WO2006026992A1

WO2006026992A1 - Altered structure of n-glycans in a fungus

Info

Publication number: WO2006026992A1
Application number: PCT/DK2005/000569
Authority: WO
Inventors: Christel Thea JØRGENSEN; Carsten Hjort
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2004-09-07
Filing date: 2005-09-07
Publication date: 2006-03-16
Anticipated expiration: 2007-03-07

Abstract

The present invention relates to methods for altering the structure of N-glycans in fun-gal cells and to fungal cells having an altered N-glycan structure.

Description

TITLE: ALTERED STRUCTURE OF N-GLYCANS IN A FUNGUS

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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. H. Freeman and Company). 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. Typically the first steps in the N-glycosylation pathway in humans and filamentous fungi, which take place in the endoplasmic reticulum (ER), both lead to the formation of the Man8GlcNAc2 or the Man9GlcNAc2 structure (Manδ or Man9, respectively). In humans Manδ and Man9 is further modified in the Golgi where the following sequential steps take place (Hamilton SR et al. (2003), Science, 301, 1244-1246):

a) Manδ or Man9 is processed by alpha-1 ,2-mannosidase 1A, 1 B and 1C to produce Man5 b) Manδ is processed by beta-1 ,2-Λ/-acetylglucosaminyltransferase I to produce GlcNAcManδ c) GlcNAcManδ is processed by Mannosidase Il to produce GlcNAcMan3 d) GlcNAcMan3 is processed by beta-1 ,2-Λ/-acetylglucosaminyltransferase Il (GnTII) to produce GlcNac2Man3

In yeast more mannose-residues are added to the Manδ or Man9 structures to create so called high mannose structures (see e.g. Hamilton et al. (2003), Science, 301 , 1244-1246). The structure of N-glycans from filamentous fungi is more diverse and only a few have been char¬ acterized. Maras M et al. (1999), Glycoconjugate Journal 16, table 2 (page 22) discloses an overview of partially or completely characterised oligosaccharides on glycoproteins from filamentous fungi. US 5,834,251 discloses methods of converting high mannose type glycosylation patterns to hybrid or complex type glycoslyation patterns.

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.

Maras M et al. (1999), FEBS Letters 452, 365-370 discloses in vivo synthesis of complex N- glycans by expression of human /V-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei.

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.

SUMMARY OF THE INVENTION

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

b) a second nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase

c) a third nucleic acid construct comprising a nucleic acid sequence encoding a mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase

d) a fourth nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase

e) a fifth nucleic acid construct comprising a nucleic acid sequence encoding a beta-

1 ,4-galactosyltransferase In a second aspect 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

b) a second nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetyl- glucosaminyltransferase

e) a fifth nucleic acid construct comprising a nucleic acid sequence encoding a beta-1 ,4-galactosyltransferase

wherein said cell has an altered N-glycan structure as compared to a parental fungal cell.

BRIEF DESCRIPTION OF DRAWINGS

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

"diamond (♦)" is a galactose residue.

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.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and short terms

The term "Man" is in the context of the present invention used as a short term for man- nose.

The term "GIcNAc" is in the context of the present invention used as a short term for N- acetylglucosamin.

The term "GaI" is in the context of the present invention used as a short term for a ga¬ lactose. The term "Man9" is used as a short term for Man9GlcNAc2, the term "Manδ" is used as a short term fro Man8GlcNAc2, the term "Man5" is used as a short term for Man5GlcNAc2 and Man3 is used as a short term for Man3GlcNAc2.

The term "ER" is used as short-term for endoplasmic reticulum. The term "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.

In the context of the present invention, the term "E. C." (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.

The term "protein" is in the context of the present invention intended to include polypep¬ tides and combinations of polypeptides. The term "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.

The term "recessive" when used in reference with a nucleic acid sequence or an allele is to be understood as an allele which is obscured in the phenotype of a heterozygote by the dominant allele, often due to inactivity or absence of the product of the recessive allele.

The term "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.

The term "open reading frame (ORF)" is in the context of the present invention to be understood with its general meaning, i.e. as a nucleic acid sequence that contains a signal for the start of translation, followed in the correct register by a sufficient length of amino acid- encoding nucleotide triplets to form a protein, followed by a signal for translation.

