US20150275222A1 - Crz1 mutant fungal cells - Google Patents

Crz1 mutant fungal cells Download PDF

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US20150275222A1
US20150275222A1 US14/437,405 US201314437405A US2015275222A1 US 20150275222 A1 US20150275222 A1 US 20150275222A1 US 201314437405 A US201314437405 A US 201314437405A US 2015275222 A1 US2015275222 A1 US 2015275222A1
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crz1
host cell
fungal host
polypeptide
endogenous
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Bo Jiang
Jun Zhuang
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Merck Sharp and Dohme LLC
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    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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    • 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
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    • 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
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature

Definitions

  • the present invention relates to CRZ1 mutant allele and fungal host cells, such as Pichia pastoris , comprising such an allele along with methods of use thereof.
  • GlycoFi has engineered Pichia to produce recombinant glycoproteins with human-like glycosylation.
  • the extensive genetic modifications have also caused fundamental changes in cell wall structures, predisposing these glyco-engineered strains to cell lysis and reduced cell robustness during fermentation.
  • These undesirable traits have resulted in substantial reductions in cell viability as well as a marked increase in intracellular protease leakage into the fermentation broth, resulting in a reduction in both recombinant product yield and quality.
  • Isolated fungal host cells such as Candida albicans; Hansenula polymorpha; Schizosaccharomyces pombe; Saccharomyces cerevisiae; Pichia pastoris , lacking functional OCH1, a polypeptide in the fungal glycosylation pathway, are known to be temperature sensitive.
  • Candida albicans och1 knock-outs are temperature sensitive at 42° C. (Bates et al., Outer Chain N-Glycans Are Required for Cell Wall Integrity and Virulence of Candida albicans , The Journal of Biological Chemistry 281: 90-98 (2006); Hansenula polymorpha och1 knock-outs are temperature sensitive at 45° C.
  • CRZ1 a zinc finger transcription factor.
  • S. cerevisiae plasma membrane and cell wall regulatory genes Cyert, Biochemical and Biophysical Research Communications 311:1143-1150 (2003). Perturbation of plasma membrane and cell wall synthesis, due to mutation of CRZ1, would have been expected to make Pichia cells less robust.
  • the published characterizations of S. cervisiae CRZ1 would have led a practitioner of ordinary skill in the art to predict that Pichia cells, lacking functional CRZ1, would be less viable and robust when placed under high temperature stress. (Matheos et al., Genes & Development 11:3445-3458 (1997); Stathopoulos et al., Genes & Development 11: 3432-3444 (1997)).
  • the present invention provides an isolated fungal host cell (e.g., Pichia such as Pichia pastoris ) lacking functional CRZ1 polypeptide, e.g., wherein the cell exhibits increased fermentation robustness and production of heterologous polypeptides, such as immunoglobulins, relative to a cell expressing functional CRZ1, e.g., wherein endogenous CRZ1 has been mutated, disrupted or partially or fully deleted; optionally comprising a heterologous polynucleotide (e.g., operably linked to a promoter such as a methanol inducible promoter) that encodes a heterologous polypeptide such as an immunoglobulin.
  • a heterologous polynucleotide e.g., operably linked to a promoter such as a methanol inducible promoter
  • endogenous CRZ1 encodes a polypeptide that comprises one or more mutations selected from the group consisting of: L33 ⁇ STOP; Q214 ⁇ STOP; L294 ⁇ STOP; S298 ⁇ STOP; E403 ⁇ G; F406 ⁇ S; F406 ⁇ L; C411 ⁇ F; and K469 ⁇ N, disruption of endogenous CRZ1, complete endogenous CRZ1 deletion, partial endogenous CRZ1 deletion (e.g., that deletes 33aa-end, 214aa-end, 294-end, 298-end of the CRZ1 polypeptide); or (ii) endogenous CRZ1 comprises one or more mutations selected from the group consisting of: a1407c; g1232t; t1216c; t1217c; a1208g; c893a; t881g; c640t; and t98a; or (iii) endogenous CR
  • the isolated fungal host cell also comprises one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) of the following characteristics: (i) wherein one or more endogenous beta-mannosyltransferase genes are mutated, disrupted, truncated or partially or fully deleted; (ii) comprising a polynucleotide encoding an alpha-1,2 mannosidase, an alpha-1,3 mannosidase, or an alpha-1,6 mannosidase; (iii) wherein one or more endogenous phosphomannosyl transferases are mutated, disrupted, truncated or partially or fully deleted; (iv) comprising a single-subunit oligosaccharyltransferase (e.g.
  • Leishmania sp. STT3D Leishmania sp. STT3D
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., ALG3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., ALG3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., ALG3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., ALG3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., ALG3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., ALG3
  • the present invention also provides a method for making the isolated fungal host cell of the present invention comprising introducing a heterologous polynucleotide into the cell which homologously recombines with the endogenous CRZ1 and partially or fully deletes the endogenous CRZ1 or disrupts the endogenous CRZ1; along with an isolated fungal host cell produced by such a method.
  • the present invention also provides an isolated polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 which comprises a mutation selected from the group consisting of: L33 ⁇ STOP; Q214 ⁇ STOP; L294 ⁇ STOP; S298 ⁇ STOP; E403 ⁇ G; F406 ⁇ S; F406 ⁇ L; C411 ⁇ F; and K469 ⁇ N; e.g., comprising a nucleotide sequence of SEQ ID NO: 2 comprising a mutation selected from the group consisting of: a1407c; g1232t; t1216c; t1217c; a1208g; c893a; t881g; c640t; and t98a.
  • Isolated vectors comprising such polynucleotides also form part of the present invention. Isolated polypeptides encoded by such polynucleotides are also part of the present invention.
  • the present invention also provides a method for producing an isolated crz1 mutant fungal host cell (e.g., Pichia such as Pichia pastoris ) having improved viability at high temperature (e.g., 32° C.) comprising introducing a mutation that encodes a polypeptide selected from the group consisting of: L33 ⁇ STOP; Q214 ⁇ STOP; L294 ⁇ STOP; S298 ⁇ STOP; E403 ⁇ G; F406 ⁇ S; F406 ⁇ L; C411 ⁇ F; and K469 ⁇ N; into the endogenous CRZ1 gene in the fungal cell.
  • the present invention also provides a method for producing one or more heterologous polypeptides (e.g., an immunoglobulin polypeptide) comprising: (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such an isolated crz1 mutant fungal host cell (e.g., Pichia such as Pichia pastoris ) (e.g., any of those discussed herein); and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell (e.g., at 24° C.) and; optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • a method for producing one or more heterologous polypeptides comprising: (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such an isolated crz1 mutant fungal host cell (e.g., Pichia such as Pichia pastor
  • the heterologous polynucleotide that encodes the heterologous polypeptide is operably linked to a methanol inducible promoter and wherein the isolated fungal host cell is cultured under conditions favorable to expression of the heterologous polypeptide in the presence of methanol.
