WO2008136564A1

WO2008136564A1 - A novel ylmpo1 gene derived from yarrowia lipolytica and a process for preparing a glycoprotein not being mannosylphosphorylated by using a mutated yarrowia lipolytica in which ylmpo1 gene is disrupted

Info

Publication number: WO2008136564A1
Application number: PCT/KR2007/006164
Authority: WO
Inventors: Jeong-Nam Park; Yunkyoung Song; Jeong-Yoon Kim; Doo-Byoung Oh; Hyun Ah Kang
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-05-04
Filing date: 2007-11-30
Publication date: 2008-11-13

Abstract

The present invention relates to a novel YlMPOl gene which plays an important role in mann osylphosphorylation of an industrial yeast Yarrowia lipolytica, and to a method for preparing a host system capable of producing recombinant glycoproteins free of mannosylphosphate by disruption of the gene. The mannosylphosphorylation is suppressed by the disruption of YlMPOl gene according to the present invention, thereby achieving humanization of glycosylation pathway of Yarrowia lipolytica.

Description

A NOVEL YLMPOl GENE DERIVED FROM YARROWIA LIPOLYTICA AND A PROCESS FOR PREPARING A GLYCOPROTEIN NOT BEING MANNO-

SYLPHOSPHORYLATED BY USING A MUTATED YARROWIA LIPOLYTICA IN WHICH YLMPOl GENE

IS DISRUPTED Technical Field

[1] The present invention relates to an YlMPOl gene (Mannosyl Phosphorylation of

Oligosaccharide) of Yarrowia lipolytica, which plays an essential role in manno- sylphosphorylation of N-linked oligosaccharide, a Yarrowia lipolytica mutant strain deficient in the MPOl gene, and a method for producing a recombinant glycoprotein using the mutant strain.

[2]

Background Art

[3] Most therapeutic proteins are glycoproteins where oligosaccharides are covalently bonded to amino acid residues as they pass through the secretory pathway. The sugar moieties are known to greatly affect biological activity and function of the glycoproteins. To date, to solve the problems such as immune response in the body, therapeutic glycoproteins are commonly produced using animal cell expression systems. However, there are drawbacks to animal cell culture systems, which include low yield, high cost, and potential viral and prion contamination. In this regard, many attempts have been made to use, as an alternative to animal cell expression systems, yeast expression systems, which are efficient and high-yield expression systems, and share the early steps of the N-linked glycosylation pathway of higher animal cells.

[4] Microbial eukaryotes, yeasts have advantages of rapidly producing a high concentration of proteins, being easily genetically engineered, and having no risk of infections by human or animal virus and prion to ensure safety. However, the final oligosaccharides synthesized in yeasts have different type of sugar moieties from those of human, and thus may cause immune responses in animal cells. To solve the above problems, there is a need for glycotechnology, by which the yeast glycosylation pathway is remodeled to express glycoproteins having oligosaccharides similar to those of human glycoproteins. [5] A traditional yeast, Saccharomyces cerevisiae has a hypermannosylated N-linked oligosaccharide composed of a series of 50 to 200 mannose residues attached to a core oligosaccharide chain and decorated with the terminal alpha 1,3-linked mannoses (Dean, Biochim. Biophys. Acta., 1426, p.309-322, 1999). In addition, manno- sylphosphate is added to the core and outer chain of oligosaccharide (Ballow, 1990, Methods Enzymol. 185: 440-470), and a glycoprotein with mannosylphosphorylated oligosaccharide was reported to induce immune responses when injected to animals (Rjsenfeld and Ballou, 1974, J. Biol. Chem. 249: 2319-2321). Thus, there is an attempt to humanize glycosylation pathway by disrupting OCHl and MNN4 genes, which mediates outer chain initiation and participates in addition of manno- sylphosphate, respectively (Jigami and Odani, Biochim. Biophys. Acta 1426, 335-345, 1999). However, mannosylphosphorylation was not completely regulated in MNN4 - disrupted strains, even though the extent was less than that in a wild- type strain (Odani et al., Glycobiology, 6, p.805-810, 1996).

[6] There is an attempt to humanize the glycosylation pathway by eliminating mannosylphosphorylation in a methylotrophic yeast, Pichia pastoris, as well as in the traditional yeast. A PNOl (Phosphorylmannosylation of N-linked Oligosacharides) gene, which plays an important role in mannosylphosphorylation in Pichia pastoris, was cloned by using a sequence of MNN4 geneof Saccharomyces cerevisiae as a probe (Miura et al., Japan), and there is a report that the mannosylphosphorylation can be controlled by the elimination of PNOl gene (WO 01/88143; Miura et al., 2004, Gene 324: 129-137).

