha-1 ,2 mannosidase.
  • Step 2 (the conversion of Man5GlcNAc2 to GlcNAcMan5GlcNAc2) is catalysed by alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase.
  • Step 3 (the conversion of GlcNAcMan5GlcNAc2 to GalGlcNAcMan5GlcNAc2) is catalysed by beta-1 ,4-galactosyltransferase.
  • Step 2b (the conversion of GlcNAcMan5GlcNAc2 to GlcNAcMan3GlcNAc2) is catalysed by mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase.
  • Step 2c (the conversion of GlcNAcMan3GlcNAc2 to GlcNAc2Man3GlcNAc2) is catalysed by alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase.
  • Step 3b (the conversion of GlcNAc2Man3GlcNAc2 to Gal(0-2)GlcNAc2Man3GlcNAc2) is catalysed by beta-1 ,4-galactosyltransferase, wherein the Gal(0-2)GlcNAc2Man3GlcNAc2 structures are described under figure 2.
  • Figure 2 shows the four different forms of Gal(0-2)GlcNAc2Man3GlcNAc2.
  • Gal(0)GlcNAc2Man3GlcNAc2 denotes GlcNAc2Man3GlcNAc2 without any GaI residues.
  • GaI(I )GlcNAc2Man3GlcNAc2 denotes GlcNAc2Man3GlcNAc2 with one GaI residue located on either the 1 ,3 arm (depicted as the upper arm) or the 1 ,6 arm (depicted as the lower arm).
  • the two version of the GaI(I )GlcNAc2Man3GlcNAc2 are considered to be equivalents.
  • Gal(2)GlcNAc2Man3GlcNAc2 denotes GlcNAc2Man3GlcNAc2 with two GaI residues; one on the 1 ,3 arm and one on the 1 ,6 arm.
  • the terms "1 ,3 arm” and “1 ,6 arm” are to be understood as referring to that the GaI residue is bound to the GIcNAc residue by a bond between carbon number 1 of the GIcNAc residue and carbon number 3 or 6, respectively of the GaI residue.
  • Man is in the context of the present invention used as a short term for man- nose.
  • GIcNAc is in the context of the present invention used as a short term for N- acetylglucosamin.
  • GaI GaI
  • Man9 is used as a short term for Man9GlcNAc2
  • Man ⁇ is used as a short term fro Man8GlcNAc2
  • Man5GlcNAc2 is used as a short term for Man5GlcNAc2
  • Man3 is used as a short term for Man3GlcNAc2.
  • ER is used as short-term for endoplasmic reticulum.
  • N-glycan is in the context of the present invention to be understood as As- paragine-linked-glycans, i.e. carbohydrates attached to an Asparagine residue in a polypep ⁇ tide.
  • E. C Enzyme Class
  • enzyme Class refers to the in ⁇ ternationally recognized enzyme classification system, Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press, Inc.
  • the term "origin” used in the context of amino acid sequences, e.g. proteins, or nucleic acid sequences is to be understood as referring to the organism from which it derives. Said sequence may be expressed by another organism using gene technology methods well known to a person skilled in the art. This also encompasses sequences which have been chemically synthesized. Furthermore, said sequences may comprise minor changes such as codon opti ⁇ mization, i.e. changes in the nucleic acid sequences which do not affect the amino acid se ⁇ quence.
  • protein is in the context of the present invention intended to include polypep ⁇ tides and combinations of polypeptides.
  • dominant when used in reference with a nucleic acid sequence or an allele is to be understood as an allele which determines the phenotype displayed in a heterozygote with another (recessive) allele.
  • parental cell is in the context of the present invention to be understood as a cell which is modified according to a method according to the present invention.
  • ORF open reading frame
  • alignments of sequences and calculation of ho ⁇ mology scores may be done using a full Smith-Waterman alignment, useful for both protein and DNA alignments.
  • the default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively.
  • the penalty for the first residue in a gap is - 12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for pro ⁇ teins and -4 for DNA.
  • Alignment may be made with the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 183:63-98).
  • the present invention relates to a method of altering the structure of N-glycans in a fun ⁇ gus comprising insertion into a fungal cell of a nucleic acid construct or a combination of nucleic acid constructs selected from the group consisting of: a) a first nucleic acid construct comprising a nucleic acid sequence encoding an alpha-
  • nucleic acid construct comprising a nucleic acid sequence encoding an al- pha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase c) a third nucleic acid construct comprising a nucleic acid sequence encoding a man- nosyl-oligosaccharide 1,3-1 ,6-alpha-mannosidase d) a fourth nucleic acid construct comprising a nucleic acid sequence encoding an al- pha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosamintransferase e) a fifth nucleic acid construct comprising a nucleic acid sequence encoding a beta- 1 ,4-galactosyltransferase
  • altered N-glycan structure is to
  • the altered N-glycan structure may be a Man5GlcNAc2, or a GlcNAcMan5GlcNAc2, or a GalGlcNAcMan5GlcNAc2, or a GlcNAcMan3GlcNAc2, or a GlcNAcMan3GlcNAc2 or a GlcNAc2Man3GlcNAc2 or a GaI(O- 2)GlcNAcMan3GlcNAc2.
  • the altered N-glycan structure may also be further modified by other enzymes naturally present in the fungal cell, which are capable of e.g. adding other sugar resi- dues to the N-glycan.
  • the altered N-glycan structure may in par ⁇ ticular comprise Man5GlcNAc2, or GlcNAcMan5GlcNAc2, or GalGlcNAcMan5GlcNAc2, or GlcNAcMan3GlcNAc2, or GlcNAc2Man3GlcNAc2 or Gal(0-2)GlcNAcMan3GlcNAc2.
  • Methods for detecting the different glycan-structures may be performed as described in Jackson P and Gallagher JT (1997), A laboratory guide to Glycoconjugate analysis, Biomethods, vol. 9, Birk- ha ⁇ ser, Basel, Switzerland.
  • a method of the present invention comprises insertion into a fungal cell of a first, or a second, or a third, or a fourth or a fifth nucleic acid construct of the present invention.
  • said method may comprise inser ⁇ tion of a combination of two or more of the above mentioned, first, second, third, fourth and fifth nucleic acid constructs.
  • any combination of said nucleic acid con ⁇ struct may be inserted into a fungal cell, the inventors of the present invention in particular foresee two main routes for altering the N-glycan structure of a fungal cell by insertion of a combination of the above mentioned, first, second, third, fourth and fifth nucleic acid con ⁇ structs. These two main routes for altering the N-glycan structure are outlined in figure 1.
  • Fig ⁇ ure 1 shows that insertion of a combination of the first and the second nucleic acid construct is the same for both routes, but after the second nucleic acid construct the routes diverge. Furthermore, both routes include as the final step the insertion of a fifth nucleic acid construct.
  • a method according to the present invention may further comprise modification of an endogenous gene encoding an enzyme ca ⁇ pable of catalyzing the binding of mannose by alpha-1 ,6-bonds to the 1 ,3-arm of Man9GlcNAc2 so that fewer mannose residues are bound by alpha-1 ,6-bonds to the Man9GlcNAc2 structure of proteins than in a parental cell.
  • the term "fewer" is to be understood so that the fraction of a particular protein which com ⁇ prises a mannose residue bound by an alpha-1 ,6-bond to Man9GlcNAc2 is lower when said protein is produced by a cell in which such an endogenous gene has been modified compared to when said protein is produced by a parental cell.
  • the presence of a mannose residue bound by an alpha-1 ,6-bond to Man9GlcNAc2 may be measured as described in Jackson P and Gal ⁇ lagher JT (1997), A laboratory guide to Glycoconjugate analysis, Biomethods, vol. 9, Birk- ha ⁇ ser, Basel, Switzerland
  • Man9GlcNAc2 The presence of a mannose residue bound by an alpha-1 ,6-bond to the 1 ,3 arm of Man9GlcNAc2 is believed to inhibit or at least partly inhibit the hydrolysis of the terminal 1 ,2- linked alpha-D-mannose in Man9GlcNAc2 to produce Man5GlcNAc2 catalysed by alpha-1 , 2- mannosidase.
