WO1994004687A1 - Modified fungal cells and method for producing recombinant products - Google Patents

Modified fungal cells and method for producing recombinant products Download PDF

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Publication number
WO1994004687A1
WO1994004687A1 PCT/EP1993/002179 EP9302179W WO9404687A1 WO 1994004687 A1 WO1994004687 A1 WO 1994004687A1 EP 9302179 W EP9302179 W EP 9302179W WO 9404687 A1 WO9404687 A1 WO 9404687A1
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Prior art keywords
fungal cell
cell according
dna
gene
sequence
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PCT/EP1993/002179
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French (fr)
Inventor
Widmar Tanner
Sabine Strahl-Bolsinger
Reinhard Fleer
Alain Fournier
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Rhone-Poulenc Rorer S.A.
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Application filed by Rhone-Poulenc Rorer S.A. filed Critical Rhone-Poulenc Rorer S.A.
Priority to CA002140894A priority Critical patent/CA2140894C/en
Priority to EP93919067A priority patent/EP0658203B1/en
Priority to AU49482/93A priority patent/AU679935B2/en
Priority to JP50589294A priority patent/JP3630424B2/en
Priority to AT93919067T priority patent/ATE245701T1/en
Priority to DE69333112T priority patent/DE69333112T2/en
Priority to US08/381,931 priority patent/US5714377A/en
Publication of WO1994004687A1 publication Critical patent/WO1994004687A1/en
Priority to FI950628A priority patent/FI121075B/en
Priority to NO19950534A priority patent/NO320349B1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

  • This invention relates to improved fungal cells and methods fo producing recombinant products of improved quality and in hig yields. More specifically, the present invention relates t fungal cells carrying specific modifications within their DN sequences which cause them to exhibit at least a reduced capa city for O-glycosylating homologous and/or heterologous pro teins, and the use of these cells as host cells to produce hig yields of recombinant products.
  • Eukaryotic hos cells include plant cells, animal cells and fungal cells.
  • bacterial host cells do not possess all the post-translational mechanisms required for maturation of mammalian polypeptides. Accordingly, said mammalian polypeptides produced in bacteria are often immature or not correctly refolded.
  • bac ⁇ terial host cells generally introduce an additional N-terminal methionine to the products.
  • Recombinant products produced in heterologous eukaryotic hosts also usually differ from their naturally-occurring counterpart in their glycosylation content. This may concern the presence versus absence of any carbohydrate structure, the localization of said carbohydrate structure on the product, as well as the nature of the carbohydrate. More specifically, it has been shown that yeast-derived recombinant products often bear additional unnatural O-glycans compared to their natural counterpart. For instance, it has been shown that, while human serum insulin-like growth factor I (IGF-I) is not glycosylated, its recombinant form produced in S.
  • IGF-I human serum insulin-like growth factor I
  • cerevisiae is O-glycosylated and, more pre ⁇ cisely, O-mannosylated [Hard et al., FEBS Letters 248 (1989), 111] .
  • human platelet- derived growth factor (PDGF) and human GM-CSF display unnatural O-mannosyl structures when produced in S. cerevisiae [Biomedic. Environ. Mass Spectrometry 19. (1990), 665; BIO/TECHNOLOGY 5. (1987) , 831] .
  • This abnormal O-glycosylation is the result of im ⁇ portant differences between the glycosylation mechanisms of mam ⁇ malian (human) cells and those of other eukaryotic cells, such as yeasts.
  • 0-glyco- sylation in fungal cells proceeds in a similar and unusual way so far not observed in any other organism.
  • the first reason is that fungal-specific glycans may introduce new immunological determinants on a protein, and a glycoprotein with such unnatural carbohydrates may therefore be antigenic when administered to humans.
  • a glycoprotein with such unnatural carbohydrates may therefore be antigenic when administered to humans.
  • proteins without appropriate carbohydrate structures may also have altered pharmacokinetic properties. It has been shown that carbohydrate structures of glycoproteins in ⁇ fluence and participate in defining their in vivo clearance rate, which is essential in determining the efficacy of a phar ⁇ maceutical. More precisely, a mannose receptor has been identi ⁇ fied on the surface of liver endothelial cells and resident macrophages which apparently represents a means for eliminating glycoproteins displaying mannose-type oligosaccharides [Stahl, Cell. Mol. Biol. 2. (1990), 317]. Therefore, the presence of un ⁇ natural, additional mannose structures on a protein may increase its clearance rate and thus decrease its plasma half life.
  • glycopro ⁇ tein has also been shown to vary with its carbohydrate content, location and nature.
  • glyco ⁇ sylation affects the biological properties of recombinant human EPO [Takeuchi et al., Proc. Natl. Acad. Sci. USA jJ6. (1989), 7819] and recombinant human tPA [Parekh et al. , Biochemistry 28. (1989) , 7644] .
  • unnatura O-glycosylation of fungal-derived recombinant products can se verely affect their immunological, biological and pharmacokine tic properties, and therefore may prevent their development fo human therapeutic use.
  • the present invention solves the problem of abnormal O-glyco sylation referred to above by providing modified fungal cell carrying genetic modification(s) within their DNA sequence which cause them to have at least a reduced capacity for O-gly cosylating native or foreign proteins.
  • modified fungal cells of the present invention can be uti lized advantageously as host cells for the production of recom binant products of high quality, having reduced or no un desirable O-glycans.
  • One object of the present invention is a fungal cell carryin genetic modification(s) within its DNA sequences which cause i to have at least a reduced capacity of O-glycosylation.
  • the fungal cell of the present invention can be chosen fro filamentous fungi and yeasts.
  • Exemplary genera of filamentou fungi contemplated by the present invention are Aspergillus Trichoder a, Mucor, Neurospora, Fusarium and the like.
  • Exemplar genera of yeasts include Kluyveromyces , Saccharomyces , Pichia Hansenula, Candida, Schizosaccharomyces and the like.
  • More pre ferred genera are those selected from the group consisting o Kluyveromyces, Saccharomyces, Pichia, Hansenula and Candida and, even more preferred, from Jluyvero-uyces and Saccharomyces
  • Exemplary strains of Kluyveromyces which constitute preferre embodiments of this invention include K. lactis, K. fragilis, waltii, K. drosophilarum and the like.
  • the preferred strain of Saccharomyces is S. cerevisiae.
  • genetic modification preferably means any suppression, substitution, deletion or ad ⁇ dition of one or more bases or of a fragment of the fungal cell DNA sequences.
  • Such genetic modifications may be obtained in vi tro (directly on isolated DNA) or in situ, for example by ge ⁇ netic engineering techniques or by exposing the fungal cells to mutagenic agents.
  • Mutagenic agents include for example physical agents such as energetic rays (X-rays, ⁇ -rays, UV, etc.) or chemical agents capable of reacting with different functional groups of DNA, such as alkylating agents (EMS, NQO, etc.) bisal- kylating agents, intercalating agents, etc.
  • Genetic modifica ⁇ tions may also be obtained by genetic disruption, for example according to the method disclosed by Rothstein et al. [Meth. Enzymol. ,194 (1991), 281-301].
  • part or all of a gene is replaced, through homologous recombination, by an in vitro modified ver ⁇ sion.
  • modified fungal cells of this invention may not provide the most useful forms of modified fungal cells of this invention since their phenoty- pical properties may not be very stable.
  • the present invention also provides a process for preparing modified fungal cells in which the modifications (and therefore the phenotypical proper ⁇ ties) are stable during segregation and/or non-reverting and/or non-leaky.
  • modified fungal cells are particularly advanta ⁇ geous as hosts for the production of recombinant products.
  • a preferred embodiment of the invention is a fungal cell carrying genetic modification(s) which are stable during segregation and/or non-reverting and/or non-leaky. These modifi ⁇ cations are generally obtained by deletion(s) or disruption(s) .
  • the genetic modification(s) carried by the fungal cells of the invention can be located either in a coding region of the DNA sequences of the cell or in a region responsible for or involved in the expression and/or transcriptional regulation of a gene. More particularly, said modification(s) will generally affect the coding region or the region responsible for or involved in the expression and/or the transcriptional regulation of one or more genes whose expression products are enzymes of the O-glyco ⁇ sylation pathway.
  • the reduced capacity of the fungal cells of the invention to O- glycosylate proteins may therefore result from the production of inactive enzymes due to structural and/or conformational chan ⁇ ges, from the production of enzymes having altered biological properties, from the absence of production of said enzymes, or from the production of said enzymes at low levels.
  • the fungal cell O-glycosylation pathway involves attachment of a first mannosyl residue to the hydroxyl group of seryl and/or threonyl amino acids of proteins or peptides, and then the ex ⁇ tension to O-linked di- and oligosaccharides by subsequent addi ⁇ tion of mannosyl residues.
  • the first mannosyl residue is trans ⁇ ferred from dolichol monophosphate mannose (Dol-P-Man) to th protein in the endoplasmic reticulum, and the additional manno ⁇ syl residues are transferred from GPD-Man in the Golgi.
  • Dol-P-Man dolichol monophosphate mannose
  • the modified funga cells carry genetic modification(s) in at least one gene whos expression product is involved in the attachment of a mannosyl residue to the hydroxyl group of seryl or threonyl amino acids.
  • the modified fungal cells carry genetic modification(s) in at least one gene whose expression product is involved in the transfer of a manno ⁇ syl residue from the Dol-P-Man precursor to the hydroxyl group of seryl or threonyl amino acids.
  • one of these genes is the gene encoding the Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase [DPM2 - also designated PMT1] whose sequence is represented in Figure 4, or any homologous gene encoding the same activity as defined below.
  • fungal cells of the invention may also carry modification(s) in the genes involved in subsequent addi ⁇ tions of mannosyl residues leading to di- or oligosaccharides, or in the synthesis of the mannosyl residues donnor (Dol-P-Man) .
  • Another object of the invention resides in a fungal cell as disclosed above in which an exogenous DNA sequence has been in ⁇ troduced.
  • exogenous DNA sequence includes any DNA sequence comprising one or more genes encoding a desired protein to be expressed and/or secreted in said cell.
  • a DNA sequence may be a complementary DNA sequence (cDNA) , an artificial DNA sequence, a genomic DNA se ⁇ quence, a hybrid DNA sequence or a synthetic or semi-synthetic DNA sequence, included in an expression cassette enabling syn ⁇ thesis in the fungal cells of said proteins.
  • the expression cas ⁇ sette preferably comprises a transcription and translation initiation region joined to the 5' end of the sequence encoding said desired protein(s) so as to direct, and optionally regu ⁇ late, the transcription and translation of said sequence. The choice of these regions may vary according to the fungal cell used.
  • these sequences are chosen from promoters and/or terminators derived from fungal cell genes, and, when ex ⁇ pression in yeast hosts is sought, from yeast genes.
  • yeast genes Of special interest are certain promoter and/or terminator regions derived from glycolytic genes of fungal cells such as, for yeasts, the genes encoding phosphoglycerate kinase (PGK) , glyceraldehyde-3- phosphate dehydrogenase (GDP) , enolases (ENO) or alcohol dehy- drogenases (ADH) . and for filamentous fungi, the genes encoding triose phosphate isomerase (tpi) .
  • PGK phosphoglycerate kinase
  • GDP glyceraldehyde-3- phosphate dehydrogenase
  • ENO enolases
  • ADH alcohol dehy- drogenases
  • tpi triose phosphate is
  • the promoter and/or terminator regions may also derive from other strongly expressed genes such as, for yeasts, the lactase gene (LAC4) . the acid phosphatase gene (PH05) . the alcohol oxidase gene (AOX) or the methanol oxi- dase gene (MOX) . and, for filamentous fungi, the cellobiohydro- lase gene (CBHI) . the alcohol dehydrogenase gene (alcA. alcC) . the glucoamylase gene (GAM) or the acetamidase gene (amds) , an the like.
  • These transcription and translation initiation regions may be further modified, e.g.
  • transcription-regulating elements such as the so-called UAS
  • transcription and translation termination region functional i the intended fungal cell may also be positioned at the 3' end o the coding sequence.