  • FIG. 1 Temperature resistant mutants displaying improved fermentation robustness and Fc titer.
  • FIG. 2( a - c ) Lineages of mutagenized Pichia pastoris strains.
  • FIG. 3 N-Glycan profiles of the temperature resistant mutants.
  • G0 N-glycan terminated by GlcNac;
  • G1 Singly galactose terminated N-glycan;
  • G2 Doubly galactose terminated N-glycan;
  • A1 Singly sialic acid terminated N-glycan;
  • A1H A1 hybrid, singly sialic acid terminated N-glycan with a hybrid structure;
  • A2 Doubly sialic acid terminated N-glycan;
  • M5 Man 5 ;
  • M6+ Man 6+ .
  • FIG. 4 Deep sequencing of temperature-resistant mutants identified multiple mutations in Pp02g02120 (CRZ1) and two mutations in Pp01g00680 (ATT1), corresponds to circled “+” symbols.
  • FIG. 5 Mutated protein Pp02g02120 was homologous to Saccharomyces cerevisiae CRZ1 zinc finger transcription factor involved in stress responses. The various isolated mutant PpCRZ1 alleles are shown. The “*” shows the location of the indicated mutation.
  • FIG. 6 Plasmid pGLY12829.
  • FIG. 7 Plasmid pGLY12832.
  • FIG. 8 DNA Constructs constructed for Pichia pastoris CRZ1 deletion and truncations.
  • FIG. 9 Pichia pastoris CRZ1 deletion and truncation mutants exhibited improved fermentation robustness at 32° C.
  • FIG. 10 Pichia pastoris CRZ1 deletion further improves fermentation robustness in an att1 mutant strain at 34° C.
  • Pichia pastoris cells lacking functional CRZ1, exhibited enhanced temperature-resistance and increased robustness.
  • a “CRZ1 wt ” fungal host cell comprises a wild-type CRZ1.
  • PpCRZ1 is Pichia pastoris CRZ1.
  • SpCRZ1 is Saccharomyces cerevisiae CRZ1.
  • High temperature with respect to the growth of isolated fungal cells such as Pichia is above 28° C., 29° C. or 30° C., e.g., 32° C.
  • a heterologous polynucleotide is a polynucleotide that has been introduced into a fungal host cell and that encodes a heterologous polypeptide.
  • a heterologous polynucleotide can encode an immunoglobulin heavy chain or an immunoglobulin light chain, e.g., comprising the light or heavy chain variable domain and, optionally, the antibody constant domain, e.g., from an antibody or antigen-binding fragment thereof, e.g., from a fully human antibody, humanized antibody, chimeric antibody, a bispecific antibody, an antigen-binding fragment of an antibody such as a Fab antibody fragment, F(ab) 2 antibody fragment, Fv antibody fragment, single chain Fv antibody fragment or a dsFv antibody fragment.
  • any such antibody can bind specifically to any epitope such as insulin-like growth factor 1 receptor, VEGF, interleukin-6 (IL6), IL6 receptor, respiratory syncitial virus (RSV), CD20, tumor necrosis factor alpha, receptor activated NF kappa B ligand (RANKL), or the RANKL receptor RANK, IgE, Her2, Her3, or the Epidermal growth factor receptor.
  • epitope such as insulin-like growth factor 1 receptor, VEGF, interleukin-6 (IL6), IL6 receptor, respiratory syncitial virus (RSV), CD20, tumor necrosis factor alpha, receptor activated NF kappa B ligand (RANKL), or the RANKL receptor RANK, IgE, Her2, Her3, or the Epidermal growth factor receptor.
  • An “endogenous” gene is a chromosomal copy of the gene.
  • a “polynucleotide” or “nucleic acid” includes DNA and RNA in single stranded form, double-stranded form or otherwise.
  • a “polynucleotide sequence” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA or RNA, and means a series of two or more nucleotides. Any polynucleotide comprising a nucleotide sequence set forth herein (e.g., crz1 mutant ) forms part of the present invention.
  • a “coding sequence” or a sequence “encoding” an expression product, such as an RNA or polypeptide is a nucleotide sequence (e.g., heterologous polynucleotide) that, when expressed, results in production of the product (e.g., a heterologous polypeptide such as an immunoglobulin heavy chain and/or light chain).
  • oligonucleotide refers to a nucleic acid, generally of no more than about 100 nucleotides (e.g., 30, 40, 50, 60, 70, 80, or 90), that may be hybridizable to a polynucleotide molecule. Oligonucleotides can be labeled, e.g., by incorporation of 32 P-nucleotides, 3 H-nucleotides, 14 C-nucleotides, 35 S-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.
  • a label such as biotin
  • a “protein”, “peptide” or “polypeptide” includes a contiguous string of two or more amino acids. Any polypeptide comprising an amino acid sequence set forth herein (e.g., Crz1 mutant polypeptide) forms part of the present invention.
  • a “protein sequence”, “peptide sequence” or “polypeptide sequence” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide or polypeptide.
  • isolated polynucleotide or “isolated polypeptide” includes a polynucleotide or polypeptide, respectively, which is partially or fully separated from other components that are normally found in cells or in recombinant DNA expression systems or any other contaminant. These components include, but are not limited to, cell membranes, cell walls, ribosomes, polymerases, serum components and extraneous genomic sequences.
  • the scope of the present invention includes the isolated polynucleotides set forth herein (e.g., crz1 mutant ) and isolated polypeptides encoded by such polynucleotides.
  • An isolated polynucleotide or polypeptide will, preferably, be an essentially homogeneous composition of molecules but may contain some heterogeneity.
  • PCR polymerase chain reaction
  • a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence to which it operably links.
  • Crz1 mutant polynucleotide operably linked to a promoter forms part of the present invention.
  • an isolated crz1 mutant fungal host cell comprising a heterologous polynucleotide (e.g., encoding an immunoglobulin polypeptide) operably linked to a promoter also forms part of the present invention.
  • a coding sequence (e.g., of a heterologous polynucleotide, e.g., reporter gene or immunoglobulin heavy and/or light chain) is “operably linked to”, “under the control of”, “functionally associated with” or “operably associated with” a transcriptional and translational control sequence (e.g., a promoter of the present invention) when the sequence directs RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which then may be RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.
  • RNA preferably mRNA
  • the present invention includes vectors or cassettes which comprise crz1 mutant polynucleotide.
  • Vectors containing a heterologous polynucleotide encoding a heterologous polypeptide can also be used in various crz1 mutant fungal host cells for production of the heterologous polypeptide (e.g., an immunoglobulin).
  • the term “vector” includes a vehicle (e.g., a plasmid) by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence.
  • Suitable vectors for use herein include plasmids, integratable DNA fragments, and other vehicles that may facilitate introduction of the nucleic acids into the genome of a host cell (e.g., Pichia pastoris ).