[7] However, it was found by GlycoFi Inc. that the disruption of the PNOl may suppress the mannosylphosphorylation, but does not completely eliminate it, leading to application of the invention (US2006/0160179). In the invention, a BLAST search was performed for the amino acid sequence of Mnn4 protein from Saccharomyces cerevisiae against the genome of Pichia pastoris (Integrated Genomics, Chicago, III). This search resulted in the identification of three genes, which were designated as MNN4A, MNN4B and MNN4C, respectively. They also found that the mannosylphosphorylation can be completely eliminated by double disruption of MNN4A and PNOl genes.

[8] A dimorphic, non-pathogenic yeast, Yarrowia lipolytica has been used on a large scale for the production of citric acid and of single-cell proteins, and is characterized by excessive secretion of extracellular proteins such as protease and lipase. Also, Yarrowia lipolytica has been considered as an excellent host system for producing therapeutic glycoproteins, since itexhibits higher protein secretion efficiency than the traditional yeast Saccharomyces, has co-translational protein modification similar in animal cells (Boisrame et al., J. Biol. Chem., 273, p.30903-30908, 1998), has a lower number of mannose attached to the core chain than Saccharomyces cerevisiae (Madzak et al., J. Biotechnol., 109, p.63-81, 2004), and has no immunogenic alpha 1,3-linked mannose. To express and secrete therapeutic glycoproteins derived from human in Yarrowia lipolytica, the glycosylation pathway of Yarrowia lipolytica has to be understood, but is still poorly understood (Jaagar et al., Yeast, 20, p.633-644, 2003, Barnay-Verdier et al., Microbiology, 150, p.2185-2195, 2004).

[9] To develop Yarrowia lipolytica as a host for secretory expression of therapeutic glycoproteins, the present inventors have conducted studies on the glycosylation pathway of Yarrowia lipolytica, and manufactured a strain comprising a disrupted YlOCHl gene which mediates outer chain initiation. However, upon disruption of the YlOCHl gene, the mannosylphosphorylation was found to be more activated.

[10]

Disclosure of Invention Technical Problem

[11] Accordingly, the present inventors performed a BLAST search for the amino acid sequence of Mnn4 protein from Saccharomyces cerevisiae, whichplays an essential role in mannosylphosphorylation, against the genome of Yarrowia lipolytica, ( http i/ZcbLlabrLfr/Genolevures/elt/YALD . resulting in identification of a novel YlMPOl gene of Yarrowia lipolytica. They also manufactured a YlMPOl disrupted strain ( YlmpolΔ), and performed analysis on secretory proteins and main ingredients of cell wall. Consequently, they found that synthesis of oligosaccharide free of manno- sylphosphate can be realized by the disruption of YlMPOl gene in Yarrowia lipolytica, thereby completing the present invention.

[12]

Technical Solution

[13] It is an orject of the present invention to provide a novel YlMPOl gene, which plays an essential role in mannosylphosphorylation in the glycosylation pathway of Yarrowia lipolytica.

[14] It is another orject of the present invention to provide a Yarrowia lipolytica mutant strain capable of producing glycoproteins free of mannosylphosphate by disruption of the YlMPOl gene. [15] It is still another ob^'ect of the present invention to provide a method for producing a recombinant glycoprotein, in which a nucleic acid molecule encoding a foreign protein is introduced into the Yarrowia lipolytica mutant strain disrupted in the YlMPOl gene to produce glycoproteins free of yeast-specific mannosylphosphate, thereby being used as a therapeutic glycoprotein.

[16]

Advantageous Effects

[17] To manufacture a yeast expression system for producing therapeutic glycoproteins derived from human by humanization of glycosylation pathway of Yarrowia lipolytica, it is essential to control the addition of yeast-specific mannosylphosphate. As described above, the results of HPLC profile analysis and alcian blue staining showed that the addition of mannosylphosphate to the core and outer sugar chains was completely suppressed in the YlMPOl disrupted strain (YlmpolA) developed according to the present invention. In Saccharomyces cerevisiae or Pichia pastoris, it was not observed that the mannosylphosphorylation is completely suppressed by only the single gene deletion.

[18] Accordingly, the method for suppressing mannosylphosphorylation by disruption of the YlMPOl gene according to the present invention can be usefully applied to development of strains producing human-type glycoproteins, with the aid of other techniques for redesigning glycosylation pathways.

[19]

Brief Description of the Drawings

[20] Fig. 1 shows a nucleotide sequence of 2,135 bp corresponding to an YlMPOl gene and a deduced amino acid sequence of YlMpol protein;

[21] Figs. 2 to 4 show a multiple alignment of amino acid sequences of Mnn4 protein, which plays an important role in mannosylphosphorylation of yeast and fungi, the corresponding proteins, and YlMpol protein of Yarrowia lipolytica by homology comparison program (http://www.ncbi.nlm.nih.gOv/B LAST/), in which the information for the species name, GeneBank Accession Number, or PCT patent number of each protein is as follows: AfMnn4p (Aspergillus fiimigatus, XP_752633); CaMnn4p (Candida albicans, AAL86704); NcLaclp (Neurospora crassa, CAB91733); PpMnn4Ap (Pichia pastoris, WO2005/060519); PpMnn4Bp (Pichia pastoris, WO2005/060519); PpMnn4Cp (Pichia pastoris, WO2005/060519); PpPnolp (Pichia pastoris, BAD06252); ScMnn4p (Saccharomyces cerevisiae, NP_012721); YlMpolp (Yarrowia lipolytica));