  • a fungal cell with an altered N-glycan structure created by a method of the present invention may in particular be used as a host for producing a protein with an altered N-glycan structure.
  • These fungal cells may in particular be used for producing a mammalian protein as the altered N-glycan structure of a fungal cell of the present invention is more similar to that found in mammalian cells than the N-glycan struc ⁇ ture of a fungal cell which has not been modified.
  • a fungal cell with an altered N-glycan structure created by a method of the present invention may in particular be used to create a heterokaryon with an altered N-glycan structure.
  • Said heterokaryon with an altered N-glycan structure may in a particular embodiment be used as a host for producing a protein with an al ⁇ tered N-glycan structure.
  • said heterokaryon may be used for producing a mam ⁇ malian protein as the altered N-glycan structure of a fungal heterokaryon of the present inven ⁇ tion is more similar to that found in mammalian cells than the unaltered N-glycan structure of a fungal heterokaryon.
  • the fungal cell with an altered N-glycan structure created by a method of the present invention is used as a host for production of a protein, then it is advantageous if the combina- tion of insertion of nucleic acid constructs into said cell creates a stepwise alteration of the N- glycan structure as outlined in figure 1.
  • the route depicted to the left in fig ⁇ ure 1 is followed (step 3) then the following combinations of nucleic acid constructs may in par ⁇ ticular be inserted into a fungal cell: i) a combination of the first and second nucleic acid construct; or ii) a combination of the first, second and fifth nucleic acid construct.
  • the fol ⁇ lowing combinations of nucleic acid constructs may in particular be inserted into a fungal cell: i) a combination of the first and second nucleic acid construct; ii) a combination of the first, second and third nucleic acid construct; iii) a combination of the first, second, third and fourth nucleic acid construct; or iv) a combination of the first, second, third, fourth and fifth nucleic acid construct.
  • nucleic acid constructs into that particular fungal cell may not necessarily be stepwise as indicated in figure 1.
  • the overall modification of the N-glycan structure of the heterokaryon may in particular be stepwise as outlined in figure 1.
  • heterokaryon comprising a first and second nuclei acid construct
  • a first nucleic acid construct may be inserted into one of the fungal cells, while the sec ⁇ ond nucleic acid construct may be inserted into the other fungal cell.
  • heterokarvon comprising a first, second and fifth nuclei acid construct
  • a first and second nucleic acid construct may be inserted into one of the fungal cells, while the fifth nucleic acid construct may be inserted into the other fungal cell.
  • a first and fifth nucleic acid construct may be inserted into one of the fungal cells, while the second nucleic acid construct may be inserted into the other fungal cell.
  • a second and fifth nucleic acid construct may be inserted into one of the fungal cells, while the first nucleic acid construct may be inserted into the other fungal cell.
  • heterokaryon comprising a first, second and third nuclei acid construct
  • a first and second nucleic acid construct may be inserted into one of the fungal cells, while the third nucleic acid construct may be inserted into the other fungal cell.
  • a first and third nucleic acid construct may be inserted into one of the fungal cells, while the second nucleic acid construct may be inserted into the other fungal cell.
  • a second and third nucleic acid construct may be inserted into one of the fungal cells, while the first nucleic acid construct may be inserted into the other fungal cell.
  • a heterokarvon comprising a first, second, third and fourth nuclei acid construct •
  • a first, second and third nucleic acid construct may be inserted into one of the fungal cells, while the fourth nucleic acid construct may be inserted into the other fungal cell.
  • a first, second and fourth nucleic acid construct may be inserted into one of the fungal cells, while the third nucleic acid construct may be inserted into the other fungal cell.
  • a first, third and fourth nucleic acid construct may be inserted into one of the fungal cells, while the second nucleic acid construct may be inserted into the other fungal cell.
  • a second, third and fourth nucleic acid construct may be inserted into one of the fungal cells, while the first nucleic acid construct may be inserted into the other fungal cell.
  • a first and second nucleic acid construct may be inserted into one of the fungal cells, while the third and fourth nucleic acid construct may be inserted into the other fungal cell.
  • a first and third nucleic acid construct may be inserted into one of the fungal cells, while the second and fourth nucleic acid construct may be inserted into the other fungal cell.
  • a first and fourth nucleic acid construct may be inserted into one of the fungal cells, while the second and third nucleic acid construct may be inserted into the other fungal cell.
  • heterokarvon comprising a first, second, third, fourth and fifth nuclei acid construct
  • a first, second, third and fourth nucleic acid construct may be inserted into one of the fungal cells, while the fifth nucleic acid construct may be inserted into the other fungal cell.
  • a first, second, third and fifth nucleic acid construct may be inserted into one of the fun ⁇ gal cells, while the fourth nucleic acid construct may be inserted into the other fungal cell.
  • a first, second, fourth and fifth nucleic acid construct may be inserted into one of the fungal cells, while the third nucleic acid construct may be inserted into the other fungal cell.
  • a first, third, fourth and fifth nucleic acid construct may be inserted into one of the fun ⁇ gal cells, while the second nucleic acid construct may be inserted into the other fungal cell.
  • a second, third, fourth and fifth nucleic acid construct may be inserted into one of the fungal cells, while the first nucleic acid construct may be inserted into the other fungal cell.
  • a first, second and third nucleic acid construct may be inserted into one of the fungal cells, while the fourth and fifth nucleic acid construct may be inserted into the other fun- gal cell.
  • a first, second and fourth nucleic acid construct may be inserted into one of the fungal cells, while the third and fifth nucleic acid construct may be inserted into the other fun ⁇ gal cell.
  • a first, second and fifth nucleic acid construct may be inserted into one of the fungal cells, while the third and fourth nucleic acid construct may be inserted into the other fungal cell.
  • a first, fourth and fifth nucleic acid construct may be inserted into one of the fungal cells, while the second and third nucleic acid construct may be inserted into the other fungal cell.
  • a first, third and fourth nucleic acid construct may be inserted into one of the fungal cells, while the second and fifth nucleic acid construct may be inserted into the other fungal cell.
  • a first, third and fifth nucleic acid construct may be inserted into one of the fungal cells, while the second and fourth nucleic acid construct may be inserted into the other fungal cell.
  • a second, third and fourth nucleic acid construct may be inserted into one of the fungal cells, while the first and fifth nucleic acid construct may be inserted into the other fungal cell.
  • a second, third and fifth nucleic acid construct may be inserted into one of the fungal cells, while the first and fourth nucleic acid construct may be inserted into the other fun ⁇ gal cell.
  • a second, fourth and fifth nucleic acid construct may be inserted into one of the fungal cells, while the first and third nucleic acid construct may be inserted into the other fun ⁇ gal cell.
  • a third, fourth and fifth nucleic acid construct may be inserted into one of the fungal cells, while the first and second nucleic acid construct may be inserted into the other fungal cell.
  • nucleic acid construct may be inserted into a fungal cell which then subsequently may be fused with one or more other fungal cells which have not be modified according to a method of the present invention.
  • heterokaryon is to comprise one or more of the first, second, third, fourth and fifth nucleic acid constructs of the present invention then the insertion of the different nucleic acid constructs may in particular be divided between the different fungal cells which are to be fused to form a heterokaryon.
  • the fungal cell in a method of the present invention in principle may be a heterokaryon, i.e.
  • the nucleic acid constructs of the present invention may in principle be inserted into a heterokaryon, the inventors of the present invention believe that it is advantageous to insert the nuclei acid constructs into the single fungal cells and then sub ⁇ sequently fuse said cells to form a heterokaryon, as the heterokaryon is probably be more un ⁇ stable than single cells thereby making introduction of nucleic acid constructs more difficult. If a heterokaryon is formed by fusion of more than two genetically different cells then the distribution of nucleic acid constructs which may be inserted into each of the genetically different cells may be different than that suggested above for two genetically different cells. The possible different combinations for insertion of the nucleic acid into each of the cells may be combined similarly as described above for two cells.