  • a signal peptide pre-sequence
  • This pre-sequence may correspond to th natural pre-sequence of the protein if this protein is naturall secreted, or it may be of another origin, e.g. obtained fro another gene, or even artificial.
  • the exogenous DNA sequence is part of a vector, which may either replicate autonomously in the fungal cell used or integrate into its own DNA sequences (chromosome) .
  • Autono ⁇ mously replicating vectors may contain autonomously replicating sequences derived from the chromosomal DNA of the fungal cell (ARS) or from naturally-occurring fungal cell plasmids such as pGKl-1 [de Louvencourt et al., J. Bacteriol.
  • Integrating vectors usually contain sequences ho ⁇ mologous to regions of the fungal cell chromosome which, after being introduced into said cell, enable integration through in vivo recombination.
  • said homologous sequences correspond to the region of the chro ⁇ mosome to be modified in the fungal cell, enabling a one-step modification-integration mechanism. Integration may also occur through non-homologous recombination.
  • the exogenous DNA sequence can be introduced into the fungal cell by any technique known in the art, and, for example, by re ⁇ combinant DNA techniques, genetic crossings, protoplast fusions, etc. Concerning recombinant DNA techniques, transformation, electroporation, or any other technique disclosed in the litera ⁇ ture may be used. More specifically, when the fungal cell is a yeast cell, the transformation may be performed according to the methods of Ito et al., [J. Bacteriol. 153 (1983), 163], Durrens et al. [Curr. Genet. 18. (1990), 7] or following the method disclosed in EP 361 991. Electroporation can be performed ac ⁇ cording to Karube et al. [FEBS Letters .82, (1985), 90] .
  • the fungal cells of the present invention can be advantageously utilized as host cells for the production of recombinant pro ⁇ ducts such as heterologous proteins having pharmaceutical and/or agro-foodstuff interest.
  • the fungal cells of this invention are particularly advantageous since they enable the production and/or secretion of high quality products, and since their gene ⁇ tic modifications do not affect the mitotic or genetic stability of said products' expression vectors.
  • the cells of this inven ⁇ tion are more particularly suitable for the production of pro- teins having human therapeutic uses and which are susceptible to O-glycosylation by the host cell.
  • a further object of this invention resides in a process for the production of recombinant products wherein a fungal cell as defined above is cultivated in conditions in which the exogenous DNA sequence is expressed and the product is recovered.
  • said product is secreted into the culture medium.
  • said product is susceptible to O-glycosylation by the host cell.
  • heterologous proteins which can be prepared with the fungal cells of the pre ⁇ sent invention: enzymes (such as superoxide dismutase, catalase, amylases, lipases, amidases, chymosine, etc., or any fragment or derivative thereof) , blood derivatives (such as human serum- albumin, alpha- or beta-globin, factor VIII, factor IX, van Wil- lebrand factor, fibronectin, alpha-1 antitrypsin, etc., or any fragment or derivative thereof) , insulin and its variants, lym- phokines [such as interleukins, interferons, colony stimulating factors (G-CSF, GM-CSF, M-CSF...), TNF, TRF, etc., or any frag ⁇ ment or derivative thereof] , growth factors (such as growth hor ⁇ mone, erythropoietin, FGF, EGF, PDGF, TGF, etc., or any fragment or derivative thereof)
  • enzymes such as superoxid
  • Another object of the invention resides in a DNA fragment encod ⁇ ing an enzyme involved in the attachment of mannosyl residues to the hydroxyl group of seryl or threonyl amino acids of proteins.
  • Applicant has provided DNA fragments encoding such enzymes for the first time. More preferably, said DNA fragment comprises the Dol-P-Man:Protein (Ser/Thr) mannosyltransferase gene whose se ⁇ quence is represented in Figure 4, any homologous gene, deriva ⁇ tive or fragment thereof.
  • homologous gene means any other gene of any fungal cell encoding an enzyme having the required activity.
  • Said other genes may be obtained, for exam ⁇ ple, by complementation of a mutant fungal cell deficient in said activity with DNA prepared from a fungal cell capable of said activity, selection of the transformants having recovered the activity, and isolating their inserted DNA sequence.
  • These other genes may also be isolated from DNA libraries by hybridi ⁇ zation with probe(s) (including PCR primers) comprising all or part of the sequence presented in Figure 4.
  • probe(s) including PCR primers
  • the term derivative means any other DNA fragment prepared by any genetic and/or chemical modification(s) of the genes mentioned above.
  • Said genetic and/or chemical modification-is) may be any suppression, substitution, deletion or addition of one or more bases or of a region of said genes, leading either to an in ⁇ creased enzyme activity or to the same activity level, or to a ⁇ decreased or null enzyme activity upon transformation in a fun ⁇ gal host cell.
  • FIG. 1 Strategy of sequencing of the PMTl gene.
  • Nucleotide sequence of the PMTl gene SEQ ID N°l
  • Amino acid sequence of the PMTl gene SEQ ID N°2
  • O-glycosylation activity of S. cerevisiae WT panel
  • Example 1 Isolation of a highly purified mannosyltransferas from S. cerevisiae and generation of peptides
  • the mannosyltransferase activity was solubilized from tota yeast membranes and purified on hydroxylapatite according t Strahl-Bolsinger and Tanner (Eur. J. Biochem. 196 (1991) , 185)
  • the protein then had to be further enriched by (NH 4 ) 2 S0 4 preci pitation before additional purification was performed via affi nity chromatography.
  • the eluted material was then separated o SDS/PAGE.
  • the resulting 92 kDa band was cut out of the gel Trypsin digestion (in the gel) yielded several non-overlappin peptides, enabling designing of probes.
  • 0.5 g freeze-dried powder of Protein A-Sepharose Cl 4B was swol len in 10 ml 100 mM NaPi, pH 7.0, for 15' and washed on a sin tered glass filter (G3) with 200 ml of the same buffer.
  • Protei A-Sepharose Cl 4B was equilibrated in 100 mM NaPi, pH 7.0. .Abou 3 to 6 ml anti-mannosyltransferase serum was dialyzed for 2 against 1 1 NaPi (100 mM) , pH 7.0. The dialyzed serum was inc bated with the column material for 16 h at 4°C. The serum w removed using a sintered glass filter (G3) .
  • the column materia was washed twice with 10 ml 100 mM NaPi, pH 8.5, and resuspende in 50 ml of the same buffer.
  • dimethylsuberimidate For covalent coupling 0.75 mg/ml dimethylsuberimidate was added The pH was adjusted to pH 8.5 by adding 5-6 drops of 1 M NaOH The material was incubated for 1 h at RT. For a second time, di methylsuberimidate was added and the pH adjusted to pH 8.5 wit 1 M NaOH. The column material was washed serially on a sintere glass filter (G3) with:
  • the material was washed and equilibrated in AB-buffer.
  • each eluted fraction wa analyzed by SDS/PAGE and Western-blot analysis as describe (Strahl-Bolsinger and Tanner, 1991) .
  • the 92 kDa protein con taining fractions fraction 2-6) were pooled and concentrated t 100 ⁇ l via microconcentrators (Centricon/Amicon) by centrifuga tion at 5000 x g. 0.9 ml 98% EtOH were added and the protei precipitated for 16 h at -20°C.
  • the precipitated protein (E.l.2.2) was resuspended in 150 ⁇ SDS-sample buffer (0.07 M Na 2 C0 3 , 0.07% ⁇ -EtSH, 2% SDS, 12% Sac charose, 0.07% bromphenolblue) .
  • SDS-gel electrophoresis ac cording to Lammli and Favre [J. Mol. Biol. .80. (1973), 575] wa carried out at 50-70 V using the BIORAD-Mini-Protean cell. Pro tein standards: HMW-Standards/Gibco BRL.
  • the 92 kDa protein band was cut o (about 10 ⁇ g of protein) .
  • the gel fragment was cut into sma pieces and shaken three times for 30' in 5 ml 50% methanol/1 acetic acid and one time for 30' in 5 ml 50% methanol.
  • the g was lyophylized for 3 h. Trypsin digestion was carried out 0.3 ml 0.2 M ammoniumhydrogen carbonate/2 ⁇ g trypsin for 16 h 37°C. Supernatant was removed.
  • Elution of the peptides was do three times for 1 h at 37°C in 0.2 ml 0.2 M ammoniumhydrog carbonate and one time for 1 h at 37°C in 0.2 ml 0.2 M ammoniu hydrogen carbonate/30% acetonitrile.
  • the eluted material w pooled, lyophylized and resolved in 0.2 ml 1M guanidinium hydr chloride/50 mM Tris/HCl, pH 7.5.
  • Peptidea were separated using reverse phase RP18 column equilibrated in 0.13% TFA.
  • Peptid were eluted by acetonitrile (0-70%) . Up to 40 different pepti peaks could be detected.
  • oligonucleotides A-C we chemically synthesized, using the codon usage of S. cerevisi (Guthrie and Abelson in: The molecular biology of the yea Saccharomyces; eds: J.N. Strathern, E.W. Jones, J.R. Broa (1982)). Oligonucleotides A-C have the following characte istics: Oligonucleotide A: peak: 23 amino acid sequence: G F D G D A Oligodeoxynucleotide: 5• -G T / c GTCACCGTCGAANCC-3• 8-fold degenerated, coding strand, 17 nucleotides
  • Amino acid sequence I S Y K P A S F I S K DNA sequence: 5'-ATTTC T / A TA T / C AA A / G CC A / T GCTTC T / A
  • the chemically synthesized oligodeoxynucleotides A-C were used to screen the plasmid library of yeast genomic DNA pCS19 (Sengstag and Hinnen, Nucl. Acids Res. 15. (1987), 233).
  • This library was prepared by partial digestion of yeast genomic DNA with Sau3A, and cloning into the Bell restriction site of the vector pCS19.
  • oligonucleotides A-C were labeled by kinase reaction, car ⁇ ried out according to Maniatis et al (T. Maniatis, J. Sambrook, E.F. Fritsch (1989) , Molecular cloning: A Laboratory manual, C.S.H. Press) . 40 pmol Oligodeoxynucleotide were labeled using 50 ⁇ Ci [ ⁇ - 32 P] -ATP. Free radioactive nucleotides were removed using "NUC Trap Push columns" (Stratagene) according to the in ⁇ struction manual of the producer. E.2.2. Screening of the library
  • the DNA-library (4992 different single colonies) was transferr from microtiterplates to nitrocellulose. Colony hybridizati was performed according to Grunstein and Hogness (PNAS (1975), 3961) in the following conditions:
  • Hybridization Filters were incubated at 44°C in 100 ml 5 Denhardt's, 6 x NET, 0.1% SDS (w/v), 0.1 mg/ml salmon sperm DN labeled oligodeoxynucleotides A and B (40 pmol each) . Hybridiz tion was performed for 16 h.
  • the filters were exposed to X-r films for 16 h, -70°C. Under these conditions, 12 positively r acting clones could be identified.
  • the 12 positive clones were analyzed in Southern blots usi three different oligodeoxynucleotides. This analysis led to t identification of one positive clone reacting with all thr oligonucleotides. This clone was called pDM3.
  • the 12 positive clones were grown in 5 ml LB medium supplement with ampicillin and their DNA was isolated according to the m thod of Birnbaum and Doly (Nucl. Acid. Res. 1_, (1979), 1513).
  • plasmids 1/10 of each isolated plasmid DNA (plasmids: pDMl-pDM12) was d gested with the restriction enzymes EcoRI-XhoI (5U each) , 1 "one for all" buffer (Pharmacia) in a total volume of 20 ⁇ l f 1 h at 37°C. DNA fragments were separated on a 1% agarose gel and blotted to nitrocellulose according to Maniatis et al. (loc. cit.) . Southern analysis was performed using oligo A and B using the same conditions as described for the library screen. The hy ⁇ bridization temperature for oligo A was 48°C, for oligo B 42°C.