For purposes of the present invention, 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).

Multiple alignments of protein sequences may be made using "ClustalW" (Thompson, J. D., Higgins, D. G. and Gibson, TJ. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). Multiple alignment of DNA se- quences may be done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence. Methods of the present invention

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-

1 ,2-mannosidase obtained from a filamentous fungi b) a second 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 The term "altered N-glycan structure" is to be understood as the structure of N- glycan(s) present on a protein produced by a fungal cell of the present invention is different from the N-glycan structure of the same protein produced by a fungal cell which differs from the former cell by having been modified by a method of the present invention and/or by com¬ prising a first, second, third, fourth or fifth or a combination of any of these nucleic acid con- struct of the present invention. In particular 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. Thus in another embodiment 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- haϋser, Basel, Switzerland.

Thus 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.

In a particular embodiment 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. Although in principle 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.

In a particular embodiment of the present invention 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. In the context of the present inven¬ tion 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- haϋser, Basel, Switzerland

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.

In one embodiment of the present invention 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.

In another embodiment of the present invention 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. Particularly, 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.

If 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. Thus in this case if 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. If the route depicted to the right in figure 1 is to be followed (steps 2b-3b) then 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.

If two or more fungal cells of the present invention are fused to create a hetero- karyon with an altered N-glycan structure the insertion into each individual cell of a combina- tion of nucleic acid constructs of the present invention is envisioned to be different from that of a single cell. For modification of single cell it is necessary to insert each of the relevant nucleic acid constructs into said cell. However, the inventors of the present invention believe that the alteration of the N-glycan structure in one fungal cell will result in the same alteration of a heterokaryon if said cell is fused with one or more other cells to create a heterokaryon. Thus if a combination of nucleic acid sequences of the present invention are to be inserted into a fungal cell and said cell is fused to another cell to create a heterokaryon the insertion of nucleic acid constructs into that particular fungal cell may not necessarily be stepwise as indicated in figure 1. However, the overall modification of the N-glycan structure of the heterokaryon may in particular be stepwise as outlined in figure 1. For example if two genetically different fungal cells of the present invention are used to create a heterokaryon which comprises two or more nucleic acid constructs of the present invention the following combinations for modification of each of the cells are envisioned (the possibility of inserting all of the nucleic acid constructs into one of the cells and none into the other also exists for each of below situations, but is not mentioned):

For a 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.

For a 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.

a 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.

a 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.

If the methods of the present invention are used to create a heterokaryon comprising only one nucleic acid construct of the present invention said 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. However, if the 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. An advantage of dividing the nucleic acid constructs between the different cells which are fused to form the heterokaryon (in contrast to inserting them all into the same cell) is that the same selection marker(s) may be used in each of the cells. Thus a smaller number of different selection markers are necessary as compared to if all nucleic acid constructs were to be inserted into the same cell. Although, 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. If 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. The nuclei of a 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.

Fungal cell

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.

Reference to "fungal cell" is 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). Representative groups of Ascomy- cota include, e.g., Neurospora, Eupenicillium (=Penicillium), Emericella (= Aspergillus), Eu- rotium (=Aspergillus), and the true yeasts listed below. Examples of 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.

In one embodiment, 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. 9, 1980. The biology of 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).

In a more particular embodiment, the yeast cell is a cell of a species of Candida, Kluy- veromyces, Saccharomyces, Schizosaccharomyces, Pichia, or Yarrowia. In a most particular embodiment, the yeast cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomy¬ ces norbensis or Saccharomyces oviformis cell. In another embodiment, the yeast cell is a Kluyveromyces lactis cell. In yet another embodiment, the yeast cell is a Yarrowia lipolytica cell. In another embodiment, 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. In a most particular embodiment, the filamentous fungal cell is an Aspergillus awamori, Asper¬ gillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most particular embodiment, 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. In still another embodiment, the filamentous fun¬ gal cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. In another particular embodiment, 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.

If 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. However, if 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. In the context of the present invention 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. In particular 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.

In particular the 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. Thus 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. Examples of 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.

Methods for formation of a heterokaryon filamentous fungus are described in US 6,543,745.