  • a method of the present invention further comprises modification of an endogenous gene encoding an enzyme capable of catalyzing the binding of mannose by alpha-1 ,6-bonds to Man5GlcNAc2 and the purpose is to obtain a heterokaryon with such a modified gene, it may be an advantage to modify said gene in each of genetically different cells which are fused to create a heterokaryon.
  • heterokaryon fungal cell does not fuse, thus if at least one of the genetically different cells which are fused to create said heterokaryons comprises such an endogenous gene which has not been modified it is envisioned that the heterokaryon will express a functional enzyme encoded by said gene, whereby binding of mannose by al- pha-1 ,6-bonds to Man5GlcNAc2 should still be possible to take place in the heterokaryon.
  • the present invention relates both to methods for altering the structure of N-glycans in a fungus and to a fungal cell comprising a first, second, third, fourth or fifth nucleic acid con ⁇ struct or a combination thereof.
  • fungal cell in the following intended to encompass both parental fun ⁇ gal cells and fungal cells of the present invention.
  • Fungi as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomy- cota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
  • Basidiomycota include mushrooms, rusts, and smuts.
  • Representative groups of Chytridiomycota include, e.g., AIIo- myces, Blastocladiella, Coelomomyces, and aquatic fungi.
  • Representative groups of Oomy ⁇ cota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya.
  • Exam- pies of mitosporic fungi include Aspergillus, Penicillium, Candida, and Alternaria.
  • Representa ⁇ tive groups of Zygomycota include, e.g., Rhizopus and Mucor.
  • the fungal cell is a yeast cell.
  • yeast as used herein includes as- cosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes).
  • the ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomy- coideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces).
  • the basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella.
  • Yeasts belonging to the Fungi lmperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S. M., and Davenport, R.R., eds, Soc. App. Bacte- riol. Symposium Series No.
  • yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, BJ. , and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts, Rose, A.H., and Harrison, J. S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathem et al., editors, 1981).
  • the yeast cell is a cell of a species of Candida, Kluy- veromyces, Saccharomyces, Schizosaccharomyces, Pichia, or Yarrowia.
  • the yeast cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomy ⁇ ces norbensis or Saccharomyces oviformis cell.
  • the yeast cell is a Kluyveromyces lactis cell.
  • the yeast cell is a Yarrowia lipolytica cell.
  • the fungal cell is a filamentous fungal cell.
  • "Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawk- sworth et al., 1995, supra). 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. In contrast, vegeta- tive growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal cell may be a cell of a species of, but not limited to, Acremo- nium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thie- lavia, Tolypocladium, or Trichoderma.
  • the filamentous fungal cell is an Aspergillus awamori, Asper ⁇ gillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell.
  • the filamentous fungal cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium ox- ysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sar- cochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell.
  • Fusarium bactridioides Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
  • the filamentous fun ⁇ gal cell is a Fusarium venenatum (Nirenberg sp. nov.) cell.
  • the filamentous host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, My- celiophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
  • a fungal cell of the present invention wherein the structure of N-glycans has been modified according to a method of the present invention, is used to create a heterokaryon
  • the fungal cell may in particular be a yeast or filamentous fungal cell.
  • said fungal cell is not fused with one or more other cells to create a heterokaryon but used e.g. for expression of a protein with an altered N-glycan structure it may in particular be a filamentous fungal cell.
  • a "heterokaryon" is to be understood as a cell with at least two genetically different nuclei.
  • Heterokaryons derive from fusion of two or more genetically different cells wherein the nuclei of said cells does not fuse resulting in a cell com ⁇ prising two or more nuclei.
  • the heterokaryon fungus may be a filamentous heterokaryon fungus or it may be a yeast heterokaryon.
  • the heterokaryon fungus may be formed naturally between two or more fungi or it may be made artificially. When two or more genetically different fungi fuse the nuclei of each of the individual cells come to coexist in a common cytoplasm.
  • One method to select for heterokaryons is to fuse two or more genetically different cells which each com- prise a genome with a characteristic which renders the survival of each cell dependent on presence of the nuclei from the other cell. For example if two genetically different cells which each depends on a particular nutrient for survival and at the same time is independent of the nutrient the other cell depends on for survival is cultured in the a medium lacking both of the nutrients this will make only cells which arise as a fusion between each of the genetically dif- ferent cells able to survive in this medium.
  • the heterokaryon filamentous fungus of the present invention may in particular contain nuclei from cells that are homozygous for all heterokaryon compatibility alleles. At least ten chromosomal loci have been identified for heterokaryon incompatibility: het-c, het-d, het-e, het- i, het-5, het-6, het-7, het-8, het-9 and het-10, and more probably exist (see e.g. Perkins et al., "Chromosomal Loci of Neurospora crassa", Microbiological Reviews (1982) 46: 462-570, at 478).
  • Formation of the heterokaryon filamentous fungus may in particular be performed by hyphal or protoplast fusion.
  • heterokaryon filamentous fungus of the present invention may be made by fusion of hyphae from two different strains of filamentous fungi, wherein the first nuclei of one the strains contains a genome that results in a characteristic which renders the fungus de ⁇ pendent on the presence of the second nucleus from the other fungus for survival under the conditions provided for fusion to form the heterokaryon, and vice versa.
  • the nucleus of each strain of filamentous fungus confers a characteristic which would result in the failure of the fungus in which it is contained to survive under the culture conditions unless the nucleus from the other filamentous fungus is also present.
  • characteristics which may be used to render the strains of filamentous fungi dependent on each other include, but are not limited to, a nutritional requirement, resistance to toxic compounds and resistance to extreme environmental conditions. For example if a first strain which requires the presence of a particu- lar nutrient is cultured on a medium lacking said nutrient along with a second strain which does not require said nutrient for survival, the nucleus of the second strain will confer the ability of a fusion of the two strains to survive even in the absence of the particular nutrient. Furthermore, if the second strain similarly requires the presence of a particular nutrient different from the nu ⁇ trient required by the first strain, then only fusions comprising a nucleus from each strain will survive in a medium lacking both of said nutrients.
  • filamentous fungi which may be fused to form a heterokaryon filamentous fungus
  • filamentous fungal host cells include those described below as filamentous fungal host cells.
  • different strains of Aspergillus e.g. A.oryzae or A.niger, Fusarium or Trichoderma may be used to form a heterokaryon filamentous fungus.
  • more than two different strains of filamentous fungi may used to form a heterokaryon, such as 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 different strains.
  • the heterokaryon filamentous fungus of the present invention is formed by fusion of two different strains of filamentous fungi.
  • characteristics which make each of the strains of fungi (that are fused to form a heterokaryon filamentous fungus) dependent on the presence of the nucleus from the other fungus for survival under the conditions provided for the fusion include the selectable markers described above.
  • said characteristic may be a characteristic that makes the fungus autotroph.
  • the culture media used for fusion of the different strains of fungi to form a heterokaryon filamentous fungus may be any media which does not complement the particu ⁇ lar characteristic of the fungi. Examples of such media are well known to a person skilled in the art as they are generally used to select for recombinant fungi.
  • fungus autotroph examples include, but are not limited to: pyrG, hemA, niaD, tpi, facC, gala, biA, lysB, sC, methG and phenA. Thus if a fungus is negative for at least one of these genes said gene may be used as a selectable marker.
  • the heterokaryon fungus is a yeast heterokaryon.
  • yeast for ⁇ mation of heterokaryons has been described as a result of a defect in karyogamy (nuclear fu ⁇ sion) during conjugation of two (or more) haploid cells (Olson BL and Siliciano PG, 2003, Yeast, 20, 893-903).
  • a mutation in the kar1 gene in one of the cells to be fused has been de ⁇ scribed as sufficient to block nuclear fusion during mating (Olson BL and Siliciano PG, Yeast, 2003, 20, 893-903).
  • At least one of the cells involved in formation of a yeast heterokaryon of the present invention comprises a mutation in the kar1 gene, e.g. a loss-of-function mutation, such as a null mutation, a non- sense or a missense mutation, whereby the nuclei of said cell can not fuse with the nuclei from another cell during mating.