  • Clones 1, 2, 3, 5, 6, 7 and 11 reacted positively with both oli ⁇ godeoxynucleotides. These seven clones were further analyzed by Southern blot analyses. Three identical blots were therefore prepared, in which the DNA of clones 1, 2, 3, 5, 6, 7 and 11 was digested with EcoRI-XhoI and blotted to nitrocellulose as de ⁇ scribed. Blots 1, 2 and 3 were prehybridized in 20 ml 5 x Den ⁇ hardt's, 6 x NET, 0.1% SDS (w/v), 0.1 mg/ml salmon sperm DNA at 50°C for 4 h.
  • Each blot was then hybridized in 10 ml 5 x Den ⁇ hardt's, 6 x NET, 0.1% SDS (w/v), 0.1 mg/ml salmon sperm DNA, 40 pmol labeled oligonucleotides for 16 h.
  • the hybridization tem ⁇ perature is indicated in Table 2, below. Washing was performe for 10' at each temperature in 50 ml 2 x SSC, 0.1% SDS (w/v) .
  • Analytic digestion with endonucleases was performed in 1 x "on for all" buffer (Pharmacia), 0.2 - 0.5 ⁇ g of DNA, 1-5 U restric ⁇ tion enzyme in a total volume of 20 ⁇ l for 1 h at 37°C.
  • Preparative digestion was performed in a total volume of 40-8 ⁇ l with 1-10 ⁇ g of DNA, 5-20 ⁇ l restriction enzyme, 1 x "one fo all" buffer for 2 h at 37°C.
  • DNA fragments were dephosphorylated with alkaline phosphatas according to Maniatis et al. (loc. cit.).
  • DNA fragments were ligated in 1 x T4-ligation buffer (50 m Tris/HCl, pH 7.5, 10 mM MgCl 2 , 5 mM DTT, 1 mM ATP) with 1 U T4 DNA ligase (total volume 10-15 ⁇ l) .
  • Molar DNA ratio of vector insert was 1:4 or 1:8.
  • the absolute amount of DNA was 20-50 ng Incubation time: 16 h at 14°C or 5 h at 25°C.
  • Competent E. coli DH5 ⁇ cells were prepared according to Hanaha (J. Mol. Biol. 166 (1983), 557). Transformation was carried ou as described by Maniatis et al. (loc. cit.) . E.4.1.7. Preparation of DNA
  • Plasmid DNA was prepared according to Birnbaum and Doly (loc cit.) .
  • DNA sequencing was done according to the method of Sanger et al (PNAS 74. (1977) , 5463) . Only plasmid DNA was sequenced. T7-DN polymerase sequencing kit (Pharmacia) was used; the radioactiv nucleotide was [ ⁇ - 35 S] -dATP (spec. act. 600 Ci/mmol) .
  • This example discloses a restriction analysis of pDM3, the iden tification of different DNA fragments recognized by oligonucleo tides A, B or C and their subcloning. Sequencing of these sub clones enabled identification of an ORF.
  • pDM3 DNA was digested with EcoRI, Xhol and EcoRI-Xhol. Souther blot analysis was performed using oligo A, B or C as a target.
  • Oligo A recognizes a 3.0 kb EcoRI fragment
  • oligo B and C re cognize a 1.1 kb EcoRI-Xhol fragment.
  • the 3.0 kb EcoRI fragmen was subcloned into pUC19 (linearized with EcoRI and dephospho rylated) .
  • the 1.1 kb EcoRI-Xhol fragment was subcloned int pUC18 (linearized with EcoRI-Sail, and dephosphorylated) .
  • Righ subclones were identified by restriction analyses and Souther blot analysis using oligo A or B/C, respectively.
  • the 3.0 kb EcoRI subclone was called pMT4, the 1.1 kb EcoRI-Xho subclone was called pMTl.
  • Further restriction analysis of pMT and pMTl was performed using a number of different restrictio endonucleases (for example: Pstl, Hindlll and Bglll) . Souther blot analysis using oligo A or B/C was carried out to define th exact region of a possible ORF.
  • the DNA inserts of plasmids pMT4 and pMTl were sequenced using the universal and reverse primers, priming nex to the polylinker of pUC19/pUC18. Also oligos A, B and C were used as sequencing primers.
  • the sequencing data resulted in a ORF of about 400 bp on both sides of the insert of pMTl.
  • Als pMT4 showed an ORF of about 200 bp when sequenced with the re verse primer. Using these sequencing data, an amino acid se quence could be deduced.
  • oligodeoxynucleotides represent parts of the newly se ⁇ quenced DNA fragments.
  • exolll/mung bean dele ⁇ tions of the vector pMT4 were made.
  • pMT4 was linearized usin Sphl (3' overlap).
  • the plasmid was then cut using BamHI (5' overlap) .
  • Exonuclease III deletion was performed according to Roberts an Lauer (Meth. Enzymol. 68. (1979), 473), Henikoff (Meth. Enzymol. 155 (1987) , 156) .
  • Overlapping ends were removed by mung bean nuclease.
  • the resul ⁇ ting plasmids were analyzed by restriction analysis usin HindiII and EcoRI.
  • Example 5 Northern blot analysis: Identification of mRNA en coding Mannosyltransferase.
  • RNA was separated using a formaldehyde agarose gel an blotted to nitrocellulose as described by Maniatis et al. (loc cit.) . E . 5 . 1.3 .
  • the 1.1 kb insert of pMTl was isolated by EcoRI-Pstl digestion. The fragment was purified using the "Gene-clean kit” (Stratagene) .
  • the nitrocellulose filter was prehybridized for 2 h at 42°C i 20 ml 5 x Denhardt's, 2 x SSC, 0.1% SDS (w/v), 50% formamid (v/v) , 0.1 mg/ml salmon sperm DNA.
  • Hybridization was performe at 42°C for 16 h in 10 ml 1 x Denhardt's, 2 x SSC, 0.1% SD (w/v), 50% formamide (v/v), 0.1 mg/ml salmon sperm DNA, 200 ⁇ [ ⁇ _32p] -dCTP labeled 1.1 kb EcoRI-Pstl fragment of pMTl.
  • Example 6 Preparation of an S. cerevisiae cell deficient in O glvcosylation activity.
  • An S. cerevisiae cell deficient in O-glycosylation activity wa prepared by gene disruption, by insertion of the URA3 gene int the Hindlll restriction site of the identified ORF, at bp 159 of the coding sequence.
  • the 1.1 kb insert of pMTl was isolated as EcoRI-Pstl fragment and subcloned into a pUC18 vector (EcoRI/Pstl linearized, de- phosphorylated, without Hindlll restriction site in the polylin- ker) .
  • the resulting vector was called pMTl.l.
  • pMTl.l was linearized with Hindlll and dephosphorylated.
  • the 1.1 kb Hindlll fragment of YEp24 (Julius et al., Cell 3 (1984), 1075) containing the URA3 gene of S. cerevisiae was isolated and subcloned into the Hindlll linearized, dephosphorylated vector pMTl.l. Clones were identified by restriction analyses and called pMTl.l/URA3 ( Figure 5) .
  • pMTl.l/URA3 has 0.24 kb PMTl coding sequence flanking one side of the URA3 gene and 0.86 kb PMTl coding sequence flanking the other.
  • CsCl-DNA of pMTl.l/URA3 was prepared according to Mania ⁇ tis et al. (loc. cit.).
  • S. cerevisiae strains SEY2101/2102 (Mat a/ ⁇ , ura3-52, leu2-3, 112 (Emr et al., loc. cit.) and SEY2101 (Mat a, ura3-52, leu2-3, 112, ade2-l) were transformed with 5 ⁇ l of the EcoRI/Sphl di gested vector pMTl.l/URA3 according to the method of Ito et al. (J. Bacteriol. 153. (1983), 163).
  • SEY2101/2102 transformants were selected on minimal media + Leu
  • SEY2101 transformants were selected on minimal media + Leu, Ade.
  • transformants could be picked and plate on the same media for a second time.
  • Genomic DNA of three haploid transformants and wild-type cells was isolated as described by Hoffmann and Winston (Gene 57 (1987) , 267) . 1 ⁇ g of the genomic DNA was digested with Xhol/EcoRI, separated on an agarose gel and blotted to nitrocel ⁇ lulose as described by Maniatis et al. (loc. cit.).
  • the blot was prehybridized in 20 ml 5 x Denhardt's, 2 x SSC, 0.1% SDS (w/v), 0.1 mg/ml salmon sperm DNA, 50% formamide (w/v) for 4 h at 42°C.
  • Hybridization was permitted in 10 ml of the same solution adding 200 ng [ ⁇ .- 32 P] -dCTP labeled 1.1 kb EcoRI/Pstl fragment of pMTl.l (see: E.5.1.3) for 16 h at 42°C. Washing was done two times at RT in 50 ml 2 x SSC, 0.1% SDS (w/v) and two times at 68°C in 50 ml 1 x SSC, 0.1% SDS (w/v) . Signal detection by X-ray films. Wild-type cells showed a single signal at 1.1 kb, reflecting the EcoRI/XhoI fragment without URA3 insertion. In the disrupte strains this signal was missing. Instead of this, a new 2.2 k fragment was recognized by the 1.1 kb target, representing th 1.1 kb EcoRI/XhoI fragment bearing the 1.1 kb URA3 insertion.
  • SEY2101 wild-type cells were grown either on YPD (10 g/1 yeas extract; 10 g/1 peptone; 20 g/1 dextrose) or on minimal media Ade, + Leu, + Ura.
  • SEY2101 PMT1::URA3 mutant cells were grow either on YPD or on minimal media + Ade, + Leu. Cells were grow at 30°C in a waterbath shaker. OD578 was measured every 30' af ter sonifying cells. Wild-type and mutant cells show nearl identical growth on both media although, in some cases, mutan cells may stick together. Nevertheless, such cells can easily b separated by sonifying (30", sonify water bath) . The growth cha racteristics of these cells are listed below:
  • Control values (without peptide) were 506 and 1031, respect ⁇ ively.
  • mutant cells show no in vitro mannosyltransferase activity.
  • Membranes (1 ⁇ l of E.7.2.1) were incubated in 20 ⁇ l SDS sample buffer for 1 h at RT. Then SDS/Page and western blot were per ⁇ formed as described by Strahl-Bolsinger and Tanner (loc. cit.). For antibody detection the Peroxidase ECL kit (Amersham) was used according to the instruction manual of the producer. Anti ⁇ bodies against the 92 kDa protein react specifically with a 92 kDa protein of wild-type membranes. In mutant membranes this 92 kDa signal is missing.
  • Wild-type and mutant cells were grown over night in minimal me ⁇ dia containing sucrose as only C-source. 7.5 OD of the culture
  • the pellet was resuspended in 1 ml 0.1 N NaOH (a 10 ⁇ l sample was used for counting radioactivity corresponding to material before ⁇ -elimination) . Incubation was maintained for 24 h at 30°C.
  • ⁇ -eliminated material was desalted via a Dowex 50WS8/H+ column (0.5cm x 6cm). The column was saturated with 0.5M mannose and equilibrated in H 2 0. The ⁇ -elimination sample was loaded onto the column and washed through with 1.5 ⁇ l H 2 0. The flow throug was collected (a 100 ⁇ l sample was used for counting radioacti ⁇ vity corresponding to ⁇ -eliminated material) and concentrated to 10 ⁇ l in the speed-vac. Thin-layer chromatography on Silicagel 60 (Merck) in acetone:butanol:H 2 0 70:15:15 was performed.
  • Mutant cells show reduced glycosylation in comparison to wild type cells.
  • the O-glycosylation in mutant cells is about 40-50 lower than in wild-type cells.
  • Example 8 Cloning of the PMTl homolog of Kluyveromyces lactis .