  • Plasmids are the most commonly used form of vector but all other forms of vectors which serve a similar function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels, et al., Cloning Vectors: A Laboratory Manual , 1985 and Supplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: A Survey of Molecular Cloning Vectors and Their Uses , 1988, Buttersworth, Boston, Mass.
  • Such vectors optionally include a secretion signal (e.g., alpha-mating factor ( ⁇ -MF) pre-pro leader sequence) operably linked to a heterologous polynucleotide.
  • a secretion signal e.g., alpha-mating factor ( ⁇ -MF) pre-pro leader sequence
  • ⁇ -MF alpha-mating factor
  • an isolated crz1 mutant fungal host cell comprising a vector that includes a heterologous polynucleotide (e.g., encoding an immunoglobulin polypeptide), e.g., operably linked to a promoter, also forms part of the present invention.
  • a polynucleotide (e.g., a heterologous polynucleotide, e.g., encoding an immunoglobulin heavy chain and/or light chain), operably linked to a promoter, may be expressed in an expression system.
  • expression system means a host cell and compatible vector which, under suitable conditions, can express a protein or nucleic acid which is carried by the vector and introduced to the host cell.
  • Common expression systems include fungal host cells (e.g., Pichia pastoris ) and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors.
  • methanol-induction refers to increasing expression of a polynucleotide (e.g., a heterologous polynucleotide) operably linked to a methanol-inducible promoter in a host cell of the present invention by exposing the host cells to methanol.
  • a crz1 mutant containing a polynucleotide operably linked to a methanol-inducible promoter forms part of the present invention.
  • Methods for inducing expression of a heterologous polynucleotide fused to such a methanol-inducible promoter by exposing a crz1 mutant fungal cell comprising the promoter construct to methanol, and culturing the cell under conditions favorable to expression of the encoded heterologous polypeptide form part of the present invention.
  • BLAST ALGORITHMS Altschul, S. F., et al., J. Mol. Biol. (1990) 215:403-410; Gish, W., et al., Nature Genet. (1993) 3:266-272; Madden, T. L., et al., Meth. Enzymol. (1996) 266:131-141; Altschul, S. F., et al., Nucleic Acids Res. (1997) 25:3389-3402; Zhang, J., et al., Genome Res. (1997) 7:649-656; Wootton, J. C., et al., Comput. Chem.
  • the present invention comprises isolated CRZ1 polynucleotides comprising a mutation (crz1 mutant polynucleotides) and polypeptides encoded by such polynucleotides (crz1 mutant polypeptides) along with isolated fungal host cells comprising endogenous CRZ1 that has been mutated in such a way (e.g., by mutation, partial or complete deletion, or disruption).
  • Such mutations in the Pichia pastoris CRZ1 polynucleotide are polynucleotides comprising the nucleotide sequence of SEQ ID NO: 2 having one or more of the following mutations: a1407c; g1232t; t1216c; t1217c; a1208g; c893a; t881g; c640t; and t98a.
  • CRZ1 polynucleotides encode CRZ1 polypeptides having the amino acid sequence of SEQ ID NO: 3 having one or more of the following mutations: L33 ⁇ STOP; Q214 ⁇ STOP; L294 ⁇ STOP; S298 ⁇ STOP; E403 ⁇ G; F406 ⁇ S; F406 ⁇ L; C411 ⁇ F; and K469 ⁇ N.
  • Such mutant polynucleotides can be introduced into the endogenous CRZ1 chromosomal locus to replace the wild-type, endogenous CRZ1 with the mutated CRZ1.
  • the present invention encompasses any CRZ1 polynucleotide comprising a mutation that encodes a CRZ1 polypeptide lacking a functional C-terminal zinc-finger domain (e.g., a non-sense mutation or deletion that truncates the zinc-finger domain). Such polypeptides are also within the scope of the present invention.
  • the zinc-finger domain of Pichia pastoris CRZ1 polypeptide comprises the amino acid sequence: SIYACSLCSKRFTRPYNLKSHLRTHADERPFQCSICGKAFARSHDRKR HEDLHSGERKYCCKGVLSDGVTTWGCEKRFARTDALGRHFKTECGKLC (amino acids 376-471 of SEQ ID NO: 3).
  • a BLASTP comparison between Saccharomyces cerevisiae CRZ1 polypeptide (SEQ ID NO: 1) and Pichia pastoris CRZ1 polypeptide (SEQ ID NO: 3) identified in the random mutatgenesis screen is as follows:
  • the present invention comprises mutant Pichia pastoris and Saccharomyces cerevisiae CRZ1 polypeptides and polynucleotides encoding such polypeptides.
  • Specific examples of Pichia pastoris and Saccharomyces cerevisiae CRZ1 polypeptides comprise one or more changes to the amino acid sequence set forth in SEQ ID NO: 1 at the locations noted with a * or ⁇ in the BLASTP comparison shown above
  • Saccharomyces cerevisiae or Pichia pastoris CRZ1 polypeptide comprises at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence similarity or identity to SEQ ID NO: 1 or 3, respectively.
  • Pichia pastoris CRZ1 polynucleotide comprises at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 2.
  • Saccharomyces cerevisiae CRZ1 wild-type polypep- tide (SEQ ID NO: 1) msfsngnmas ymtssngeeq sinnkndidd nsayrrnnfr nssnsgshtf qlsdldldvd mrmdsansse kisknlssgi pdsfdsnvns llspssgsys adlnyqslyk pdlpqqqlqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqtp tlkveqsdtf qwdddiltpad nqhrpsltnq f
  • the present invention includes isolated crz1 mutant fungal host cells which may include additional mutations in its genetic background.
  • a “crz1 mutant ” fungal host cell haploid or diploid
  • the endogenous chromosomal CRZ1 genes have been mutated, disrupted or partially or fully deleted, or expression of CRZ1 protein has been reduced in any way (e.g., by anti-sense RNA, interfering RNA such as small interfering RNA (SiRNA)), or the activity of the CRZ1 polypeptide has been chemically inactivated (e.g., by small molecule inhibitors), and thus, the cell partially or fully lacks functional CRZ1 polypeptide levels and/or CRZ1 activity to any degree relative to an isolated fungal host cell wherein CRZ1 has not been mutated or interfered with or the like.
  • siRNA small interfering RNA
  • the crz1 mutant fungal host cell is more viable (e.g., in a fermentor or bioreactor) at high temperature or at 24° C. than a fungal host cell comprising the full level of CRZ1, e.g., at 32° C., e.g., for up to about 90-110 hours of induction at 32° C.