Examples of filamentous fungi which may be fused to form a heterokaryon filamentous fungus include those described below as filamentous fungal host cells. In particular different strains of Aspergillus, e.g. A.oryzae or A.niger, Fusarium or Trichoderma may be used to form a heterokaryon filamentous fungus. In principle, 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. In particular the heterokaryon filamentous fungus of the present invention is formed by fusion of two different strains of filamentous fungi.

Examples of 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. In particular 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. In the case of fusion of different fungi, however, at least two different characteristics/markers are used for the selection. Exam¬ ples of characteristics or markers which may be used include those described above as se¬ lectable markers useful for the nucleic acid construct. Examples of genes which may make a fungus autotroph 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.

In another embodiment the heterokaryon fungus is a yeast heterokaryon. In 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). Thus in a particular embodiment of the present invention 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. The cloning of kar1 in yeast is described in Rose MD and Fink GR, Cell, 1987, 48, 1047-1060. Methods of transformation of fungi are well known and may be performed as described below for the fungal host cells. 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.

In a particular embodiment 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.

If 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.

In a particular embodiment of the present invention 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-glycosylation enzymes and nucleic acid constructs

It is an object of the present invention to alter the structure of 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. However, 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. Cells, in particular eukaryotic cells are compartmentalized with different processes taking place in different compartments. The formation of the 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.

In the context of the present invention the term "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. In a particular embodiment 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. In another embodiment 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. Thus the 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.

In another embodiment 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. In another embodiment 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. For a correct functioning of the alpha-1 ,2-mannosidase, i.e. catalysing the hydrolysis of terminal 1 ,2-linked alpha-D-mannose residues in the oligosaccharide Man9GlcNAc2 so as to produce Man5GlcNAc2, it is an advantage if said enzyme is located in the ER.

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. In a particular embodiment of the present invention 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. gel- electrophoresis or by measuring the activity of the enzyme. Such methods are known to a per¬ son skilled in the art. The amount of Man5GlcNAc2 may be measured as described in Jackson P and Gallagher JT (1997), A laboratory guide to Glycoconjugate analysis, Biomethods, vol. 9, Birkhaϋser, Basel, Switzerland

As the alpha-1 ,2-mannosidase in particular may be located in the ER 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. Thus 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. To over-express the alpha-1 ,2-mannosidase 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.

Second nucleic acid construct

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). Alpha-1 , 3-mannosyl-glycoprotein 2-beta-N- acetlyglucosaminyltransferases have been classified as belonging to E.G. 2.4.1.101 which are enzymes capable of catalyzing the following reaction: UDP-N-acetyl-D-glucosamine + 3- (alpha-D-mannosyl)-beta-D-mannosyl-R -> UDP + 3-(2-[N-acetyl-beta-D-glucosaminyl]-alpha- D-mannosyl)-beta-D-mannosyl-R; wherein R represents the remainder of the N-linked oligo¬ saccharide in the glycoprotein acceptor and UDP is uridine diphosphate. In the context of the present invention the short term GnT1 may be used for alpha-1 , 3-mannosyl-glycoprotein 2- beta-N-acetlyglucosaminyltransferase.

The function of GnT1 with regard to the modification of N-glycans is to catalyse the ad¬ dition of N-acetylglucosamine to Man5GlcNAc2 to produce GlcNAcMan5GlcNAc2.

In a particular embodiment of the present invention the GnT1 may be of a mammalian origin as only mammalian GnT1 's have been identified so far. However, 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. For example, 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. In another embodiment 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. In another embodiment 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. As it is not trivial to express mammalian proteins in fungi 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. However, in a particular embodiment the sequence may have been modified so that it is more similar to the sequence encoding proteins naturally ex¬ pressed by fungi. For example 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 reason for this is that 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. Thus 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. Thus to obtain a particular amino acid at a given position it may be advantageous to change the codon of the mammalian sequence to a codon which the fungus use more frequently to. 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.

Another example of minor changes which may be made to the mammalian sequence includes removal of cryptic introns, e.g. as described in WO 97/49821. 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. Thus by modification of the nucleic acid sequence it is possible to avoid that 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.