  • recent years have shown that the productivit of bakers yeast is often limited, especially when the secretio of the product to the culture medium is required, The use o fungal systems other than S. cerevisiae is therefore preferabl in a number of cases [c.f. Romanos et al., Yeast 8. (1992) 423
  • the present invention is no limited to bakers yeast, since the sequence of the PMTl gen isolated from S. cerevisiae can be advantageously used for th identification of genes encoding similar enzymatic activities i other fungal species. Furthermore, the sequence informatio revealed in the present invention may also be used for th identification of related mannosyltransferase encoding genes i
  • the region of the PMTl nucleotide sequence corresponding to th central, hydrophilic region of the mannosyltransferase protei was chosen to design PCR primers [Polymerase-catalyzed Chai Reaction, Saiki et al., Science 230(1985) 1350-1354; Mullis Faloona, Meth. Enzymol. 155 (1987) 335-350] for the amplifica tion of homologous genes. Amplification requires hybridization also termed annealing, of these synthetic oligonucleotides wit its target DNA.
  • the specificity with which individual regions o genomic DNA are amplified depends on the conditions of the P reaction and the degree of homology between the primers and t nucleotide sequence to be amplified. Subsequent to the anneali step, the primers are extended using a thermostable D polymerase. Once the complementary strand has been polymerize the two strands are separated by heat denaturation and a ne cycle of annealing and polymerization may begin.
  • oligonucleotides takes int account that several codons may contain the information for th incorporation of the same amino acid into a nascent polypeptid chain, most often varying in the third ("wobble") position of triplet.
  • Each primer therefore represents a mixture of oligo nucleotides where Y signifies C or T, R signifies A or G, and signifies A, C, G or T.
  • Genomic DNA of K lactis strain CBS2359 was prepared as describe by Sherman et al. ["Methods in Yeast Genetics", Cold Sprin Harbor Laboratory Press (1986) p 127] . lOng of genomic DNA was used in a standard PCR reaction [Sambrock et al., "Molecula Cloning - A Laboratory Manual", second edition, Cold Sprin Harbor Laboratory Press (1989)] in the presence of 1 ⁇ g of eac of the primers and 5% deionized formamide. The amplification wa performed using a "DNA Thermal Cycler" (Perkin Elmer Cetus) an “AmpliTag DNA Polymerase” (Perkin Elmer Cetus, 5 units pe reaction tube) .
  • DNA Thermal Cycler Perkin Elmer Cetus
  • AmpliTag DNA Polymerase Perkin Elmer Cetus, 5 units pe reaction tube
  • the 400 bp fragment amplified with the primer combinatio Sq3908+Sq3910 was subcloned into the vector pCRII (TA CloningTM Invitrogen Corp.) following the indications of the supplier, an partially sequenced according to the method described i E.4.1.9. using the universal primer.
  • Sequence comparison between the S cerevisiae PMTl gene and the fragment isolated from K. lacti genomic DNA by PCR amplification reveals 75% and 80.5% identit on the nucleotide and amino acid level, respectively ( Figure 8)
  • the amplified 400 bp DNA fragment may be used to target selectable marker gene to the K.
  • lactis PMTl chromosomal locu in analogy to the experiment described under E.6. leading to a disrupted gene. This will yield a K. lactis strain with reduce Ser/Thr specific mannosyltransferase activity.
  • the amplified fragment may be used as homologous hybridization probe for the cloning of the entire K. lactis PMTl gene using standar procedures.

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Abstract

This invention relates to improved fungal cells and methods for producing recombinant products of improved quality and in high yields. More specifically, the present invention relates to fungal cells carrying specific modifications within their DNA sequences which cause them to exhibit at least a reduced capacity for O-glycosylating homologous and/or heterologous proteins, and the use of these cells as host cells to produce high yields of recombinant products.

Description

MODIFIED FUNGAL CELLS AND METHOD FOR PRODUCING RECOMBINANT PRODUCTS
This invention relates to improved fungal cells and methods fo producing recombinant products of improved quality and in hig yields. More specifically, the present invention relates t fungal cells carrying specific modifications within their DN sequences which cause them to exhibit at least a reduced capa city for O-glycosylating homologous and/or heterologous pro teins, and the use of these cells as host cells to produce hig yields of recombinant products.
The development of recombinant DNA technology has made possibl the production of foreign products in host cells in which exoge nous DNA sequences coding for those products have been intro duced. The advantage of this technology is that products can b produced in high yields, in highly purified form, with no ris of contamination such as viral contamination (AIDS, hepatitis B etc.) . These recombinant techniques have been widely used fo the production of recombinant proteins in prokaryotic as well a eukaryotic host cells. Prokaryotic host cells include E. col [Nagata et al., Nature 284 (1980), 316; EP 001 929], Bacill sύbtilis [Saunders et al., J. Bacteriol. 169 (1987), 2917] Strep omyces, and Co-rynebacteriiun (EP 433 117) . Eukaryotic hos cells include plant cells, animal cells and fungal cells.
However, the large-scale production of recombinant products these techniques is still limited, due to problems of expressi efficiency of these exogenous DNA sequences, due also to vect instability and to intracellular degradation of the recombina products by the host cell in which they are made. Concerning e pression efficiency, efforts have been made to isolate stro promoters, leading to increased expression levels of exogeno DNA sequences, and therefore to increased production levels recombinant products. Various systems have also been develop in order to increase the stability of the vectors within t host cells, the most frequently used of which consisting in t insertion on the vector of an antibiotic resistance gene enabl¬ ing recombinant host cells to survive and grow in a selective medium. With respect to intracellular degradation, several mutant cells lacking or having a reduced protease activity have been disclosed, thereby limiting the capacity of said cells to degrade recombinant products.
However, additional problems still limit large-scale production and pharmaceutical use of recombinant products. One of these arises from the fact that recombinantly produced products are often different from their natural counterparts. For example, bacterial host cells do not possess all the post-translational mechanisms required for maturation of mammalian polypeptides. Accordingly, said mammalian polypeptides produced in bacteria are often immature or not correctly refolded. Furthermore, bac¬ terial host cells generally introduce an additional N-terminal methionine to the products.
Recombinant products produced in heterologous eukaryotic hosts also usually differ from their naturally-occurring counterpart in their glycosylation content. This may concern the presence versus absence of any carbohydrate structure, the localization of said carbohydrate structure on the product, as well as the nature of the carbohydrate. More specifically, it has been shown that yeast-derived recombinant products often bear additional unnatural O-glycans compared to their natural counterpart. For instance, it has been shown that, while human serum insulin-like growth factor I (IGF-I) is not glycosylated, its recombinant form produced in S. cerevisiae is O-glycosylated and, more pre¬ cisely, O-mannosylated [Hard et al., FEBS Letters 248 (1989), 111] . In the same way, it has been shown that human platelet- derived growth factor (PDGF) and human GM-CSF display unnatural O-mannosyl structures when produced in S. cerevisiae [Biomedic. Environ. Mass Spectrometry 19. (1990), 665; BIO/TECHNOLOGY 5. (1987) , 831] . This abnormal O-glycosylation is the result of im¬ portant differences between the glycosylation mechanisms of mam¬ malian (human) cells and those of other eukaryotic cells, such as yeasts. In this respect, it has been observed that 0-glyco- sylation in fungal cells (including yeasts and filamentous fungi) proceeds in a similar and unusual way so far not observed in any other organism.
The occurrence of this undesirable O-glycosylation on fungal- derived recombinant products constitutes an important drawback to this technology for the production of pharmaceuticals.
The first reason is that fungal-specific glycans may introduce new immunological determinants on a protein, and a glycoprotein with such unnatural carbohydrates may therefore be antigenic when administered to humans. In this respect, it is known for example that most humans have antibodies directed against N- linked yeast annan chains [Feizi and Childs, Biochem. J. 245 (1987) , 1] .
Another reason is that proteins without appropriate carbohydrate structures may also have altered pharmacokinetic properties. It has been shown that carbohydrate structures of glycoproteins in¬ fluence and participate in defining their in vivo clearance rate, which is essential in determining the efficacy of a phar¬ maceutical. More precisely, a mannose receptor has been identi¬ fied on the surface of liver endothelial cells and resident macrophages which apparently represents a means for eliminating glycoproteins displaying mannose-type oligosaccharides [Stahl, Cell. Mol. Biol. 2. (1990), 317]. Therefore, the presence of un¬ natural, additional mannose structures on a protein may increase its clearance rate and thus decrease its plasma half life.
Still another reason is that biological activity of a glycopro¬ tein has also been shown to vary with its carbohydrate content, location and nature. For example, it has been shown that glyco¬ sylation affects the biological properties of recombinant human EPO [Takeuchi et al., Proc. Natl. Acad. Sci. USA jJ6. (1989), 7819] and recombinant human tPA [Parekh et al. , Biochemistry 28. (1989) , 7644] . For the reasons mentioned above, it is clear that the unnatura O-glycosylation of fungal-derived recombinant products can se verely affect their immunological, biological and pharmacokine tic properties, and therefore may prevent their development fo human therapeutic use.
The present invention solves the problem of abnormal O-glyco sylation referred to above by providing modified fungal cell carrying genetic modification(s) within their DNA sequence which cause them to have at least a reduced capacity for O-gly cosylating native or foreign proteins.
Applicant has found that it is possible to obtain geneticall modified fungal cells having reduced capacity of O-glycosylatio which are still viable and show good growth characteristics i industrial fermentation conditions. Unexpectedly, Applicant ha also shown that said genetic modifications do not affect stabi lity of these fungal cells when transformed with exogenous DNA The modified fungal cells of the present invention can be uti lized advantageously as host cells for the production of recom binant products of high quality, having reduced or no un desirable O-glycans.
One object of the present invention is a fungal cell carryin genetic modification(s) within its DNA sequences which cause i to have at least a reduced capacity of O-glycosylation.
The fungal cell of the present invention can be chosen fro filamentous fungi and yeasts. Exemplary genera of filamentou fungi contemplated by the present invention are Aspergillus Trichoder a, Mucor, Neurospora, Fusarium and the like. Exemplar genera of yeasts include Kluyveromyces , Saccharomyces , Pichia Hansenula, Candida, Schizosaccharomyces and the like. More pre ferred genera are those selected from the group consisting o Kluyveromyces, Saccharomyces, Pichia, Hansenula and Candida and, even more preferred, from Jluyvero-uyces and Saccharomyces Exemplary strains of Kluyveromyces which constitute preferre embodiments of this invention include K. lactis, K. fragilis, waltii, K. drosophilarum and the like. The preferred strain of Saccharomyces is S. cerevisiae.
In the meaning of the present invention, genetic modification preferably means any suppression, substitution, deletion or ad¬ dition of one or more bases or of a fragment of the fungal cell DNA sequences. Such genetic modifications may be obtained in vi tro (directly on isolated DNA) or in situ, for example by ge¬ netic engineering techniques or by exposing the fungal cells to mutagenic agents. Mutagenic agents include for example physical agents such as energetic rays (X-rays, χ-rays, UV, etc.) or chemical agents capable of reacting with different functional groups of DNA, such as alkylating agents (EMS, NQO, etc.) bisal- kylating agents, intercalating agents, etc. Genetic modifica¬ tions may also be obtained by genetic disruption, for example according to the method disclosed by Rothstein et al. [Meth. Enzymol. ,194 (1991), 281-301].
According to this method, part or all of a gene is replaced, through homologous recombination, by an in vitro modified ver¬ sion.
Genetic modifications can also be obtained by any mutational in¬ sertion on DNA sequences, such as transposons, phages, etc.
In addition, it is known that certain modifications such as point mutations can be reversed or attenuated by cellular mech¬ anisms. Such modifications may not provide the most useful forms of modified fungal cells of this invention since their phenoty- pical properties may not be very stable. The present invention also provides a process for preparing modified fungal cells in which the modifications (and therefore the phenotypical proper¬ ties) are stable during segregation and/or non-reverting and/or non-leaky. Such modified fungal cells are particularly advanta¬ geous as hosts for the production of recombinant products.
Accordingly, a preferred embodiment of the invention is a fungal cell carrying genetic modification(s) which are stable during segregation and/or non-reverting and/or non-leaky. These modifi¬ cations are generally obtained by deletion(s) or disruption(s) .