  • an isolated crz1 mutant fungal host cell (e.g., in a fermentor or bioreactor) comprising a heterologous polynucleotide, e.g., encoding an Fc polypeptide, expresses significantly more heterologous polypeptide (e.g., 4 times or 5 times more), e.g., Fc polypeptide, than a CRZ1 wild-type fungal host cell comprising such a heterologous polynucleotide encoding an Fc polypeptide (such crz1 mutant fungal host cells are within the scope of the present invention).
  • a heterologous polynucleotide e.g., encoding an Fc polypeptide
  • an isolated crz1 mutant fungal host cell (e.g., in a fermentor or bioreactor) comprising a heterologous polynucleotide, e.g., encoding a heterologous Fc polypeptide, express heterologous polypeptide, e.g., Fc polypeptide, with an N-glycan profile essentially identical to that of a CRZ1 Wt fungal host cell, e.g., are able to effectively modify their N-glycans, e.g., Fc N-glycans, with high levels of terminal sialic acids, and/or with A2 levels ranging from 77 to 84%, and/or A1 levels from 4 to 7% (such crz1 mutant fungal host cells are within the scope of the present invention).
  • the isolated crz1 mutant fungal host cell of the present invention comprises endogenous mutant CRZ1 polypeptide, e.g., which comprises the amino acid sequence of SEQ ID NO: 3 having one or more of the following mutations: L33 ⁇ STOP; Q214 ⁇ STOP; L294 ⁇ STOP; S298 ⁇ STOP; E403 ⁇ G; F406 ⁇ S; F406 ⁇ L; C411 ⁇ F; and K469 ⁇ N; e.g., in an embodiment of the invention, the mutant endogenous CRZ1 polynucleotide of the isolated crz1 mutant fungal host cell comprises a nucleotide sequence of SEQ ID NO: 2 having one or more of the following mutations: a1407c; g1232t; t1216c; t1217c; a1208g; c893a; t881g; c640t; and t98a.
  • endogenous mutant CRZ1 polypeptide e.g., which comprises the
  • endogenous CRZ1 in an isolated crz1 mutant fungal host cell of the present invention, is mutated such that it lacks a functional C-terminal zinc-finger domain, e.g., due to mutation or truncation.
  • the fungal host cell endogenous CRZ1 has been replaced with a mutant CRZ1, e.g., any of those set forth herein such as a partial deletion mutant, a complete deletion mutant or a mutant comprising a nonsense mutation.
  • An endogenous CRZ1 gene in an isolated crz1 mutant fungal host cell may be partially deleted, thus leaving only part of the CRZ1 coding sequence in the chromosomal locus where CRZ1 would naturally occur (e.g., wherein the CRZ1 zinc finger domain is partially or fully deleted); fully deleted, thus leaving no CRZ1 coding sequence in the chromosomal locus wherein CRZ1 would naturally occur (e.g., wherein CRZ1 is fully deleted and replaced with another polynucleotide such as an auxotrophic marker); disrupted, thus inserting a heterologous sequence into the chromosomal CRZ1 gene; mutated at one or more points in the chromosomal gene; mutated so as to lower CRZ1 expression levels or activity in the cell (e.g., wherein a partially or fully inactivating mutation is introduced into the CRZ1 zinc finger domain) as compared to a cell wherein CRZ1 has not been so mutated;
  • CRZ1 expression can be lowered or eliminated in any way, e.g., by interference with expression using anti-sense CRZ1 molecules, SiRNA CRZ1 molecules, or by enhancing CRZ1 protein degradation, or by chemical inhibition using small molecule inhibitors.
  • isolated crz1 mutant fungal host cells are part of the present invention.
  • the scope of the present invention encompasses isolated crz1 mutant fungal host cells that are “viable” at high temperature, and are more robust during fermentation, as well as uses of such cells as discussed herein.
  • Isolated fungal host cell viability in a liquid cell culture, within a bioreactor/fermentor environment, for example, at high temperature such as 32° C. is, in an embodiment of the invention, determined by measuring cellular lysis in the cell culture.
  • crz1 mutant fungal host cellular lysis is, in an embodiment of the invention, evaluated microscopically or by determining the double stranded DNA content of the culture medium. Microscopic evaluation is done to score the amount of cellular debris that is observed in the culture medium.
  • Cellular debris in the culture medium is a result of cell lysis and, thus, a marker for cell lysis and a means by which to determine cell viability in the culture.
  • a score of 1, 2, 3, 4 or 5 is given; with 5 representing the most lysis, i.e., greater than 90% cellular lysis.
  • crz1 mutant fungal host cells exhibited less than a 5 score for lysis for between 90 and 110 hours following induction at 32° C.
  • the culture medium containing crz1 mutant fungal host cells induced at 32° C. had 30 micrograms/milliliter or less double standed DNA for between 90 and 110 hours.
  • Double stranded DNA can be determined using any of several methods known in the art including by determining the amount of fluorescent dye, with an affinity for double stranded DNA (e.g., bisbenzimide, an indole-derived stain such as Hoechst 33342, Hoechst 33258 or 49,6-diamidino-2-phenylindole; a phenanthridinium stain such as ethidium bromide or propidium Iodide; or a cyanine dye such as PicoGreen, YOYO-1 iodide, SYBR Green I or SYBR Gold; see, for example, Cosa et al., Photochemistry and Photobiology 73(6):585-599 (2001)), bound to double stranded DNA in the
  • the quantity of double stranded DNA in the culture can then be determined on this basis. Accordingly, cells in culture with a microscopic lysis score of less than 5 and/or a double stranded DNA content of 30 micrograms/milliliter or less are considered “viable”.
  • Isolated fungal host cell viable for about 90 to about 110 hours after induction (e.g., in a bioreactor or fermentor) at 32° C. may be referred to herein as a “temperature-resistant” or “temperature-resistance” phenotype.
  • the present invention includes such host cells comprising a heterologous polynucleotide encoding a heterologous polypeptide (e.g., a reporter or immunoglobulin heavy and/or light chain) wherein the heterologous polynucleotide may be operably linked to a promoter; as well as methods of use thereof, e.g., methods for expressing the heterologous polypeptide in the fungal host cell.
  • a heterologous polypeptide e.g., a reporter or immunoglobulin heavy and/or light chain
  • the present invention includes methods for making one or more heterologous polypeptides in an isolated crz1 mutant fungal host cell (e.g., Pichia ) comprising, optionally, one or more further changes (e.g., mutations to endogenous genes and/or expression of one or more other genes; e.g., as discussed herein, for example, to produce modified glycosylation of expressed polypeptides) comprising (i) introducing a polynucleotide encoding the heterologous polypeptide into the crz1 mutant fungal host cell and (ii) culturing the crz1 mutant fungal host cell under conditions favorable to expression of the heterologous polypeptide in the cell and, optionally, (iii) isolating the heterologous polypeptide from the crz1 mutant fungal host cell.
  • an isolated crz1 mutant fungal host cell e.g., Pichia
  • further changes e.g., mutations to endogenous genes and/or expression of one or more other genes;
  • a crz1 mutant fungal host cell also comprises a mutation in ATT1.