For the GnT1 to function optimally, i.e. to add N-acetylglucosamine to Man5GlcNAc2, it is an advantage if said enzyme is located in the Golgi, in particular in the early Golgi, wherein "early Golgi" refers to that part of the Golgi which a protein enters from the ER in contrast to the "late Golgi" which is that part of the Golgi which an protein enters before it is e.g. secreted. As described above it is an advantage if the GnT1 reside in the Golgi. 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. Thus in a particu¬ lar embodiment the second nucleic acid construct may further comprise a Golgi retention sig- nal. In particular, 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. by Vervecken W et al. (2004), Applied and Environ- mental Microbiology, p.2639-2946. In particular 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.

In a particular embodiment 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.

Third nucleic acid construct

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.

In a particular embodiment of the present invention the Mannosidase Il may be of a mammalian origin as only mammalian homologous of this enzyme has been identified so far. However, 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. More particularly, the Mannosidase Il may be of human or rat origin. More particularly, 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. In another embodiment 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. Furthermore, as described above for GnT1 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.

In another embodiment 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. In another embodiment 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. Furthermore, as also described for GnT1; 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. Thus 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. In particular 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.

In a particular embodiment 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.

Fourth nucleic acid construct

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. 2.4.1.143, which are enzymes capable of catalyzing the following reaction: UDP-Λ/-acetyl-D-glucosamine + 6- (alpha-D-mannosyl)-beta-D-mannosyl-R -> UDP + 6-(2-[Λ/-acetyl-beta-D-glucosaminyl]-alpha- D-mannosyl)-beta-D-mannosyl-R, wherein R represents the remainder of the Λ/-linked oligo¬ saccharide in the glycoprotein acceptor and UDP is uridine diphosphate. The function of GnT2 with regard to the modification of N-glycans is to catalyze the binding of GIcNAc to GlcNAcMan3GlcNAc2 to produce GlcNAc2Man3GlcNAc2.

In a particular embodiment of the present invention the GnT2 may be of a mammal¬ ian origin as only mammalian homologous of this enzyme has been identified so far. However, the present invention is not limited to GnT2 of mammalian origin as it is envisioned that GnT2 from other organisms may be identified. More particularly, the GnT2 may be of human or rat origin. More particularly, 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. In another embodiment 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. In another embodiment 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.

Furthermore, as described above for GnT1 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.

Furthermore, as also described for GnT1 ; 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.

Thus in a particular embodiment 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. In particular 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.

In a particular embodiment 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.

Fifth nucleic acid construct

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:

UDP-galactose + N-acetyl-beta-D-glucosaminylglycopeptide -> UDP + beta-D- galactosyl-1 ,4-N-acetyl-beta-D-glucosaminylglycopeptide.

The function of 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. As shown in figure 2 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). In a particular em¬ bodiment of the present invention 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. Fur¬ thermore, 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.

In contrast to the above situation GaIT only catalyze binding of GaI to the 1 ,3 arm of GIcNAcMan5GlcNAc2. Thus for this reaction only two glycans are formed, i.e. GO and G1 , wherein the formed denotes GlcNAcMan5GlcNAc2 (no GaI residue was bound) and GalGlcNAcMan5GIcNAc2 (one GaI residue was bound) with the GaI residue bound to the 1 ,3 arm.

In a particular embodiment of the present invention the GaIT may be of a mammalian origin as only mammalian homologous of this enzyme has been identified so far. However, the present invention is not limited to GaIT of mammalian origin as it is envisioned that GaIT from other organisms may be identified. More particularly, the GaIT may be of human or rat origin. More particularly, 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. In another embodiment 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. In another embodiment 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. In another embodiment 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. In another embodiment 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.

Furthermore, as described above for GnT1 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).

Furthermore, as also described for GnT1 ; 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. In particular 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.

In a particular embodiment 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.

Alpha-1 ,6-mannosyltransferase

Some 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.

Thus in a particular embodiment of the present invention 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.

In Saccharomyces cerevisiae an alpha-1 ,6-mannosyltransferase called MNN9 has been identified, the amino acid sequence of which is shown in SEQ ID NO: 12. Thus in a par- ticular embodiment 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.

Methods for inactivation of endogenous genes are well known to a person skilled in the art and may be performed as described by e.g. Miller et al., 1985, Molecular and Cell Biology, 5, pp. 1714-1721 or Fincham JR, 1989, Microbiolgocial reviews, 53 (1), pp. 148-170. In par¬ ticular modification of such genes may result in that at least 50%, such as 60% or 70% or 80% or 90% or 95% of the N-glycans in the fungal cell does not contain any mannose residues bound to Man9GlcNAc by an alpha-1 ,6 bond. 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, Birkhaϋser, Basel, Switzerland.