The genetic modification(s) carried by the fungal cells of the invention can be located either in a coding region of the DNA sequences of the cell or in a region responsible for or involved in the expression and/or transcriptional regulation of a gene. More particularly, said modification(s) will generally affect the coding region or the region responsible for or involved in the expression and/or the transcriptional regulation of one or more genes whose expression products are enzymes of the O-glyco¬ sylation pathway.
The reduced capacity of the fungal cells of the invention to O- glycosylate proteins may therefore result from the production of inactive enzymes due to structural and/or conformational chan¬ ges, from the production of enzymes having altered biological properties, from the absence of production of said enzymes, or from the production of said enzymes at low levels.
The fungal cell O-glycosylation pathway involves attachment of a first mannosyl residue to the hydroxyl group of seryl and/or threonyl amino acids of proteins or peptides, and then the ex¬ tension to O-linked di- and oligosaccharides by subsequent addi¬ tion of mannosyl residues. The first mannosyl residue is trans¬ ferred from dolichol monophosphate mannose (Dol-P-Man) to th protein in the endoplasmic reticulum, and the additional manno¬ syl residues are transferred from GPD-Man in the Golgi. I contrast, higher eukaryotic (non-fungal) cells O-glycosylat following a different mechanism, in that the initial step is th covalent attachment of N-acetyl-galactosamine to seryl or threo nyl amino acids, no lipid-coupled oligosaccharide donor is in volved in this first reaction, the initial step occurs in th Golgi, the structures of carbohydrates are different, etc.
In a preferred embodiment of the invention, the modified funga cells carry genetic modification(s) in at least one gene whos expression product is involved in the attachment of a mannosyl residue to the hydroxyl group of seryl or threonyl amino acids.
In a more preferred embodiment of the invention, the modified fungal cells carry genetic modification(s) in at least one gene whose expression product is involved in the transfer of a manno¬ syl residue from the Dol-P-Man precursor to the hydroxyl group of seryl or threonyl amino acids.
Still more preferably, one of these genes is the gene encoding the Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase [DPM2 - also designated PMT1] whose sequence is represented in Figure 4, or any homologous gene encoding the same activity as defined below.
In addition to modification(s) in one gene involved in the at¬ tachment of mannosyl residues to the hydroxyl group of seryl or threonyl amino acids, fungal cells of the invention may also carry modification(s) in the genes involved in subsequent addi¬ tions of mannosyl residues leading to di- or oligosaccharides, or in the synthesis of the mannosyl residues donnor (Dol-P-Man) .
Specific examples of such fungal cells are disclosed in the ex¬ amples.
Another object of the invention resides in a fungal cell as disclosed above in which an exogenous DNA sequence has been in¬ troduced.
In the meaning of the present invention, the term exogenous DNA sequence includes any DNA sequence comprising one or more genes encoding a desired protein to be expressed and/or secreted in said cell. Such a DNA sequence may be a complementary DNA sequence (cDNA) , an artificial DNA sequence, a genomic DNA se¬ quence, a hybrid DNA sequence or a synthetic or semi-synthetic DNA sequence, included in an expression cassette enabling syn¬ thesis in the fungal cells of said proteins. The expression cas¬ sette preferably comprises a transcription and translation initiation region joined to the 5' end of the sequence encoding said desired protein(s) so as to direct, and optionally regu¬ late, the transcription and translation of said sequence. The choice of these regions may vary according to the fungal cell used. Generally, these sequences are chosen from promoters and/or terminators derived from fungal cell genes, and, when ex¬ pression in yeast hosts is sought, from yeast genes. Of special interest are certain promoter and/or terminator regions derived from glycolytic genes of fungal cells such as, for yeasts, the genes encoding phosphoglycerate kinase (PGK) , glyceraldehyde-3- phosphate dehydrogenase (GDP) , enolases (ENO) or alcohol dehy- drogenases (ADH) . and for filamentous fungi, the genes encoding triose phosphate isomerase (tpi) . The promoter and/or terminator regions may also derive from other strongly expressed genes such as, for yeasts, the lactase gene (LAC4) . the acid phosphatase gene (PH05) . the alcohol oxidase gene (AOX) or the methanol oxi- dase gene (MOX) . and, for filamentous fungi, the cellobiohydro- lase gene (CBHI) . the alcohol dehydrogenase gene (alcA. alcC) . the glucoamylase gene (GAM) or the acetamidase gene (amds) , an the like. These transcription and translation initiation regions may be further modified, e.g. by in vitro mutagenesis, by intro¬ duction of additional control elements or synthetic sequences, or by deletions. For example, transcription-regulating elements, such as the so-called UAS, originating from another promoter ma be used to construct hybrid promoters which enable the growt phase of the fungal cell culture to be separated from the phas of expression of the desired protein(s) encoding sequence(s). transcription and translation termination region functional i the intended fungal cell may also be positioned at the 3' end o the coding sequence. In addition, at the N-terminus of the pro tein sequence, a signal peptide (pre-sequence) may be introduce so as to direct the nascent protein to the secretory pathway o the fungal cell used. This pre-sequence may correspond to th natural pre-sequence of the protein if this protein is naturall secreted, or it may be of another origin, e.g. obtained fro another gene, or even artificial. Preferably, the exogenous DNA sequence is part of a vector, which may either replicate autonomously in the fungal cell used or integrate into its own DNA sequences (chromosome) . Autono¬ mously replicating vectors may contain autonomously replicating sequences derived from the chromosomal DNA of the fungal cell (ARS) or from naturally-occurring fungal cell plasmids such as pGKl-1 [de Louvencourt et al., J. Bacteriol. 154 (1982), 737], pKDl (EP 241 435), 2μm plasmid (Broach, Cell 28. (1982), 203-204) and the like. Integrating vectors usually contain sequences ho¬ mologous to regions of the fungal cell chromosome which, after being introduced into said cell, enable integration through in vivo recombination. In a specific embodiment of the invention, said homologous sequences correspond to the region of the chro¬ mosome to be modified in the fungal cell, enabling a one-step modification-integration mechanism. Integration may also occur through non-homologous recombination.
The exogenous DNA sequence can be introduced into the fungal cell by any technique known in the art, and, for example, by re¬ combinant DNA techniques, genetic crossings, protoplast fusions, etc. Concerning recombinant DNA techniques, transformation, electroporation, or any other technique disclosed in the litera¬ ture may be used. More specifically, when the fungal cell is a yeast cell, the transformation may be performed according to the methods of Ito et al., [J. Bacteriol. 153 (1983), 163], Durrens et al. [Curr. Genet. 18. (1990), 7] or following the method disclosed in EP 361 991. Electroporation can be performed ac¬ cording to Karube et al. [FEBS Letters .82, (1985), 90] .
The fungal cells of the present invention can be advantageously utilized as host cells for the production of recombinant pro¬ ducts such as heterologous proteins having pharmaceutical and/or agro-foodstuff interest. The fungal cells of this invention are particularly advantageous since they enable the production and/or secretion of high quality products, and since their gene¬ tic modifications do not affect the mitotic or genetic stability of said products' expression vectors. The cells of this inven¬ tion are more particularly suitable for the production of pro- teins having human therapeutic uses and which are susceptible to O-glycosylation by the host cell.
Accordingly, a further object of this invention resides in a process for the production of recombinant products wherein a fungal cell as defined above is cultivated in conditions in which the exogenous DNA sequence is expressed and the product is recovered. In a preferred embodiment, said product is secreted into the culture medium. In another preferred embodiment, said product is susceptible to O-glycosylation by the host cell.
The following proteins are cited as examples of heterologous proteins which can be prepared with the fungal cells of the pre¬ sent invention: enzymes (such as superoxide dismutase, catalase, amylases, lipases, amidases, chymosine, etc., or any fragment or derivative thereof) , blood derivatives (such as human serum- albumin, alpha- or beta-globin, factor VIII, factor IX, van Wil- lebrand factor, fibronectin, alpha-1 antitrypsin, etc., or any fragment or derivative thereof) , insulin and its variants, lym- phokines [such as interleukins, interferons, colony stimulating factors (G-CSF, GM-CSF, M-CSF...), TNF, TRF, etc., or any frag¬ ment or derivative thereof] , growth factors (such as growth hor¬ mone, erythropoietin, FGF, EGF, PDGF, TGF, etc., or any fragment or derivative thereof) , apolipoproteins, antigenic polypeptides for the preparation of vaccines (hepatitis, cytomegalovirus, Eppstein-Barr, herpes, etc.), or any fusion polypeptide such as, for example, fusions comprising an active moiety linked to a stabilizing moiety.
Another object of the invention resides in a DNA fragment encod¬ ing an enzyme involved in the attachment of mannosyl residues to the hydroxyl group of seryl or threonyl amino acids of proteins. Applicant has provided DNA fragments encoding such enzymes for the first time. More preferably, said DNA fragment comprises the Dol-P-Man:Protein (Ser/Thr) mannosyltransferase gene whose se¬ quence is represented in Figure 4, any homologous gene, deriva¬ tive or fragment thereof. In the meaning of the present invention, homologous gene means any other gene of any fungal cell encoding an enzyme having the required activity. Said other genes may be obtained, for exam¬ ple, by complementation of a mutant fungal cell deficient in said activity with DNA prepared from a fungal cell capable of said activity, selection of the transformants having recovered the activity, and isolating their inserted DNA sequence. These other genes may also be isolated from DNA libraries by hybridi¬ zation with probe(s) (including PCR primers) comprising all or part of the sequence presented in Figure 4. In this respect, it is also an object of this invention to use the DNA fragments provided, or any part thereof, as hybridization probe(s) or for the complementation of mutant phenotypes, for the obtention of homologous genes of fungal cells.
The term derivative means any other DNA fragment prepared by any genetic and/or chemical modification(s) of the genes mentioned above. Said genetic and/or chemical modification-is) may be any suppression, substitution, deletion or addition of one or more bases or of a region of said genes, leading either to an in¬ creased enzyme activity or to the same activity level, or to a~ decreased or null enzyme activity upon transformation in a fun¬ gal host cell.
LEGEND OF THE FIGURES
Figure 1 Restriction map of plasmids pDM3, pMT4 and pMTl.
Figure 2 Subcloning of plasmid pDM3.
Figure 3 Strategy of sequencing of the PMTl gene. Nucleotide sequence of the PMTl gene (SEQ ID N°l) . Amino acid sequence of the PMTl gene (SEQ ID N°2) . Construction and restriction map of pMTl.l/URA3. O-glycosylation activity of S. cerevisiae WT (panel
A) and MT (panel B) .
Partial nucleotide sequence of the K. lactis PMTl gene (SEQ ID No. 3) .
Figure imgf000013_0001
Nucleotide (Panel A) and predicted amino acid (Panel
B) sequence comparison between the S. cerevisiae PMTl gene (upper sequences) and the K. lactis homolog
(lower sequences) isolated by PCR amplification of K. lactis genomic DNA. Dots represent sequence identity, question marks indicate sequence ambiguity. Th nucleotide sequence complementary to the prime Sq3910 is underlined.
EXAMPLES
Example 1; Isolation of a highly purified mannosyltransferas from S. cerevisiae and generation of peptides
The mannosyltransferase activity was solubilized from tota yeast membranes and purified on hydroxylapatite according t Strahl-Bolsinger and Tanner (Eur. J. Biochem. 196 (1991) , 185) The protein then had to be further enriched by (NH4)2S04 preci pitation before additional purification was performed via affi nity chromatography. The eluted material was then separated o SDS/PAGE. The resulting 92 kDa band was cut out of the gel Trypsin digestion (in the gel) yielded several non-overlappin peptides, enabling designing of probes.
E.l.l. ( Hfl^SO^ precipitation
100 ml of fractions of the hydroxylapatite column containin mannosyltransferase activity was mixed with (NH4)2S04 up to final concentration of 30% (w/v) and stirred gently for 1 h i an ice/salt bath. The mixture was centrifuged for 30' (8000 g) . The resulting pellet was resuspended in 8 ml AB-buffer (1 mM Tris/HCl, pH 7.5, 15% glycerol (vol%) , 0.1% lubrol (vol%) 150 mM NaCl) and dialyzed for 1 h against the same buffer Storage: -20°C.