  • a crz1 mutant fungal host cell does not comprise a mutation in ATT1, e.g., endogenous ATT1 is wild-type, (e.g., the cell comprises wild-type, functional ATT1 polypeptide)—such cells and their uses, as discussed herein, are part of the present invention.
  • Isolated fungal host cells of the present invention are cells belonging to the Fungi kingdom, for example, in an embodiment of the invention, the fungal host cell is any yeast such as a budding yeast and/or a fission yeast. In an embodiment of the invention, the host cell is any methylotrophic yeast.
  • Methylotrophic yeasts are a small group of yeast species capable of utilizing methanol as the sole source of carbon and energy. Examples of methylotrophic yeast include Pichia pastoris, Pichia angusta ( Hansenula polymorpha ), Pichia methanolica , and Candida boidinii .
  • the host cell is selected from the group consisting of any Pichia cell, such as Pichia pastoris, Pichia angusta ( Hansenula polymorpha ), Pichia flnlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta ( Ogataea minuta, Pichia lindneri ), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis or Pichia methanolica; Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus or
  • the isolated fungal host cell is glycoengineered.
  • such a cell has been genetically engineered to produce glycoproteins where the N- or O-linked glycosylation are modified from their native form, e.g., either through inactivation or deletion of genes involved in N-glycosylation such as OCH1, ALG3, PNO1, and/or BMT1, BMT2, BMT3, BMT4, or genes involved in O-glycosylation such as PMT1, PMT2 and/or PMT4 or though heterologous expression of glycosyltransferases such as GnTI, GnTII, GalT, and/or SialT, or glycosidases such as MNSI and/or MNSII.
  • a glycoengineered isolated fungal host cell comprises any one or more of the following characteristics:
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., Alg3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., Alg3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., Alg3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., Alg3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., Alg3
  • an endogenous dolichol-P-Man dependent alpha-1,3-mannosyltransferase e.g., Alg3
  • OCH1 is mutated, disrupted, truncated or partially or fully deleted; (x) comprising a polynucleotide encoding galactosyltransferase; (xi) comprising a polynucleotide encoding nucleotide sugar transporter; (xii) comprising a polynucleotide encoding sialyltransferase; (xiii) comprising a polynucleotide encoding acetylglucosaminyl transferase and/or (xiv) wherein one or more endogenous proteases (e.g., PEP4 and PRB1) are mutated, disrupted, truncated or partially or fully deleted.
  • endogenous proteases e.g., PEP4 and PRB1
  • Mutation of CRZ1 in glycoengineered isolated fungal host cells has been shown, herein, to reverse temperature sensitivity, to enhance cell robustness during fermentation, and to reverse poor production, i.e., to increase production, of heterologous polypeptides, such as immunoglobulins, at least in part, in such cells.
  • Such glycoengineered isolated fungal host cells are part of the present invention.
  • Methods for reversing temperature sensitivity and/or enhancing cell robustness during fermentation of a glycoengineered isolated fungal host cell by mutating CRZ1 or otherwise decreasing expression of CRZ1 polypeptide are also part of the present invention.
  • N-glycan and “glycoform” are used interchangeably and refer to an N-linked oligosaccharide, e.g., one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide.
  • N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.
  • Predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl-neuraminic acid (NANA)).
  • GalNAc N-acetylgalactosamine
  • GlcNAc N-acetylglucosamine
  • sialic acid e.g., N-acetyl-neuraminic acid (NANA)
  • N-glycans have a common pentasaccharide core of Man 3 GlcNAc 2 (“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to N-acetyl; GlcNAc refers to N-acetylglucosamine).
  • Man refers to mannose; “Glc” refers to glucose; and “NAc” refers to N-acetyl; GlcNAc refers to N-acetylglucosamine).
  • N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man 3 GlcNAc 2 (“Man 3 ”) core structure which is also referred to as the “trimannose core”, the “pentasaccharide core” or the “paucimannose core”.
  • N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid).
  • a “high mannose” type N-glycan has five or more mannose residues.
  • a “complex” type N-glycan typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a “trimannose” core.
  • Complex N-glycans may also have galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residues that are optionally modified with sialic acid or derivatives (e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl).
  • Gal galactose
  • GalNAc N-acetylgalactosamine residues
  • sialic acid or derivatives e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl
  • Complex N-glycans may also have intrachain substitutions comprising “bisecting” GlcNAc and core fucose (“Fuc”).
  • Complex N-glycans may also have multiple antennae on the “trimannose core,” often referred to as “multiple antennary glycans.”
  • a “hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core.
  • the various N-glycans are also referred to as “glycoforms.”
  • PNGase or “glycanase” refer to peptide N-glycosidase F (EC 3.2.2.18).
  • an isolated crz1 mutant fungal host cell such as a Pichia cell (e.g., Pichia pastoris ) is genetically engineered to include a nucleic acid that encodes an ⁇ -1,2-mannosidase that has a signal peptide that directs it for secretion.
  • the crz1 mutant host cell is engineered to express an exogenous ⁇ -1,2-mannosidase enzyme having an optimal pH between 5.1 and 8.0, preferably between 5.9 and 7.5.
  • the exogenous enzyme is targeted to the endoplasmic reticulum or Golgi apparatus of the host cell, where it trims N-glycans such as Man 8 GlcNAc 2 to yield Man 5 GlcNAc 2 . See U.S. Pat. No. 7,029,872.
  • the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , ⁇ -1,2-mannosidase + host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • the invention also encompasses a method for producing a heterologous recombinant glycoprotein comprising an N-glycan structure that comprises a Man 5 GlcNAc 2 glycoform in a crz1 mutant fungal host cell that does not display alpha-1,6 mannosyltransferase (e.g.
  • the method comprising the step of introducing into the crz1 mutant och1 ⁇ fungal host cell, a polynucleotide encoding the heterologous recombinant glycoprotein, and a polynucleotide encoding an alpha-1,2 mannosidase enzyme selected to have optimal activity in the ER or Golgi of said host cell, the enzyme comprising: (a) an alpha-1,2 mannosidase catalytic domain having optimal activity in said ER or Golgi at a pH between 5.1 and 8.0; fused to (b) a cellular targeting signal peptide not normally associated with the catalytic domain selected to target the mannosidase enzyme to the ER or Golgi apparatus of the host cell; and culturing the fungal host cell under conditions favorable to expression of the heterologous recombinant glycoprotein, whereby, upon expression and passage of the heterologous recombinant glycoprotein through
  • Isolated crz1 mutant fungal host cells of the present invention such as Pichia host cells (e.g., Pichia pastoris ) are, in an embodiment of the invention, genetically engineered to eliminate glycoproteins having alpha-mannosidase-resistant N-glycans by deleting or disrupting one or more of the ⁇ -mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, and/or BMT4) (See, U.S. Pat. No. 7,465,577) or abrogating translation of RNAs encoding one or more of the beta-mannosyltransferases using interfering RNA, antisense RNA, or the like.