[22] Fig. 5 is the results of analysis of identity and similarity (Fig. 5a) between the

YlMpol protein of Yarrowia lipolytica and other proteins, which are presumed to play an important role in mannosylphosphorylation of yeast and fungi, and a tree diagram (Fig. 5b) showing relationships between the proteins (performed by using the online program ( http://align. genome.jp/):

[23] Fig. 6 is a schematic diagram showing DNA recombination for disruption of the

YlMPOl gene of Yarrowia lipolytica (Fig. 6a) and the result of Southern blotting for the YlMPOl disrupted strain (Fig. 6b), in which each lane represents the following strains; Lane 1: wild-type strain (YlMPOl), Lane 2: Ylmpol disrupted strain popped out of selectable marker YIURA3 (Ylmpol ::tc -YlU RA3-tc), Lane 3: Ylmpol disrupted strain (Ylmpol ::tc);

[24] Fig. 7 is a schematic diagram showing the construction of recombinant expression vector which expresses endoglucanase I (EGI) derived from Trichoderma reesei; and

[25] Fig. 8 is the result of analyzing the characteristics related to glycosylation of Ylmpol disrupted strains. Fig. 8a is the result of HPLC analysis on N-linked oligosaccharides of glycoproteins (Endoglucanase I, EGI) secreted from Ylmpol disrupted strains, manufactured by using the wild-type strain and YlOCHl disrupted strain as a mother strain (a) wild-type strain, (b) Ylmpol disrupted strain, (c) Ylochl disrupted strain, (d) Ylmpol/Ylochl doubledisrupted strain). Fig. 8b is the result of HPLC analysis on N- linked oligosaccharides of mannoproteins derived from the wild-type strain and YlMPOl disrupted strain, in which EGI and mannoproteins isolated and purified from each media and cell wall were treated with a peptide JV-glycosidase F (PNGase F) to cleave the sugar chains, and the sugar chains were labeled with a fluorescent compound at their reducing end. A retention time of standard sugar chain is marked with an arrow (M7, Man GIcNAc -PA; M8, Man GIcNAc -PA; M9, Man GIcNAc -

7 2 8 2 9 2

PA; MlO, Man GIcNAc -PA), and peaks corresponding to sugar chains with manno- sylphosphate are marked with *: * corresponds to an M8 type sugar chain with mono-

2 mannosylphosphate, and * corresponds to an M9 type sugar chain with monoman- noyslphosphate. Fig. 8c is the result of alcian blue staining of Ylmpol disrupted strain ( Ylmpol::tc) and wild-type strain, in which the strains grown to stationary phase were reacted with 0.1% alcian blue at room temperature for 30 min, and then the staining level was scanned to compare the color difference between two strains. Since the extent of alcian blue staining is correlated with the amount of mannosylphosphate attached to sugar chain of mannoprotein in cell wall, it can be seen that the amount of mannosylphosphate is remarkably reduced in the Ylmpol disrupted strain. [26]

Best Mode for Carrying Out the Invention

[27] The present invention relates to a novel YlMPOl gene which plays an essential role in mannosylphosphorylation of an industrial yeast, Yarrowia lipolytica, and to a method for preparing a host system capable of producing a recombinant glycoprotein free of mannosylphosphate by disruption of the gene.

[28] As used herein, the term "glycoprotein", refers to a protein that is glycosylated on one or more asparagines, or one or more serine or threonine residues, or is glycosylated on asparagine and serine or threonine residues. As used herein, the term "mannosylphosphate" refers to a non-human sugar residue found in yeast and fungi, and is transferred to both the core and outer sugar chains of glycoproteins (Jigami Y & Odani T, Biochimica et Biophysica Acta 1426, p.335-345, 1999).

[29] The present inventors performed a BLAST search for the amino acid sequence of

Mnn4 protein from Saccharomyces cerevisiae, whichplays an essential role in mannosylphosphorylation, against the genome of Yarrowia lipolytica ( http i/ZcbLlabrLfr/Genolevures/elt/YALD . resulting in identification of a novel YlMPOl gene of Yarrowia lipolytica. A base sequence of the gene is described in SEQ ID NO. 1, and an amino acid sequence of deduced Ylmpol protein is described in SEQ ID NO. 2.

[30] In one aspect, the present invention provides an Ylmpol protein having the amino acid sequence represented by SEQ ID NO. 2, which plays an important role in mannosylphosphorylation.

[31] In another aspect, the present invention provides a nucleic acid molecule encoding the Ylmpol protein, and a nucleic acid molecule which has 75% or higher, preferably 85%, and more preferably 90% or higher homology therewith and encodes a polypeptide which exhibits an activity of the Ylmpol protein. Preferably, the nucleic acid molecule is a nucleic acid molecule having a base sequence of SEQ ID NO. 1, and includes an analogue thereof or a fragment thereof.