  • a loss-of-function mutation such as a null mutation, a non- sense or a missense mutation
  • Conditions for culturing a heterokaryon fungus are similar to those for culturing the fungi that it arises from with the exception that the heterokaryon is cultured in a medium selecting for at least two different characteristics.
  • the selection for at least two different characteristics needs at least to be present during formation of the heterokaryon but usually it also an advantage to keep this selection pressure, i.e. the selection for at least two characteristic during subsequent culturing to ensure the stability of the heterokaryon.
  • Methods for culturing fungi are well known to a person skilled in the art.
  • the fungal cell may comprise one of the following combinations of nucleic acid constructs of the present invention: i) a combination of the first and second nucleic acid construct; or ii) a combination of the first, second and fifth nucleic acid construct Or if the other route of altered N-glycan structure is to be obtained it may comprise one of the following combinations: i) a combination of the first and second nucleic acid construct; ii) a combination of the first, second and third nucleic acid construct; iii) a combination of the first, second, third and fourth nucleic acid construct; or iv) a combination of the first, second, third, fourth and fifth nucleic acid construct. If as mentioned previously in the section of "Methods of the invention" the fungal cell is used for creation of a heterokaryon then the combination of nucleic acid constructs described in that section may in particular be present in such a fungal cell.
  • the fungal cell of the present invention is a heterokaryon and if it comprises more than one nucleic acid construct of the present invention it may either comprise the nucleic acid constructs in the same nuclei or it may in particular comprise the nucleic acid constructs in dif- ferent nuclei.
  • the combination of such nucleic acid constructs in each of the nucleic may as described in the "Methods" section.
  • At least one endogenous gene of the fungal cell has been modified so that fewer mannose residues are bound by alpha-1 ,6- bonds to the 1,3 arm of the Man9GlcNAc2 structure of proteins than in a parental cell. More particularly, said endogenous gene may be Mnn9.
  • N-glycans in a fungal cell, such as a filamentous fungus or in a heterokaryon fungus, wherein the latter in particular may be a filamentous heterokaryon fungus or a yeast heterokaryon.
  • the structure of N-glycans is different in fungi compared to mammalian cells.
  • fungal cells are widely used for industrial production of proteins and it would therefore be an advantage if the N-glycan struc ⁇ ture in the fungal cell could be altered so that it resembles that found in mammalian cell.
  • Figure 1 shows an outline of the different steps of the glycosylation pathway which it is an object of the present invention to introduce and/or modify one or more of in a fungal cell. Each step shows the processing of a particular N-glycan structure by a N-glycosylation en ⁇ zyme to another N-glycan structure.
  • the present invention relates both to methods for altering the N-glycan structure of a fungal cell and to such fungal cell, e.g. to fungal cell with an altered N-glycan structure.
  • the N-glycan structure in a fungal cell is in the present invention altered by insertion of a nucleic acid construct comprising a nucleic acid sequence encoding at least one of en ⁇ zymes shown in figure 1 , i.e. which are selected from the group consisting of alpha-1 ,2- mannosidase, alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, man- nosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase, alpha-1 ,6-mannosyl-glycoprotein 2-beta-N- acetylglucosaminyltransferase and beta-1 ,4-galactosyltransferase.
  • a nucleic acid construct comprising a nucleic acid sequence encoding at least one of en ⁇ zymes shown in figure 1 , i.e
  • N-glycan structure is believed to take place both in the ER and Golgi, thus for a correct formation of the N-glycan structure it is important that the N-glycosylation enzymes involved in formation of said structure are located in the compartment where the reaction they catalyze takes place.
  • First nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 , 2- mannosidase
  • the first nucleic acid construct of the present invention comprises a nucleic acid se ⁇ quence encoding an alpha-1 ,2-mannosidase obtained from a filamentous fungi.
  • alpha-1 ,2-mannosidase is to be un- derstood as enzymes capable of catalyzing the hydrolysis of the terminal 1 ,2-linked alpha-D- mannose residues in the oligosaccharide Man9GlcNAc2, so as to produce Man5GlcNAc2.
  • the alpha-1 ,2-mannosidase used in the present invention derives from a filamentous fungi.
  • it may derive from a strain of Aspergillus, e.g. A.nidulans or A.oryzae. More particularly it may comprise the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 2.
  • it may consist of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 2.
  • nucleic acid sequence encoding the alpha-1 ,2-mannosidase may in particular comprise the nucleic acid sequence shown in SEQ ID NO:1 , or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% iden ⁇ tity with the nucleic acid sequence shown in SEQ ID NO: 1. In another embodiment it may consist of the nucleic acid sequence shown in SEQ ID NO: 1 or an nucleic acid sequence hav ⁇ ing at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence shown in SEQ ID NO: 1.
  • it may comprise the CDS shown in SEQ ID NO:1 , or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 1.
  • it may con ⁇ sist of the CDS shown in SEQ ID NO: 1 or an nucleic acid sequence having at least 70% iden- tity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 1.
  • Alpha-1 ,2-mannosidases are present in filamentous fungal cells but there they are usually not localized in the ER and will thus only be present in the ER transiently during secre ⁇ tion of the alpha-1 ,2-mannosidase. So the alpha-1 ,2-mannosidase is generally not present in the ER in large enough quantities to enable the conversion of all Man9GlcNAc2 structures to Man5GlcNAc2.
  • an ER retention signal hav ⁇ ing the amino acid sequence His-Asp-Glu-Leu or Lys-Asp-Glu-Leu may be fused to the car- boxy terminal of the alpha-1 ,2-mannosidase, which means that the amount of alpha-1 ,2,- mannosidase in the ER or the fraction of glycosylated proteins comprising the Man5GlcNAc2 structure is higher in a fungal cell of the present invention (wherein a first nucleic acid construct has been inserted) than in a parental cell.
  • the amount of alpha-1 , 2-mannosidase may be measured by e.g. isolating the enzyme from the cell and measuring the amount by e.g.
  • Man5GlcNAc2 may be measured as described in Jackson P and Gallagher JT (1997), A laboratory guide to Glycoconjugate analysis, Biomethods, vol. 9, Birkha ⁇ ser, Basel, Switzerland
  • the first nucleic acid construct may in particular further comprise a nucleic acid sequence encoding the amino acid sequence HDEL (His-Asp-Glu-Leu) or KDEL (Lys-Asp-Glu-Leu).
  • An ER retention signal is a signal present in a protein which makes said protein resides in the ER, so far only short amino acid sequences located at the 3'-end of the protein has been identified as being ER re ⁇ tention signals.
  • said ER retention signal may particularly be located downstream of the 3'-end of the nucleic acid sequence encoding an alpha-1 , 2-mannosidase, typically the nucleic acid sequence encoding the ER retention signal may be located right next to the nucleic acid sequence encoding a protein as it is translated as part of the protein.
  • the first nucleic acid construct may in par ⁇ ticular comprise a constitutive promoter, such as one of those mentioned below, more particu ⁇ larly the tpiA or the gpd promoter.
  • the tpiA promoter has been described in McKnight GL et al, 1986, Cell, 46 (1), pp. 143-147 and the gpd promoter has been described in Punt PJ et al, 1990, Gene, 93, pp. 101-109.
  • the second nucleic acid construct of the present invention comprises a nucleic acid sequence encoding an alpha-1 ,3-mannosyl-glycoprotein 2-beta-N- acetylglucosaminyltransferase (GnT1).
  • GnT1 alpha-1 ,3-mannosyl-glycoprotein 2-beta-N- acetylglucosaminyltransferase
  • Alpha-1 , 3-mannosyl-glycoprotein 2-beta-N- acetlyglucosaminyltransferases have been classified as belonging to E.G.
  • GnT1 with regard to the modification of N-glycans is to catalyse the ad ⁇ dition of N-acetylglucosamine to Man5GlcNAc2 to produce GlcNAcMan5GlcNAc2.
  • the GnT1 may be of a mammalian origin as only mammalian GnT1 's have been identified so far.