E.1.2. Affinity chromatography
E.1.2.1. Preparation of the affinity chromatography column
0.5 g freeze-dried powder of Protein A-Sepharose Cl 4B was swol len in 10 ml 100 mM NaPi, pH 7.0, for 15' and washed on a sin tered glass filter (G3) with 200 ml of the same buffer. Protei A-Sepharose Cl 4B was equilibrated in 100 mM NaPi, pH 7.0. .Abou 3 to 6 ml anti-mannosyltransferase serum was dialyzed for 2 against 1 1 NaPi (100 mM) , pH 7.0. The dialyzed serum was inc bated with the column material for 16 h at 4°C. The serum w removed using a sintered glass filter (G3) . The column materia was washed twice with 10 ml 100 mM NaPi, pH 8.5, and resuspende in 50 ml of the same buffer.
For covalent coupling 0.75 mg/ml dimethylsuberimidate was added The pH was adjusted to pH 8.5 by adding 5-6 drops of 1 M NaOH The material was incubated for 1 h at RT. For a second time, di methylsuberimidate was added and the pH adjusted to pH 8.5 wit 1 M NaOH. The column material was washed serially on a sintere glass filter (G3) with:
a) 50 ml 100 mM NaPi, pH 8.0 b) 25 ml 100 mM NaPi, pH 8.0, 3 M ammonium rhodanide c) 100 ml 100 mM NaPi, pH 8.0
The material was washed and equilibrated in AB-buffer.
E.l.2.2. Purification of the 92 kDa protein
8 ml of the (NH4)2S04 precipitated and dialyzed protein (E.l.l.) was incubated with the affinity column material (E.1.2.1) for 1 h at 4°C with gentle shaking. A column (2 cm x 0.5 cm) wa filled and washed with 15 ml AB-buffer. The column was elute with 100 mM glycine/HCl pH 3.0, 0.05% lubrol (vol%) , 15% glyce- rol (vol%) . Fractions of 0.9 ml were collected and neutralize immediately with 1 M Tris (15 μl/0.9 ml fraction) .
To detect the 92 kDa protein, 40 μl of each eluted fraction wa analyzed by SDS/PAGE and Western-blot analysis as describe (Strahl-Bolsinger and Tanner, 1991) . The 92 kDa protein con taining fractions (fraction 2-6) were pooled and concentrated t 100 μl via microconcentrators (Centricon/Amicon) by centrifuga tion at 5000 x g. 0.9 ml 98% EtOH were added and the protei precipitated for 16 h at -20°C. The precipitated protein wa pelleted by centrifugation for 30' at 10,000 x g.
E.1.3. SDS-PAGE
The precipitated protein (E.l.2.2) was resuspended in 150 μ SDS-sample buffer (0.07 M Na2C03, 0.07% β-EtSH, 2% SDS, 12% Sac charose, 0.07% bromphenolblue) . SDS-gel electrophoresis ac cording to Lammli and Favre [J. Mol. Biol. .80. (1973), 575] wa carried out at 50-70 V using the BIORAD-Mini-Protean cell. Pro tein standards: HMW-Standards/Gibco BRL.
Protein was detected by staining with 0.05% Coomassie R25 (w/v) , 25% isopropanol (vol%) , 10% acetic acid (vol%) and de staining in 7.5% acetic acid (vol%) . E.1.4 Trvpsin digestion and designing of oligonucleotides
After SDS-PAGE (E.1.3.) the 92 kDa protein band was cut o (about 10 μg of protein) . The gel fragment was cut into sma pieces and shaken three times for 30' in 5 ml 50% methanol/1 acetic acid and one time for 30' in 5 ml 50% methanol. The g was lyophylized for 3 h. Trypsin digestion was carried out 0.3 ml 0.2 M ammoniumhydrogen carbonate/2μg trypsin for 16 h 37°C. Supernatant was removed. Elution of the peptides was do three times for 1 h at 37°C in 0.2 ml 0.2 M ammoniumhydrog carbonate and one time for 1 h at 37°C in 0.2 ml 0.2 M ammoniu hydrogen carbonate/30% acetonitrile. The eluted material w pooled, lyophylized and resolved in 0.2 ml 1M guanidinium hydr chloride/50 mM Tris/HCl, pH 7.5. Peptidea were separated using reverse phase RP18 column equilibrated in 0.13% TFA. Peptid were eluted by acetonitrile (0-70%) . Up to 40 different pepti peaks could be detected. Five of the main peaks were sequenc via automated sequence analysis according to Edman (G. Allen i Sequencing of proteins and peptides, Laboratory Techniques Biochem. and Mol. Biol. 9_ ed. : Burdon, R.H. & Knippenberg, P.H Elsevier (1989)). Among the sequences thereby obtained, thr were suitable for designing oligonucleotides, which are prese ted in Table 1, below.
Table 1
Figure imgf000017_0001
On the basis of these sequences, oligonucleotides A-C we chemically synthesized, using the codon usage of S. cerevisi (Guthrie and Abelson in: The molecular biology of the yea Saccharomyces; eds: J.N. Strathern, E.W. Jones, J.R. Broa (1982)). Oligonucleotides A-C have the following characte istics: Oligonucleotide A: peak: 23 amino acid sequence: G F D G D A Oligodeoxynucleotide: 5• -GT/cGTCACCGTCGAANCC-3• 8-fold degenerated, coding strand, 17 nucleotides
Oligonucleotide B: Peak: 34
Amino acid sequence: E P H V Y E DNA sequence: 5'-c/TTCGTAGACG/ATGA/TGGT/cTC-3' 16-fold degenerated, coding strand, 18 nucleotides
Oligonucleotide C: Peak: 15
Amino acid sequence: I S Y K P A S F I S K DNA sequence: 5'-ATTTCT/ATAT/CAAA/GCCA/TGCTTCT/A
TTT/AAAA-3• 128-fold degenerated, coding strand, 33 nucleotides
Example 2; Screening of a plasmid library of vβast genomic DNA
The chemically synthesized oligodeoxynucleotides A-C (E.1.4.) were used to screen the plasmid library of yeast genomic DNA pCS19 (Sengstag and Hinnen, Nucl. Acids Res. 15. (1987), 233). This library was prepared by partial digestion of yeast genomic DNA with Sau3A, and cloning into the Bell restriction site of the vector pCS19.
E.2.1. Labeling of Oligodeoxynucleotides
The oligonucleotides A-C were labeled by kinase reaction, car¬ ried out according to Maniatis et al (T. Maniatis, J. Sambrook, E.F. Fritsch (1989) , Molecular cloning: A Laboratory manual, C.S.H. Press) . 40 pmol Oligodeoxynucleotide were labeled using 50 μCi [γ-32P] -ATP. Free radioactive nucleotides were removed using "NUC Trap Push columns" (Stratagene) according to the in¬ struction manual of the producer. E.2.2. Screening of the library
The DNA-library (4992 different single colonies) was transferr from microtiterplates to nitrocellulose. Colony hybridizati was performed according to Grunstein and Hogness (PNAS (1975), 3961) in the following conditions:
- Prehybridization: Filters were incubated at 44°C in 200 ml x Denhardt's, 6 x NET, 0.1% SDS (w/v), 0.1 mg/ml salmon spe DNA, for at least 4 h (5 x Denhardt's: 0.1% ficoll, 0.1% pol vinylpyrrolidone, 0.1% BSA; 6 x NET: 0.9 M NaCl, 90 mM Tris-HC pH 8.3, 6 mM EDTA, pH 8.0).
- Hybridization: Filters were incubated at 44°C in 100 ml 5 Denhardt's, 6 x NET, 0.1% SDS (w/v), 0.1 mg/ml salmon sperm DN labeled oligodeoxynucleotides A and B (40 pmol each) . Hybridiz tion was performed for 16 h.
- Washing conditions: Filters were washed three times in 50 6 x SSC, 0.1% SDS (w/v) at 0°C for 15'.
To detect positive colonies, the filters were exposed to X-r films for 16 h, -70°C. Under these conditions, 12 positively r acting clones could be identified.
Example 3: Southern Analysis of the 12 positive clones
The 12 positive clones were analyzed in Southern blots usi three different oligodeoxynucleotides. This analysis led to t identification of one positive clone reacting with all thr oligonucleotides. This clone was called pDM3.
The 12 positive clones were grown in 5 ml LB medium supplement with ampicillin and their DNA was isolated according to the m thod of Birnbaum and Doly (Nucl. Acid. Res. 1_, (1979), 1513).
1/10 of each isolated plasmid DNA (plasmids: pDMl-pDM12) was d gested with the restriction enzymes EcoRI-XhoI (5U each) , 1 "one for all" buffer (Pharmacia) in a total volume of 20 μl f 1 h at 37°C. DNA fragments were separated on a 1% agarose gel and blotted to nitrocellulose according to Maniatis et al. (loc. cit.) . Southern analysis was performed using oligo A and B using the same conditions as described for the library screen. The hy¬ bridization temperature for oligo A was 48°C, for oligo B 42°C. Clones 1, 2, 3, 5, 6, 7 and 11 reacted positively with both oli¬ godeoxynucleotides. These seven clones were further analyzed by Southern blot analyses. Three identical blots were therefore prepared, in which the DNA of clones 1, 2, 3, 5, 6, 7 and 11 was digested with EcoRI-XhoI and blotted to nitrocellulose as de¬ scribed. Blots 1, 2 and 3 were prehybridized in 20 ml 5 x Den¬ hardt's, 6 x NET, 0.1% SDS (w/v), 0.1 mg/ml salmon sperm DNA at 50°C for 4 h. Each blot was then hybridized in 10 ml 5 x Den¬ hardt's, 6 x NET, 0.1% SDS (w/v), 0.1 mg/ml salmon sperm DNA, 40 pmol labeled oligonucleotides for 16 h. The hybridization tem¬ perature is indicated in Table 2, below. Washing was performe for 10' at each temperature in 50 ml 2 x SSC, 0.1% SDS (w/v) .
Table 2
Figure imgf000020_0001
Clone 3 was the only clone reacting with oligo A, B and C. Th clone was called pDM3 and further analyzed. Byam lft 4t Analysis of pDM3
E.4.1. Methods
E.4.1.1 Digestion with restriction endonucleases
Analytic digestion with endonucleases was performed in 1 x "on for all" buffer (Pharmacia), 0.2 - 0.5 μg of DNA, 1-5 U restric¬ tion enzyme in a total volume of 20 μl for 1 h at 37°C.
Preparative digestion was performed in a total volume of 40-8 μl with 1-10 μg of DNA, 5-20 μl restriction enzyme, 1 x "one fo all" buffer for 2 h at 37°C.
E.4.1.2. DNA-gelelectrophoresis
Separation of DNA fragments was performed according to Maniati et al. (loc. cit.) .
E.4.1.3. Isolation of DNA fragments _
After separation, DNA fragments were isolated using the "Gene clean kit" (Stratagene) according to the instruction manual—o the producer.
E.4.1.4. Treatment with alkaline phosphatase
DNA fragments were dephosphorylated with alkaline phosphatas according to Maniatis et al. (loc. cit.).
E.4.1.5. Ligation
DNA fragments were ligated in 1 x T4-ligation buffer (50 m Tris/HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT, 1 mM ATP) with 1 U T4 DNA ligase (total volume 10-15 μl) . Molar DNA ratio of vector insert was 1:4 or 1:8. The absolute amount of DNA was 20-50 ng Incubation time: 16 h at 14°C or 5 h at 25°C.
E.4.1.6. Transformation of E. coli
Competent E. coli DH5α cells were prepared according to Hanaha (J. Mol. Biol. 166 (1983), 557). Transformation was carried ou as described by Maniatis et al. (loc. cit.) . E.4.1.7. Preparation of DNA
Plasmid DNA was prepared according to Birnbaum and Doly (loc cit.) .
E.4.1.8. Southern blot analysis
Southern blot analysis was performed using the same condition a described in E.3.