[32] The term "homology", as used for the YlMPOl gene derived from Yarrowia lipolytica, is intended to indicate the degree of similarity to the base sequence of a wild type, and includes a base sequence having an identity of preferably 75% or higher, more preferably 85% or higher, even more preferably 90% or higher, and most preferably 95% or higher, with the base sequence of the YlMPOl gene of the present invention. It will be appreciated by those skilled in the art that a nucleic acid molecule which has the homology in the above range and encodes a polypeptide having the same activity can be readily prepared using a method known in the art such as recombinant DNA technology, resulting from substitution, addition or deletion of one or more base sequences of the YlMPOl gene according to the present invention. This homology comparison may be performed by using a commercially available comparison program. A commercially available computer program may express homology between two or more sequences in a percentage, and a homology (%) may be calculated for adjacent sequences.

[33] A novel gene and its resulting protein may be analyzed by sequence comparison with homologous protein families present in various organisms to predict its function. The sequence comparison may be performed by using a commercially available analysis software or a web based analysis system. In the present invention, a protein resulting from the novel gene of Yarrowia lipolytica was analyzed using a web based analysis system to compare identity and similarity with homologous protein families.

[34] The term "identity", as used for the YlMPOl protein derived from Yarrowia lipolytica, means that a position in the compared sequence is occupied by the same amino acid residue, upon sequence comparison of proteins derived from various organisms, and expresses the percentage (%) of same amino acid residues being present at a given position. In addition, the term "similarity" means that a position in the compared sequence is occupied by the amino acid residue with a similar chemical property, upon sequence comparison of proteins, and expresses the percentage (%) of amino acid residue with a similar chemical property being present at a given position.

[35] To prepare a more preferred host capable of producing human-type glycoproteins by regulating the mannosylphosphorylation of Yarrowia lipolytica, the present inventors performed polymerase chain reaction to obtain the YlMPOl gene which plays an essential role in mannosylphosphorylation, and then constructed an YlMPOl disruption vector by using the PCR product. Then, they transformed Yarrowia lipolytica with the YlMPOl disruption vector to manufacture a YlOCHl disrupted strain (Yarrowia lipolytica mpolA) (Example 3), which was deposited at KCTC (Korean Collection for Type Cultures; Korea Institute of Bioscience and Biotechnology, 52, Ueun-dong, Yusunggu, Daejeon, Korea) on March 27, 2007 under accession number KCTC 11102BP.

[36] In still another aspect, the present invention relates to a mutant strain producing glycoproteins free of mannosylphosphate, prepared by disruption of the YlMPOl gene which plays an essential role in mannosylphosphorylation, and the mutant strain is preferably a Yarrowia lipolytica mutant strain YlmpolΔ, deposited under accession number KCTC 11102BP.

[37] The specific disruption, that is, inactivation of a target gene on the genome may be easily achieved by those skilled in the art using a method established in the art, and the method is not particularly limited. The present inventors first constructed an YlMPOl disruption vector using the YlMPOl gene, and transformed Yarrowia lipolytica withthe vector to induce a homologous recombination between the genome and the vector. Selection markers useful for the construction of the YlMPOl disruption vector are not particularly limited, but include markers providing selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface protein expression. In the practice of the present invention, YIURA3 was used as a selection marker.

[38]

[39] An HPLC analysis and alcian blue staining were performed to analyze sugar chains attached to glycoproteins which were expressed in the prepared YlMPOl deleted stain (YlmpolA) (see Fig. 8). From the result of oligosaccharide profile analysis on secretory proteins and cell wall proteins expressed in the YlmpolA strain, neutral sugars were found to show the same pattern as those of a wild-type, and acidic sugars produced by mannosylphosphorylation were not observed. In addition, it was found that the YlmpolA strain was hardly stained with alcian blue, as compared to the wild-type strain, indicating that the addition of mannosylphosphate to the sugar chain of cell wall proteins which were expressed in the YlmpolA strain was suppressed.

[40] The above results indicate that binding of mannosylphosphate to both the core and outer sugar chains can be suppressed by deletion of only the YlMPOl gene of Yarrowia lipolytica. Upon the deletion of ScMNN4 gene, a traditional yeast, Sac- charomyces cerevisiae showed a remarkable decrease in alcian blue staining. However, from the result of structural analysis on its sugar chains, it was found that the addition of mannosylphosphate to the core sugar chain was not completely suppressed. In Pichia pastoris, four homologous genes including PpPNOl, PpMNN4A, PpMNN4B and PpMNN4C exist, and the mannosylphosphorylation was completely suppressed by a double deletion of PpPNOl and PpMNN4B. In particular, the disruption of only the PpPNOl gene did not cause a reduction in alcian blue staining. Thus, the PpPNOl gene is considered not to play an essential role in addition of mannosylphosphate to outer sugar chains.