  • ⁇ -mannosyltransferase genes e.g., BMT1, BMT2, BMT3, and/or BMT4
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , ⁇ -mannosyltransferase ⁇ (e.g., bmt1 ⁇ , bmt2 ⁇ , bmt3 ⁇ , and/or bmt4 ⁇ ) host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • ⁇ -mannosyltransferase ⁇ e.g., bmt1 ⁇ , bmt2 ⁇ , bmt3 ⁇ , and/or bmt4 ⁇
  • Isolated crz1 mutant fungal host cells e.g., Pichia , e.g., Pichia pastoris
  • Isolated crz1 mutant fungal host cells also include those that are genetically engineered to eliminate glycoproteins having phosphomannose residues, e.g., by deleting or disrupting one or both of the phosphomannosyl transferase genes PNO1 and MNN4B (See for example, U.S. Pat. Nos. 7,198,921 and 7,259,007), which can include deleting or disrupting one or more of the phosphomannosyltransferases or abrogating translation of RNAs encoding one or more of the phosphomannosyltransferases using interfering RNA, antisense RNA, or the like.
  • such fungal host cells produce glycoproteins that have predominantly an N-glycan selected from the group consisting of complex N-glycans, hybrid N-glycans, and high mannose N-glycans wherein complex N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 3 GlcNAc 2 , GlcNAC (1-4) Man 3 GlcNAc 2 , NANA (1-4) GlcNAc (1-4) Man 3 GlcNAc 2 , and NANA (1-4) Gal (1-4) Man 3 GlcNAc 2 ; hybrid N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 5 GlcNAc 2 , GlcNAcMan 5 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 , and NANAGalGlcNAcMan 5 GlcNAc 2 ; and high mannose N-glycans are, in an embodiment of the group consisting of complex N
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , phosphomannosyl transferase ⁇ (e.g., pno1 ⁇ and/or mnn4b ⁇ ) host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • phosphomannosyl transferase ⁇ e.g., pno1 ⁇ and/or mnn4b ⁇
  • Isolated crz1 mutant fungal host cells such as Pichia host cells (e.g., Pichia pastoris ) of the present invention include those that are genetically engineered to include a nucleic acid that encodes the Leishmania sp. single-subunit oligosaccharyltransferase STT3A protein, STT3B protein, STT3C protein, STT3D protein, or combinations thereof such as those described in WO2011/06389.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , ( Leishmania STT3A + , Leishmania STT3B + , Leishmania STT3C + , and/or Leishmania STT3D + ) host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • Isolated crz1 mutant fungal host cells e.g., Pichia pastoris
  • Isolated crz1 mutant fungal host cells also include those that are genetically engineered to eliminate nucleic acids encoding dolichol-P-Man dependent alpha(1-3) mannosyltransferase, e.g., ALG3, such as described in U.S. Patent Publication No. US2005/0170452.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , alg3 ⁇ host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • Isolated crz1 mutant fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris ) expressing a polypeptide having an endomannosidase activity (e.g., human (e.g., human liver), rat or mouse endomanosidase) that is targeted to a vesicular compartment within the host cell are part of the present invention.
  • Pichia cells e.g., Pichia pastoris
  • a polypeptide having an endomannosidase activity e.g., human (e.g., human liver), rat or mouse endomanosidase
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , endomannosidase + host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • Isolated crz1 mutant fungal host cells such as Pichia cells (e.g., Pichia pastoris ) of the present invention are, in an embodiment of the invention, engineered for producing a recombinant sialylated glycoprotein in the host cell, e.g., wherein the host cell is selected or engineered to produce recombinant glycoproteins comprising a glycoform selected from the group consisting of Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2 , e.g., by a method comprising: (a) transforming, into the crz1 mutant fungal host cell, one or more polynucleotides encoding a bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, an N-acetylneuraminate-9-phosphate synthase, and a CMP-sialic acid synthase; (b) transforming into the host cell
  • a recombinant sialylated glycoprotein comprising a glycoform selected from the group consisting of NANA (1-4) Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2 glycoform is produced.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a czr1 mutant , bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase + , N-acetylneuraminate-9-phosphate synthase + , CMP-Sialic acid synthase + , CMP-sialic acid transporter + , 2,6-sialyltransferase + fungal host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • isolated czr1 mutant fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris ), are, in an embodiment of the invention, engineered for generating galactosylated proteins, e.g., having a terminal galactose residue and essentially lacking fucose and sialic acid residues on the glycoprotein.
  • the isolated czr1 mutant fungal host cell comprises an isolated nucleic acid molecule encoding 3-galactosyltransferase activity and at least a polynucleotide encoding UDP-galactose transport activity, UDP-galactose C4 epimerase activity, galactokinase activity or galactose-1-phosphate uridyl transferase, e.g., wherein the host cell is genetically engineered to produce N-linked oligosaccharides having terminal GlcNAc residues and comprising a polynucleotide encoding a fusion protein that in the host cell transfers a galactose residue from UDP-galactose onto a terminal GlcNAc residue of an N-linked oligosaccharide branch of an N-glycan of a glycoprotein, wherein the N-linked oligosaccharide branch is selected from the group consisting of Glc
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • an isolated czr1 mutant fungal host cell of the present invention such as Pichia cells (e.g., Pichia pastoris ) lacks functional OCH1 protein, e.g., wherein endogenous OCH1 is mutated.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , och1 ⁇ host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • Isolated crz1 mutant fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris ) expressing a galactosyltransferase e.g., an alpha 1,3-galactosyltransferase or a beta 1,4-galactosyltransferase are part of the present invention.
  • Pichia cells e.g., Pichia pastoris
  • a galactosyltransferase e.g., an alpha 1,3-galactosyltransferase or a beta 1,4-galactosyltransferase are part of the present invention.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , galactosyltransferase + host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • Isolated crz1 mutant fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris ) expressing a nucleotide sugar transporter are part of the present invention.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , nucleotide sugar transporter + host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • Isolated crz1 mutant fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris ) expressing a sialyltransferase are part of the present invention.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , sialyltransferase + host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • Isolated crz1 mutant fungal host cells of the present invention such as Pichia cells (e.g., Pichia pastoris ) expressing an acetylglucosaminyl transferase, e.g., GNT1 or GNT2 or GNT4 are part of the present invention.
  • Pichia cells e.g., Pichia pastoris
  • an acetylglucosaminyl transferase e.g., GNT1 or GNT2 or GNT4 are part of the present invention.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant , acetylglucosaminyl transferase + host cell and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • the term “essentially free or as it relates to lack of” a particular sugar residue, such as fucose, or galactose or the like, on a glycoprotein is used to indicate that the glycoprotein composition is substantially devoid of N-glycans which contain such residues.