  • the present invention is not limited to GnTI's of mammalian origin as it is envisioned that GnTI's from other organ ⁇ isms may be identified.
  • the GnT1 may of a human or rat origin. More particularly GnT1 may comprise the amino acid sequence shown in SEQ ID NO: 4 or an amino acid se ⁇ quence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 4. In another embodiment it may consist of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 4.
  • the nucleic acid sequence encoding the GnT1 may in particular comprise the nucleic acid sequence shown in SEQ ID NO:3, or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence shown in SEQ ID NO: 3. In another embodiment it may consist of the nucleic acid sequence shown in SEQ ID NO: 3 or an nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence shown in SEQ ID NO: 3.
  • it may comprise the CDS shown in SEQ ID NO:3, or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 3.
  • it may con ⁇ sist of the CDS shown in SEQ ID NO: 3 or an nucleic acid sequence having at least 70% iden- tity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 3.
  • a synthetic gene encoding a mammalian GnT1 may in particular be used.
  • the sequence of the synthetic gene may be iden ⁇ tical to e.g. that of the cDNA of a mammalian such as the mRNA or CDS sequence of the hu ⁇ man GnT1 shown in SEQ ID NO: 3.
  • the sequence may have been modified so that it is more similar to the sequence encoding proteins naturally ex ⁇ pressed by fungi.
  • said sequence may have been codon optimized which means that codon(s) of the nucleic acid sequence enccoding the GnT1 may be changed so that they resemble the codon(s) used by the fungal cell into which the second nucleic acid construct is introduced into.
  • the frequency with which the different nucleic acid codons are used to encode a particular amino acid differs between different species, e.g. be ⁇ tween fungi and mammalian cells.
  • a particular codon may in mammalian cells encode one amino acid while the same condon in a fungus may encode a different amino acid or it may not even be recognized by the fungal cell because e.g. the frequency of nucleic acid se ⁇ quence comprising this codon may be low and instead a different codon may be recognized by the fungal cell to encode that particular amino acid.
  • Codon optimization is a well-known phenome ⁇ non for a person skilled in the art and may e.g. be performed as described in e.g. Sharp PM et al, 1988, Nucleic Acid Res, 16, pp. 8207-8211 or Ramakrishna L et al, 2004, Journal of Virol- ogy, 78 (17), pp. 9174-9189.
  • Cryptic introns are se ⁇ quences which are not recognized as an intron by the cell which naturally express the protein, e.g. in the present case a mammalian cell, but which is recognized as an intron by the cell into which the nucleic acid is introduced to produce the protein, e.g. in the present case a fungal cell.
  • the fungal cell recognises part(s) of the nucleic acid sequence as an intron if it is not recognised as an intron by e.g. the mammalian cell from which it derives.
  • the human GnT1 is known to comprise a Golgi retention signal within its amino acid sequence; however, it is unknown whether or not this retention signal is recognized in a fungal cell.
  • the second nucleic acid construct may further comprise a Golgi retention sig- nal.
  • the Golgi retention signal may be one normally present in a fungal protein so that said signal is recognised as being a Golgi retention signal by the fungal cell.
  • An example of a suitable Golgi retention signal includes but is not limited to the Kre2 signal, in particular it may be the first 100 amino acids of the KRE2 gene from Saccharomyces cerevisiae, which is shown in SEQ ID NO: 7, as describe e.g.
  • said Golgi retention signal may be located up ⁇ stream of the 5'-end of the nucleic acid sequence encoding the GnT1 , in particular it may lo ⁇ cated right next to said 5'-end in the open reading frame (ORF) encoding the GnT1, so that the Golgi retention signal is translated together with the GnT1 as one amino acid sequence.
  • the second nucleic acid construct may further comprise a constitutive promoter, such as one of those mentioned above, more particularly the tpiA or the gpd promoter.
  • the third nucleic construct of the present invention comprises a nucleic acid sequence encoding a mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase also known as Mannosi- dase II.
  • Mannosidase Il have been classified as belonging to E.G. 3.2.1.114 and they are en ⁇ zymes capable of catalysing the hydrolysis of the terminal 1 ,2-linked alpha-D-mannose resi ⁇ dues in the oligosaccharide GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2.
  • the Mannosidase Il may be of a mammalian origin as only mammalian homologous of this enzyme has been identified so far.
  • the present invention is not limited to Mannosidase Il of mammalian origin as it is envisioned that Mannosidase Il from other organisms may be identified.
  • the Mannosidase Il may be of human or rat origin.
  • the Mannosidase Il may com ⁇ prise the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 9.
  • it may consist of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 9.
  • a synthetic gene encoding a mam ⁇ malian Mannosidase Il may in particular be used, e.g. a synthetic gene which has been modi- fied to codon optimize and/or to remove cryptic intron(s).
  • the nucleic acid sequence encoding the Mannosidase Il may in particular comprise the nucleic acid sequence shown in SEQ ID NO:8, or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nu ⁇ cleic acid sequence shown in SEQ ID NO: 8. In another embodiment it may consist of the nu- cleic acid sequence shown in SEQ ID NO: 8 or an nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence shown in SEQ ID NO: 8.
  • it may comprise the CDS shown in SEQ ID NO:8, or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 8.
  • it may con ⁇ sist of the CDS shown in SEQ ID NO: 8 or an nucleic acid sequence having at least 70% iden ⁇ tity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 8.
  • the Mannosidase Il may in particular be lo ⁇ cated in Golgi, and those Golgi retention signal described for GnT1 may also be used to local- ise the Mannosidase Il in Golgi.
  • the third nucleic acid construct may further comprise a Golgi retention signal, such as the Kre2 signal described above, in particular it may be the first 100 amino acids of the KRE2 gene from Saccharomyces cerevisiae (shown in SEQ ID NO: 7) as describe e.g. by Vervecken W et al. (2004), Applied and Environmental Microbiology, p.2639-2946.
  • said Golgi retention signal may be located upstream of the 5'-end of the nucleic acid sequence encoding the Mannosidase II, in particular it may be located right next to said 5'-end in the open reading frame (ORF) encoding the Mannosidase II, so that the Golgi retention signal is translated together with the Mannosidase Il as one amino acid se ⁇ quence.
  • ORF open reading frame
  • the third nucleic acid construct may further comprise a con- stitutive promoter controlling the expression of mannosyl-oligosaccharide 1 ,3-1 ,6-alpha- mannosidase, such as one of those mentioned above, more particularly the tpiA or the gpd promoter.
  • a con- stitutive promoter controlling the expression of mannosyl-oligosaccharide 1 ,3-1 ,6-alpha- mannosidase, such as one of those mentioned above, more particularly the tpiA or the gpd promoter.
  • the fourth nucleic acid construct of the present invention comprises a nucleic acid se- quence encoding an alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, which is also known as GnT2 and these terms may be used interchangeably in the context of the present invention.
  • GnT2's have been classified as belonging to E.C.
  • the GnT2 may be of a mammal ⁇ ian origin as only mammalian homologous of this enzyme has been identified so far.
  • the present invention is not limited to GnT2 of mammalian origin as it is envisioned that GnT2 from other organisms may be identified.
  • the GnT2 may be of human or rat origin.
  • the GnT2 may comprise the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 11.
  • it may consist of the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% iden ⁇ tity with the amino acid sequence shown in SEQ ID NO: 11.
  • the nucleic acid sequence encoding the GnT2 may in particular comprise the nucleic acid sequence shown in SEQ ID NO: 10, or it may be comprise a nucleic acid sequence hav- ing at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence shown in SEQ ID NO: 10. In another embodiment it may consist of the nucleic acid sequence shown in SEQ ID NO: 10 or an nucleic acid sequence having at least 70% iden ⁇ tity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence shown in SEQ ID NO: 10.
  • it may comprise the CDS shown in SEQ ID NO: 10, or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 10. In another embodiment it may consist of the CDS shown in SEQ ID NO: 10 or an nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NOMO.
  • a synthetic gene encoding a mammalian GnT2 may in particular be used, such as a synthetic gene which has been modified to codon optimize and/or to remove cryptic intron.