[41] As described above, since the mannosylphosphorylation can be suppressed by disruption of only one gene, Yarrowia lipolytica has a better advantage, as compared to Saccharomyces cerevisiae or Pichia pastoris. Such advantage is more beneficial for hu- manization of glycosylation pathway in yeast, which requires deletion of yeast- specific genes and introduction of various new genes.

[42] On the other hand, in Yarrowia lipolytica, the yeast- specific glycosylation can be completely eliminated by a double disruption of YlOCHl and YlMPOl genes, which mediates outer chain initiation and participates in mannosylphosphorylation, respectively.

[43] In still another aspect, the present invention provides a method for preparing a mutant strain capable of producing various human-compatible oligosaccharides, in which the YlMPOl disrupted strain (YlmpolA) is additionally redesigned to have a double disruption of YlMPOl and YlOCHl gene as a preferable example. In the present invention, a Yarrowia lipolytica mutant strain (ochl Δ/mpolΔ) was manufactured by a double disruption of YlMPOl and YlOCHl genes (Example 4), which was deposited at KCTC (Korean Collection for Type Cultures; Korea Institute of Bioscience and Biotechnology, 52, Ueun-dong, Yusunggu, Daejeon, Korea) on April 26, 2007 under accession number KCTC 11126BP.

[44] In still another embodiment, the present invention relates to a method for producing various human-type glycoproteins using the Yarrowia lipolytica mutant strain, and to human-type glycoproteins produced according to the same method.

[45] Since glycoproteins prepared according to the present invention, which are expressed in the Yarrowia lipolytica mutant strain deficient in YlMPOl gene or both YlMPOl and YlCHOl genes, have humanized sugar chains to be less immunogenic in humans, and are identical or similar to proteins produced in humans with respect to solubility, sensitivity to proteases, trafficking, transport, secretion, recognition by other proteins or factors, or the like, they may be suitable for therapeutic use.

[46] A produced glycoprotein may be purified by an ordinary method, and the purification protocol may be determined according to the properties of the specific protein to be purified. This determination is considered as an ordinary skill to those skilled in the art. For example, a target protein may be purified by a typical isolation technique, such as precipitation, immunoadsorption, fractionation, and various chromatographic methods.

[47] Glycoproteins capable of being produced according to the present invention are exemplified by cytokines (e.g., EPO, interferon-alpha, interferon-beta, interferon-gamma, G-CSF, etc.), clotting factors (e.g., VIII factor, IX factor, human protein Q, antibodies for therapeutic use (e.g., immunogloblulins, Fab, double specific antibodies, monovalent antibodies, diabody, etc.) and Fc fusion proteins, therapeutic enzymes (e.g., glucocerebrosidase, alpha-galactosidase, alpha-L-iduronidase, alpha-glucosidase, etc.), endothelial growth factor, growth hormone releasing factor, Typanosoma cruzi trans-sialidase, HIV envelope protein, influenza virus A haemagglutinin, influenza neuraminidase, bovine enterokinase activator, bovine herpes virus type- 1 glycoprotein D, human angiostatin, human B7-1, B7-2 and B-7 receptor CTLA-4, human tissue factor, growth factors (e.g., platelet-derived growth factor), human alpha-antitrypsin, tissue plasminogen activator, plasminogen activator inhibitor- 1, urokinase, plasminogen, and thrombin, but are not limited thereto.

[48]

Mode for the Invention

[49] Hereinafter, a better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[50]

[51] Example 1: Identification of YlMPOl gene

[52] A BLAST search was performed using an amino acid sequence of Mnn4 protein from Saccharomyces cerevώ/αe,whichplays a crucial role in mannosylphosphorylation, against the genome of Yarrowia lipolytica to identify the YlMPOl gene of Yarrowia lipolytica of the present invention, and the homologous gene encodes a protein consisting of 644 amino acids (Fig. 1).

[53] The present inventors also found the MNN4 gene and corresponding gene families in eukaryotes including Aspergillus fumigatus , Candida albicans, Neurospora crassa, Pichia pastoris, Saccharomyces cerevisiae, and Yarrowia lipolytica, and they compared amino acid sequences encoding them with each other (Figs. 2 to 4). The YlMpol protein does not contain a repeating sequence

(Lys-Lys-Lys-Lys-Glu-Glu-Glu-Glu) consisting of four lysines (Lys) and four glutamic acids (GIu), found in the Mnn4 protein of Saccharomyces cerevisiae.