  • essentially free means that the amount of N-glycan structures containing such sugar residues does not exceed 10%, and preferably is below 5%, more preferably below 1%, most preferably below 0.5%, wherein the percentages are by weight or by mole percent.
  • glycoprotein composition “lacks” or “is lacking” a particular sugar residue, such as fucose or galactose, when no detectable amount of such sugar residue is present on the N-glycan structures.
  • glycoprotein compositions produced by host cells of the invention will “lack fucose,” because the cells do not have the enzymes needed to produce fucosylated N-glycan structures.
  • a composition may be “essentially free of fucose” even if the composition at one time contained fucosylated N-glycan structures or contains limited, but detectable amounts of fucosylated N-glycan structures as described above.
  • the scope of the present invention encompasses a diploid isolated fungal host cell wherein only one endogenous chromosomal CRZ1 gene has been mutated, disrupted, truncated or partially or fully deleted and the other endogenous chromosomal CRZ1 gene has not been mutated, disrupted, truncated or partially or fully deleted and encodes a functional CRZ1 polypeptide.
  • Homogeneous diploids lacking functional CRZ1 polypeptide e.g., because both endogenous chromosomal copies of the CRZ1 gene have been mutated, disrupted, truncated or partially or fully deleted are also part of the present invention.
  • the scope of the present invention includes methods for producing one or more heterologous polypeptides comprising (i) introducing a polynucleotide encoding the heterologous polypeptide(s) into such a crz1 mutant host cell (e.g., as discussed herein) and (ii) culturing the host cell under conditions favorable to expression of the heterologous polypeptide(s) in the cell, for example, for as long as the cells are viable, and, optionally, (iii) isolating the heterologous polypeptide(s) from the host cell.
  • Methods for expressing heterologous polypeptides in fungal host cells is generally known and conventional in the art.
  • the present invention encompasses any isolated fungal host cell discussed herein suspended in a liquid culture medium. Any lysate of an isolated fungal host cell discussed herein is also within the scope of the present invention.
  • the culture conditions used for a fungal host cell expression system can be varied depending on the particular conditions at hand.
  • fungal host cells can be grown in liquid culture medium in shaken-flasks or in fermentors (e.g., 1 L, 2 L, 5 L, 10 L, 20 L, 30 L, 50 L, 100 L, 200 L, 500 L, 1000 L, 10,000 L volume).
  • Various growth mediums may be used to culture fungal host cells.
  • the medium is at a pH of between pH 3 and 7 (e.g., 3, 4, 5, 6 or 7); in an embodiment of the invention, pH is increased with a base such as ammonium hydroxide.
  • the temperature is maintained at about 24° C. or 26° C.
  • the growth medium contains yeast nitrogen base (e.g., with ammonium sulfate; with or without essential amino acids), peptone and/or yeast extract.
  • yeast nitrogen base e.g., with ammonium sulfate; with or without essential amino acids
  • peptone e.g., peptone
  • yeast extract e.g., peptone, peptone and/or yeast extract.
  • Various supplements may be added to an growth medium such as biotin, dextrose, methanol, glycerol, casamino acids, L-arginine-hydrochloride, ammonium ions (e.g., in the form of ammonium phosphates).
  • the growth medium is minimal medium containing yeast nitrogen base, water, a carbon source such as dextrose, methanol or glycerol, biotin and histidine.
  • the cell culture comprises trace minerals/nutrients such as copper, iodine, manganese, molybdenum, boron, cobalt, zinc, iron, biotin and/or sulfur, e.g., CuSO 4 , NaI, MnSO 4 , Na 2 MoO 4 , H 3 BO 3 , CoCl 2 , ZnCl 2 , FeSO 4 , biotin and/or H 2 SO 4 .
  • the cell culture comprises an anti-foaming agent (e.g., silicone).
  • the present invention encompasses methods for making a heterologous polypeptide (e.g., an immunoglobulin chain or an antibody or antigen-binding fragment thereof) comprising introducing, into an isolated fungal crz1 mutant host cell (e.g., Pichia , such as Pichia pastoris ) a heterologous polynucleotide encoding said polypeptide, e.g., that is operably linked to a promoter, e.g., a methanol-inducible promoter and culturing the host cells,
  • a promoter e.g., a methanol-inducible promoter
  • a batch phase e.g., a glycerol batch phase
  • a non-fermentable carbon source such as glycerol
  • additional non-fermentable carbon source e.g., glycerol
  • methanol fed-batch phase wherein the cells are grown in the presence of methanol and, optionally, additional glycerol.
  • methanol concentration is set to about 2 grams methanol/liter to about 5 grams methanol/liter (e.g., 2, 2.5, 3, 3.5, 4, 4.5 or 5).
  • an initial seed culture is grown to a high density (e.g., OD 600 of about 2 or higher) and the cells grown in the seed culture are used to inoculate the initial batch phase culture medium.
  • a high density e.g., OD 600 of about 2 or higher
  • the host cells are grown in a transitional phase wherein cells are grown in the presence of about 2 ml methanol per liter of culture.
  • the cells can be grown in the transitional phase until the methanol concentration reaches about zero.
  • Heterologous polypeptides that are isolated from a fungal host cell are, in an embodiment of the invention, purified. If the heterologous polypeptide is secreted from the fungal host cell into the liquid growth medium, the polypeptide can be purified by a process including removal of the fungal host cells from the growth medium. Removal of the cells from the medium may be performed using centrifugation, discarding the cells and retention of the liquid medium supernatant. If the heterologous polypeptide is not secreted, the liquid medium can be discarded after separation from the fungal host cells which are retained. Thereafter, the fungal host cells may be lysed to produce a crude cell lysate from which the heterologous polypeptide may be further purified.
  • Heterologous polypeptide purification is, in an embodiment of the invention, performed by chromatography, e.g., column chromatography.
  • Chromatographic purification can include the use of ion exchange, e.g., anion exchange and/or cation exchange, protein-A chromatography, size exclusion chromatography and/or hydrophobic interaction chromatography. Purification can also include viral inactivation of the composition comprising the polypeptide, precipitation and/or lyophilization.
  • UV mutagenesis was performed as previously described (Barnard et al. 2010; Jiang et al. 2011; Potgieter et al. 2009; Winston F 2008). Except otherwise specified, all 1 L Bioreactor fermentation runs were scheduled to end after 100-120 hours of MeOH induction. However, a fermentation run was terminated prematurely if excess cell lysis was observed. Cell lysis was determined either by microscopic examination, or by measuring the amount of nuclear DNA released into the supernatant (Barnard, 2010). Excess cell lysis was defined by either greater than 90% cells lysed by microscopic examination, or greater than 30 microgram/ml DNA concentration in the supernatant determined by Picogreen assay.