  • the GnT2 may in particular be located in Golgi, and those Golgi retention signal described for GnT1 may also be used to localise the GnT2 in Golgi.
  • the fourth nucleic acid construct may further comprise a Golgi retention signal, such as the Kre2 signal described above, in particular it may be the first 100 amino acids of the KRE2 gene from Saccharomyces cerevisiae (shown in SEQ ID NO: 7) as describe e.g. by Vervecken W et al. (2004), Applied and Environmental Microbiol ⁇ ogy, p.2639-2946.
  • a Golgi retention signal such as the Kre2 signal described above, in particular it may be the first 100 amino acids of the KRE2 gene from Saccharomyces cerevisiae (shown in SEQ ID NO: 7) as describe e.g. by Vervecken W et al. (2004), Applied and Environmental Microbiol ⁇ ogy, p.2639-2946.
  • said Golgi retention signal may be located upstream of the 5'- end of the nucleic acid sequence encoding the GnT2, in particular it may located right next to said 5'-end in the open reading frame (ORF) encoding the GnT2, so that the Goigi retention signal is translated together with the GnT2 as one amino acid sequence.
  • ORF open reading frame
  • the fourth nucleic acid construct may further comprise a constitutive promoter controlling the expression of the alpha-1 ,6-mannosyl-glycoprotein 2-beta- N-acetylglucosaminyltransferase, such as one of those mentioned above, more particularly the tpiA or the gpd promoter.
  • the fifth nucleic acid construct of the present invention comprises a nucleic acid se- quence encoding a beta-1 ,4-galactosyltransferase (GaIT), which are enzymes that have been classified as belonging to E.G. 2.4.1.38 and are capable of catalyzing the following reaction:
  • GaIT beta-1 ,4-galactosyltransferase
  • GaIT with regard to the modification of N-glycans is to catalyse the binding of galactose (GaI) to GlcNAcMan5GlcNAc2 to produce GalGlcNAcMan5GlcNAc2 or to bind GaI to GlcNAc2Man3GlcNAc2 to produce Gal(0-2)GlcNAc2Man3GlcNAc2.
  • GaI galactose
  • the binding of GaI to GlcNAc2Man3GlcNAc2 typically results in the formation of four different N-glycan structures; GO which denotes GlcNAc2Man3GlcNAc2 (no GaI residue was bound), G1 which denotes GalGlcNAc2Man3GlcNAc2 (one GaI residue was bound) where the GaI residue is bound either to the 1 ,3 arm or the 1 ,6 arm of GlcNAc2Man3GlcNAc2 and G2 which denotes Gal2GlcNAc2Man3GlcNAc2 (two GaI residues were bound, one GaI residue bound to the 1 ,3 arm and one to the 1 ,6 arm of GlcNAc2Man3GlcNAc2).
  • At least 10%, such as between 10-50%, 10-40%, 10-30%, 15-50%, 15-40%, 15-35%, 20-40%, 25-35% of the Gal(0-2)GlcNAc2Man3GlcNAc2 structures are GaI(I )GlcNAc2Man3GlcNAc2, wherein GaI(I )GlcNAc2Man3GlcNAc2 includes both of the above mentioned forms with the GaI bound to the 1 ,3 arm or the 1 ,6 arm, respectively.
  • the fraction of Gal(2) GlcNAc2Man3GlcNAc2 structures may in particular be less than 15%, such as between 0-15%, 0-10%, 0-5%, 0-3% or 1-5%. This may in particular be relevant if e.g. a mammalian antibody is expressed in a fungal cell of the present invention as approximately 30% of the molecules of a particular antibody expressed in humans has the GaI(I )GlcNAc2Man3GlcNAc2 structure while the other 70% has the Gal(0)GlcNAc2Man3GlcNAc2.
  • GaIT only catalyze binding of GaI to the 1 ,3 arm of GIcNAcMan5GlcNAc2.
  • GaIT only catalyze binding of GaI to the 1 ,3 arm of GIcNAcMan5GlcNAc2.
  • GlcNAcMan5GlcNAc2 no GaI residue was bound
  • GalGlcNAcMan5GIcNAc2 one GaI residue was bound
  • the GaIT may be of a mammalian origin as only mammalian homologous of this enzyme has been identified so far.
  • the present invention is not limited to GaIT of mammalian origin as it is envisioned that GaIT from other organisms may be identified.
  • the GaIT may be of human or rat origin.
  • the GaIT may comprise the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 6.
  • it may consist of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the amino acid sequence shown in SEQ ID NO: 6.
  • the nucleic acid sequence encoding the GaIT may in particular comprise the nucleic acid sequence shown in SEQ ID NO:5, or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence shown in SEQ ID NO: 5. In another embodiment it may consist of the nucleic acid sequence shown in SEQ ID NO: 5 or an nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence shown in SEQ ID NO: 5.
  • it may comprise the CDS shown in SEQ ID NO:5, or it may comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 5.
  • it may con ⁇ sist of the CDS shown in SEQ ID NO: 5 or an nucleic acid sequence having at least 70% iden ⁇ tity, such as 75% or 80% or 85% or 90% or 95% identity with the CDS shown in SEQ ID NO: 5.
  • it may comprise the nucleic acid sequence encoding the ma ⁇ ture peptide shown in SEQ ID NO:5, or it may be comprise a nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence encoding the mature peptide shown in SEQ ID NO: 5. In another embodiment it may consist of the nucleic acid sequence encoding the mature peptide shown in SEQ ID NO: 5 or an nucleic acid sequence having at least 70% identity, such as 75% or 80% or 85% or 90% or 95% identity with the nucleic acid sequence encoding the mature peptide shown in SEQ ID NO: 5.
  • a synthetic gene encoding a mammalian GaIT may in particular be used including a synthetic gene, such as a synthetic gene which has been modified for the purpose of codon optimization and/or to remove cryptic intron(s).
  • the GaIT may in particular be located in Golgi, and those Golgi retention signal described for GnT1 may also be used to localise the GaIT in Golgi, such as the Kre2 signal described above, in particular it may be the first 100 amino acids of the KRE2 gene from Saccharomyces cerevisiae (shown in SEq ID NO: 7) as described e.g. by Vervecken W et al. (2004), Applied and Environmental Microbiology, p.2639- 2946.
  • said Golgi retention signal may be located upstream of the 5'-end of the nu- cleic acid sequence encoding the GaIT, in particular it may be located right next to said 5'-end end in the open reading frame (ORF) encoding the GaIT, so that the Golgi retention signal is translated together with the GaIT as one amino acid sequence.
  • ORF open reading frame
  • the fifth nucleic acid construct may further comprise a con ⁇ stitutive promoter, such as one of those mentioned above, more particularly the tpiA or the gpd promoter.
  • yeast cells have been found to comprise an endogenous gene encoding alpha-
  • 1 ,6-mannosyltransferase which are enzymes capable of binding a mannose residue by an al ⁇ pha-1 , 6-bond to the 1 ,3 arm of Man9GlcNAc2. If this reaction takes place in a fungal cell of the present invention it may be an advantage to inactivate such endogenous enzymes as the pres ⁇ ence of a mannose residue on the 1,3 arm of Man9GlcNAc2 bound by an alpha-1, 6-bond is believed to inhibit the action of the alpha-1 ,2-mannosidase and thereby inhibit the processing of Man9GlcNAc2 to Man5GlcNAc2.
  • At least one endogenous gene encoding an alpha-1 ,6-mannosyltransferase capable of catalyzing the binding of mannose by alpha-1 ,6-bonds to Man9GlcNAc2, in a fungal cell of the present invention has been modified so that said cell forms fewer alpha-1 , 6 mannose bonds than a parental fungal cell.
  • an alpha-1 ,6-mannosyltransferase called MNN9 has been identified, the amino acid sequence of which is shown in SEQ ID NO: 12.
  • an endogenous gene encoding an alpha-1 ,6-mannosyltransferase which has at least 40%, e.g. 50% or 60% or 70% or 80% or 90% identity with the sequence of SEQ ID NO: 12 has been modified in a fungal cell of the present invention so that said cell forms fewer alpha-1 ,6 mannose bonds than a parental fungal cell.