[54] Fig. 5 is the results of analysis of identity and similarity (Fig. 5a) and a tree diagram

(Fig. 5b) which show relationships between the proteins from each species. The Mpol protein of Yarrowia lipolytica exhibited a 40% identity with that of Saccharomyces cerevisiae, a 34% identity with that of Candida albicans, a 33% identity with the Pnol protein of Pichia pastoris, a 32% identity with the Mnn4A protein of Pichia pastoris, a 27% identity with the Mnn4B protein of Pichia pastoris, a 33% identity with the Mnn4C protein of Pichia pastoris, a 41% identity with that of Neurospora crassa, and a 37% identity with that of Aspergillus fumigatus. As shown in the tree diagram of Fig. 5b, the YlMpol protein was found on the different branch from the Mnn4 protein of Saccharomyces cerevisiae or the Pnol protein of Pichia pastoris, and taxonomically similar to the Mnn4 proteins derived from fungus such as Neurospora crassa and Aspergillus fumigatus. Thus, it can be thought that the YlMpol protein belongs to a protein group, which is responsible for mannosylphosphorylation in yeast and fungus, but exhibits different properties from the Mnn4 protein of Saccharomyces cerevisiae, or the Pnol and Mnn4A, Mnn4B, Mnn4C proteins of Pichia pastoris, of which properties are known.

[55]

[56] Example 2: Construction of YlMPOl disruption vector

[57] To disrupt the YlMPOl gene in Yarrowia lipolytica, a disruption vector was constructed using an YIURA3 selectable marker cassette of Fig. 6a, as follows. A genomic DNA of Yarrowia lipolytica SMS397A (MATA adel ura3 xpr2) strain was subjected to polymerase chain reaction (PCR) using a Pfu polymerase (Neurotics, Korea) and two pairs of primer sets, YlMPOl-Fl (C AAC AC ACC ATCGG AG AQ (SEQ ID NO. 3) and YlMPOl-Rl fCCATGGATCCGTAGATCT CTGCC- GAAAATCAGACAG) (SEQ ID NO. 4), and YlMPO 1-F2 ( AGATCTACG- GATCCATGGATCCAGGAGAGACCAGAG) (SEQ ID NO. 5) and YlMPO 1-R2 (GTTGCGTCATTCTCTCCA) (SEQ ID NO. 6) to obtain two fragments of 571 bp and 477 bp, respectively. Then, fusion polymerase chain reaction (fusion PCR) was performed using two fragments as a template, a primer set of YlMPOl-Fl and YlMPO 1-R2, and an Ex Taq polymerase (Takara, Japan) to obtain a fused fragment (1,066 bp) fused by a linker sequence (18 bp; AGATCTACGGATCCATGG) (SEQ ID NO. 7). The fused fragment was subcloned into a pGEM T easy vector (Promega, USA) to construct a pT-YlMP01+ vector (4,084 bp). A tc-YlURA3-tc selectable marker was cleaved from a pYLUB vector (Song et al., J. Microbiology, 41, pl21-128, 2003) using restriction enzymes, BamHl and BgHI, and the product (2,783 bp) was cloned into a BgIII site of the pT-YlMP01+ vector to construct an Ylmpol::tc-YlURA3-tc disruption vector, a pT- YlMPOlD vector (6,867 bp).

[58]

[59] Example 3: Establishment of Ylmpol disrupted Yarrowia lipolytica strain

[60] The YlMPOl gene of Yarrowia lipolytica was disrupted using the gene disruption vector constructed in Example 2. An YlMPOl disrupted strain (M4D3) and a M4D3P1 strain, in which the YIURA3 selectable marker was removed therefrom, were manufactured as follows. First, to disrupt the YlMPOl gene, the Ylmpol : :tc-YlURA3-tc disruption vector, the pT-YlMP01D vector were cleaved with a restriction enzyme, Notl and a wild type SMS397A was transformed with the resulting fragment (3,849 bp). Transformants were selected on SC-URA selection media (2% glucose, 0.67% yeast nitrogen base w/o amino acid, DO supplement-URA). Then, the selected transformants were subjected to polymerase chain reaction to select YlMPOl disrupted strains, designated as M4D3 { Ylmpol ::tc -YlU RA3-tc). The selected M4D3 strain was cultured in media supplemented with 5-fluorootic acid (5-FOA, 0.675 g/liter), and the YIURA3 selectable marker cassette was popped out of the M4D3 strain. The resulting strain was designated as M4D3P1 ( Ylmpol A, Ylmpol ::tc). All of the strains were subjected to Southern blotting, and the results are shown in Fig. 4B (lane 1: SMS397A, lane 2: M4D3P1, lane 3: M4D3).

[61]