E.4.1.9. DNA sequence analysis
DNA sequencing was done according to the method of Sanger et al (PNAS 74. (1977) , 5463) . Only plasmid DNA was sequenced. T7-DN polymerase sequencing kit (Pharmacia) was used; the radioactiv nucleotide was [α-35S] -dATP (spec. act. 600 Ci/mmol) .
E.4.2. Identification of the ORF
This example discloses a restriction analysis of pDM3, the iden tification of different DNA fragments recognized by oligonucleo tides A, B or C and their subcloning. Sequencing of these sub clones enabled identification of an ORF.
E.4.2.1. Subcloning of pDM3 DNA fragments hybridizing with olig A, B or C.
pDM3 DNA was digested with EcoRI, Xhol and EcoRI-Xhol. Souther blot analysis was performed using oligo A, B or C as a target.
Oligo A recognizes a 3.0 kb EcoRI fragment, oligo B and C re cognize a 1.1 kb EcoRI-Xhol fragment. The 3.0 kb EcoRI fragmen was subcloned into pUC19 (linearized with EcoRI and dephospho rylated) . The 1.1 kb EcoRI-Xhol fragment was subcloned int pUC18 (linearized with EcoRI-Sail, and dephosphorylated) . Righ subclones were identified by restriction analyses and Souther blot analysis using oligo A or B/C, respectively.
The 3.0 kb EcoRI subclone was called pMT4, the 1.1 kb EcoRI-Xho subclone was called pMTl. Further restriction analysis of pMT and pMTl was performed using a number of different restrictio endonucleases (for example: Pstl, Hindlll and Bglll) . Souther blot analysis using oligo A or B/C was carried out to define th exact region of a possible ORF.
Restriction maps of pDM3, pMT4 and pMTl are shown on Figure 1.
E.4.2.2. Sequence analysis
From both ends, the DNA inserts of plasmids pMT4 and pMTl wer sequenced using the universal and reverse primers, priming nex to the polylinker of pUC19/pUC18. Also oligos A, B and C wer used as sequencing primers. The sequencing data resulted in a ORF of about 400 bp on both sides of the insert of pMTl. Als pMT4 showed an ORF of about 200 bp when sequenced with the re verse primer. Using these sequencing data, an amino acid se quence could be deduced. This AA-sequence showed peptide sequen ces known from the peptide analysis of the 92 kDa protei (peptides corresponding to peaks 15, 23, 34 were found) . Ac cording to these data, the 5'/3' orientation of the gene coul be predicted.
Several other subclones were constructed and sequenced using th universal and reverse primers of pUC18/l9 (Figure 2) .
The following oligodeoxynucleotides were also used for sequen cing:
Figure imgf000023_0001
These oligodeoxynucleotides represent parts of the newly se¬ quenced DNA fragments.
For sequencing the 5' region of the gene, exolll/mung bean dele¬ tions of the vector pMT4 were made. pMT4 was linearized usin Sphl (3' overlap). The plasmid was then cut using BamHI (5' overlap) .
Exonuclease III deletion was performed according to Roberts an Lauer (Meth. Enzymol. 68. (1979), 473), Henikoff (Meth. Enzymol. 155 (1987) , 156) .
Overlapping ends were removed by mung bean nuclease. The resul¬ ting plasmids were analyzed by restriction analysis usin HindiII and EcoRI.
Sequence analysis of the clones was carried out using the re verse primer of pUC19. The sequence strategy is shown in Figur 3. Sequence data are given in Figure 4.
Example 5; Northern blot analysis: Identification of mRNA en coding Mannosyltransferase.
E.5.1. Methods
E.5.1.1. Isolation of RNA
Total RNA was isolated from yeast strain SEY2101 (Mat a, ade2-l, leu2-3, 112, ura3-52 (Emr et al. PNAS .80. (1983), 7080) accordin to Domdey et al. (Cell 39. (1984), 611).
E.5.1.2. Northern blot
Total RNA was separated using a formaldehyde agarose gel an blotted to nitrocellulose as described by Maniatis et al. (loc cit.) . E . 5 . 1.3 . The DNA- target
The 1.1 kb insert of pMTl was isolated by EcoRI-Pstl digestion. The fragment was purified using the "Gene-clean kit" (Stratagene) .
200 ng of the DNA fragment were labeled with [α-32P] -dCTP (50 μCi) using the "megaprime" labeling kit (Amersham) according t the instruction manual of the producer.
E.5.2. Results
The nitrocellulose filter was prehybridized for 2 h at 42°C i 20 ml 5 x Denhardt's, 2 x SSC, 0.1% SDS (w/v), 50% formamid (v/v) , 0.1 mg/ml salmon sperm DNA. Hybridization was performe at 42°C for 16 h in 10 ml 1 x Denhardt's, 2 x SSC, 0.1% SD (w/v), 50% formamide (v/v), 0.1 mg/ml salmon sperm DNA, 200 μ [α_32p] -dCTP labeled 1.1 kb EcoRI-Pstl fragment of pMTl. Washin was done twice at RT and two times at 50°C in _50 ml 0.1 x SSC, 0.1% SDS (w/v) . Hybridization of the target was detected by ex posure to X-ray film (-70°C, 16 h) . A single mRNA with the siz of 3 kb was detected. "~
This procedure may be easily repeated by the person skilled i the art with other probes derived from the sequence of Figure and with RNA from other sources (other fungal cells) .
Example 6: Preparation of an S. cerevisiae cell deficient in O glvcosylation activity.
An S. cerevisiae cell deficient in O-glycosylation activity wa prepared by gene disruption, by insertion of the URA3 gene int the Hindlll restriction site of the identified ORF, at bp 159 of the coding sequence. E.6.1. Construction of the plasmid used for the gene disruption
The 1.1 kb insert of pMTl was isolated as EcoRI-Pstl fragment and subcloned into a pUC18 vector (EcoRI/Pstl linearized, de- phosphorylated, without Hindlll restriction site in the polylin- ker) . The resulting vector was called pMTl.l.
pMTl.l was linearized with Hindlll and dephosphorylated. The 1.1 kb Hindlll fragment of YEp24 (Julius et al., Cell 3 (1984), 1075) containing the URA3 gene of S. cerevisiae was isolated and subcloned into the Hindlll linearized, dephosphorylated vector pMTl.l. Clones were identified by restriction analyses and called pMTl.l/URA3 (Figure 5) .
pMTl.l/URA3 has 0.24 kb PMTl coding sequence flanking one side of the URA3 gene and 0.86 kb PMTl coding sequence flanking the other. CsCl-DNA of pMTl.l/URA3 was prepared according to Mania¬ tis et al. (loc. cit.).
E.6.2. Transformation of veast
40 μg of pMTl.l/URA3 CsCl-DNA was digested with Sphl/EcoRI. T check that the digestion was complete, part of the digested DN was analyzed on a DNA agarose gel. The digest was then pheno- lized and the DNA precipitated with 98% EtOH (Maniatis et al., loc. cit.) . DNA was resolved in 10 μl TE, pH 8.0.
S. cerevisiae strains SEY2101/2102 (Mat a/α, ura3-52, leu2-3, 112 (Emr et al., loc. cit.) and SEY2101 (Mat a, ura3-52, leu2-3, 112, ade2-l) were transformed with 5 μl of the EcoRI/Sphl di gested vector pMTl.l/URA3 according to the method of Ito et al. (J. Bacteriol. 153. (1983), 163).
SEY2101/2102 transformants were selected on minimal media + Leu SEY2101 transformants were selected on minimal media + Leu, Ade.
After 3-4 days at 30°C, transformants could be picked and plate on the same media for a second time. E.6.3. Genomic Southern blot of the transformants
Genomic DNA of three haploid transformants and wild-type cells was isolated as described by Hoffmann and Winston (Gene 57 (1987) , 267) . 1 μg of the genomic DNA was digested with Xhol/EcoRI, separated on an agarose gel and blotted to nitrocel¬ lulose as described by Maniatis et al. (loc. cit.).
The blot was prehybridized in 20 ml 5 x Denhardt's, 2 x SSC, 0.1% SDS (w/v), 0.1 mg/ml salmon sperm DNA, 50% formamide (w/v) for 4 h at 42°C.
Hybridization was permitted in 10 ml of the same solution adding 200 ng [α.-32P] -dCTP labeled 1.1 kb EcoRI/Pstl fragment of pMTl.l (see: E.5.1.3) for 16 h at 42°C. Washing was done two times at RT in 50 ml 2 x SSC, 0.1% SDS (w/v) and two times at 68°C in 50 ml 1 x SSC, 0.1% SDS (w/v) . Signal detection by X-ray films. Wild-type cells showed a single signal at 1.1 kb, reflecting the EcoRI/XhoI fragment without URA3 insertion. In the disrupte strains this signal was missing. Instead of this, a new 2.2 k fragment was recognized by the 1.1 kb target, representing th 1.1 kb EcoRI/XhoI fragment bearing the 1.1 kb URA3 insertion.
Example 7: Characterization of the mutant
E.7.1. Growth
SEY2101 wild-type cells were grown either on YPD (10 g/1 yeas extract; 10 g/1 peptone; 20 g/1 dextrose) or on minimal media Ade, + Leu, + Ura. SEY2101 PMT1::URA3 mutant cells were grow either on YPD or on minimal media + Ade, + Leu. Cells were grow at 30°C in a waterbath shaker. OD578 was measured every 30' af ter sonifying cells. Wild-type and mutant cells show nearl identical growth on both media although, in some cases, mutan cells may stick together. Nevertheless, such cells can easily b separated by sonifying (30", sonify water bath) . The growth cha racteristics of these cells are listed below:
Figure imgf000028_0001
In a logarithmically growing culture, 54.7% of wild-type cells and 56% of mutant cells show buds. After growing for 24 h on YPD wild-type cells reached an OD578 of 11.4 and mutant cells of 12.3.
E.7.2. Jn vitro mannosyltransferase activity and Western blot
E.7.2.1. Preparation of crude membranes
SEY2101 was grown in 100 ml minimal media + Ade + Leu + Ura to OD578 = 0.5. SEY2101 PMT1::URA3 was grown in 100 ml minimal me¬ dia + Ade, + Leu to OD578 = 0.5.
Two preparations of each strain were carried out. Work was per¬ formed on ice; all buffers were at 4°C. 40 OD of cells were pel¬ leted and washed in 25 ml TMA (50 mM Tris/HCl, pH 7.5, 7.5 mM MgCl2) . Cells were resuspended in 100 μl TMA and transferred to a violax tube. 0.3 g of glass beads were added and cells broken on a vortex four times for 30" (cooling on ice between breaking intervals) . The extract is removed from glass beads using a pasteur pipette. Glass beads are washed three times with 250 μl TMA. All washing solutions are pooled in an Eppendorf cup. The solution is centrifuged for 15" (10,000 x g) . Supernatant is re¬ moved and the pellet resuspended in 40 μl TMA (1 OD = 1 μl)
E.7.2.2. Mannosyltransferase assay ( in vi tro)
1 and 5 μl of the crude membranes (E.7.2.1) were tested for en¬ zyme activity as described by Strahl-Bolsinger and Tanner (loc. cit.) . Two parallel samples from wild-type and mutant cells wer measured. Mean values of these two independent measurements ar shown.
Figure imgf000029_0001
!) Control values (without peptide) were 506 and 1031, respect¬ ively.
In contrast to wild-type cells, mutant cells show no in vitro mannosyltransferase activity.
E.7.2.3. Western blot analysis
Membranes (1 μl of E.7.2.1) were incubated in 20 μl SDS sample buffer for 1 h at RT. Then SDS/Page and western blot were per¬ formed as described by Strahl-Bolsinger and Tanner (loc. cit.). For antibody detection the Peroxidase ECL kit (Amersham) was used according to the instruction manual of the producer. Anti¬ bodies against the 92 kDa protein react specifically with a 92 kDa protein of wild-type membranes. In mutant membranes this 92 kDa signal is missing.
E.7.3. In vivo O-glvcosylation
To investigate in vivo glycosylation, wild-type and mutant cells were grown in the presence of [3H] -mannose. Then a crude cell wall plus membrane fraction was isolated and O-glycosylated ma¬ terial released by β-elimination.