General 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.

Expression includes transcription and translation of the nucleic acid sequence into a polypeptide sequence. For secreted polypeptides this usually also includes secretion of said polypeptide.

Expression vectors

The choice of expression vector will often depend on the host cell into which it is to be introduced. Examples of 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. Examples of 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).

Alternatively, 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. The term, "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. Examples of 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 protease (as described in U.S. Patent No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly, 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. Further suitable 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.

Examples of suitable 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. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488. In a mammalian host cell, useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV). 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 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. Particularly, for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, 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.

The procedures used to ligate the nucleotide sequences coding for the modified poly- peptide or the parent polypeptide of the present invention, the promoter and optionally the ter¬ minator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al.). 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.

Transformation of a fungal cell

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.

In a particular embodiment the protein may be of mammalian origin. In the context of the present invention the term "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.

In a particular embodiment 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. 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. Thus in a par¬ ticular embodiment 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. If it comprises a Gal(0-2)GlcNAc2Man3GlcNAc2 N-glycan structure approximately 30% of the heavy chains may comprise a Gal1GlcNAc2Man3GlcNAc2 N-glycan structure while approximately 70% of the heavy chains may comprise a GalOGIcNAc2Man3GlcNAc2 N-glycan structure.

MATERIALS AND METHODS EXAMPLES

Example 1

Trimming of high mannose glvcans to Man5GlcNac2.

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.

Having confirmed the desired glycosylation pattern, 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). Example 2

Adding N-acetyl glucosamine to Man5GlcNac2.

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. 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.

The effect of the transformation on glycosylates is analysed the same way as in ex- ample 1 using the Thermomyces lanuginosus lipase. It is confirmed that the N linked structure of the lipase consist of GlcNAcMan5GlcNAc2.

Having confirmed the desired glycosylation pattern, pyrG mutants are selected as in example 1. The selected strain is termed Aspergillus oryzae GnT1.

Example 3

Trimming GlcNacMan5GlcNac2 to GlcNacMan3glcNac2

The human mannosidase Il (Mannosyl-oligosaccharide 1 ,3-1,6-alpha-mannosidase), gene is expressed in Aspergillus oryzae in the following way:

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 effect of the transformation on glycosylates is analysed the same way as in ex¬ ample 1 using the Thermomyces lanuginosus lipase. It is confirmed that the N-linked structure of the lipase consist of GlcNAcMan3GlcNAc2. Having confirmed the desired glycosylation pattern, pyrG mutants are selected as in example 1. The selected strain is termed Aspergillus oryzae Man2.

Example 4

Adding N acetyl glucosamine to GlcNacMan3GlcNac2.

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.

The effect of the transformation on glycosylations is analysed the same way as in ex- ample 1 using the Thermomyces lanuginosus lipase. It is confirmed that the N-linked structure of the lipase consist of primarily GlcNAc2Man3GlcNAc2.

Having confirmed the desired glycosylation pattern, pyrG mutants are selected as in example 1. The selected strain is termed Aspergillus oryzae GnT2. Example 5

Adding Galactose to GlcNac2Man3GlcNac2.

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.

The effect of the transformation on glycosylates is analysed the same way as in ex¬ ample 1 using the Thermomyces lanuginosus lipase. It is confirmed that the N-linked structure of the lipase consist of a significant amount of GalGlcNAc2Man3GlcNAc2 and some Gal2GlcNAc2Man3GlcNAc2

Having confirmed the desired glycosylation pattern, pyrG mutants are selected as in example 1. The selected strain is termed Aspergillus oryzae GaIT.

Claims

1. Method of altering the structure of N-glycans in a fungus comprising insertion into a fungal cell of a nucleic acid construct or a combination of nucleic acid constructs selected from the group consisting of:

e) a fifth nucleic acid construct comprising a nucleic acid sequence encoding a beta- 1 ,4-galactosyltransferase.

2. A method according to claim 1 , wherein the first nucleic acid construct further comprises a nucleic acid sequence encoding an ER retention signal.