  • Man9GIcNAc2 The presence of a mannose residue bound by an alpha-1 ,6-bond to Man9GIcNAc2 may be measured as described by Jackson P and Gallagher JT, 1997, A laboratory guide to Glycoconjugate analysis, Biomethods, vol. 9, Birkha ⁇ ser, Basel, Switzerland.
  • the nucleic acid constructs of the present invention may in particular be expression vectors capable of facilitating expression of one of the N-glycosylation enzymes of the present invention (the alpha-1 ,2-mannosidase, alpha-1 ,3-mannosyl-glycoprotein 2-beta-N- acetylglucosaminyltransferase, mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase, alpha- 1 ,6-mannosyI-glycoprotein 2-beta-N-acetylglucosamintransferase and beta-1 ,4- galactosyltransferase) which is encoded by the nucleic acid sequence comprised in nucleic acid construct, when said nucleic acid construct is inserted into a fungal cell.
  • the alpha-1 ,2-mannosidase alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-
  • Expression includes transcription and translation of the nucleic acid sequence into a polypeptide sequence.
  • this usually also includes secretion of said polypeptide.
  • the choice of expression vector will often depend on the host cell into which it is to be introduced.
  • a suitable vector include a linear or closed circular plasmid or a virus.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an ex- trachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromo ⁇ some.
  • the vector may contain any means for assuring self-replication.
  • origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARSl
  • the origin of replication may be one having a mutation which makes it function as temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).
  • the vector may be one which, when introduced into the host cell, is inte ⁇ grated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • Vectors which are integrated into the genome of the host cell may contain any nu ⁇ cleic acid sequence enabling integration into the genome; in particular it may contain nucleic acid sequences facilitating integration into the genome by homologous or non-homologous re ⁇ combination.
  • the vector system may be a single vector, e.g. plasmid or virus, or two or more vectors, e.g. plasmids or virus', which together contain the total nucleotide sequence to be in ⁇ troduced into the genome of the host cell, or a transposon.
  • the vector may in particular be an expression vector in which the nucleic acid se- quence encoding the polypeptide is operably linked to additional segments or control se ⁇ quences required for transcription of the DNA.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. tran ⁇ scription initiates in a promoter and proceeds through the nucleotide sequence encoding the modified polypeptide or the parent polypeptide.
  • Additional segments or control sequences in ⁇ clude a promoter, a leader, a polyadenylation sequence, a propeptide sequence, a signal se- quence and a transcription terminator. At a minimum the control sequences include a promoter and transcriptional and translational stop signals.
  • the promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for use in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei as- partic proteinase, Aspergillus niger neutral alpha amylase, Aspergillus niger acid stable alpha- amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like proteas
  • promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral (alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters.
  • promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099), the tpiA promoter or the gpd promoter.
  • the tpiA pro ⁇ moter has been described in McKnight GL et al, 1986, Cell, 46 (1), pp. 143-147 and the gpd promoter has been described in Punt PJ et al, 1990, Gene, 93, pp. 101-109.
  • promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawa ⁇ saki, J. MoI. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPH (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters. Further useful promoters are obtained from the Saccharomyces cerevisiae enolase
  • ENO-1 gene the Saccharomyces cerevisiae galactokinase gene (GAL1 ), the Saccharomy ⁇ ces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene.
  • GAL1 Saccharomyces cerevisiae galactokinase gene
  • ADH2/GAP Saccharomy ⁇ ces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes
  • Saccharomyces cerevisiae 3-phosphoglycerate kinase gene Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.
  • useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
  • SV40 Simian Virus 40
  • RSV Rous sarcoma virus
  • BPV bovine papilloma virus
  • the nucleic acid sequence encoding a polypeptide may also, if necessary, be operably connected to a suitable terminator.
  • the recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which pro ⁇ vides for prototrophy or auxotrophs.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrex
  • 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 (phosphinothricin acetyltrans-ferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance mark ⁇ ers, as well as equivalents from other species.
  • amdS acetamidase
  • argB ornithine carbamoyltransferase
  • bar phosphinothricin acetyltrans-ferase
  • hygB hygromycin phospho
  • amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.
  • selection may be accomplished by co- transformation, e.g., as described in WO 91/17243, where the selectable marker is on a sepa ⁇ rate vector.
  • An expression vector typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and optionally a repressor gene or various activator genes.
  • Filamentous fungal host cells may be transformed by a process involving protoplast for- mation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.
  • Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023, EP 184,438, and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81 :1470-1474.
  • a suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147-156 or in co-pending US Serial No. 08/269,449. Proteins
  • the present invention also relates to a method of producing a protein with an altered N- glycan structure comprising culturing a fungal cell of the present invention comprising a nucleic acid sequence encoding the protein and recovering the protein.
  • An altered N-glycan structure is to be understood as an N-glycan structure which is different from the N-glycan structure of said protein when expressed naturally.
  • Said protein may be a protein which is naturally ex ⁇ pressed by the cell or it may in particular be a protein which is not naturally expressed by the cell, such as a protein of mammalian origin.
  • the protein may be of mammalian origin.
  • mammalian origin is intended to encompass amino acid se ⁇ quences, e.g. proteins, or nucleic acid sequences which have been isolated from a mammalian cell. However, it is also intended to encompass such sequences which are identical to a se ⁇ quence isolated from a mammalian cell but which has been chemically synthesised or ex ⁇ pressed by a non-mammalian cell using e.g. well-known methods of gene technology.
  • Fur- thermore it is also intended to encompass sequences which comprise minor differences as compared to the mammalian counterpart such as codon optimization, i.e. changes in the nu ⁇ cleic acid sequences which do not affect the amino acid sequence and/or the removal of cryp ⁇ tic introns.
  • the protein may be a monoclonal antibody (mAb), e.g. a human mAb or antibody fragments, such as Fab, single chain antibodies, diabodies or triabod- ies.
  • mAb monoclonal antibody
  • the heavy chain of a human IgG antibody contains only one N-linked glycan in the con ⁇ stant domain, known to be important for the effect and/or function of antibodies.
  • a human mAb wherein one or both of heavy chains of said mAb comprises a Man5GlcNAc2, or a GlcNAcMan5GlcNAc2, or GalGlcNAcMan5GlcNAc2, or a GlcNAcMan3GlcNAc2, or a GlcNAc2Man3GlcNAc2a or a Gal(0-2)GlcNAc2Man3GlcNAc2 N- glycan structure may be produced in a fungal cell of the present invention.
  • the heavy chains may comprise a Gal1GlcNAc2Man3GlcNAc2 N-glycan structure while approximately 70% of the heavy chains may comprise a GalOGIcNAc2Man3GlcNAc2 N-glycan structure.
  • An Aspergillus oryzae Mnn9 homologue is identified.
  • the mnn9 gene product is in- volved in alfa-1 ,6 mannose bond formation.
  • the mnn9 gene is disrupted from HowB425 (WO 98/12300) by standard gene disruption methods applying the pyrG gene as selective marker.
  • Disruptants are identified by Southern blot analysis.
  • a pyrG mutant is selected by first selecting for growth on minimal plates containing uridine and 5-flouroorotic acid (5-FOA), and secondly demonstrate that the selected strains can be complemented by transformation with the Asper- gillus oryzae pyrG gene.
  • the selected strain is termed Aspergillus oryzae mnn9-A.
  • the A. nidulans mannosidase 1C gene (SEQ ID NO: 1) is cloned into an Aspergillus oryzae expression vector down-stream of the gapdh (glycer aldehyde phosphate dehydro ⁇ genase) promoter.
  • the mannosidase is C-terminally fused to a sequence encoding the amino- acid sequence His-Asp-Glu-Leu. These four amino acids constitute an ER retention signal that will localize the mannosidase to the ER.
  • the expression vector further more harbors the A. oryzae pyrG gene as a selective marker flanked by an upstream sequence for the A.