E.7.3.1 Treatment with [3H] -mannose
Wild-type and mutant cells were grown over night in minimal me¬ dia containing sucrose as only C-source. 7.5 OD of the culture
(OD578 = 1-2) were pelleted and washed with 5 ml H20 (prewarme 30°C) . The cells were grown in 5 ml YP/0.5% sucrose/250 μCi
[3H] -mannose in a waterbath shaker for 2 h at 30°C.
E.7.3.2. Isolation of crude cell wall and membrane fraction
5 OD of the [3H] -mannose treated cells were centrifuged an washed three times with 1 ml TMA. Cells were resuspended in 20 μl TMA and broken with glass beads as described in E.7.2.1 (1 μl sample was used for counting radioactivity corresponding to total incorporation) . The extract was then centrifuged for 15' (10,000 x g) and the supernatant was removed (100 μl sample was used for counting radioactivity corresponding to soluble mate¬ rial) .
E.7.3.3. β-elimination
The pellet was resuspended in 1 ml 0.1 N NaOH (a 10 μl sample was used for counting radioactivity corresponding to material before β-elimination) . Incubation was maintained for 24 h at 30°C.
E.7.3.4. Analysis of β-eliminated material
β-eliminated material was desalted via a Dowex 50WS8/H+ column (0.5cm x 6cm). The column was saturated with 0.5M mannose and equilibrated in H20. The β-elimination sample was loaded onto the column and washed through with 1.5 μl H20. The flow throug was collected (a 100 μl sample was used for counting radioacti¬ vity corresponding to β-eliminated material) and concentrated to 10 μl in the speed-vac. Thin-layer chromatography on Silicagel 60 (Merck) in acetone:butanol:H20 70:15:15 was performed. Stan¬ dards: mannose, sucrose, stachyose, raffinose. The chromatogra- phic run was repeated once. Sugars were detected with 0.5 KMn04 in 100 ml IN NaOH. Radioactivity was detected by a thin- layer scanner (Berthold) (see Figure 6) .
Figure imgf000030_0001
Mutant cells show reduced glycosylation in comparison to wild type cells. The O-glycosylation in mutant cells is about 40-50 lower than in wild-type cells. Example 8: Cloning of the PMTl homolog of Kluyveromyces lactis .
The yeast S. cerevisiae used to be the system of choice when th production of a heterologous protein in a fungal host cell wa desired. However, recent years have shown that the productivit of bakers yeast is often limited, especially when the secretio of the product to the culture medium is required, The use o fungal systems other than S. cerevisiae is therefore preferabl in a number of cases [c.f. Romanos et al., Yeast 8. (1992) 423
488; Fleer, Curr. Opinion Biotechnol. 3. (1992) 486-496] . One o the alternative yeast hosts is represented by the genu
Kluyveromyces for which superior secretion yields have bee found with respect to several proteins of commercial interes
[e.g. Van den Berg et al., Bio/Technology 8. (1990) 135-139]. Th following example demonstrates that the present invention is no limited to bakers yeast, since the sequence of the PMTl gen isolated from S. cerevisiae can be advantageously used for th identification of genes encoding similar enzymatic activities i other fungal species. Furthermore, the sequence informatio revealed in the present invention may also be used for th identification of related mannosyltransferase encoding genes i
S. cerevisiae.
E.8.1. Design of degenerate PCR primers for the amplification o PMTl related genes
The region of the PMTl nucleotide sequence corresponding to th central, hydrophilic region of the mannosyltransferase protei was chosen to design PCR primers [Polymerase-catalyzed Chai Reaction, Saiki et al., Science 230(1985) 1350-1354; Mullis Faloona, Meth. Enzymol. 155 (1987) 335-350] for the amplifica tion of homologous genes. Amplification requires hybridization also termed annealing, of these synthetic oligonucleotides wit its target DNA. The specificity with which individual regions o genomic DNA are amplified depends on the conditions of the P reaction and the degree of homology between the primers and t nucleotide sequence to be amplified. Subsequent to the anneali step, the primers are extended using a thermostable D polymerase. Once the complementary strand has been polymerize the two strands are separated by heat denaturation and a ne cycle of annealing and polymerization may begin.
Four examples of oligonucleotides suitable as primers for PC amplification of PMTl homologs are presented in Table 2 below.
Table 2
Figure imgf000032_0001
The design of these "degenerate" oligonucleotides takes int account that several codons may contain the information for th incorporation of the same amino acid into a nascent polypeptid chain, most often varying in the third ("wobble") position of triplet. Each primer therefore represents a mixture of oligo nucleotides where Y signifies C or T, R signifies A or G, and signifies A, C, G or T.
E.8.2. PCR amplification of K. lactis genomic DNA
Genomic DNA of K lactis strain CBS2359 was prepared as describe by Sherman et al. ["Methods in Yeast Genetics", Cold Sprin Harbor Laboratory Press (1986) p 127] . lOng of genomic DNA wer used in a standard PCR reaction [Sambrock et al., "Molecula Cloning - A Laboratory Manual", second edition, Cold Sprin Harbor Laboratory Press (1989)] in the presence of 1 μg of eac of the primers and 5% deionized formamide. The amplification wa performed using a "DNA Thermal Cycler" (Perkin Elmer Cetus) an "AmpliTag DNA Polymerase" (Perkin Elmer Cetus, 5 units pe reaction tube) . The conditions for denaturation, annealing, a polymerization (30 cycles) were 91°C (1 min) , 42°C (2 min) , a 72°C (3 min) , respectively, except for the first cycle whe denaturation was for 5 min. The results of the PCR amplifica¬ tions using the primers disclosed above are presented in Table 3 below.
Table 3
Primer Combinations Approx. size of amplified DNA DNA fragments (bp) expected for PMTl observed homolog
Sq3908+Sq3910 400 400
Sq3908+Sq3911 600 600
Sq3909+Sq3910 170 170 300
Sq3909+Sq3911 400 400 800
These results show that it is not only possible to obtai fragments exhibiting the same size as that expected for the K. lactis homolog of the S. cerevisiae PMTl gene but that, "ϊ addition, DNA fragments can be amplified with high specificit that most likely correspond to another gene coding for a closel related enzymatic activity.
E.8.3 Partial sequence characterization of the K. lactis PMT homolog
The 400 bp fragment amplified with the primer combinatio Sq3908+Sq3910 was subcloned into the vector pCRII (TA Cloning™ Invitrogen Corp.) following the indications of the supplier, an partially sequenced according to the method described i E.4.1.9. using the universal primer. The sequence obtained i presented in Figure 7. Sequence comparison between the S cerevisiae PMTl gene and the fragment isolated from K. lacti genomic DNA by PCR amplification reveals 75% and 80.5% identit on the nucleotide and amino acid level, respectively (Figure 8) The amplified 400 bp DNA fragment may be used to target selectable marker gene to the K. lactis PMTl chromosomal locu in analogy to the experiment described under E.6. leading to a disrupted gene. This will yield a K. lactis strain with reduce Ser/Thr specific mannosyltransferase activity. In addition, the amplified fragment may be used as homologous hybridization probe for the cloning of the entire K. lactis PMTl gene using standar procedures.

Claims

1. Fungal cells carrying genetic modification(s) within their DNA sequences which cause them to have at least a reduced capacity of O-glycosylation.
2. Fungal cell according to claim 1 wherein said modifica- tion(s) comprise any suppression, substitution, deletion, addition, disruption and/or mutational insertion.
3. Fungal cell according to claim 2 wherein said modifica- tion(s) are stable during segregation and/or non-reverting and/or non-leaky.
4. Fungal cell according to any of claims 1 to 3 wherein said modification(s) are located in one or more coding regions of the DNA sequences of the cell.
5. Fungal cell according to any of claims 1 to 3 wherein said modification(s) are located in one or more regions respon¬ sible for or involved in the expression and/or transcrip¬ tional regulation of a gene.
6. Fungal cell according to claim 4 or 5 wherein said gene i a gene whose expression product is an enzyme of the O-gly¬ cosylation pathway.
7. Fungal cell according to claim 6 wherein the reduced capa city of O-glycosylation results from the production of in active enzymes, from the production of enzymes having al tered biological properties, from the absence of productio of said enzymes, or from the production of said enzymes a low levels.
8. Fungal cell according to claim 6 wherein said gene is gene whose expression product is involved in the attachmen of mannosyl residues to the hydroxyl group of seryl o threonyl amino acids.
9. Fungal cell according to claim 8 wherein said gene is a gene whose expression product is involved in the transfer of mannosyl residues from the Dol-P-Man donor to the hy¬ droxyl group of seryl or threonyl amino acids.
10. Fungal cell according to claim 9 wherein said gene is the gene encoding the Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase [PMTl] whose sequence is represented in Figure 4, or any homologous gene encoding the same activity.
11. Fungal cell according to any of claims 8 to 10, furthe comprising modification(s) in one or more genes involved i subsequent additions of mannosyl residues, or in the syn¬ thesis of the mannosyl residues donor (Dol-P-Man) .
12. Fungal cell according to any of the preceding claims i which an exogenous DNA sequence has been introduced.
13. Fungal cell according to claim 12 wherein the exogenous DN sequence comprises one or more genes encoding a desire protein to be expressed and/or secreted in said cell.
14. Fungal cell according to claim 13 wherein said DNA sequenc is included in an expression cassette comprising a tran scription and translation initiation region joined to th 5' end of said DNA sequence encoding the desired pro tein(s) .
15. Fungal cell according to claim 14 wherein said transcrip tion and translation initiation region is chosen from pro moters derived from fungal cell genes.
16. Fungal cell according to claim 14 wherein said expressio cassette further comprises a transcription and translatio termination region at the 3' end of the DNA sequence en coding the desired protein(s) .
17. Fungal cell according to claim 14 wherein said expression cassette further comprises a signal peptide (pre-sequence) at the N-terminus of the desired protein sequence so as to direct the nascent protein to the secretory pathway of said fungal cell.
18. Fungal cell according to any of claims 12 to 17 wherein the exogenous DNA sequence is part of a vector which may either replicate autonomously in said fungal cell or integrate into its own DNA sequences (chromosome) .
19. Fungal cell according to any of the preceding claims wherein it is chosen from filamentous fungi and yeasts.
20. Fungal cell according to claim 19 wherein the filamentous fungi is chosen from the group consisting of Sspergillus, Trichoderma, Mucor, Neurospora, Fusarium, and the like.
21. Fungal cell according to claim 19 wherein the yeast is cho¬ sen from the group consisting of Kluyveromyces, Saccharomy¬ ces, Pichia, Hansenula, Candida, Schizosaccharomyces and the like.
22. Fungal cell according to claim 21 wherein the yeast is cho¬ sen from the group consisting of iQuyvero-Tiyσes, Saccharomy¬ ces, Pichia, Hansenula, and Candida, and, more preferably, from Jluyvero-nyces and Saccharomyces.
23. Process for the production of recombinant products wherei a fungal cell according to claims 12-22 is cultivated i conditions in which the exogenous DNA sequence is ex pressed, and the product is recovered.
24. Process according to claim 23 wherein said product is se creted into the culture medium.
25. Process according to claims 23 or 24 wherein said produc is susceptible to O-glycosylation by the fungal cell.
26. DNA fragment encoding an enzyme involved in the attachmen of mannosyl residues to the hydroxyl group of seryl o threonyl amino acids of proteins.
27. DNA fragment according to claim 26 comprising the Dol-P Man:Protein (Ser/Thr) Mannosyl Transferase gene whose se quence is represented in Figure 4, or any fragment or deri vative thereof.
28. DNA fragment according to claim 26 comprising all or par of the sequence presented in Figure 7.
29. The use of a DNA fragment according to claim 26 or 27 o any part thereof as hybridization probe(s) or for the com plementation of mutant phenotypes for the obtention of ho mologous genes of fungal cells.
30. DNA fragments Sq3908, Sq3909, Sq3910 and Sq3911.
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