  • YGLY29010 Five of the temperature-resistant mutants (YGLY29010, YGLY29030, YGLY29031, YGLY29042, and YGLY29012) were derived from YGLY27890 ( FIG. 2 ), which expresses a human Fc fragment.
  • YGLY27890 FIG. 2
  • YGLY29010 contained a single mutation within a gene, Pp02g02120, which showed a high-level of sequence homology to the CRZ1 gene of Saccharomyces cerevisiae .
  • distinct mutations in the same PpCRZ1 gene were also detected in YGLY28997, YGLY28998, YGLY17159, YGLY28999, YGLY29030, and YGLY29012.
  • ScCRZ1 is a calcineurin-responsive zinc finger transcription factor involved in stress responses.
  • the zinc finger domains are very well conserved between PpCRZ1 and ScCRZ1, and are both located at the c-terminal end (see sequence alignment above).
  • the four non-sense, stop-codon mutations found from the temperature-resistant mutants were located upstream of the zinc-finger, thus all giving rise to truncated PpCRZ1 fragments without the zinc-finger domain.
  • the remaining 5 mutants they contain mis-sense, amino-acid substitution mutations, all of which located within the zinc-finger domain, with 4 of them clustered within 25 nucleotides.
  • Pichia pastoris mutant CRZ1 isolated from yGLY28997 (encoding the mutation: L33 ⁇ STOP, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTT
  • Pichia pastoris mutant CRZ1 isolated from yGLY29012 (encoding the mutation: Q214 ⁇ STOP, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGGGTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTCATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTTTTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATCGGAGCTG TCAACTCATTCCCATATTACCCCTCCA
  • Pichia pastoris mutant CRZ1 isolated from yGLY29010 (encoding the mutation: L294 ⁇ STOP, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGGGTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTCATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTTTTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATCGGAGCTG TCAACTCATTCCCATATTACCCCTC
  • Pichia pastoris mutant CRZ1 isolated from yGLY29042 (encoding the mutation: S298 ⁇ STOP, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGGGTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTCATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTTTTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATCGGAGCTG TCAACTCATTCCCATATTACCCCTC
  • Pichia pastoris mutant CRZ1 isolated from yGLY28998 (encoding the mutation: E403 ⁇ G, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGGGTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTCATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTTTTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATCGGAGCTG TCAACTCATTCCCATATTACCCCTC
  • Pichia pastoris mutant CRZ1 isolated from yGLY17159 (encoding the mutation: F406 ⁇ S, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGGGTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTCATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTTTTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATCGGAGCTG TCAACTCATTCCCATATTACCCCTCCA
  • Pichia pastoris mutant CRZ1 isolated from yGLY28999 (encoding the mutation: F406 ⁇ L, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGGGTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTCATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTTTTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATCGGAGCTG TCAACTCATTCCCATATTACCCCTCCA
  • Pichia pastoris mutant CRZ1 isolated from yGLY29030 (encoding the mutation: C411 ⁇ F, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGGGTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTCATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTTTTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATCGGAGCTG TCAACTCATTCCCATATTACCCCTCCA
  • Pichia pastoris mutant CRZ1 isolated from yGLY29031 (encoding the mutation: K469 ⁇ N, mutated nucleotide is in bold font)
  • ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGGGTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTCATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTTTTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATCGGAGCTG TCAACTCATTCCCATATTACCCCTCCA
  • CRZ1 ORF was either completely deleted, or the endogenous CRZ1 gene was replaced with the truncated versions shown in FIG. 8 , in YGLY21203, which is a non-mutagenized ura5 auxtroph Pichia strain derived from YGLY17108 by 5-FOA counter-selection.
  • Plasmid pGLY12829 ( FIG. 6 ) was constructed by cloning a 1.5 kb genomic DNA fragment immediately upstream of the CRZ1 ORF in front of the ALG3 terminator, followed by the lacZ-URA5-lacZ URAblaster, and then connected to a 2 kb genomic DNA fragment containing the last 234 by of the CRZ1 ORF plus 1.7 kb of the downstream region. After SfiI digestion, this CRZ1-upstream-URAblaster-CRZ1-downstream DNA fragment was transformed into a non-mutagenized host strain (e.g., YGLY17108).
  • a non-mutagenized host strain e.g., YGLY17108
  • this URAblaster-cassette replaced the endogenous CRZ1 gene, deleting 85% of CRZ1's coding region, thus generating a complete CRZ1 knock-out mutant.
  • genomic DNA polymerase chain reaction (PCR) assays were conducted using the following oligonucleotides as PCR primers: GTATGCGATATAGTGTGGA (SEQ ID NO: 4, 1545 by upstream of CRZ1 start) and TGGGGAGAAGGTACCGAAG (SEQ ID NO: 5, within the ALG3 terminator) to confirm the 5′ junction of the gene-replacement; CACTACGCGTACTGTGAGCC (SEQ ID NO: 6, within the lacZ) and AGGATATCAAACCCGACCAG (SEQ ID NO: 7, 2045 by downstream of the CRZ1 stop codon) to confirm the 3′ junction of the gene-replacement; plus “GACACATGCGAAATGTCCTG” (SEQ ID NO:8, 120 by downstream of the CRZ1 stop codon) and “TTGAGTTGGCAGCTTCTCAG” (SEQ ID NO: 9, within the CRZ1 ORF, 1075 by after the start) to confirm the absence
  • PCR primers GTATGCGATATAGT
  • Plasmid pGLY12832 ( FIG. 7 ) was constructed by cloning a 1.5 kb DNA fragment (0.6 kb upstream region, the 1st 879 by of the CRZ1 ORF, plus 2 stop codons) in front of the ALG3 terminator sequence, followed by the lacZ-URA5-lacZ URAblaster, and then connected to a 2 kb genomic DNA fragment containing the last 234 by of the CRZ1 ORF plus 1.7 kb of the downstream region. Homologous recombination-mediated double-crossovers between the SfiI-fragment of pGLY12832 ( FIG. 7 ) and the chromosomal CRZ1 region replaced the endogenous CRZ1 ORF with a truncated version of CRZ1 with only the first 294 amino acid residues.
  • the truncation and deletion mutants were subjected to standard DasGip MeOH fed-batch fermentation runs (Hopkins et al., 2011) to determine whether they would also display increased fermentation robustness at 32° C.
  • the YGLY17108 control strain displayed heavy lysis and was not viable within 65 hours of MeOH induction at 32° C.
  • the strains harboring the complete deletion and truncations of the CRZ1 gene showed a remarkable increase in fermentation robustness and successfully completed more than 130 hours of MeOH induction.
  • the phenotypes exhibited by these directed gene-replacement strains closely resembled those displayed by the corresponding UV-induced mutants, illustrating that the mutations within the CRZ1 gene were responsible for the improved thermal tolerance and fermentation robustness observed from the UV-induced mutants.

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