  • oryzae alkaline protease alp (SEQ ID NO: 3 of WO 98/12300), the 5' end of the gapdh promoter up ⁇ stream the pyrG gene and flanked by the mannosidase expression cassette and the down ⁇ stream sequence for the alkaline protease downstream the pyrG gene.
  • the DNA constructs are liniarized so that the alkaline protease flanking sequences are at the ends of the plasmid are transformed into Aspergillus oryzae mnn9-A.
  • Transformants are analyzed by sourthem blot analysis, and a transformant of each mannosidase construct where the pyrG gene and the mannosidase expression cassette replaced the alkaline protease is se ⁇ lected.
  • the effect of the transformation on glycosylates is analysed using the Thermomyces lanuginosus lipase (the nucleic-and amino acid sequence of which is shown in US 5869438) as a reporter protein as this contains one N-glycosylation site.
  • This lipase gene is transformed into the selected strains using amdS based expression plasmids and the TAKA promoter as described in patent WO 91/17243. It is confirmed that the N-linked structure of the lipase consist of Man5GlcNAc2.
  • pyrG mutants are selected by first selecting for growth on minimal plates containing uridine and 5-FOA, and secondly demon ⁇ strate that the selected strain can be complemented by transformation with the Aspergillus oryzae pyrG gene.
  • the selected strain is termed Aspergillus oryzae AnMan (for the Aspergillus nidulans mannosidase strain).
  • the human GnT 1 (alpha-1 ,3-mannosyl-glycoprotein beta-1 , 2-N- acetylglucosaminyltransferase) gene is expressed in Aspergillus oryzae in the following way: A synthetic gene codon optimized for Aspergillus oryzae encoding the human GnT 1 gene (the amino acid sequence shown in SEQ ID NO: 2) is designed and cloned into an Aspegillus oryzae expression vector downstream of the tpi (those phosphate isomerase) pro ⁇ moter.
  • tpi those phosphate isomerase
  • the mannosidase is N-terminal fused to an alpha-1 ,2-mannosyl-transferase (Kre2p) homologue Golgi retention signal.
  • the expression vector further more harbors the A. oryzae pyrG gene as a selective marker flanked by an upstream sequence for the A. oryzae Neutral metalloprotease I (SEQ ID NO: 2 of WO 98/12300), the 5' end of the tpi promoter upstream the pyrG gene and flanked by the GnT 1 expression cassette and the downstream sequence for the neutral metallo protease downstream the pyrG gene.
  • the DNA constructs are liniarized so that the neutral metallo protease flanking se- quences are at the ends of the plasmid before they are transformed into Aspergillus oryzae AnMan. Transformants are analyzed by Sourthem blot analysis, and a transformant where the pyrG gene and the GnT 1 expression cassette has replaced the neutral metallo protease is selected.
  • pyrG mutants are selected as in example 1.
  • the selected strain is termed Aspergillus oryzae GnT1.
  • Mannosidase Il Mannosyl-oligosaccharide 1 ,3-1,6-alpha-mannosidase
  • a synthetic gene codon optimized for Aspergillus oryzae encoding the human man ⁇ nosidase Il gene (the amino acid sequence is shown in SEQ ID NO: 9) is designed and cloned into an Aspegillus oryzae expression vector downstream of the tpi (triose phosphate isom ⁇ erase) promoter.
  • the mannosidase is N-terminal fused to an alpha-1 ,2 mannosyl-transferase (Kre2p) homologue Golgi retention signal.
  • the expression vector furthermore harbours the A. oryzae pyrG gene as a selective marker flanked by an upstream sequence for the A.
  • oryzae pepE gene (SEQ ID NO: 3 in WO 97/22705), the 5' end of the tpi promoter upstream the pyrG gene and flanked by the mannosidase Il expression cassette and the downstream sequence for the pepE gene downstream the pyrG gene.
  • the DNA constructs are liniarized so that pepE flanking sequences are at the ends of the plasmid and are transformed into Aspergillus oryzae GnT1. Transformants are analyzed by Sourthern blot analysis, and a transformant where the pyrG gene and the mannosidase Il ex ⁇ pression cassette replaced the pepE gene is selected.
  • the human GnT 2 (Alpha-1,6-mannosyl-glycoprotein 2-beta-N- acetylglucosaminyltransferase), gene is expressed in Aspergillus oryzae in the following way:
  • a synthetic gene codon optimized for Aspergillus oryzae encoding the human GnT 2 gene (the amino acid sequence is shown in SEQ ID NO: 11) is designed and cloned into an Aspegillus oryzae expression vector downstream of the tpi (triose phosphate isomerase pro ⁇ moter.
  • the mannosidase is N-terminal fused to an alpha-1 ,2-mannosyltransferase (Kre2p) homologue Golgi retention signal.
  • the expression vector further more harbors the A. oryzae pyrG gene as a selective marker flanked by an upstream sequence for the A.
  • oryzae pepC gene (SEQ ID NO: 1 in WO 97/22705)), the 5' end of the tpi promoter upstream the pyrG gene and flanked by the GnT 2 expression cassette and the downstream sequence for the pepC gene downstream the pyrG gene.
  • the DNA constructs are liniarized so that pepC flanking sequences are at the ends of the plasmid are transformed into Aspergillus oryzae GnTl Transformants are analyzed by Sourthern blot analysis, and a transformant where the pyrG gene and the GnT2 expression cassette replaced the pepC gene is selected.
  • pyrG mutants are selected as in example 1.
  • the selected strain is termed Aspergillus oryzae GnT2.
  • the human galT (beta-1,4-galactosyltransferase) gene is expressed in Aspergillus oryzae in the following way: A synthetic gene codon optimized for Aspergillus oryzae encoding the human galT gene (the amino acid sequence is shown in SEQ ID NO: 6) is designed and cloned into an Aspegillus oryzae expression vector downstream of the tpi (triose phosphate isomerase) pro ⁇ moter. The mannosidase is N terminal fused to the kexB Golgi retention signal. The expression vector further more harbors the A. oryzae pyrG gene as a selective marker flanked by an up- stream sequence for the A. oryzae AMG gene, the 5' end of the tpi promoter upstream the pyrG gene and flanked by the galT expression cassette and the downstream sequence for the AMG gene downstream the pyrG gene.
  • the DNA constructs are liniarized so that AMG flanking sequences are at the ends of the plasmid are transformed into Aspergillus oryzae GnTL Transformants are analyzed by Sourthem blot analysis, and a transformant where the pyrG gene and the galT expression cas ⁇ sette replaced the AMG gene is selected.
  • pyrG mutants are selected as in example 1.
  • the selected strain is termed Aspergillus oryzae GaIT.

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Abstract

L'invention concerne des procédés de modification de la structure de N-glycanes dans des cellules fongiques, et des cellules fongiques à structure de N-glycanes modifiée.
PCT/DK2005/000569 2004-09-07 2005-09-07 Structure de n-glycanes modifiee dans un champignon WO2006026992A1 (fr)

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WO2012069593A3 (fr) * 2010-11-24 2013-01-03 Novartis International Pharmaceutical Ltd. Enzymes de fusion
AU2015333921B2 (en) * 2014-10-13 2018-12-13 Dow Agrosciences Llc Copi coatomer gamma subunit nucleic acid molecules that confer resistance to coleopteran and hemipteran pests
CN110121365A (zh) * 2016-12-29 2019-08-13 财团法人生物技术开发中心 制备糖蛋白-药物偶联物的方法
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012069593A3 (fr) * 2010-11-24 2013-01-03 Novartis International Pharmaceutical Ltd. Enzymes de fusion
US9399764B2 (en) 2010-11-24 2016-07-26 Novartis International Pharmaceutical, Ltd. Fusion enzymes
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi
AU2015333921B2 (en) * 2014-10-13 2018-12-13 Dow Agrosciences Llc Copi coatomer gamma subunit nucleic acid molecules that confer resistance to coleopteran and hemipteran pests
CN110121365A (zh) * 2016-12-29 2019-08-13 财团法人生物技术开发中心 制备糖蛋白-药物偶联物的方法

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