3. A method according to claim 2, wherein the nucleic acid sequence encoding the ER retention signal is located at the 3'-end of the nucleic acid sequence encoding the alpha-1 ,2- mannosidase.

4. A method according to any of claims 2-3, wherein the ER retention signal has the amino acid sequence HDEL.

5. A method according to any of the preceding claims, wherein the first nucleic acid construct further comprises a constitutive promoter, e.g. tpi or gpd promoter.

6. A method according to any of the preceding claims, wherein the nucleic acid sequence encoding the alpha-1 ,2-mannosidase comprises the nucleic acid sequence shown in SEQ ID NO: 1 or a sequence which has at least 70% identity with the sequence of SEQ ID NO: 1.

7. A method according to any of the preceding claims, wherein the nucleic acid sequence encoding an alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyl- transferase is of mammalian origin.

8. A method according to any of the preceding claims, wherein the second nucleic acid construct further comprises a constitutive promoter, e.g. the tpi or gpd promoter.

9. A method according to any of the preceding claims, wherein the second nucleic acid construct further comprises a nucleic acid sequence encoding a Golgi retention signal, such as the amino acids 1-100 of SEQ ID NO: 7 located at the 5'-end of the nucleic acid sequence encoding the alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase.

10. A method according to any of the preceding claims, wherein the nucleic acid sequence encoding a mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase is of mammalian origin.

11. A method according to any of the preceding claims, wherein the third nucleic acid construct further comprises a nucleic acid sequence encoding a Golgi retention signal, such as the amino acids 1-100 of SEQ ID NO: 7 located at the 5'-end of the nucleic acid sequence encoding the mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase.

12. A method according to any of the preceding claims, wherein the nucleic acid sequence encoding an alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyl- transferase is of mammalian origin.

13. A method according to any of the preceding claims, wherein the fourth nucleic acid construct further comprises a nucleic acid sequence encoding a Golgi retention signal, such as the amino acids 1-100 of SEQ ID NO: 7 located at the 5'-end of the nucleic acid sequence encoding the alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyl-transferase.

14. A method according to any of the preceding claims, wherein the third, fourth or fifth nucleic acid construct further comprises a constitutive promoter, e.g. the tpi or gpd promoter.

15. A method according to any of the preceding claims, wherein the nucleic acid sequence encoding a beta-1 ,4-galactosyltransferase is of mammalian origin.

16. A method according to any of the preceding claims, wherein the fifth nucleic acid construct further comprises a nucleic acid sequence encoding a Golgi retention signal, such as the amino acids 1-100 of SEQ ID NO: 7 located at the 5'-end of the nucleic acid sequence encoding the beta-1 ,4-galactosyltransferase.

17. A method according to any of the preceding claims, wherein the fungal cell is a filamentous fungal cell, such as a strain of Aspergillus, e.g. A.oryzae.

18. A method according to any of the preceding claims, wherein at least one endogenous gene encoding an alpha-1 ,6-mannosyltransferase capable of catalyzing the binding of mannose by alpha-1 ,6-bonds to the 1 ,3 arm of Man9GlcNAc2 has been modified so that fewer mannose residues are bound by alpha-1 ,6-bonds to the 1 ,3 arm of Man9GlcNAc2 structure of proteins than in a parental cell.

19. A method according to claim 18, wherein the endogenous gene encodes an enzyme having at least 40% identity with the sequence of SEQ ID NO: 12.

20. A method of preparing a heterokaryon fungus comprising an altered structure of N- glycans comprising fusion of at least two genetically different fungal cells under conditions suitable for formation of a heterokaryon, wherein at least one of the genetically different cells have been altered according to any of claims 1-19.

21. A fungal cell comprising a nucleic acid construct or a combination of nucleic acid constructs selected from the group consisting of:

b) a second nucleic acid construct comprising a nucleic acid sequence encoding an alpha-1 ,3-mannosyl-gIycoprotein 2-beta-N-acetyl- glucosaminyltransferase

c) a third nucleic acid construct comprising a nucleic acid sequence encoding a mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase d) a fourth nucleic acid construct comprising a nucleic acid sequence encoding an alpha-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,

22. A fungal cell according to claim 21 , wherein the first nucleic acid construct further comprises a nucleic acid sequence encoding an ER retention signal.