[62] Example 4: Establishment of Ylochl/Ylmpol double disrupted

Yarrowia lipolvtica strain

[63] To establish an YlOCHl disrupted Yαrrowiα lipolyticα, an YlOCHl disruption vector was constructed using an YIURA3 selectable marker cassette in the same manner as in Example 2. The genomic DNA of Yαrrowiα lipolyticα SMS397A strain was subjected to polymerase chain reaction using two pairs of primer sets, YlOCHl-Fl (ACTTTTTGCATCTGCGGAQ (SEQ ID NO. 8) and YlOCHl-Rl ( CCATGGATC- CGTAGATCTAGGAGTTCGAAGACGTTG) (SEQ ID NO. 9), and Y10CH1-F2 ( AGATCTACGGATCCATGGGACCGACTCTGTCTTCGA) (SEQ ID NO. 10) and YlOCH 1-R2 (CATCCTCCTGATATACGQ (SEQ ID NO. 11) to obtain fragments containing a portion of N- and C-terminus of YlOCHl gene. Then, fusion polymerase chain reaction was performed using two fragments as a template and a primer set of YlOCHl-Fl and YlOCH 1-R2 to obtain a fused fragment fused by a linker sequence of SEQ ID NO. 7 used in Example 2. The fused fragment was subcloned into a pGEM T easy vector to construct a pT-Y10CHl+ vector. A tc-YlURA3-tc selectable marker was cleaved from a pYLUB vector, and the fragment was cloned into the pT- YlOCH 1+ vector to construct an Ylochl::tc-YlURA3-tc disruption vector, a pT-Y10CHlD vector. To disrupt the YlOCHl gene, the pT-Y10CHlD vector was cleaved with a restriction enzyme, Notl and a wild type Yαrrowiα lipolyticα was transformed with the resulting fragment. Transformants were selected on SC-URA selection media. Then, the selected transformants were subjected to polymerase chain reaction to select YlOCHl disrupted strains. The selected strain was cultured in media supplemented with 5-fluorootic acid, and the Y1URA3 selectable marker cassette was popped out of the strain to give a strain ( YlochlA, Ylochl::tc). In addition, the strain (YlochlA) was subjected to genetic recombination as performed in Example 3 using the pT- YlMPOlD vector prepared in Example 2 to manufacture a Ylochl Δ/Ylmpol A double disrupted Yarrowia lipolytica strain (Ylochl::tc Ylmpol::tc).

[64]

[65] Example 5: Construction of model glvcoprotein-expressing host

[66] To analyze glycoproteins expressed in a wild-type Yarrowia lipolytica and strains disrupted in glycosylation-related genes, strains expressing endoglucanase I (EGI), derived from Trichoderma reesei, were manufactured as follows. An EGI expressing vector, pXCSIn(Ffis) (Park et al. Appl Biochem Biotechnol, 87, 1-15, 2000) was treated with EcoPJ and CIaI, so as to isolate an EcoPJ/Clal fragment of about 3.5 kb containing a promoter of XPR2 gene encoding AEP (alkaline extracellular protease) derived from Yarrowia, an endoglucanase (EGI) gene tagged with 6 histidine residues at the C-terminus, and a terminator sequence of XPR2 gene. The fragment was introduced into a pAUX-1 vector (pIMR53, Sohn et al., J Bacteriol, 180, 6736-42, 1998) treated with EcoRI and CIaI to construct a pAUXEGI vector (Fig. 7).

[67] Then, a one-step transformation method (Chen et al., Appl. Microbiol Biotechnol.

48, p.232-235, 1997) was performed to introduce the vector into Yarrowia lipolytica. That is, a wild-type strain and an YlmpolΔ strain were smeared on YPD solid media, respectively and cultured for 16 to 24 hrs. Then, a loop of 5x10 cell was suspended in 100 [d of one-step buffer [50%(w/v) PEG 4000; 2M DTT; 2M lithium acetate(pH6.0); single-strand carrier DNA(IO βg/β&)], and 500 ng or more of the recombinant vector pAUXEGI were added thereto, followed by mixing well. The transformed cells were incubated in a water bath at 39⁰C for 1 hr, spread on selective minimal media, and then cultured at 28⁰C for 3 to 4 days to obtain transformants.

[68]

[69] Example 6: Structural analysis of N-linked oligosaccharide of

Ylmpol disrupted strains

[70] To analyze the structure of oligosaccharides which were attached to secretory glycoproteins expressed in a wild-type Yarrowia lipolytica, and Ylmpol A, YlochlA and Ylochl A/Ylmpo IA mutant strains, the following method was employed. First, the strains precultured in YPD (1% yeast extract, 2% peptone, 2% glucose) media were inoculated in YPDm (1% yeast extract, 1% proteose peptone 0.1% glucose, 50 mM sodium phosphate buffer solution (pH6.8)), and cultured at 28⁰C for 30 hrs. The secreted EGI proteins in culture media were recovered using a cellulose membrane (YM-30, Millipore), and passed through a nickel column to selectively isolate EGI proteins tagged with 6 histidine residues at the C-terminus. The isolated EGI proteins were treated with a peptide-N-glycosidase F (PNGase-F) to cleave the N-linked oligosaccharides on the EGI proteins. Then, the oligosaccharides were isolated and purified using PGC (porous graphite column), and then labeled with a fluorescent compound, 2-aminopyridine (PA) at their reducing end. An HPLC analysis was performed to compare the profile of the N-linked oligosaccharide between two strains. The HPLC analysis (Waters, USA) was performed using an amine column (Asahipak NH2P-504E, Showa denko, Japan), and a linear gradient of a solvent A (0.2 M tri- ethylamine-acetic acid:acetonitrile =1:9, pH 7.3) and a solvent B (0.2 M triethylamine- acetic acid:acetonitrile=9: 1, pH 7.3) at a ratio of 8020 to 5:95 and a flow rate of 1 ml/ min for 52 min. The oligosaccharides labeled with a fluorescent compound (PA) were detected by fluorescence (excitation wavelength=320 nm and emission wavelength=400 nm) using a fluorescence detector connected to HPLC. [71] In addition, in order to analyze cell wall mannoproteins derived from the wild- type