23. A fungal cell according to any of claims 21-22, wherein the nucleic acid sequence encoding the ER retention signal is located at the 3'-end of the nucleic acid sequence encoding the alpha-1 ,2-mannosidase.

24. A fungal cell according to any of claims 22-23, wherein the ER retention signal has the amino acid sequence HDEL.

25. A fungal cell according to any of claims 21-24, wherein the first nucleic acid construct further comprises a constitutive promoter, e.g. tpi or gpd promoter.

26. A fungal cell according to any of claims 21-25, wherein the alpha-1 ,2-mannosidase is obtained from a strain of Aspergillus, e.g. A.oryzae or A.nidulans.

27. A fungal cell according to any of claims 21-26, wherein the nucleic acid sequence encoding the alpha-1 ,2-mannosidase comprises the nucleic acid sequence shown in SEQ ID NO: 1 or a sequence which has at least 70% identity with the sequence of SEQ ID NO: 1.

28. A fungal cell according to claim 21-27, wherein the second nucleic acid construct further comprises a constitutive promoter, e.g. the tpi or gpd promoter.

29. A fungal cell according to any of claims 21-28, wherein the nucleic acid sequence encoding a alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase is of mammalian origin.

30. A fungal cell according to any of claims 21-29, wherein the second nucleic acid construct further comprises a nucleic acid sequence encoding an Golgi retention signal, such as such as the amino acids 1-100 of SEQ ID NO: 7 located at the 5'-end of the nucleic acid sequence encoding the alpha-1 ,3-mannosyl-glycoprotein 2-beta-N-acetyl- glucosaminyltransferase.

31. A fungal cell according to any of claims 21-30, wherein the nucleic acid sequence encoding a mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase is of mammalian origin.

32. A fungal cell according to any of claims 21-31 , wherein the third nucleic acid construct further comprises a nucleic acid sequence encoding a Golgi retention signal, such as the amino acids 1-100 of SEQ ID NO: 7 located at the 5'-end of the nucleic acid sequence encoding the mannosyl-oligosaccharide 1 ,3-1 ,6-alpha-mannosidase.

33. A fungal cell according to any of claims 21-32, wherein the nucleic acid sequence encoding an alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosamintransferase is of mammalian origin.

34. A fungal cell according to any of claims 21-33, wherein the fourth nucleic acid construct further comprises a nucleic acid sequence encoding a Golgi retention signal, such as such as the amino acids 1-100 of SEQ ID NO: 7 located at the 5'-end of the nucleic acid sequence encoding the alpha-1 ,6-mannosyl-glycoprotein 2-beta-N-acetylglucosamin- transferase.

35. A fungal cell according to any of claims 21-34, wherein the fifth nucleic acid construct further comprises a constitutive promoter, e.g. the tpi or gpd promoter.

36. A fungal cell according to any of claims 21-35, wherein the nucleic acid sequence encoding a beta-1 ,4-galactosyltransferase is of mammalian origin.

37. A fungal cell according to any of claims 21-36, wherein the fifth nucleic acid construct further comprises a nucleic acid sequence encoding a Golgi retention signal, such as such as the amino acids 1-100 of SEQ ID NO: 7 located at the 5'-end of the nucleic acid sequence encoding the beta-1 ,4-galactosyltransferase.

38. A fungal cell according to any of claims 21-37, wherein 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.

39. A fungal cell according to claim 38, wherein the endogenous gene encodes an enzyme having at least 40% identity with the sequence of SEQ ID NO: 12.

40. A fungal cell according to any of claims 21-39, wherein the fungal cell is a filamentous fungal cell, such as a strain of Aspergillus, e.g. A.oryzae.

41. A fungal cell according to any of claims 21-40, wherein the fungal cell is a heterokaryon.

42. A fungal cell according to claim 41 , wherein the heterokaryon is a filamentous heterokaryon fungus.

43. A fungal cell according to claim 41 , wherein the heterokaryon is a yeast heterokaryon.

44. A method of producing a protein with an altered glycan structure comprising culturing a fungal cell of any of claims 21-43, comprising a nucleic acid sequence encoding the protein and recovering the protein.

45. A method according to claim 44, wherein the protein is a monoclonal antibody.