Yarrowia lipolytica and YlmpolΔ strain, the strains were grown to stationary phase, and then their oligosaccharides from cell wall mannoproteins were analyzed as follows. The strains precultured in YPD media were inoculated in 200 ml of YPD media, and grown to stationary phase. Two strains were recovered by centrifugation, and then cell wall mannoproteins were suspended in a citric acid buffer (pH 7.0), followed by sterilization under high pressure (121⁰C, 1 hr). Then, ethanol was added thereto, and cell wall proteins were recovered by centrifugation. The recovered cell wall proteins were treated with a peptide-N-glycosidase F (PNGase-F) to cleave the oligosaccharides, and the oligosaccharides were isolated and purified using PGC (porous graphite column). Then, the oligosaccharides were labeled with a fluorescent compound, 2-aminopyridine (PA). An HPLC analysis was performed under the same conditions to compare the profile of the oligosaccharide between two strains. The HPLC analysis was performed using an amine column (Asahipak NH2P-504E, Showa denko, Japan), and a linear gradient of a solvent A (100% acetonitrile) and a solvent B (0.2 M triethylamine-acetic acid, pH 7.0) at a ratio of 6040 to 25:75 and a flow rate of 1 ml/min for 60 min. The N-linked oligosaccharides labeled with a fluorescent compound (PA) were detected by fluorescence (excitation wavelength=315 nm and emission wavelength=380 nm). [72] As shown in Figs. 8a and 8b, the profile of neutral sugar chain of YlmpolΔ strain exhibited the same pattern as that of wild-type. However, the profile of acidic sugar chain with mannosylphosphate (region marked with *) was not observed. The results indicate that the mannosylphosphorylation of N-linked oligosaccharides can be completely suppressed by a single disruption of YlMPOl gene.

[73]

[74] Example 7: Alcain blue staining

[75] A cationic phthalocyanine dye, alcian blue is characterized by binding to anionic cell surface, and the extent of alcian blue staining is correlated with the amount of mannosylphosphate attached to mannoprotein in cell wall. Therefore, the wild-type strain and YlmpolΔ strain(M4D3Pl) were subjected to alcian blue staining, and compared to each other to determine the amount of mannosylphosphate exposed on the cell wall.

[76] First, the strains were cultured in YPD media at 28⁰C for 16 hrs, and grown to stationary phase. Then, the cells were recovered, and washed with a 0.02 N hydrochloric acid solution (pH 3.0). The cells were reacted with a 0.02 N hydrochloric acid solution containing 0.1% alcian blue (Sigma, USA) at room temperature for 30 min, and then washed with distilled water. The cells were transferred to 96-well tissue culture plates, and color difference was observed. As shown in Fig. 8c, it was found that the YlmpolΔ strain was not well stained with alcian blue, as compared to the wild- type. The result indicates that the mannosylphosphorylation of oligosaccharides of cell wall proteins of YlmpolΔ strain was suppressed.

[77]

Industrial Applicability

[78] In the present invention, Yarrowia lipolytica was developed as a host system for producing therapeutic glycoproteins derived from human, thereby being used in medical field as a host system capable of producing therapeutic glycoproteins of high quality and yield.

[79]

Claims

[1] A polypeptide which has an amino acid sequence of SEQ ID NO. 2 or 90% or higher homology therewith, and exhibits mannosylphosphorylation activity. [2] The polypeptide according to claim 1, wherein the polypeptide having an amino acid sequence of SEQ ID NO.

2 is derived from Yarrowia lipolytica.

[3] A nucleic acid molecule encoding the polypeptide of claim 1.

[4] The nucleic acid molecule according to claim 3, having a base sequence of SEQ

ID NO. 1 or 90% or higher homology therewith. [5] A Yarrowia lipolytica mutant strain which has a disrupted YlMPOl gene of

Yarrowia lipolytica represented by SEQ ID NO. 1 and produces glycoproteins lacking mannosylphosphate. [6] The Yarrowia lipolytica mutant strain according to claim 5, which is identified by accession number: KCTC 11102BP. [7] The Yarrowia lipolytica mutant strain according to claim 5, wherein an YlOCHl gene is further disrupted to eliminate yeast-specific glycosylation. [8] The Yarrowia lipolytica mutant strain according to claim 7, which is identified by accession number: KCTC 11026BP. [9] A method for producing glycoproteins lacking yeast specific-manno- sylphosphate, wherein a nucleic acid molecule encoding a foreign protein is introduced into the mutant strain according to any one of claims 5 to 7.